CA2112799A1 - Method and assay system for neurotrophin activity - Google Patents

Abstract

The present invention describes a method of treating neurotrophin-expressing tumors comprising interrupting the autocrine survival loop by administering a pharmaceutically effective amount of a substance capable of interrupting the autocrine loop. As examples, antisense nucleic acids and K252a or its derivatives may be used in pharmaceutically acceptable compositions, to interrupt the autocrine loop of a tumor cell which depends on the neurotrophin it expresses for its survival. A model system for identifying other means of interrupting autocrines loop is also described.

Description

METHOD AND ASSAY SYSTEM FOR NEUROTROPH~N ACTIVITY

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 utili~e an autocrine loop mechanism for survival. More specifically, this invention relates to methods of interrupting the signal transduction pathway of the brain~derived neurotrophic factor ("BDNF"~ and causing cell death in BDNF-expressing cells.
Background of the InventiQn A. Neural Crest Derived Tumors The term neuroblastoma is used to designate the spestrum of neurogenic neoplasms derived from embryonic sympathetic neuroblasts, neural ~est oells and the mantle layer of the neural tube. Neuroblastoma tumors, the most commonly diagnosed neoplasms in infants under 1 year of age, occur with a frequency of 1 out of 10,000 live births. Neuroblastoma is considered the third most common malignancy of childhood, accounting for approximately 10~ of all pediatric neoplasms, and at least 1~% of all cancer-related deaths in children.
Biochemically, neuroblastomas often contain elements of both adrenergic and cholinergic neurotransmitter pathways. They exp~ess neilron-specific enolase and neuro~ilarnent proteins and exhibit substrate adherent cell gr~w~ in culture with neurite formation. Perhaps the most salient 2 5 feature of huIslan neuroblastoma is ampliffcation of ~e N-myc oncogene,whi~h has been identified ;n 19 of 22 neuroblastoma oell lines and in approximately 31% of tumor tissues from patients ~nth stage m and stage IV
neuroblastoma [Kohl et al, Science 226:1335 (19~4)]. The human neuroblastoma cell line SK-N-SH and its derivative SH-SY5Y are of thoracic WO 93/00909 PCl'/US92/03392 origin rather than neural crest, and do not express amplified N-mvc but do express N-ras.
Another class of tumors, small cell lung carcinoma (SCLC) share a common developmental lineage with neuroblastomas, both apparently being derived from neural progenitor oells of neural crest origin [Carney, Cancer Res., 45:2913 ~1985); Gazdar et al, Cancer Res., 45:2924 (1985)1-Small cell lung carcinoma represents approximately one third of all lung cancers. While several neuroblastomas are known to express amplified levels of N-myc, SCLC are generally charaeteri~ed by activated levels of either 1 0 N-m~ or L- myc._Small cell carcinomas represent approximately 20-25% of all pulmonary malignancies, yielding an incidence of approximately 25,000-30,000 cases per year in the United States, and are by far the most aggressive of pulmonary malignancies. SCLC is frequently (70%-90%) metastatic at preæn~ation. Small cell carcinomas are neuroectodermal in origin. These turnors possess properties of amine precursor uptake and decarboxylation (APUD), and other neuronal characteristics, such as the production of neuroactive peptides. Paraneoplastic syndromes, such as subacute sensory neuropathy, occur with greater frequency among vicdms of small cell than other lung cancers.
B. Neurotrophins and Their Receptors The development and function of the nervous system depends on proteins, termed neurotrophic factors, originally defined by their ability o support the survival of neuronal cells. In addition to promoting neuronal survival, these hctors can influence prolifera~ve and differentiative processes within the nervous system and may also have ac~ons outside the -nervous system. Much has been learned about the prototypical neuronal survival molecule, nerve growth factor (NG~), and more recently, its two structural relatives, brain-derived neurotrophic factor (~DNF) and neurotrophin-3 (NT-3). These three proteins along with the recently wo s3/oosos 2 1 1 2 7 9 9 PCI~USg~/03392 .. . .

identified neurotrophin-4 lHollbook et al, Neuron, 6:84~58 (1991)] comprise a family (designated "neurotrophins"), each member of which shares about 55% to 60% amino acid sequence identity with the others. Understanding the biological roles of these neurotrophic factors requires characterization of the receptor and signal transduction pathways they use to exert their effects.
The trk farnily of protein tyrosine kinase receptors has been identified as biologically functional receptors for the neurotrophins. BDNF is a neuronal survival molecule which is capable of binding to the trk cell-surface receptor known as trkB, which has in~insic protein kinase activity and is mainly expressed in the nervous sys~em lKaplan et al, Natur~, 350:158 (1991); Klein et al, Cell. 65:189 (1991); Hempstead et al, Natur~, 350:678 (1991)].
The trkB gene encodes protein that binds and rnediates functional responses to both BDNF and NT-3 lPCI International Application WO91/03568; Squinto et al, ~, 65:885 (1991); Glass et al, Cell. 66:405~13 (1991); Soppet et al, Ce!l, 1 5 65:$95 (1991)l-The recent introduction of the trk receptors into fibroblasts has enabled the creation of cells that display biological responses to neurotrophic factors that mimic the actions of traditional growth factors. Introduction of the trlcB
receptor into NIH 3T3 cells that req~re oertain grow~ factQrs ~FGF or PDGF) 20 ~ for proliferation and survival resulted in cells capable of surviving on exogenous, physiologically appropriate levels of BDNF or NT-3. Such mo~ls pro~nde a powerful assay system that can be used to detect and/~r measure neurotrophin acti~rity, to iden~fy agents that ~bi~ neurotrophin-like ae~iYity and to identify anta~orusts which block binding of ligands to 2 5 neuro~rophin receptors [Glass et al, ~3.
The utilization of reoeptor tyrosine kinases by the family of NGF-related neurotrophic factors suggests that the signal ~ansduc~on mechanisms u~lized by neurons fundarnentally resemble those utilized by other cell types in response to mitogenic hetors. This finding is consistent with recent data W O 93/00909 PC~r/US92/03392 that neurotrophic factors can act as mitogens in certain contexts [Cattaneo and McKay, Nature 347:762 (1991); Glass et al, s~pral. BDNF and NT-3 might serve as survival and/or mitogenic factors for neuronal precursors that have not yet achieved a post-mitotic phenotype.
Along wi~ the trk family of protein tyrosine kinases which have been identified as biologically functional receptors for the neurotrophins, the ERK
kinases represent a reoently identifiecl and molecularly cloned family of extracellular signal-regulated protein kinases [Boulton et al, ~, 65:663-75 (1991)]. ERK activity is rapidly activated in response to growth factor (i.e., insulin and NGF) s'dmulation of oells and represents a dass of intracellular Ser/Thr kinases ~at are themselves phosphorylated on tyrosine IBoulton et al, supral. Tyrosine phosphorylation of the ERKs has been shown in vitro to greatly enhanoe their kinase activity.
C. Antisense Technolo~y Understanding the molecular events that guide the regulation of cell proliferation, differentiation and survival should ultimately lead to the rational design of specifically targeted drugs for the treatment of various diseases including cancer, immunodeficiency, and neurodegeneration.
Recently, a powerful experimental tool has emerged that allows for a selective 2 0 and efficient means for inhibiting the expression of key gene products known to be involved in the control of eukaryo~c cell proliferation, differentiation' and survival. T~e technique involves ~e use of antisense DNA~r RN;A
molecules designed to provide translation arrest of ~ese key cellular regulatory proteins. By crea~i g a null mutant for a speafic gene product, this 2 5 tedtnology has allowed for the direct assessment of the specific function of a single gene product during important cellular transitional periods.
Currently, there are two primary approaches to achieving antisense-di~ected translational arrest of protein synthesis. The first method makes use of stably-transfected promoter-directed gene constructs designed to WO 93/00909 PCI'/US92/03392 constitutively synthesize compleme~t~ry~ ~i~se mRNA sequences. This technology generally results in hybridization arrest of protein translation for a given Bene product. It has recently been suggested that RNase may play a role in this mechanism by deaving RNA/DNA duplexes forrned between S antisense mRNA and DNA. Other possible mechanisms include impaired nuclear processing or the inability of the RNA/DNA duplex to be efficiently translated. An alternative approach to vector-driven antisense translational arrest involves the synthesis of short 5' or 3' synthetic antisense oligodeoxyribonucleotides (ar.tisense DNA~. Several reports have recently demonstrated the ability of antisense DNA to arrest the translation of selected RNAs when added to eukaryotic cells in vitro. (reviewed in Van der Krol et al, Biotechniques, 6:95~973 (1988).
Several approaches have been taken to use oligonudeotides that are complernentary to selected cellular or viral target nudeic acid sequences to modulate the expressi~)n of the target nucleic acid sequence. There have been several reports on the use of specific nucleic aad sequences to irlhibit viral replication ~see for example Goodchild et al, Proç. Natl. Acad. Sci. IISA, 85.5507-5511 (1988~; Wickstrom et al, Proc. Natl. Acad. Sci. ~, 85:1028-1032 (1988); and Kawasaki, ~aÇ~- Aads Res., 13:4991 (1985)].
Several laboratories have àttempted to develop modified oligonucleotides that are relatively membrane permeable and nuclease resistant. One approach involYes the development of nonionic - -oligonu~leoffde analogs. Examples of such analogs include methylphosphonates ~Smith et al, oc. Natl. Acad. Sa. USA. 83:2787-2791 Z 5 (1986); Agris et al, Bi~hemistry 2~:6268 6D5 (1986~; Jayaraman et al, oc.
Natl. Acad. ~ . USA, 78:1537-1541 (19813]; phosphorothioates Agarwal et al, Pro~. ,Natl. Acad. ~. I.JSA, 83:414~4146 (1988); Matsukura et al, ~p~. ~.
Acad. Sci. USA, 84:7706-7710 (1987); ~arcus-Selcura et al, Nucl. Acids Res., wo 93/00909 Pc~/Us92/03392 2tl~799 15:5749 5763 (1987)]; and phosphorarr~idates lAgarwal et al, Proc. Natl. Acad.
Sa. USA, 83:4143~146 (1988)].
It has been speculated that phosphorothioates may, in addition to binding to complementary target nucleic acid sequences, also direct the inhibition of primer binding to HIV reverse transcriptase [Matsukura et al, Proc. Natl. Acad. Sci. USA, 84:770~7710 (1987)~. Antitemplate inhibition has also been described using polynucleotides, including partially thiolated polycytidylic acid [reviewed in Stein and Cohen, Cancer Res..48:2659-2668 (1988)].
1 0 Another approach has involved conjugating the oligonucleotide to a molecule that will increase the effiaency of uptake of the oligonudeotide by the cell. Examples of such conjugates include cholesteryl-conjugated oligonucleotides l~etsinger et al, Proc. Natl. Acad. SC1. USA, 86:6553-6556 (1989)] and a poly-L-lysine conjugate [Lemaitre et al, Proc. Natl. Acad. Sci.
1 5 USA, 84:64~652 (1987)]. Another example includes an oligonucleotide joined t~rough a linking arm to a group that imparts amphophilic character to the final product in order to increase the efficiency of membrane transport lPCT Publication No. WO 88/09810, published December 15, 1988l.
Another approach that has been taken involves the use of reactive oligonucleotides, i.e. antisense oligonucleotides linked to reactive agents thatare able to modify the target nucleic acid. One such group of reacting agents are intercalating agents whid~ can bind to the duplex by internal lI~isertion between adjacent base pairs or bind to external nucleoside and phosphate elements respec~vely. Examples of intercalators that have been attac~ed to oligonudeotides and oligonucleotide analogs include acridine, anthridium, and photoactivatable psoralen [reviewed in Zon, Pharm. Res., 5:539-549 ~1988)1. Another such group of reactive groups coupled to oligomers include metal complexes such as EDTA-Fe(II), o-phenanthroline~u(I), or porphyrin-Fe(I~) lreviewed in Krol et al, BioTechnique_ 6:958-976 (1988)]. These WO 93/00909 P~/US92/03392 21127g9 compounds can generate hydroxyl radicals in the presence of molecular oxygen and a reducing agent. The resulting radicals can cleave the complementary strand following attack on the target nucleic aad backbone.
There have been many recent publications dealing with inhibition by antisense oligonudeotides. For example, proliferation of human malignant melanomas has been inhibited in vitro by antisense oligonucleotides directed against basic fibroblast growth factor [Becker et al, EMB 1-, 8:3685 (1989)]. The generation of RNA antisense to part of the human N-myc gene via an episomally replicating expression vector has been observed to blocl~
transdifferentiation of neuroectodermal tumor cell lines lWhitesell et al, Mol.
Cell. Biol.. 11t3):1360-1371 (1991)]. Oligonucleotides antisense to the gene forthe neuronal microtubule associated protein tau, when added to culture -~
media, inhibited neurite polarity in primary cerebellar neurons [Caceres and Kosik, Nature, 343:461 (1990)]. An antisense oligonucleotide to transfDrming growth hctor beta 3 inhibited epithelial-mesenchymal transformation of embryonic cardiac endothelial oells in explant cultures lPot~s et al,Proc. Natl.Acad. Sci. !JSA, 88:151~1520 (1991)1 D. Autocrine Loops The involvement of the autocrine growth mechanism in neoplasia 2~) was first identified in 1980 tsporn and Todaro, N. Engl. I. Med., 308:878-80 (1980)]. Autocrine loops have been observed for ~arious growth factor molecules and tumor ~ell lines. Certain tumor cells are Icnown to~synthesize and respond to growth hctors ~at are required for normal cellular growth and division. Via autocrine signaling, the cells respond to substances they 2 ~ them3elve~ produce. Autocrine loops might serve to accelerate or amplify a cellular respor~se in twnor 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 the disruption of the autocrine loop. In other W093/00909 211.2799 PCI`/US92/03392 words, the mere ability of an oligonudeotide that is antisense to a particular factor to adversely effect cellular growth is indicative that the cell is synthesizing that factor and the factor is required for growth or survival of the cell. In such situations, the existence of cellular receptors for the factor or even the release of the growth factor into the environment may not be detectable.
Antisense oligonucleotides have been used to demonstrate that transforming growth factor-beta serves as an autocrine cell differentiation hctor responsible for the transformation of epithelial cells to mesenchymal 1 0 cells lPotts et al, Proc. Natl. Acad Sci. US~, 88:1516 (1991)~. Antisense approaches have been used to demonstrate that basic fibroblast growth factor appears to be required for the autocrin~stimulated proliferation of bDth human melanomas [Bed~er et al, EMB0 1-, 8:3685 (1989)] and transformed human astrocytes EMorrison et al, J. Biol. Chem., 266:728 (1991)~. Antisense 1~ oligonucleotides against growth hormone inhibit lymphocyte proliferation lWeigent et al, Endocrinology, 128:2053 (1991)].
E. Protein Kinas~ Inhibitors Specific protein phosphorylation inhibitors have been used for studying the effect of a number of kinases and their actions ~n the phosphorylation of key cellular proteins for ~e biological activity of nerve growth factor on its target cells.
The kinase inhibitor ~S2a, isolated from ~he culture broth-of Nocardiosis sp. and i~ derivatives, are described in U..S. Patent Nos.
5,402; 4,877,7;'6; and 4,923,986, which documents are incorporated herein by re~erence. K252a and staurosporine were iNtially characterized as potent protein kinase C (PKC) and cyclic nucleotid~dependent kinase inhibitors in vitro lKase et al, Biochem. Bioph~. Res. ~Q~ 142:436 440 (1987)], but are now known to have broader actions that include inhibition of tyrosin~
specific protein kinases ~Fujita-Yamaguchi and Kathuria, Biochem. Biophvs.

