Note: Descriptions are shown in the official language in which they were submitted.
WO 93/00909 PCI /US92/03392
2112799
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
711 2799
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
211Z799
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.
1 0
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~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
1 1
WO 93/00909 PCI/US92/03392
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
1 3
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%.
1 4
WO 93/00909 PCI`/US92/03392
21 12799
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.
1 5
.. ....... . . ... .. .. ~.. . . - . . . ... ~ .. . ~ . . . .. .
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
16
WO 93/00909 PCr/US92/03392
2112799
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)~.
1 8
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,
1 9
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.
22
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
24
~ 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
2112 1 99
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
26
,`1 wo 93/0~)909 PCI/US92/03392
2112799
,~
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.
27
,
:
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
28
WO 93/00909 PCI/US92/03392
` 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
29
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.
31
.
_
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 +
2 5 CHP-4Q4
` C~-234 +
C~D?-134 +
CHP-12~ +
NMB +
3 0 KCNR +
NGP +
BE2 +
KAN
GI +
3Ej AS
32
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
CHP-100
A4573 +
5838
EW-1
71
6647
N1050
32
DW
SK-N-L0 +
Ll
Non-neuronal Cell Lines
Y79
F01
BU2
Mol
.
C0L~3~0
HELA
3 6 U~37
K562
MCF7
.
33
.-
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
34
.
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)
36
~ 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
37
~ 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.
41
'.~
;
,,
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
42
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:~
43
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
44
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
46
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~.
49
wo 93/00gog PCr/lJSg2/03392
~IZ7~9 `
TABLE 3
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
51
~ .
wo ~3/00909 PCr~US92/~33g2
2112799
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
1 0
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:
53
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
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(C) Sl~ANI:)EDNES~: single
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(x~) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GAAAAGGATG GTCATCAC
~4
WO 93~00909 PCr/US92/033g2
211279~
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(~a) SEQUENCE DESCRI}rIlON: SEQ ID NO:~:
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1 6
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WO 93/00909 Pcr/uss2/o3392
21 ~ 2799
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1 0
(xi) SEQUENCE DESCRIPI ION: SEQ ID NO:4:
CGCCAGTCTG ACCCTGCC
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WO 93/00909 PCI`/US9~/03392
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l~GACCAl'rA A~AGGGGA
57