wo 93/00909 ~ 1 ~ 2 7 ~ !~ Pcr/uss2/o3392 Res. Comm. 157:955 962 (1988); O'Brian and Ward, L Natl. Canc. Instit. 82: 1734-1734 (1990)]. Nanomolar concentrations of K252a and its derivatives in vitro have been found to inhibit, in a somewhat selective fashion, protein kinase C, cyclic AMP-and cyclic G~P-dependent protein kinases, myosin light chain kinase, and calmodulin-dependent phosphodiesterase ~Kase et al, Biochem.
Biophys. Res. Commun., 142:43640 (1987); Nakanishi, I. E~iol. Chem., 263:6215-19 (1988); Kase et al, L Antibiotiç 39:1059~0 (1986)1. The mechanism of the selective kinase inhibition appears related to a competition for the adenosine 5' triphosphate (ATP)-binding site on the en7yme.
1 O Although K252a and staurosporine do not seem to diminish FGF or EGP responses in PC12 cells, they are able to block the earliest detectable signalling processes induced by NGF, induding NGF induced tyrosine phosphorylation. ~252a also has been shown to inhibit the NGF induced outgrowth of neurites from primary cultures of embryonic dorsal root ganglion ~xplants as well as to completely block the survival activity of NGF
on primary cultures of embryonic ~hick sympathetic neurons [Matsuda and Fukuda, Neurosci. Lett., 87~ 17 (1988); Borasio, Neurosci. Lett., 108:207-12 (1990)].
Another class of tyrosine protein kinase inhibitors, the thiazolidine-2 O dione class of inhibitors, has demonstrated specific epidermal growth factor (EG~:~ induced reoeptor autophosphorylation and have been shown to inhib;t EG~dependent cells. ~Geis~ler et al, "Thiazolidin~Diones," L BioL Chem., 265:2225~22261 (1990)3. These inhibitors are analogous to K252a in their specif;c mechanism of interruptin~ grow~h factor mediated cell changes.
2 5 A need e~asts in the art for methods and pha~naceutical compositions capable of ~tailing ~e growth of BDN~-expressing tumor cells in vivo, such as neuroblastoma, and inhibiting tumor progression. In particular, a need exists for means to interrupt the BDNF autocrine survival loop of BDNF-expressing tumor cells.

wo s3/oosos 2 1 1 2 7 9 9 Pcr/US92/03392 Summarv of the Invention The present invention is directed to a method of treating mammals bearing a tumor cell of a type characterized by expression of a BDNF. In particular, the present invention relates to the identification of an autocrine survival loop in BDNF-expressing tumor cells and means for interrupting the autocrine loop in order to cause cell death.
The present invention is further directed to recombinant cells that serve as a model system for cells, induding tumor cells, that are dependent on an autocrine loop for survival. Such recombinant cells provide a means for screening compounds for therapeutic efficacy in the treatment of tumors that utilize autocrine survival loops.
In one aspect of the invention, recombinant cells ~at express both ~ BDNF and the trkB receptor, and thus depend upon a BDNF autocrine loop for survival, are utilized to identify agents which interrupt the BDNF
aLtocrine loop and which can be used to treat tumor cells that similarly depend upon such autocrine loops for survival and/or proliferation.
In one aspect, the invention is direc~ed to nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding BDNF or a portion thereof. "Antisense" as used herein re~ers to a nucleic acid ~apable of 2 0 hybridi7ing to a portion of a BDNF RNA (preferably mRNA~ by virh~e of some sequence complementari~y.
The antisense nucleic acids of the invention which are us~d to-interrupt a BDNF autocrine sur~nval loop may be oligonudeotid~s that ~re doubl~stranded or single-stranded, R~JA or DNA or a modification or Z 5 deAvative ~ereof, which can be directly administered to a cell, or which can ~e produced intracellularly by transcription of exogenous, introduced sequences.

wo g3/00909 PCr/uS92/03392 ~112799 In another aspect, this invention is directed to the use of staurosporine, K252a or its derivatives, or other protein kinase inhibitors to interrupt a BDNF autocrine survival loop.
The BDNF antisense nudeic acids and the K252a or its derivatives provided by the instant invention can be used for the treatment of tumors, the cells of which tumor type can be demonstrated to express BDNF.
In one embodiment, the invention is directed to methods for inhi~iting the expression of a BDNF nucleic acid sequence in a eucaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense BDNF nucleic acid of the invention.
In another embodiment of this invenffon, staurosporine, K;~2a or its derivatives or other protein kinase inhibitors may be used to interrupt the BDNF autocrine loop at the level of the cell surface receptor by inhibiting phosphorylation of the BDNF receptor.
In another embodiment, the identification of cells expressing func~onal BDNP or other neurotrophin receptors can be carried out by observing the ability of a neurotrophin to "rescue" such cells from the cytotoxic effects of a BDNF antisense nucleic acid.
Another aspect of the invention provides for the diagnosis of human neuro~lastoma or small cell lung carcinoma by detec~ing BDNF expression in cells obtained from pa'dents.
The invention further provi~es phsrmaceutical compositi-ons comprising an effective amount of the BDNF antisense nucleic acids of the invention in a pharmaceutically acceptable carrier. Methods for treatment of various diseases and disorders comprising admirustering the pharmaceutical compositions of ~e invention are slso provided.
In another aspect, there is provided a pharmaceutical composition which comprises as its active ingredient, K252a, staurosporine, or a related compound in a pharmaceutically acceptable carrier. This composition may 2~12799 be used in the ~eatment of various diseases and disorders related to BD~F-expressing t~n~r cells.
A further aspect of this invention is a pharmaceutical composition which comprises as one active ingredient, K252a, staurosporine, or a related compound and as a second act~ve ingredient, the BDNF antisense nucleic acids of the invention. Alternatively, the staurosporine, K252a or other protein kinase inhibitor may be combined with any other conventional pharmaceutieal agent useful in the treatrnent or prevention of disorders associated with BDNF-expressing tumor cells.
Still a further aspect of this invention is a method for treating patients having neuroblastoma or small cell lung carcinoma by administering an effec~dve amount af the matenals and compositions described above.
Yet another aspect of this invention is a method of stimulating neurite outgrowth by administering particularly low doses of a protein kinase inhibitor such as K252a.
O~er aspects and advantages of the present invention are described further in the following detailed description of preferred embodiments of the present invention.
DesiptiQn of the Figures 2 0 Figure 1. Inhibition of 8DNF synthesis by antisense oligonucleotides in a wheat germ lysate in vitro translation system. BDNF mRNA was synthesized in vitro using the plasnud expression construct pC8h-~, [see PCT
Publica~on No. W0 91/03568 published March 21, 1991] whi :h contains the 17 bacterial promoter for effi~ent in vitro ~ranscription by the 17 RNA
2 5 polymerase~ as per the manufacturer's instructions (Promeg~, Madison, WI).
BDNF mRNA was purified and then placed into a wheat genn lysate ~n vitro translation system (Promega) in the absence or presence of BDNF
oligonucleotides. The synthesis of BDNF protein was followed by using 35S-Wo 93/00909 PCr/US92~03392 ~112799 methione in the reaction. Control oligonucleotide refers to the use of a random 18-mer unrelated to the sequence of human BDNF.
Figure 2. Effects of 3'-AS-BDNF upon cell viability in culture. The percentage of cell viability (y axis) is shown for different concentrations of 3'-AS-BDNF (x axis, micromolar) added to the cultured neuroblastoma cells. A:
L;A-N-5 oells; B: LA-N-I cells; C: SK-ES cells; D: SH-SY5Y cells.
Figure 3. Effect of the c~addition of various neurotrophins with 3'-AS-BDNF upon neuroblastoma cell lines. The indicated neuroblastoma cell lines were simultaneously incubated with 5011M 3'-AS-BDNF and either no neurotrophin (open squares with center dot), BDNF (closed diamonds), NT-3 (open squares), or NGF (open diamond~. Y axis: peroentage cell viability; x 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~04 oells.
Figure 4. Northern blot analysis of total oellular RNA (10 llg per lane) derived from small ceil lung carcinoma cell lines or adult rat brain (lane 1).
The northern blot was hybridized to a human BDNF probe. Small cell lung carcinoma oell lines are as follows: H82 (lane 2), H209 (lane 3), H345 (lane 4),H378 (lane 5), H510 (lane 6), and N417 (lane 7).
~igure 5. Northern (RNA) blot comparisons of BDNF expression in both human and rodent tumor oells lines. Total RNA (10 ~lg) from each cell, line was frac~onated, transferred to membranes and hybridized with 32p BDNF as previously described lMaisonpier~e, et al. Science 247:1446 (1990).
Neurobl~stoma cell lines in panels B and C are represented by LAN5, SY5Y
and N18TG2.
2 5 Figure 6. Morphological effects of antisense and sense BDNF oligomers - on LA-N-5 neuroblastoma. Light photomicrographs of I,A-N-5 neuroblastoma cells either untreated (Panel A), treated with 10 uM 3'-AS-BDNF oligomer (Panel B), with 10 uM 3'-S-BDNF oligomer (Panel C) or with bo~ 10 ~M 3'-AS-BDNF and 100 ng/ml of human recombinant BDNF (Panel wo 93/oosos Pcr/US92/03392 ~ 7 9 !1 D): sirnilar results to those shown in Panel B were obtained with the other antisense BDNF oligomer, PS-AS-BDNF. LA^N-5 neuroblastoma cells were seeded into 6-well Costar plates at a density of 3 x 105 cells per well in RPMI
1640 (Irvine Scien~dfic) supplemented with 10% fetal bovine serum (FBS~, 1%
S penicillin, 1% streptomycin (P/S) and 2 mM glutanune. Eighteen hours after seeding, the cells were transferred into serum-free defined media [Zhan et al, Mol. Cell~ Biol. 6:3541 (1986)]and treated for 72 hours with the reagents described above. Engineered BDNF was produced in CHO cells and purified from CHO cell conditioned media to homogeneity as previously described 1 0 [Squinto et al, Cell 65:885 (1991)~.
F;gure 7. Dual-staining flow cytometric assay to quantitate both DNA
and protein content of LA-N-5 neuroblastoma cells. LA-N-5 neuroblastoma cells were seeded into 10 cm plates at a density of 1 x 106 cells per plate and were cultured as described in Figure 6. Cells were either untreated (Panel A) 1 !j or treated for 48 hours with 10 ~lM 3'-A~BDNF alone (Panel B3, 3'-A~BDNF
with 100 ng/rnl of BDNF (Panel C) or with high concentrations (100 ~lM) of control 3'-~BDNF (Panel D). Following these treatments, cells were harvested and resuspended in PB~versene (PBS with glucose and EDTA) to o~tain single cell suspensions. Cells were stained with both 1 ~lg/ml of DAPI
(for DNA content - left side of panels) and with 10 ~g/ml of sulforhodamine ~01 (for protein content.- right side of panels) and analyzed by flow cytometry as described [Del Bino, et al. Exp. Ce~l Res. 193:27(1991); Jakobisiak, et al. Proc.
Natl. Acad. Sa. U~A :3628 (1991)]. At least 5 x 103 cells were counted for each analysis. 'rhe line drawn through the pro~ein profiles highlights the 2 ~ decrease in fluorescence observed in Panel D (right side) relative to Panels A-C (right side3. The arrow in B indicates an apoptotic population of cells. Cell cyde phaæs are indicated. The peroentage of cells in S phase were as follows:
Panel A-25.5%; Panel B-16.2%; Panel C-30.1%; Panel D-24.9%.

WO 93/00909 PCI`/US92/03392 Figure 8. Dos~response killing curves for an~sense (AS) and sense (S) BDNF oligomers on human neuroblastoma and recombinant autocrine 3T3 fibroblasts. Human neuroblastoma cells (LA-N-5, panel A; SH-SY5Y, panel B) were cultured as described for Figure 6 while BDNF autocrine 3T3 cells (Panel C) were cultured in growth factor-deficient media as described lGlass et al, Cell 66:405 (1991); Zhan et al, Mol. Cell. Biol., ~1. All cells were plated at a density of 2 x 104 cells per well in a 24-well Costar plate. Various concentrations (0, 1, 5, 10, 50, 100, and 250 ~lM) of the 3'-A~iBDNF or control 3'-~BDNF oligomers were added to the cultures for 4 hours in the absence or presence of human recombinant BDNF (100 ng/ml) using serum-free EMEM
(Panels A and B) or growth-factor deficient media (Panel C) to allow for oligo nucleotide uptake into the cells. Insulin, transferrin, and selenium (lTS) were then immediately added to the cultures and cell viability was assessed 72 hours later by determining the concentration of glucose remaining in the culture media. Solid squares-3'AS; Open squares-3'AS and BDNF; Solid circles-3'S; Open circles-3'S and BDNF.
Figure 9. Identification of constitutively autophosphorylated trk receptors in neuroblastoma cell lines. Panel A, Anti-phosphotyrosine immunoblot of autophosphorylated trkB reoeptors that were specifically 2 0 immunoprecipitated from total protein lysates prepared from approxima~ely, 3x106 NIH3T3 cells expressing trkB 13T3(trkB)] and treated with BDNF or from 2-5x106 untreated neuroblastoma cells. Panel B, N18TG2 neuroblastoma cells were untreated or pretreated with 200 nM K~52a prior to the preparation of cell lysates and ~k-specific immunopreapitation. Panel C, Anti-2 5 phosphotyrosine inununoblot of total protein lysates prepared from ~IH3T3 cells (3T3), 3T3(trkB) oells treated with BDNF, or untreated 3T3(autocrine) cells. Position of trkB is noted with an arrow while molecular weight standards (kD) are indicated on the left side of the figure. Solid squares-3'AS;C)pen squares-3'AS and BDNF; Solid cirdes-3'S; Open circles-3'S and BDNF.

.. ....... . . ... .. .. ~.. . . - . . . ... ~ .. . ~ . . . .. .

WO 93/0090~ P~/US92~03392 211~79!1 Figure 10. Identification of constitutively autophosphorylated trk receptors in neuroblastoma cell lines. Panel A. Anti-phosphotyrosine immunoblot of autophosphorylated trkB receptors that were specifically immunoprecipitated from total protein lysates prepared from approx~mately 3 X 106 NIH3T3 oells expressing trkB and treated with BDNF or from 2-5 X 106 untreated neuroblastoma cells. Panel B, N18TG2 neuroblastoma cells were untreated or pretreated with 200 nM K^252a prior to the preparation of cell lysates and trk-specific immunoprecipitation. Panel C, Anti-phosphotyrosine immunoblot of total protein lysate prepared from NIH3T3 cells (3T3), NIH3T3(trkB) cells treated with BDNF, or untreated 3T3-autocrine cells.
Position of trkB is noted with an arrow while molecular weight standards (kD) are indicated on the left side of the figure.
Figure 11. Differential effect of K252a on neuroblastoma (Panel A) and 3T3 oell lines (Panel B). whose survival either depends on or is unrelated to a BDNF autocrine survival loop. Neuroblastoma cells (Panel A) and 3T3 cells (Panel B) were seeded into 24-well plates as described in Figure 7. After seeding, all cells were transferred to growth factor-deficient media. Parental 3T3 cells were maintained in 50 pM FGF. The cells were treated for 48 hours with various concentrations of K252a (ranging from 0 to 250 nM). Cell 2 0 viability for all cell lines was determined with the glucose utilization assay o,n duplicate samples. Panel A; Open squares-SH-SY5Y (without BDNF); Closed squares~ N-5 (with BDNF); Closed circles-N18TG2 (with BDNF). Panel B;
Open squares- 3I3 (with FG~); Closed squares-3T3 autocrine ~with BDNF~.
Figure 12. Differential effect of K252a on small cell luIIg carcinoma 2~ (NCI-H69), lung adenocarcinoma (Calu-3), 3T3~autocrine) and neuroblastoma ~J18TG2) cells. Cells were seeded into 24-well plates as described in Figure 7.
The cells were treated for 48 hours with 0, 50 and 100 nM K252a. Cell viability for all cell lines was determined with the glucose utilization assay on duplicate samples. Histograms (left to right) are indicated as follows: Solid WO 93/00909 PCr/US92/03392 histogr~rn; NCI-H69; Bold diagonally hatched histogram; N18TG~; dotted histogram; 3T3~autocrine); ~ine diagonally hatched histogsam; Calu-3.

Detailed Description of the Invention S The present invention provides methods and pharmaceutical compositions for therapeutically treating marnmals bearing tumor cells which express neurotrophins to inhibit or interfere with the growth of the tumor cells and their progeny. The compositions and methods of the present -invent;on involve administering to the affected mammal an effective amount of a substance which interferes with the tumor cells' autocrine survival loop.
More specifically, two examples of mechanisms which may be used to interfere with a BDNF-autocrine survival loop and thereby cause cell death in - the BDNF-expressing tumor cell, are provided.
One mechanism for practicing the present invention involves the use of nu~leic aads of at least six nudeotides that are antisense to 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 a BDNF RNA (preferably mRNA) by virtue of some sequence complementarty.
Another mechanism involves the use of the compound staurosporine, K252a or its derivatives, which are described in Murakata et al, U. S. Patent No. 4,923,986 ar.d European Pa~nt Applications Nos. 303,697, published ~ebruary 22, 1989, and No. 323,171, published July 5, 1989, or other protein Icinase inhibitors.
2~; In addition~ the invention provides a recombinant autocrine loop cell model that embodies many features of tumor cells (such as neuroblastomas and SCl.C's) that utilize autocrine survival loops. A cell utilizing an autocrine survival loop, as used herein~ refers to a cell which expresses a molecule that is necessary for its own survival.

WO 93/00909 Pcr~uss2/o3392 - 211~799 ~y~li~r~ ol A~ltocrine Loop A. Inhibition of BDNF ExE~ession The antisense nucleic acids of the invention interrupt the BDNF-autocrine loop on an intercellular level, by preventing the synthesis of BDNF
by the cell which depends on BDNF expression for survival. The mechanism of K252a and related compounds to interrupt the BDNF-autocrine loop occurs at the level of the BDNF receptor, by preventing the activation and phosphorylation of the trk B receptor. Another class of tyrosine protein kinase inhibitors, the thiazolidinedione class of inhibitors [Geissler et al, L
Biol. Chem., 265:22255-22261 (1990)~ may act in a similar manner to K252a.
Other protein kinase inhibitors, such as calphostin C, staurosporine, I~252b, KT5720, ~5823, and KT5926 (Kamiya Biomedical Company, Thousand Oaks, California) may also be used. Other mechanisms for interrupting the BDNF
autocrine loop are also encompassed by this invention.
The antisense nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced 2 0 sequences.
The BDNF antisense nucleic acids provided by the instant invention can be used for the ~eatment of tumors, the cells of which turnor type can be demonsi~rated (~ vitro or in yàyQ) to express the BDNF geIle~ Such demonstration can be by detection of BDNF RNA or of BDNP protein~
2~ According to the invention, BDNF antisense oligomers not only prevent g~owth of such tumors, but can also result in dea~ of tumor cells by an unusual mechanism involving programmed or J'apoptotic" death, which is characterized by loss of DNA prior to loss of cellular protein. ~Arends et al, An~. I P~thol.13 :593 ~1990)~.

wo 97/oosos 2 1 1 2 7 9 9 t I ,, PCI`/US92/03392 The invention further provides pharmaceutical compositions comprising an effective amount of the BDNF antisense nudeic acids of the invention in a pharmaceutically acceptable c~rrier. Methods for treatment of various diseases and disorders comprising administering the pharmaceutical compositions of ~e invention are also provided.
In another embodiment, the invention is directed to methods for inhibiting the expression of a BDNF nucleic acid sequence in a eucaryotic cell comprising providing the cell with an effective amount of a composition compAsing an antisense BDNF nucleic acid of the invention.
1 0 In another embodiment, the identification of cells expressing functional BDNF or other neurotTophin receptors can be carried out by observing the ability of a neurotrophin to "rescue" such cells from the cytotoxic effects of a BDNF antisense nudeic acid.
Another aspect of the invention provides for the diagnosis of human 1 5 neuroblastoma or small cell lung carcinoma by detecting BDNF expression in cells obtained from patients. Such detection can be carried out by detecting BDNF RNA or protein expression.
The antisense nucleic acids of the invention are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 nucleotides). The oligonucleotides can be DNA or RNA or chimeric mixture,s or derivatives or modified versions thereof, single-stranded or doubl~
stranded. The oligonudeotide cal~ be modified at the base moîety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport aoss the 2 5 oell membrane lsee e.g. Letsinger et al, Proc Natl. Acad. Sci. USA. 86:6553-6556 (1989); Lemaitre et al, Proc. Natl. Acad. Sci. ~, 84:648-652 (19B7); PCI
Publication No. WO88/09810, publi~hed December 15, 1988] or blood-brain barrier lsee e.g. PCT Publica~on No. WO 89/10134, published April 25, 19B8], hybridization-triggered deavage agents [see e~g. Krol et al, BioTechniques, wo s3/oosos 2 1 1 2 7 9 9 Pcr/uss2/o3392 6:958-976 (1988)] or intercalating agents [see e.g. Zon, Pharm. Res., 5:539-549 (1988)].
In a preferred aspect of the invention, a BDNF antisense oligonucleotide is provided, preferably of single-stranded DNA. In a most preferred aspect, such an oligonucleotide comprises a sequence antisense to the last 6 codons of human BDNF. The oligonucleotide may be modified at any position on its structure with substituents generally 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-fluorouracil, ~bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, ~(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, l-methylinosine, ~,2-dimethylguanine, 2methyladenine, 2-methylguarune, 3-methylcytosine, 5 methylcytosine, N6-adenine, 7-methylguanine, 5methylaminomethyluracil, 5-methoxyaminomethyl-2thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2methylthio-N6-isopentenyladenine, uracil-~oxyacetic acid (v), wybutoxosin~, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouradl, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-~o~cyacetic acid methylester" uracil-5-oxyacetic acid (v), 5-methyl-2 thiouracil, ~(3-amin~3-N-2-carboxypropyl) uracil, (acp3jw, and 2, 6-.. . .
alamlnopurme.
In another embodiment, the oligonucleotide comprises at leàst one 2 5 modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the oligonudeotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a 15 WO 93/00909 2 1 1 2 7 9 9 PCl /US92/03392 phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the oligonucleotide is an ~-anomeric oligonucleotide. An o~-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ~-units, the strands run parallel to each other [Gau~der et al, Nucl. Acids Res., 5:6625 6641 (1987)].
The oligonucleotide may be conjugated to another molecule, e.g., a peptide hybridization triggered cross-linking agent, transport agent hybAdization-triggered cleavage agent, etc.
Oligonucleo~des of the invention may be syn~esized by standard methods known in the art, e.g. by use of an automated DNA synthexizer (such as are commercially available from Biosearch, Applied Biosystems, 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 by use of controlled pore glass polymer supports [Sarin et al, Proc. Natl. Acad. ~ci. USA~ 85:744~7451 (1988)1 etc.
In a specific embodiment, the BDNF antisense oligonucleotide comprises catalytic RNA, or a ribozyme [see, e.g., PCT International Publication WO 90/~1364, published October 4, 1990; Sarver et al, Science 247:1222-1225 (1990)]. ln another embodiment, the oligonucleotide is a 2'-~
me~ylAbonucleotide lInoue et al, Nucl. Acids Res., 15:6131 6148 (198~)~, or a chime~ic RNA-DNA analogue lInoue et al, FEBS Lett., 215:327-330 ~1987)1.
In an alternative embodiment, the BDNF antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequenoe. For example, a vector can be introduced In vivo such that it is taken up by a cell, ~nthin which cell 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 nudeic acid. Such a W093/00909 2112799 Pcr/US92/03392 vector can remain episomal or ~ome chromosomally integrated, as lorlg as it can be ~anscribed to produce ~e desired antisense RNA. Such vectors can be constTucted by recombinant DNA technology methods standard in ~e art.
Vectors can be plasmid, viral, or o~ers knowsl ;n the art, used for repticatlon 5 and e~ression in mammalian cells. Expression of the sequenoe encoding the BDNF antiænse RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be induc~ble or ~onstitutive. Such promoters include but are not limited to: th~ SV40 early promot~ region lBernoist and Ch;lmbon, Nature, 290:304-310 (1981)l, the 10 promoter ~ontained in the 3' long tenninal repeat of Rous sarcoma virus IYamamoto et al, Cell, 22:787-~g7 (1980)~ e h~pes thymidine kinase promoter [Wagner et al, ~ Acat. ~ USA, ~s.i441-i44s (1981)], ~e regulatory sequenoes of ~e metallo~ion~ gene ~Brinster et a~, Naturç, 29~42 (198~]; etc The antisense nucleic acids of ~e ~nvention ~omprise complementa~y to least a portion of a RNA tr~ipt of a BDNF ~ene, preferably a hwnan 8DNF gene. However, absolute a~mple~entarily, although prefe~ed~ is not req~red. A sequenoe ~complementa~y to at least a portion of an RNA~
referred to herein, mean~ a ~equence ha~ing suff~dent ~omplementarily to be 20 able ~o hybn~e ~th the RNA, h~mdng a stable duplex (or triplex, in ~e case of double-stranded BD~ antisense nucleic aads). The abili~ to hybridiæ
will depend on bo~ the degree of complementarily and ~e len~ of ~ie an~se n-l~leic aa~ Generally, t~e langer ~he hybr~diz~ng nu~leic aad, ~e more base mismatches wi~ a BDNF RNA it may ~on~ain and still form a 25 stable duplex (or triplex, as ~e case may be). One sldll~d in ~e art can asoertain a tolerable degree of mismat~ by u~e of standard prooedures to determine the melting point of the hybridized complex.
B. ~nterruption of Receptor Phosphorvlation The compound known as K2~2a ;s commerci~lly available from Kamiya Biomedical Company in Thousand Oaks, California and is otherwise cies~ibed by the referenoes cited above.

wo 93/009o9 2 1 1 2 7 9 9 Pcr/lJS92/03392 The physiol~cally active substance K252a is a derivative of a substanoe K~S2 which was produced by culturing a microorgalusm of ~e genus Nocardiosis lMal~;uda et al, U. S. Patent No. 4,555,40~]. K252 is de~nedin Murakata et al, U. S. Patent No. 4,877,776 as a compound 5 represented by the fonnula: H
,~N >5 1 0 R'~ ~ 'C~
H~C~
yo X
wherein Rl and R2 are H or OH; X is l::OOH, COOR or CH2OH; Y is H, R or COR, and Z is OH, OR or SR, where R is a lower aLcy~ ~252 has ~een shown ~o inhi~it ~e grow~ of human uterine canoer ~eLa cells, h~lman breast canoer oeLls MCF7, human ~lon adenocar~oma oells COLQ320DM, human lun~ caranoma oells PC10 by means of protein kinase inhibitory aetivity.
Deriva~ves of K252 are shown in Muraka~a e~ al, U.~S. Patent No.

- 4,923,9~6 as compounds of the formula. R4 W1~N~!50 2S M~

wherein Wl, W~, R~ 3, R4, X and Y represent vario~ substituents.
Without being bound by theory or mechanism, our data indicates that staurosporine and its derivatives and K252a and its derivatives operate by interfering with the phosphorylation of the neurotrophin receptor. More 2.3 ~r ~ r ~~

wo g3/00909 2 1 1 2 7 ~ 9; Pcr/l~sg2/033g2 specifically, by interrupting the BDNF autocrine loop at the level of the cell surface receptor, the trk B tyrosine l<inase receptor is inactivated. Suppression of the phosphorylation of cellular proteins is believed to be due to the direct effect of K252a or staurosporine or their derivatives to specifically interfere w~th BDNF mediated cellular responses. Other protein kinase inhibitors, such as thiazolidine-diones, which inhibit EGF-induced receptor phosphorylation, may act similarly to interfere w~th BDNF-mediated cellular responses.

0 Therapeutiç Utility The materials of this invention may be used to treat tumors, of a type which has been shown to express BDNP. Such tumors include but are not limited to neuroblastoma, small cell lung carcinoma, and some neuroepithelial tumors. In cne embodiment, a single stranded DNA
antisense BDNF oligonucleotide is used in the treatment of neuroblastoma.
In another embodiment, staurosporine or ~252a is used in the treatment of neuroblastoma.
Other tumor types which express BDNF RNA can be identified by various methods known in the art. Such methods include but are not O limited to hybridization with a BDNF-specific nucleic acid, e.g. by Northern hybridization, dot blot hybridization, by observing ~e ability of RNA to be translated in vitro into BDNF, etc. In a preferred aspect, primary tumor tissue from a pa~ent can be assayed for BDN~ expression pnor to treatment.
Pharmaceutical compositions of the invention, comprising an effective ' 5 amount of a subst~nce which interferes with the BDNF autocrine survival loop in a pharmaceutically acceptable carrier, can be administered to a patient having a tumor which is of a type that expresses BDNF RNA.
The therapeutic and pharmaceutical compositions of the present invention for inhibiting the growth of BDNF-expressing tumor cells ~ wo 93/00909 2 1 ~ ~ 7 !~ 9 PCr/US92/0339~
'~ .
therefore comprise a therapeutically effective amount of a substance capable of interfering with a ~DNF-autocrine loop in admixture with a . pharmaceutically acceptable carrier. The ph~rmaceutical compositions having tumoriadal activity may be utilized in conventional type formulations such as, e.g., solutions, syrups, emulsions, injectables, tablets, capsules, Ol suppositories.

Suitable calTiers are well known to those of skill in the art of pharmacology [see, e.g., Remingtons Prac~ice of Pharmacy, 9th, 10th and 11th Ed.l Exemplary carriers include sterile saline, lactose, sucrose, calcilum phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, squalene and water. Additionally, the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone or with a wax. Optionally, suitable cherNcal stabilizers may be used to improve the stability of the pharmaceutical preparation. Suitable chemical stabili~ers are well known to ~ose of skill in the art and include, for example, citric aad and other agents to adjust pH, chelating or sequestering agents and antiox~ants.
The formulations of the pharmaceutical composition~containing K252a, staurosponne, or a deriva~ve thereof, or any other protein kinase inhibitor may conveniently be presented in wut dosage form and may be prepared by any of the conventional methods. Alternatively, the composition may be in a form adapted for slow release in ~Q, as is known - in ~he art. All methods include ~e step c)f lbringing into association the active ingredient with the carrier which m~y constitute one or more accessory ingredients.
The amount of the substance which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be deterrnined by standard clinical techniques. In one wo 93/oosos Pcr/US92/03392 embodiment of this invention, it would be desirable to determine the antisense cytotoxicity of the tumor type to be treated in vitro e.g. in the assay systems described in the example~ infra. and then in 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 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.
Further, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion 1 5 during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
The invention also provides for pharrnaceutical compositions comprising substances which interfere with a BDNF-autocrine loop adrninistered via liposomes, microparticles, or microcapsules. In various embodiments of the inYention, it may be useful to use such compositions to achieve sustained release of the substanoes. In a specific embodiment, it may be desirable to utilize liposomes targeted via antibodies to speafic identifiable tumor antigells (e.g., oell surhce antigens selective for neuroblastoma or 2 5 SCLC) lLeoneffl et al, Proe. Natl. Acad. i. USA. 87:2448-2451 (1990);
Renneisen et al, I. Biol. Çhem., 265:16337-16342 (1990)].
K252a or staurosporine or their derivatives, as well as other protein kinase inhibitors may also be employed in accordance with the methods and compositions of this invention, alone or in combination with other ,`1 wo 93/0~)909 PCI/US92/03392 ,~
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 an~-sense nucleic acids of this invention. Other agents, e.g., antimetabolites, alkylating agents, vinca allcaloids, antineoplastic antibiotics, platinum derivatives, substituted ureas,adrenocortico steroids, cytokines, interleukins, interferons or antibodies, may also be employed in conjunction with such kinase inhibitors to treat a variety of canoers characterized by BDNF-expressing cells and related diseases.
The dosage regimen involved in administering an effective amount 1 O ` of, for example, K252a in a method for treating the below-desibed conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of the tumor, time of administration and other clinical factors. The dosage of the compositions of the invention used to treat the specific disease condition described herein may be varied depending on the particular disease and the 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 age~t, such as cyclophosphamide, cytokines, interleukins, interferons or antibodies, as mentioned above. It is especially contemplated ~at the antisense oligonucleotides of this invention may be combined with pharmaceutical compositions containing protein kinase inhibitors. When these agents are combined in a pharma~eutical composition, it is anticipated that each active 2 5 ingredient will be present in the combined composition in the same concentration or slightly lower concentration than if this agent was administered alone.

, :

wo 93/oosos Pcr/us~2/o33g2 ` 21127~9`

The therapeutic mechanism of the compositions and methods of the psesent invention differs in principle from that of the large majority of drugs in use for treatment of tumors associated with BDNF-expression in use at the S present time. Alone or in combination with other known tumoricidal agents, substances which interfere with the BDNF-autocrine survival loop display highly specific activity so that patients do not suffer the many disadvantages of conventional canoer therapy.
BDNF-expressing tumors susceptible to treatment by the present method and compositions include, but are not limited to, neuroblastoma, small cell lung carcinoma, and some neuroepithelial tumors. Other tumor types which express BDNP can be identified by various methods known in - the art. According to the method of the present invention, where desired, primary tumor tissue from a patient can be assayed for BDNP expression prior to treatment.
In addition to treating the ma~malian disorders described hereinabove, the methods and compositions of this invention may be utili~ed for veterinary purposes in the treatment of BDNF-expressing tumors that aMict horses, swine, cattle and fowl, for example. These~disorders may be treated using quantities of the compound that may be used in treating the mammalian disorders described herein above.
.
Diagn~stic Utility Most human neuroblastoma cell lines express some level of human BDNF mRNA. BDNF mRNA expression appears to be unique to neuroblastoma with only a few exeeptions (such as small cell lung carcinoma and a few neuroepithelial tumor cells). These results suggest that BDNF
mRNA expression may serve as a useful marker clinically for human neuroblastoma, as well as SCLC and some neuroepithelial tumors. Given ` 21~ 279~3 that the best clinical marker for neuroblastoma to date is N-myc amplification and that N-myc is only amplified in approximately 30% of all lat~stage neuroblastoma (Stages m and IV), BDNF mRNA expression may be a more useful and broad ranging clinical marker for both early and late stage neuroblastoma.

Identification of Cells Expressing Functional BDNF or C)ther Neurotrophm Receptors Some neuroblastsma cell lines can be effectively rescued from the 1 0 cytotoxic effects of antisense BDNF oligonucleotides by the additiosl of either BDNF, NGP and NT-3 (see Section 6, infra). Thus, one may predict that some neuroblastomas express functional receptors for BDNF, NGF or NT-3 based on the ability of these individual ligands to rescue such a cell type from 5 antisense cytotoxicity. For example, our data (Example 1., section 1.13 suggest that LA-N-5 neuroblastoma cells express functional receptors for NGF, BDNF, and NT-3, while LA-N-1 cells express only functional BDNF receptors, and that CHP-134 and CHP~04 neuroblastoma cells express both NGF and BDNF
receptors but lack NT-3 receptors.

2 0 Genetic En~ineerin~ of a Model Cell S~stem That Mimics Autocrine-Loop Dependent TumQr Cells Oespite the identification of a BDNF-dependent autoaine surv~val loop in neuro~lastomas, the properties of such cells reflecting such a survival loop such as detectable levels of Ir~NA for ~kB, or detectable levels of a Z 5 constituitively phosphorylated trk receptor, were not readily detected.
Previous studies of autocrine loops involving conventional mitogenic factors have demonstrated that these loops can function in the absence of detectably secreted factor, with receptor activation occasionally occurring intracellularly.
[Zhan and Goldfarb, Mol. Cell. Biol. 6:3541 (1986)]. Furthermore, chronic wo s3/o~sos 2 1 1 ~ 7 9 9 Pc~r/uss2/o3392 J autocrine stimulation can result in the substantial down-regulation of receptor autophosphorylation as well as rapid turnover of the involved receptor, both of which can make it difficult or impossible to detect constitutively phosphorylated receptors. Similarly, continuous exposure of neuronal cells to NGF, while required for survival, eventually results in substantial down-regulation of the activated trkA receptor. lKaplan et al, Science 252:554 (1991)].
To overcome these problems and to provide a model cell utilizing a BDNF autocrine loop in which interruption of such a loop can be readily detected in vitro thus providing a useful system to screen for compounds with the ability to interrupt such an autocrine loop, a recombinant cell system was created. This system utili2ed a BDNF/trk~ mediated autocrine loop.
This system is based on a variant NIH 3T3 fibroblast cell line whose growth and survival in defined media normally requires either fibroblast growth factor ~FGF3 or platelet-derived growth hctor (PDGF) [Lee, and Dono~;hue, I-Cell B;ol. 113:361 (1~91)l; death of these fibroblasts due to factor depAvation is also apoptotic. lErnfors et al, Neuron ~:S11 (1990)]. When these oells are stably transfected with the trkB receptor, BDNF can substitute for FGF or PDGF. tGlass et al, Cell 66:405 (1991?]. C~transfection of these cells with both2 0 trkB and BDNP leads to a NIH3T3 cell lreferred to as 3T3(autocrine) or MBx]
which can survive in defined media without the addition of exogenous growth hctor (i.e., they become autocrine for BDNF). Strikingly, t~se autocrine NIH3T3 cells are in many ways similar to neuroblastomas dependent upon a BDNF autoaine suIvival loop. Por example, they display 2 5 a similar sequence-specific susceptibility to BDNF antisense oligomer, which can be overcome by exogenous BDNF. Furthe~nore, these cells do not secrete detectable levels of BDNF into the media, nor do they display detectable levels of a constitutively phosphorylated trkB receptor.

~ W O 93/00909 $ ;. ! ; PC~r/US92/03392 2 1 ~ 2 '~
In addition, K2S2a and staurosporine act on BDNF/trkB-transfected NIH3T3 oells grown in defined media in a manner which is very similar to their effect on neuroblastomas, thus confirming that such cells provide a useful assay system for identifying agents that can be used to destroy autocrineloop dependent tumor cells through disruption of the autocrine loop. Other autocrine loop model systems, which are engineered to encode and express a particular factor, as well as the receptor for that factor, may also be created and used, as contemplated herein, to identify agents that interrupt such autocrine loops. Such factors include, but are not limited to, nerve growth factor, neurotrophin-3, neurotrophin~, and ciliary neurotrophic factor.
~ order that the invention described herein may be more fully understood, the following examples are set forth. It should 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 Findings:_Neuroblastoma As described herein, we have shown that antisense oligonucleotides ~O directed against BDNF are cytotoxic in vitro to neuroblastoma cell lines, thus, demonstrating that human neuroblastoma cells require BDNF as an autocrine survival molecule and that these neuroblastoma cells can be rescue~ from ~e cytotoxie effects of antisense BDNF by administering exogenous BDNF. Since most of these neuroblastoma cells do not express 2 5 detechble levels of trk B mRNA, our data imply that additional reoeptors for BDNF might exist.

.
_ wo s3tooso9 2 1 1 Z 7 9 9 Pcr/US92/03392 1.1 Human and Rodent Neuroblastoma Cell Lines Express ~DNF mRNA
We screened a panel of human and rodent tumor cell lines for the expression of BDNF mRNA by Northern blotting approaches [Maisonpierre et al, Science, 247:1446-1451 (1990)~. Table I and Figure 5 summarize these S results and indicate ~at most neuroblastoma cell lines (17 of 18 tested) express BDNF mRNA and that expression of BDNF mRNA appears to be somewhat unique to neuroblastoma. For exarnple, only 3 of 12 neuroepi~elioma or E~nng's sarcomas express BDNF mRNA. No non-neural h~mor tested was found to express 8DNF mRNA. The non-neuronal tumor cell lines tested included retinoblastomas, melanomas, carcinomas (cervical and breast) and leukemias. The single neuroblastoma cell line that did not express BDNF mRNA was SH-SY5Y which is unique in that it is of thoracic - as opposed to neural crest-derived origin. Dr. Mark Israel (UCSD) provided RNA blots of some of these neuroblastoma cell lines.
1 5 Table I
Expression of BDNF mRNA in Human Cell Lines~

Cell Line BDNF Expression 2 0 Neuroblastoma 382 +
GICAN +
NB-69 +

` C~-234 +
C~D?-134 +
CHP-12~ +
NMB +
3 0 KCNR +
NGP +
BE2 +
KAN
GI +
3Ej AS

wo 93/oosos 2 1 1 2 7 9 9 Pcr/US92/0~392 Table I (Cont'd.) LA-N-1 +
LA-N-5 +
IMR-32 +
SH-SYSY

Neuroepithelioma/Ewing's l 0 SK-N-MC

A4573 +

DW
SK-N-L0 +
Ll Non-neuronal Cell Lines Mol .
C0L~3~0 HELA
3 6 U~37 .

.-W093/0090g æll2799 Pc~r/US92/03392 ï

~Data are a summary of Northern blot~dng results of human cell line RNAs probed with human BDNF. (+) indicates positive expression of hBDNF mRNA and (-) indicates a lack of ~DNF mRNA. (+/-) indicates a very low level of expression. (Actual Northern blotting data is shown in the BDNF patent).
__ -1.2 Antisense Oligonucleotides Inhibit the in vitro Translation ofBDNF
mRNA
Three antisense oligonucleotides were synthesized complementary to various regions of the humsn 5 BDNF gene [see PCT International Publication No. WO91/03568, published March 21,1991~ as set forth in Table II below. Each oligonucleotide ("oligo") was made as an 18-mer and the complementary sense oligos served as controls in all experirrents.
The S' antisense oligQ (5'-AS BDNF; SEQ ID NO: 1) consisted of human BDNF DNA sequence beginning 3 nucleotides upstream of the presumptive ATG start codon and extending 4 codons downstream from this initiating methionine. l~e second antisense oligo corresponded to BDNF DNA sequence around the dibasic residue processing site ~PS AS
BDNF; SEQ ID NO: 3) and the third antisense oligo (the 3'-AS-BDNF; SEQ
ID NO: 5) corresponded to the last 6 codons of human BDNF.

2 5 Table II - -Antisense BDNF Oligonucleotides ~'-A~BD~F 5'~AA AAG GAT GGT CAT CAC-3' 1 -129 to -124 3 0 S-S-BDNF 5'~TG ATG ACC ATC CTI l~C-3' 2 -129 to -124 PS A~BDNF 5'-GGC AGG GTC AGA GTG GCG-3' 3 -1 to ~5 .

w093/009u9 2112 Pcr/uss~/o33s~

Table II Cont'd PS-~BON~5'-CGC CAG TCTGAC CCI GCC-3' 4 -1 to ~5 (Arg) (~ys) 3'-A~BDNF5'~TA TCC ccr TIT AAT GGT-3' 5+113 to +119 3'-~BDNF5'-l~G ACC ATI AAA AGG GGA-3' 6+113 to +119 ..
.

AS - refers to antisense sequenoe; S refers to sense sequence; PS- refers to dibasic amino acid processing site. The presumptive ATG start codon is highlighted in the 5'-S-BDNP sequence. The Arg-Lys dibasic residue codons are indicated in the PS-~BD~F sequence. All oligonudeotides were synthesized on an Applied Biosystems nucleic acid synthesizer.

1 5 Eac~ antisense BDNF oligonucleo~de was first tested for its ability to inhibit the synthesis of BDNF using a wheat germ Iysate in vitro translation system (Figure 1). I~e synthesis of BDNF (~/- antisenæ or ~F;
.~ sense oligonucleotide) was followed in this assay system ~y metabolic labeling with 3~methionine, polyacrylamide gel electrophoresis, and fluorography (Figure 1). It was observed that a random 1~mer (control oligo) had no inhibitory effect on 8DNF synthesis in ~ at both 1 and 6~1M concenb~ations. The 5'~ BDNF (SEQ ID NO: 1~ oligo had a slight . inhibitory effect on BDNF synthesis in VitlO at a concen~ation of 111M and prohund inhibitory e~fects at 6~1M. l~e 3'-~BDNF (SEQ ID NO: 5) and 2 5 ~he P~BDNF ~EQ ID NO: 3) bo~ effectively inhibited BDNI: synthesis at concentrations of 1~M and 6~1M. The complementary sense oligos had little to no effect on ~e in vitro synthesis of BDNF at 111M but did have some inhibitory activity at very high concentrations (6,~) (representative data shown for the 5'-~BDNF oligo (SEQ ID NO: 2); Figure 1).

.~.
:

i WO s3/~osos , Pcr/uss2/o339~
'` 1279'g' ' ' ..
.~
:.
1.3 3' Ant sense BDNF, But Not 5'-Ant ense or Sense BDNF
Oligonucleotides are (: ~totoxic for BDNF-Expressing Neuroblastoma Cells S We tested the effects of the antisense BDNF oligonucleotides (5'-AS-BDNF, PS-AS-BDNF, and 3'-AS-BDNF~ on oell viability when added directly to cultures of human neuroblastoma cells in vitro.
Human neuroblastoma cells were cultured in Eagle's modified essential medium (EME~) with 10~o fetal bovine serum (FBS), 2mM
glutamine, and 1% each of penicillin and streptomycin (complete media).
For ~ntisense assays, the cells were plated into 2~well Costar p,lates at a seeding density of 2 x 104 cells/well. An~sense oligonucleotide uptake was carried out by adding antisense oligos directly to the cells ;n EMEM
without serum. The concentration of antisense or sense oligonucleotide 1 5 added to the cell culture media ranged ~rom 0.1 to 50,iM (Flgure 2). ~fter a 4 hour incubation period wi~ the respective oligonucleotide, lTS
supplement (insulin, transferrin and selenium) was added to the cell culture wells and cell viability was assessed at 9~ hours after oligo addition. Duplica~e wells were assayed and averaged. Cell viability was 2 0 assessed by trypan blue staining. (Figure 2). The morphological effects of sense and antisense BDNF oligomers on LAN-5 neuroblastoma cells are shown in ~he light pho~omicrographs in E:igure 6. As shown in Figure 2, the LA-N-5 neuroblast~ma cell line was the most sensitive to the cytoto~ae effec~; of ~e 3'-A~BONP (SE~Q ID NO: 5) oligonucleotide.
2 5 Greater than 80% of these cells were killed with ~M 3'-A~BDNF within 96 hours. LA-N-l neuroblastoma cells were somewhat less sensitive than LAN-5 oells, as only 40% of these cells were kîlled with ll,M 3'-AS-BDNF at 96 hours. Interestingly, LA-N-l cells express less BDNF mRNA than LA-N-5 cells. SK-ES and SH-SY5Y cells (which do not express BDNF mRNA) ~ Wo 93/00909 PCr/u~92/033s2 :'' 2~ 9 were resistant to the cytotoxic effects of the 3'-A~BDNF even at concentrations of 50"M. Some loss of cell viability was observed at 5(~M
concentrations of 3'-AS-BDNF for these resistant cells lines but the same loss of cell viability was o~served when these cells were treated with 50~M
of the control 3'-sense-BDNF (3'-~BDNF) oligonucleotide. In fact, no greater than 30% cell loss was observed on any of the four cell lines when treated for 96 hours in serum-free media with 5~M 3'-~BDNF. Finally, no loss of cell viability was observed on any of the neuroblastoma cell lines when treated with the 5'-A~BDNF oligo (SEQ ID NO: 1) but the PS-AS
BDNF (SEQ ID N0: 3) gave results virtually identical with the 3'-A~
BD~ (SEQ ID NO: 5).
BDNF an!dsense oligomers, but not the control oligomers, had profound effects on cellular morphology when added to neuroblastoma alltures at low conoentrations (Figure 6A~).. As would be expected if 1~ these effects were derived from the disruption of a BDNF autocrine loop, antisens-mediated shanges in cell morphology could be prevented by the addition of exogenous BDNF (Figure 6D~.

1.4 BDNF-Expressing Neuroblas~pma Cells can b~ Selectively Rescued 2 0 from the Cytotoxic Effects of the 3'-AS-8DNF Oligonlldeotide bv the C~
Addition of Neurotrophins to ~e Cell Cu ture $ystem Figure 3 (A - E) shows the results of coaddition experiments where e~er 8DNP, NGF, or neurotTophin 3 ~-3~ (100 ng/ml of each purified ~ re~mbinant factor, obt~ined from CH0 oells transfected with the 2 5 respective neurotrophin gene lsee PCI International Publication No. WO
91/03568] was added simult~neously with the 3'-A~BDNF (SEQ ID N0: 5) oligonucleotide (5û uM) to various n~uroblastoma cells ~i.e., SH-SY5Y ~A), LA-N-1 (B), LA-N-~ (C), CHP-134 (D), C~04 (E)). Cell viability was determined on duplicate wells of a 2~well plate by trypan blue staining at ~ wog3/oosos ZllZ799 PCr/US92/03392 24 hour intervals after the addition of 3'-A~BDNF ~/- neurotrophic factor. Cell number was also recorded with a hemocytometer. As , described above, the oligonudeotide uptake was carried out by adding the oligo directly to the cell culture system under serum-free conditions for 4 S hours. The data in Figure 3A demonstrate that the 3'-AS-BDNF has no cytotoxic effects on SH-SY5Y cells which are negative for BDNF mRNA.
Both C~-134 and CHP-404 cells (Figure 3D and E, respectively) are :t' sensitive to the cytotoxic effects of the 3'-AS BDNF and each of these cell lines can be rescued (7~90%) by the c~addition of either BDNF and NGF
but not NT-3. LA-N-1 cells are only rescued from 3'-A~BDNF cytoto~acity by BDNF (Figure 3B) while LA-N-5 cells are rescued by all three neurotrophins (Figure 3C). Although not shoum in Figure 3, we observed that the 3'-AS-BDNF oligonucleotide was cytostatic as well as ~totoxic on LA-N-1, LA-N-5, CHP-134, and CHP-4~ cells but not on SH-SY5Y cells.
1 !j 1.~ Treatment of Neuroblastomas With 8DNF Antisense Oligomers ` Causes Apoptotic Death In contrast to the effects resulting from the disruption of previously described growth factor autocrine loops [Becker, et al. EMB0 I. 8: 3685 (1989); Morrison, I Biol. Chem. 266:728(1991); El-Badry, et al. I. Clin. Invest.87:648(1991); Selinfreund, et al. 1. Cell Biol. 111:2021 ~1990), even those known to be operative in neuroblastoma, the BDNF antisense oligomers not only prevented neuroblastoma growth,, but al50 resulted in the death - ~ of neuroblastoma tumor cells ~ ure 6B). If BDNF is ind~d functioning 2 5 in ~ese cells as a neuronal sur~nval molecule, it would be expected that the death due to disrup~on of a BDNF autocrine loop might occur by mechanisms similar to those described previously for nellronal cell death .
follo~nng neurotrophic factor deprivation lMartin, et al. L Cell Biol.
106:829 (19883; Scott, et al. I. Neurobiol. 21:630 t19903; Batistatou, et al. I ~ell , ~8 wo 93/oosos 2 1 1 2 7`~ 9 Pcr/uss2/o3392 Biol. 115: 461 (1991); Rukenstein, et al. I. Neurosci. 11: 2552 ~1991)~.
Although the morphological patterns displayed by neurons undergoing naturally occurring cell death may vary 1 Server, et al. in .Apoptosis. l?~e Molecul~r 8asis of Cell Death, pp. 263-279 (1991)], neuronal death may generally be marked by some of the same biochemical changes that characterize progra~uned cell death in other systems such as the thymus and the prostate (Batistatou, ~; Rukenstein, supra, Wylie, et al. Int.
Rev. Cytol. 68: 251(1980)1. In particular, the activation of the endogenous calciusn-dependent endonuclease that results in the loss of DNA prior to 1 0 the loss of cellular protein, may be a general feature of programmed or "apoptotic" death lArends, et al. Am T. ;Pathol. 136: 593 (1990)]. We took advantag of a double-staining (i.e., for DNA and protein) flow cytometric assay to distinguish between apoptosis and necrosis [Del 8ino, et al. ~.
Cell Res. 193:27 (1991); Jakobisiak, et al. Proc. Natl. Acad. Sci. USA 88: 3628 (1991)]. The DNA profile of LA-N-5 human neuroblastoma cells is typical of many norsnally cyding 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 either S phase or in G2+M (Figure 7A). Treatment with antisense BDNF oligomers results in the appearance of a~ apoptotic 2 0 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 ~phase, as usually seen in apoptotic populations (Del Bino, supra). BDNP rescue of antisens~
treated autocrine cells, as previously observed in Figure 6D, prevented the 2 5 appearance of the apoptotic population (Figure 7C). Although neuroblastoma cells were not suscepJdble to low concentrations of BDNF
sense or random sequence oligomers, these oligomers resulted in neuroblastoma cell death, as well as death of cells not dependent on BDNF
autocrine loops, when present at high concentrations (see belcw). In ~vog3/oosos 11'2,79~ PCI/US92/1~3392 contrast to the apoptotic profile exhibited by neuroblastoma cells treated with low concentrations of BDNF antisense oligomers, high concentrations of sense oligomers resulted in DNA and protein profiles consistent with necrosis - loss of cellular protein is apparent without an S effect on DNA content or percentage of cells in S phase (Figure 7D).
To verify that BDNP antisense oligomers operate in a sequenc~
dependent manner to speafically kill neuroblastoma cells requiring a BDNF autocrine loop, we compared the viability of neuroblastoma cell lines when exposed to varying concentrations of either sense or antisense oligomers. Dos~response studies revealed that antisense BDNF
oligomers were strikingly more potent than control oligomers in their ability to kill a BDNF-expressing neuroblastoma, LA-N-5 (Figure ~A).
Conversely, antisense and control oligomers were eo~ually ineffective in killing the only BDNF-negative neuroblastoma, SY5Y (Figure 8B).
Although the addition of exogenous BDNF did not alter the effects of either sense or antisense oligomers on SY5Y cells (Figure 8B), exogenous BDNF markedly shifted the antisense oligomer kill curve on LA-N-5 cells so that it matched that of the control sense oligomer ~Figure 8A). While the BDNF-negative Ewing's sarcoma oell line, SK-ES, was similar to SY5Y
in its insensitivity to antisense BDNF oligomers, exarnination of four additional BDNF-positive cell lines (CHP-134, N18TG2, CHP~04 and LA-~-1) revealed an exquisite susceptibility to antisense BDNF oligomers, as well as an ability to be rescued by BDNF, that was essentially indis~inguishable from that of LA-N-~ oells (data not shown).
2 5 Thu~ the an~sense BDNF oligomer aets in a sequence-specific manner and only OIt E~DN~:-expressing neuroblastomas. Furthermore, the toxicity of the antisense oligomer is reduced to the level of control oligomers by the addition of exogenous BDNF. Together with our observation ~at neuroblastoma death caused by antisense BDNF

: WO 93/00909 PCl/US9~/033~2 211 '~7!3 .
oligomers occurs by apoptosis, while death due to higher concentrations of control oligomers is necrotic, these data unequivocally demonstrate that BDNF antisense oligomers selectively activate apoptotic cell death in neuroblastomas by disrupting an autocrine survival loop dependent on the continued synthesis of BDNF.

1.6 Qnclusion Most human neuroblastoma cell lines express some level of human BDNF mRNA and BDNF mRNA expression appears to be unique to i neuroblastoma with only a ~ew exceptions (such as small cell l-mg carcinoma and a few neuroepithelial tumors). These results suggest that BDNF mRNA expression may serve as a useful marker dinically for human neuroblastoma. Given that the best clinical marker for neuroblastoma to date is N-myc amplification and that N-myc is only amplified in approximately 30~O of all lat~stage neuroblastoma (Stages m and IO, BDNP r~A expression may be a more useful and broad ranging clinical marker for both early and late stage neuroblastoma.
3'-A~BDNF (SEQ ID NO: 5~ and P~AS BDNF ~SEQ ID NO: 3) oligonudeotides are cytostatic and cytotoxic on only those human 2 0 neuroblastoma cells that express BDN~: mRNA. These results imply that BDN~: mRNA-positive neuroblastomas require a BDNF autocrine loop for their own proli~eration and survival. Our results suggest that ~t least some neuroblastomas may be effe~ve~y and sp~dfi~ly killed by trea~nent with antisense BDNF oligonucleotides.
2 5 Some neuroSlastoma cell lines can be effectively rescued from the cytoto~ac effects of antisense BDNF oligonucleotides by the addition of ei~er BDNF, NGF or NT-3.

'.~
;
,, wo 93/0~909 PCr/US92/03392 ` ~1127~

Example 2 Experimental Findings: Small Cell Lung Caranoma 2.1 Small Cell,Lung Carcinoma Cell lines,Express BDNF mRNA
With the aim of gaining insight into the potential role of BDNF as an autocrine survival factor for small cell lung carcinoma tumors, we utilized a Northern blotting approach to exa~nine the expression of BDNF
mRNA in several small cell lung carcinoma cell lines.
Total RNA samples prepared from six difhrent small cell lung carcinoma cell lines were obtained from Dr. Jim Battey's laboratory at the NIH. The cell lines shown in Pigure 4 are as follows: H82 (lane 2), H209 (lane 3), H345 ~lane 4), H378 (lane 5~, HS10 (lane 6), and N417 (lane 7). 10 ug of each of the cell line RNAs were used for the Northern blot, and the level of BDNF rnRNA was compared directly with adult rat brain (lane 1).
We found that all six small cell lung carcinomas expressed some BDNF
mRNA. As demonstrated previously for tissues and neuroblastoma cell lines lMaisonpielTe et al, Science. 247:1446-1451 (1990)], two transcripts were detected. The small cell lung carcinoma cell line H378 (lane 5) ex~pressed particularly high levels of BDNF mRNA: approximately 2 to 3 times that expressed in adult rat brain (lane 1).

2.2 3 _ Antisense BD~JP, But Not 5, An~sens~or Sense BDNF
' Oligonucleotides Are Cytotoxic For BDNF-Expressing Small Cell Lung 2 5 Caranoma ~ells Antisense BDNP nucleotides prepared as set forth in Example 1.2 were added 'direc~y to cultures of SCLC cells (H345 and H378) in yitro.
Assays were conducted as set forth in Example 1.3. The concentration of antisense or sense oligonucleotide added to the oell culture media ranged wo 93/00909 Pcr/uS92/03392 ~ 7 ~ '~
from 0.1 to 50 uM (Figure 8). After a 4 hour incubation period with the respective oligonucleotide, lTS supplement (insulin, transferrin and selenium) was added to the cell culture wells and cell viability was assessed at 96 hours after oligonucleotide addition. Duplicate wells were assayed by trypan blue staining. As shown ~n Figure 9, both the H345(8A) and H378(8B) cells were extremely sensitive to the cytotoxic effects of the 3 antisense BDNF(SEQ. ID NO:5) oligonucleotide, but not to 5 or sense oligonucleotide.

2.3 BDNF-Expressing SCLC Cells Can Be Selectivelv Rescued From the Cytotoxic Effect of the 3-A~BDNF Oligonudeotide By the C~Addition Of BDNF to the Cell Culture System Figure 9(A ~ 8) shows the result of c~addition expeAments where BDNF (lOOng/ml, obtained from CHO cells transfected with the respec~ive neurotrophin gene) was added simultaneously with the 3'-AS-8DNF (SEQ
ID NO:5) oligonucleotide to ~45 and H378 SCLC cell cultures. As shown in Figure 9, both cell lines were rescued by the co addition of 8DNF.

2.4 ~a5~
In addition to human neuroblastomas, SCLC cell lines express some level of hu nan BDNF mRNA. These results suggest that BDNF mRNA
expression may serve as a useful marker clinically for SCLC.
3 -A~BDNF (SEQ ID NO:5) and PS AS-BDNF (SEQ ID NO: 3) oligonucleotides are cytoshtic and cytotoxic on both SCLC oell lines tested.
These results imply that BDNP mRNA-positive SCLC lines require a BDNF autocrine loop for their owsl proliferation and survival and that such oells may be effectively killed by treatment with antisense BDNF
oligonucleotides .

j:~

wo 93/oosos 2 1 1 2 7 9 9 PCl`~US92~03392 Example 3 3Jl K252a Blocks NGF but not FGF Signal Transduction Pathways in PC12 Cells In order to asoertain that K252a can act to specifically block neurotrophin-mediated cellular responses, we examined the tyrosine phosphorylation profile of total protein lysates prepared from PC-12 cells that have been stimulated wi~ NGF or FGF either in the absence or presenoe of ~ree structurally related protein kinase inhibitors: K252a (isolated from the microbacterium Norcardiopsis sp.), staurosporine (isolated from Streptomyces sp.) or H-7 (1-(5- Isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride).
We compared the tyrosine phosphorylation in total lysates prepared from PC-12 cells that were pretreated with kinase inhibitors K252a, shurosporine or H-7 for 15 minutes and then administered either NGF or 1 5 FGF for 5 minutes. PC-12 cells were grown to approximately 70%
confluency in 100 mm t;issue culture dishes 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 into the same medium and administered to the cells in 200 microliter aliquots.
Pollowing incubation, we washed the cells twice at 4 C with phosphate buffered saline containing lrr~ sodium orthovanadate.
Complete aspira~on of ~e wash buffer was followed by cell lysis using 500 ~ microliters of supplemented RIPA buffer (phosphated buffered saline without calaum and magnesium but containing 1% NP40, 0.5% DOC, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM P~ISF, 0.14 l~U/mg aprotinin). We mLxed the plates using a Van-mixer at 4 C for 15 minutes to lyse the cells. The cell lysate was transferred to a 2.2 ml Eppendorf tube and microfuged for 15 minutes at 4 C. We removed and discarded ~e wo 93/00909 2 1 :1 2 7 9 9 Pcr/US92/03392 pellet on ice using a sterile too~hpick. The supernatant represented the total lysate and was made 1x with 5x protein loading dye. The Iysate was boiled at 95 C for 3 to 5 minutes, separated on a 10~o SD~polyacrylamide gel, transferred to Immobilon (Millipore) and then challenged with 1:1000 anti-phosphotyrosine antibodies (UBI). One microliter/ml Goat anti-mouse IgG-'25I conjugate was used for detection by autoradiography. (SA =
1~.C/ml) Analysis of ~e resulting gel electrophoresis autoradiograph showed that in comparison with untreated PC-12 cells, 0.01% or 0.02% DMSO was not toxic to the cells and did not alter the phosphorylation pattern. In addition, the electrophoresis was run on samples of each of the following:
cells which were treated with inhibitors alone I100 nM and 200 nM K252a, 100 nM staurosporine, and 25.uM H-71; cells which were treated with factors alone 100 ng/rnl NGF; 10,50,100 and 200 ng/ml FGF]; cells which were pretreated with inhibitors followed by administration of 100 ng/ml NGF
[100 nM and 200 nM K252a, 100 nM staurosporine, 25 nucroM H-73 and cells which had been pretreated with inhibitors, followed by administration of 50 ng/ml FGF administration l100 nM and 200 nM
K252a, 100 nM staurosponne, 25 microM H-71. The inhi~itors had been resuspended from ~e manufacturer in DMSO with final concentration added to oells not exceeding 0.02%.
Compared to the untreated con~aol, administration of N~;F (100 ng/ml) to PC12 cells for 5 minutes resulted in ~e rapid tyrosine ` phosphorylation of ERK1 (43 kd) and ERX2 ~41 kd) along wi~ a high 2 5 molecular weight protein (140 lcd) presumed to be the TrkA reoeptor~
Stimulation of PC12 cells with 1~100 ng/ml of FGF resulted only in detectable ERK2 tyrosine phosphorylation while 200 ng/ml of FGF
inhibited this effect. Treatment with the inhibitors alone or with DMSO
(vehicle control) did not affect the phosphorylation profile. H^7 did not wo 93/oosos Pcr/us92/o33s2 ~ 9 9 block the signal transductio~ pathways of either NGF or FGF.
Significantly, staurosporine and K252a blocked the NGF pathway as reflected in loss of TrkA, ERK1 and ERK2 tyrosine phosphorylation but not the FGF p~thway since ERK2 phosphorylation remained at the control level.
In conclusion, we observed that NGF and FGF stimulate early cellular responses in PC12 cells via independent signal transduction pathways differen!dated by the spedficity of staurosporine and K252a NGF-mediated responses.

Example 4 4.1 K252a Blocks BDNF Stimulation of trkB and ERK ,T~rosine - Phosphorylation To deternune whether the ac~on of K252a could also block the ~ignal transduction pathway of BDNF, we engineered 3T3 cells to express a functional trkB receptor. We have previously demonstrated that these trkB expressing 3T3 cells are dependent on BDNF for their survival and proliferation in defined media ID. J. Glass et al, Cell, 66:405~13 (1991)3. The same panel of inhibitors used in the PC12 cell assays were adminis~ered to 2 O 3T3 cells expressing trkB.
We compared tyrosine phosphorylation in total lysates of neuroblastoma (N18TG2) cells and 3T3 cells expressing trkB tha~were ' pretreated wi~ ~e inhibitors used in Example 3 for 15 minutes but then ` were administered 100 ng~ml BDNF for 5 minutes. We processed andimmunoblotted the Iy~ates wi~ antiphosphotyrosine antibodies as described in Example 3. Lysates from untreated oells and those treated with BDNF were compared with cells which had ~een pretreated with inhibitors alone [25 mi~oM H-7, 100 nM K252a, and 100 nM
staurosporine] and cells which had been pretreated with inhibitors and W O 93/00909 21 127 9 9 PC~r/US9?./03392 then administered 100 ng/ml BDNF 125 microM H-7, 100 nM K252a, and 100 nM staurosporinel-~nmunoblots of autophosphorylated trkB receptors immunoprecipitated from total protein Iysates of 3T3 (autocrine) cells S treated with BDNF 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 kkB and ERK2 as compared ~ith the untreated control. In agreement with the PC-12 cell data presented in Exarnple 3, the tyrosine phosphorylation pattern revealed that pretreatrnent of cells with K252a and staurosporine, but not H7, abolished BDNF-stimulated tyrosine phosphorylation of trkB. The inhibitors alone did not appear to change the tyrosine phosphorylation profile. Staurosporine completely blocked ERK~ phosphorylation but at 100 nM K252a, ERK2 tyrosine phosphorylation was still readily detectable. Since 100 nM ~;252a blocked appro~nately 50% of the ERKl and ERK2 phosphorylation in 20 PC-1~
cells, minor stimulation of the trkB receptor by BDNF (undetectable with this assay) might ~e sufficient to transmit an intracellular response.

2 0 Exa~n~le 5 5.1 K252a Interrupts Trk Signal Transduction and Causes Death in Cell _ines Dependent on Trk Rece~ptor Activation for Their Surviv~l Our findings with ~kl3 expressing 31~ cells indicate that K252a could disrupt a BDNF aut~e survival loop by inhibiting phosphoryla~on and activation of the trkB receptor in response to BDNF.
We examined this hypothesis using 4 human neuroblastoma cell lines as well as a 3T3 fibroblast model cell system, 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 deperdent on FGP for wo 93/oosos , Pcr/uss2/o3392 21127~9 survival-in serum-free defined media. We have previously demonstrated that 3T3 cells expressing trkA survive in defined media supplemented with NGF while ~kB expressing 3T3 cells survive in defined media supplemented with BDN~ [Glass et al, Cell, 66:405~13 (1991)]. 3T3 cells S expressing both trkB and BDNF survive in defined media alone 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-S and SK-N-LO cells express BDNF mRNA while SH-SY5Y and SK-ES
cells do not express BDNF mRNA. However, trk~ expression at the mRNA level waç only detectable in the LA-N-5 cell line.
In order to examine the effects of the protein kinase inhibitor K252a on the survival of the human neuroblastoma cells, the small cell lung carcinoma cells and the trkB~xpressing 3T3 cells, we developed a cell viability assay based on glucose utilizatic~n. The underlying principle of 1~ this assay derives from the fact that viable cells will metabolize the glucose provided in their gro~th media while dead cells will not. Therefore, the glucose conoentration in the growth meclia is inversely related to ~e number of viable cells in the culture.
Cells were seeded into 24-well plat~s at an approximate densi~y of 2 2 0 x 10J cells per well. Human neuroblastoma oells. SCLC cells and lung adenocarcinoma oells were cultured in RPM~ 1640 with fetal bovine serum, while 3T3 oells were cultured in serum-free defined media with the appropriate neurotrophin (i.e., FGP, 8DN~ or NGP at 500 pM) [Glass et al, 5~, supral. TrkB/BDNF autocrine 3T3 cells (MBx) were cultured in 2~; def~ned media alone. All cell culture media contained 450 mg/dl of glucose at the time of cell seeding. 36 hours after seeding, K252a was added (solubiliæd in DMS0 - see Example 1) at concentrations of 0,10, 50, 100, 250, 500,1000, and 2000 nM. All assays were performed in triplicate.
Cell morphology was monitored visually and cell viability was assessed by 48.

wo 93~00909 ;~ 1 l 2 7 9 9 PCr/US92/033g2 the glucose utilization assay after 6 days in culture. Glucose concentration (mg/dl) was determined by transferring a 50 ul aliquot of the growth media to blood glucose strips and then readirlg these strips 2 minutes later in a blood glucose monitor.
Glucose readings were averaged and then plotted versus the conoentration of K252a to estimate the LDso for each drug on each cell line. Table 3 shows the LDso values for K252a for each cell line. Our data (Figure 11) demonstrate that K252a is cytocidal for human neuroblastoma cells and that this tyrosine kinase receptor inhibitor is efficacious for neuroblastoma cell lines requiring a BDNF autocrine loop for their survival in culture (i.e., SK-N-LO and LA-N-~). For exarnple, LA-N-5 cells are approximately 20 to 15~fold more sensitive to the cytocidal effects of K252a than SK-ES or SH-SY5Y cells, respectively. SK-N-LO cells are also more sensitive to X252a (2.5- to 19-fold) than either SK-ES or SH-SY5Y
cells. Since SK-N-L0 cells apparen~y do not express trkB, our data also suggest that these oells may express a unique ~oe52a-sensitlve ~k-like BDNF receptor or, alternatively, that ~he level of trkB expression in these cells is below the detect~ble liIsuts. Further, our data demonstrate~ that protein kinase inhibitors such as K252a ase cytocidal for small cell lung 2 û cardnoma cells but not cytocidal for lung adenocarcinoma cells. A
comparison of the effect of KZ52a on 3T3-auto~ine, neuroblastoma cells N18TG2), small cell lung carcinoma (NCI-H69) and lung adenocaranoma cells (Calu-3) is shown In Figure 1~.

wo 93/00gog PCr/lJSg2/03392 ~IZ7~9 `

Cell ~
Neurite Outgrowth Neuroblastoma ~50 (5 davs) SK-ES 200nm NE
SH-SYSY 1500 nm NE
LA-N-5 10 nm ~25 nm SK~N-LO 80 nm NE
1 5 Fibrobl~t Cells MBx 25 nm MG87 (+FGF) 175 nm MG87 trkA (+NGF) 30 nm MG87 trkB (+Br)N~:) 3û nm Small Cell Lun~Carcinoma oells Na-H69 100 nm Lun~Adenocarcinoma cells CaLu-3 7~0 nm M(;87 cells are 3T3 eells which require FGF for survival in defined medium.
MG87 trk-A cells require NGP.
3 5 MG87 trk-B cells require BDNF~
NE = no effect We have also shown that K~252a and staurosporine ase more efficaaous (approximately 6-fo~d) ~or 3T3 fibroblasts expressing trk receptor ~cinases (MBx, MG87 trkA, and MG87 trkB) which require neurvtrophins for survival in defined media rela~hve to the parental 3T3 cells (MG87), WO 93/0090~
2 ~ 1 2 7 9 9 ~ Pcr/Us92/03392 which survive in defined media supplemented with FGF. These data with the transfected 3T3 cells provide a convirlcing argument that the cytocidal effects of K252a and staurosporinè a~e more pronounced for the trk family of receptors relative to the ~G~ receptor tyrosine kinase.
Cumulatively, our data support the hypothesis that K25Za selectively disrupts the neurotrophin/t~ receptor signal transduction pathway and that K252a and staurosporine are effective inhibitors of the trlcB/BDN~
autocrine su~vival loop.
Finally, we observed that at con~entrations of K252a of appro~amately 25 nM or less, a pronounced antiproliferative effect as well ,as significant neurite outgtowth ~ould be detected in LA-N-5 neuroblastoma c~lls at around 4 to 5 days in culture. These morphological changes are reminiscent of NG~ induction of LA-N-5 cell differentiation. These observations with respect to low concentrations of K252a suggest that this 1~ drug may function as a par~al agonist of the trk receptor signal transduction pathway.
As described herein we have shown that K252a may be an important selective antagon-st of the neurotrophin~t~k receptor signal ~ansduction pathway and, therefore, might be potentially useful therapeutically for the 2 0 killing of neurally-deri~red hmor c~lls dependent on a BDN~ autocrine survival loop. We expect ~at other cancer oell lines which express BDN~
would be similarly affect~d by K252a. For example, some neuroepitheIial tumors would l~e expected to be adYersely affected by K25~a and o~er ~ pro~ein kinase inhibi~ors.
2~ e certain embodiments of the iIlYention have been particularly described, it will be apparent to those skilled in the art that many modifications and Yariations may be made. Therefore, the present invention is not to be construed as lirnited by any of the particular ~ .

wo ~3/00909 PCr~US92/~33g2 embodiments shown, ratller its scope will l)e defined onl~ by llle Cl~lilnS
wllicll follow.

DEPOSITS

The following cell line has been deposited with tlle ~merican Type Cul~ure Collection, 12301 Parklawn Drive, Rockville, Maryland 20852:
DEPOSIT ACÇESSION NUMBER
MBx WO 93/00909 PCr/US9~/~3392 ~11.'2199 SEQUENCE LISTING

(1) GENERAL INFORMATION:

5 ~ (i) APPLICANT: Squinto, Stephen P.
Yancopoulos, George D.
Nye, Steven H.

(ii) TlTLE OF INVENTION: Method for Inhibiting 1 0 Neurotrophin Activity (iii) NUMBEROF SEQUENCES: 6 (iv) CO~ESPONDENCE ADDRESS
(A) ADDRESSEE: Howson and Howson (B~ STREET: 321 Norristown Road, Box457 (C) Cl~ Spring House (D) STATE: PA
(E) COUNTRY: U.S.A.
(F) ZIP: 1~477 2 0 (v) COMPUTER READABLE FORM:
(A) MEDIUM lYPE: Floppy di~k ,, (B~ CC)MPUI~: IBM PC compatible -(C) OPI;~IING SYSTEM: PC~;/M~DO~;
(D~ 50FI~W~: Patent In Release #1.0, Version #1.~5 ~5 (vi~ CURRENT APPl;~CA'rI~N D~TA:
(A) APPLICATIVN ~M~ER: US
(13) FILlNG DATE:

WO 93/~10909 2 1 1 2 7 9 9 PCI/I 592/03392 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:
(A) APPLICAnON NUMBER: US 07/728,784 (13) FILING DATE: 03~ 1991 ~viii) Al~ORNEY/AGENT INFORMATION:
(A) NAME: Bak, Mary E.
(B) REGISTRATION NUMBER: 31,215 (C) REFERENCE/DOCKEI NUMBER: RPIP-USl ~ix) TELECOMMUNICATION INFORMAllON:
(A) TELEPHONE: (215)540-9206 tB) TELEFAX: (215) $4~5818 1 5 (C) TELEX: 91~250 6892 (V lNFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18basepairs (B) TYPE: nucleic aad (C) Sl~ANI:)EDNES~: single (D~ TOPOLOGY: unknown (ii) MC)LECULETYPE: cDNA

(x~) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GAAAAGGATG GTCATCAC

~4 WO 93~00909 PCr/US92/033g2 211279~

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) lYPE: nucleicacid (C) S~RANDEDNESS: single (D) TOPOLOGY: unknown 1 0 (ii) MOLECULETYPE: cD~A

(~a) SEQUENCE DESCRI}rIlON: SEQ ID NO:~:

GTGATGACCA TCCITl~C

~2) INFORMATIQN FOR SEQ ID NO:3:

(i) SEQUEN5;~E~ CHARAClERlSTICS:
(A) LENGTH: 18 ~ase pairs ~B) TYPE: nudeicacid (C) STRANDEDNESS: single (D) TOPOLOGY: unlcnown (ii) MOLECULE TYPE: cDNA
(xi~ SEQUENCE DESC~ION: SEQ ID NO:3:

GGCAGC,GTCA GAGI~;GCG

WO 93/00909 Pcr/uss2/o3392 21 ~ 2799 (~) ~FORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nudeic aad (C) STRANDEI)NESS: single (D) TOPOLOGY: unlcnown (ii) MOLECULETYPE: cDNA

(xi) SEQUENCE DESCRIPI ION: SEQ ID NO:4:

CGCCAGTCTG ACCCTGCC

(V ~J~OPMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACI'ERISIICS:
(A) LENGTH: 18 base pairs ~B) TYPE: nucleic acid 2 0 (C~ STRANDEI)NESS: single (D) TQPOLOGY: unkno~

(ii) MOIECULE IYPE: cDNA

2 S (xi) SEQUlENCE DESCRII~I70N: SEQ ID NO:5:

CTATCCC~Cl~ ITAATGGI

(2) INFORMATIC)N FOR 5EQ ID NO:6:

5~

WO 93/00909 PCI`/US9~/03392 2 1 ~ 2 7 9 ! ~

(i) SEQUENCE CHARA(: TE~lSTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nudeicacid S (C) Sll~ANDEDNE5S: single (D) TOPOLOGY: unknown (u) MOLECUI~ETYPE: cDNA

(xi) SEQUENCE DESCE~IPIION: SEQ ID NO:6:

l~GACCAl'rA A~AGGGGA

Claims

What is claimed:

1. A method for treating a patient with a tumor, of a tumor type characterized by expression of a brain-derived neurotrophic factor gene in which brain- derived neurotrophic factor promotes survival of cells of the tumor, comprising administering to the patient an effective amount of an oligonucleotide, which oligonucleotide (a) consists of at least six nucleotides; (b) comprises a sequence complementary to at least a portion of a RNA transcript of the brain-derived neurotrophic factor; (c) is hybridizable to the RNA transcript, and (d) reduces the expression of brain-derived neurotrophic factor protein in a cell to which it is administered.

2. The method according to claim 1 in which the patient is a human, and the brain-derived neurotrophic factor gene is a human gene.

3. The method according to claim 1 in which the tumor is a neuroblastoma.

4. The method according to claim 1 in which the tumor is a small cell lung carcinoma.

5. The method according to claim 2 in which the tumor is a neuroblastoma.

6. The method according to claim 2 in which the tumor is a small cell lung carcinoma.

7. The method according to claim 2 in which the oligonucleotide consists of at least 18 nucleotides.

8. The method according to claim 5 in which the oligonucleotide consist of at least 18 nucleotides.

9. The method according to claim 6 in which the oligonucleotide consists of at least 18 nucleotides.

10. The method according to claim 8 in which the oligonucleotide is selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 5.

11. The method according to claim 2 in which the oligonucleotide contains a sequence complementary to a portion of the brain-derived neurotrophic factor RNA
containing the codons encoding the Arg-Lys processing site.

12. The method according to claim 5 in which the oligonucleotide contains a sequence complementary to a portion of the brain-derived neurotrophic factor RNA
containing the codons encoding the Arg-Lys processing site.

13. The method according to claim 6 in which the oligonucleotide contains a sequence complementary to a portion of the brain-derived neurotrophic factor RNA
containing the codons encoding the Arg-Lys processing site.

14. The method according to claim 2 in which the oligonucleotide contains a sequence complementary to a portion of the brain-derived neurotrophic factor RNA
lying within the last 6 condons of the RNA.

15. The method according to claim 5 in which the oligonucleotide contains a sequence complementary to a portion of the brain-derived neurotrophic factor RNA
lying within the last 6 codons of the RNA.

16. The method according to claim 6 in which the oligonucleotide contains a sequence complementary to a portion of the brain-derived neurotrophic factor RNA
lying within the last 6 codons of the RNA.

17. The method according to claim 2 in which the oligonucleotide contains at least one modified nucleotide.

18. The method according to claim 5 in which the oligonucleotide contains at least one modified nucleotide.
19. The method according to claim 6 in which the oligonucleotide contains at least one modified nucleotide.

20. The method according to claim 17 in which the oligonucleotide has at least one modified base moiety.

21. The method according to claim 17 in which the oligonucleotide has at least one modified sugar moiety.

22. The method according to claim 17 in which the oligonucleotide has at least one modified phosphate backbone.

23. An isolated oligonucleotide consisting of at least six nucleotides, and comprising a dequence complementary to at least a portion of a RNA
transcript of a brain-derived neurotrophic factor gene, which oligonucleotide is (i) hybridizable to the RNA transcript and (ii) modified so as to have increased plasma membrane permeability or increased resistance to nuclease degradation.

24. The oligonucleotide of claim 23, in which the brain-derived neurotrophic factor gene is a human gene.

25. The oligonucleotide of claim 24 which consists of at least 18 nucleotides.

26. The oligonucleotide of claim 25 which is selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 5.

27. The oligonucleotide of claim 24 which contains a sequence complementary to a portion of the brain- derived neurotrophic factor RNA containing the codons encoding the Arg-Lys processing site.

28. The oligonucleotide of claim 24 which contains a sequence complementary to a portion of the brain- derived neurotrophic factor RNA lying within the last 6 codons of the RNA.

29. The oligonucleotide of claim 24 which contains at least one modified nucleotide.

30. The oligonucleotide of claim 29 which has at least one modified base moiety.

31. The oligonueleotide of claim 29 which has at least one modified sugar moiety.

32. The oligonucleotide of claim 29 which has at least one modified phosphate backbone.

33. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 23; and a pharmaceutically acceptable carrier.

34. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 24, and a pharmaceutically acceptable carrier.

35. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 25; and a pharmaceutically acceptable carrier.

36. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 26; and a pharmaceutically acceptable carrier.

37. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 27; and a pharmaceutically acceptable carrier.

38. A pharmacautical composition comprising a therapeutically effective amount of the oligonucleotide of claim 28; and a pharmaceutically acceptable carrier.

39. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 29; and a pharmaceutically acceptable carrier.

40. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 30; and a pharmaceutically acceptable carrier.

41. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 31; and a pharmaceutically acceptable carrier.

42. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of claim 32; and a pharmaceutically acceptable carrier.

43. A method of inhibiting the expression of a nucleic acid sequence encoding brain-derived neurotrophic factor in a cell comprising exposing the cell to an effective amount of oligonucleotide of claim 23.

44. A method of inhibiting the expression of a nucleic acid sequence encoding brain-derived neurotrophic factor in a cell comprising exposing the cell to an effective amount of the oligonucleotide of claim 24.

45. A method for diagnosing the presence of a small cell lung carcinoma in a patient, in which the small cell lung carcinoma produces detectable brain-derived neurotrophic factor mRNA, comprising detecting the expression of a brain-derived neurotrophic factor gene in patient cells suspected of being small cell lung carcinoma cells, in which the expression is detected by detecting the production of brain-derived neurotrophic factor RNA or protein.

48. A method of identifying a cell type exhibiting a brain-derived neurotrophic factor autocrine loop comprising:
(a) exposing a first cell in vitro to (i) an amount of an oligonucleotide effective in inhibiting the expression of brain-derived neurotrophic factor protein, said oligonucleotide consisting of at least six nucleotides, comprising a sequence complementary to at least a portion of a RNA transcript of a brain-derived neurotrophic factor gene, and being hybridizable to the RNA
transcript, and (ii) an amount of brain-derived neurotrophic factor protein sufficient to compensate for the amount of inhibition in brain-derived neurotrophic factor expression produced by the oligonucleotide;
(b) after step (a), detecting the survival of the first cell;
(c) exposing in vitro a second cell, of the same cell type as the first cell, to (i) said amount of the oligonucleotide, and (ii) substantially no brain-derived neurotrophic factor protein; and (d) after step (c), detecting death of the second cell, in which (i) the survival of the first cell in the presence of both the oligonucleotide and brain-derived neurotrophic factor protein, and (ii) the death of the second cell in the presence of the oligonucleotide and the substantial absence of brain-derived neurotrophic factor protein, indicates that the cell type exhibits a brain-derived neurotrophic factor autocrine loop.

49. A method of identifying a cell type exhibiting a neurotrophin-3 autocrine loop comprising:
(a) exposing a first cell in vitro to (i) an amount of an oligonucleotide effective in inhibiting the expression of neurotrophin-3 protein, said oligonucleotide consisting of at least six nucleotides, comprising a sequence complementary to at least a portion of a RNA transcript of a neurotrophin-3 gene, and being hybridizable to the RNA
transcript, and (ii) an amount of neurotrophin-3 protein sufficient to compensate for the amount of inhibition in neurotrophin-3 expression produced by the oligonucleotide;
(b) after step (a) detecting the survival of the first cell;
(c) exposing in vitro a second cell, of the same cell type as the first cell, to (i) said amount of oligonucleotide, and (ii) substantially no neurotrophin-3 protein; and (d) after step (c), detecting death of the second cell, in which (i) the survival of the first cell in the presence of both the oligonucleotide and neurotrophin-3 protein, and (ii) the death of the second cell in the presence of the oligonucleotide and the substantial absence of neurotrophin-3 protein, indicates that the cell type exhibits a neurotrophin-autocrine loop.

50. A method of identifying a cell type exhibiting a nerve growth factor autocrine loop comprising:
(a) exposing a first cell in vitro to (i) an amount of an oligonucleotide effective in inhibiting the expression of nerve growth factor protein, said oligonucleotide consisting of at least six nucleotides, comprising a sequence complementary to at least a portion of a RNA transcript of a nerve growth factor gene, and being hybridizable to the RNA transcript, and (ii) an amount of nerve growth factor protein sufficient to compensate for the amount of inhibition in nerve growth factor expression produced by the oligonucleotide;
(b) after step (a), detecting the survival of the first cell;
(c) exposing in vitro a second cell, of the same cell type as the first cell, to (i) said amount of the oligonucleotide, and (ii) substantially no nerve growth factor protein; and (d) after step (c), detecting death of the second cell, in which (i) the survival of the first cell in the presence of both the oligonucleotide and nerve growth factor protein, and (ii) the death of the second cell in the presence of the oligonucleotide and the substantial absence of nerve growth factor protein, indicates that the cell type exhibits a nerve growth factor autocrine loop.

51. A method of causing cell death in a BDNF
expressing tumor cell comprising exposing the cell to an effective amount of a substance capable of intexrupting a BDNF-autocrine survival loop.

52. A method of interrupting a BNDF autocrine loop in a BDNF-expressing tumor cell comprising exposing the cell to effective amount of the oligonucleotide of claim 23.

53. A method of interrupting a BDNF autocrine loop in a BDNF-expressing tumor cell comprising exposing the cell to an effective amount of K252a or its derivative.

54. A method of interrupting a BDNF autocrine loop in a BDNF-expressing tumor cell comprising exposing the cell to an effective amount of thiazolidine-diones or their derivatives.

55. A method of inhibiting the growth of a BDNF
stimulated tumor in a subject comprising administering to the subject a pharmaceutical composition to block the phosphorylation and activation of BDNF receptors on the tumor.

56. The method according to claim 55 wherein the pharmaceutical composition comprises K252a or its derivative.

57. The method according to claim 55 wherein the pharmaceutical composition comprises thiazolidinediones or their derivatives.

58. A method for treating a mammal bearing a tumor of a type characterized by expression of brain-derived neurotrophic factor, comprising administering to a mammal a pharmaceutically acceptable composition comprising a pharmaceutically effective amount of K252a or its derivative.

59. The method according to claim 58 in which the mammal is a human.

60. The method according to claim 58 in which the tumor is a neuroblastoma.

61. The method according to claim 58 in which the tumor is a neuroepithelial tumor.

62. The method according to claim 58 in which the tumor is a small cell lung carcinoma.

63. The method according to claim 58 wherein the K252a is administered in combination with at least one other chemotherapeutic agent.

64. The method according to claim 63 wherein the chemotherapeutic agent is an isolated oligonucleotide consisting of at least six nucleotides, and comprising a sequence complementary to at least a portion of a RNA transcript of a brain-derived neurotrophic factor gene, which oligonucleotide is (i) hybridizable to the RNA transcript and (ii) modified so as to be (a) capable of entering a living cell; and (b) able to reach a concentration effective in reducing expression of brain-derived neurotrophic factor protein.

65. The method according to claim 63 wherein the chemotherapeutic agent is selected from the group consisting of antimetabolites, alkylating agents, vinca alkaloids, antineoplastic antibiotics, platinum derivatives, substituted ureas, adrenocortico steroids, cytokines, interleukins, interferons and antibodies.

66. A pharmaceutically acceptable composition which comprises as active ingredient, a pharmaceutically effective amount of K252a with at least one substance selected from the group consisting of pharmaceutical carriers, diluents, excipients and adjuvants, for the treatment of BDNF-expressing tumors.

67. The composition according to claim 66 which is adapted for oral, parenteral, rectal or topical administration.

68. The composition of claim 66 which additionally comprises at least one other chemotherapeutic agent selected from the group consisting of antimetabolites, alkylating agents, vinca alkaloids, antineoplastic antibiotics, platinum derivatives, substituted ureas, adrenocortico steroids, cytokines, interleukins, interferons and antibodies.

69. A method of stimulating neurite outgrowth in a tumor cell, expressing a trkB receptor comprising exposing the tumor cell to K252a or its derivatives in a dosage range of less than 25 nM.

70. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the tumor cell to an effective amount of the oligonucleotide of claim 23.

71. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the cell to an effective amount of the oligonucleotide of claim 24.

72. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the cell to an effective amount of the oligonucleotide of claim 25.

73. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the cell to an effective amount of the oligonucleotide of claim 26, 27, or 28.

74. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the cell to an effective amount of the oligonucleotide of claim 29, 30, or 31.

75. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the cell to an effective amount of the oligonucleotide of claim 32.

76. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the tumor cell to an effective amount of K252a or its derivative.

77. A method of causing cell death in a BDNF-expressing tumor cell comprising exposing the tumor cell to an effective amount of a thiazolidine-dione or its derivative.

78. A method according to claim 51 in which the tumor is a neuroblastoma.

79. The method according to claim 70 in which the tumor is a neuroblastoma.

80. The method according to claim 73 in which the tumor is a neuroblastoma.

81. The method according to claim 51 in which the tumor is a small cell lung carcinoma.

82. The method according to claim 70 in which the tumor is a small cell lung carcinoma.

83. The method according to claim 73 in which the tumor is a small cell lung carcinoma.

84. A method of identifying an agent capable of inhibiting the growth or causing the death of a cell comprising:
(a) exposing a first and a second cell to an agent, wherein said first cell expresses a recombinant receptor for a neurotrophic factor and survival of the first cell depends on the presence of the neurotrophic factor, and the second cell is of the same cell 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 said decrease indicates the ability of the agent to inhibit or cause the death of the cell.

85. The method according to claim 84 wherein said neurotrophic factor is BDNF.

86. The method according to claim 84 wherein said first and second cell types are fibroblasts.

87. The method according to claim 86 wherein said fibroblasts are NIH3T3 cells.

88. The method according to claim 84 wherein said neurothrophic factor is present by exogenous addition 89. The method according to claim 84 wherein said neurotrophic factor is present by endogenous production by cell.

90. A method of reducing the growth or causing the death of a BDNF-expressing tumor cell comprising exposing the cell to a pharmaceutically effective dose of an agent identified by the method of claim 84.

91. A composition comprising the oligonucleotide of claim 23 for use in a method of reducing the growth or causing the death of a BDNF-expressing tumor cell.

92. Use of a composition comprising K252a or its derivative, staurosporine or its derivative, or a thiazolidinedione or its derivative for the manufacture of a medicament for reducing the growth or causing the death of tumor cells expressing BDNF
receptor.

93. A cell line that is capable of surviving in serum free medium and that is prepared by a process comprising transfecting a cell that is incapable of surviving in serum-free medium with a recombinant nucleic acid encoding a neurotrophic factor and a recombinant nucleic acid encoding a receptor for that factor; and determining that the transfected cell survives in serum-free mediums.

94. The cell line of claim 93 wherein said neurotrophic factor is selected from the group consisting of BDNF, ciliary neurotrophic factor, nerve growth factor, neurotrophin-3 and neurotrophin-4.

95. The cell line of claim 93 wherein said neurotrophin factor is BDNF.

96. The cell line of claim 95 wherein said receptor is trkB.

97. The cell line MBx deposited in the American Type Culture Collection as Accession Number CRL 11025.

98. The method according to claim 84 wherein said first cell type is MBx.