U.S. patent application number 10/146058 was filed with the patent office on 2003-02-27 for antisense-oligonucleotides for the treatment of immuno-suppressive effects of transforming growth factor-beta (tgf-beta).
This patent application is currently assigned to Biognostik Gesellschaft Fur Biomolekulare Diagnostik mbH. Invention is credited to Bogdahn, Ulrich, Brysch, Wolfgang, Schlingensiepen, Karl-Hermann, Schlingensiepen, Reimar, Sclingensiepen, Georg-Fredinand.
Application Number | 20030040499 10/146058 |
Document ID | / |
Family ID | 26133181 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030040499 |
Kind Code |
A1 |
Sclingensiepen, Georg-Fredinand ;
et al. |
February 27, 2003 |
Antisense-oligonucleotides for the treatment of immuno-suppressive
effects of transforming growth factor-beta (TGF-beta)
Abstract
Antisense-oligonucleotides or effective derivatives thereof
hybridizing with an area of a gene coding for transforming growth
factor-.beta. (TGF-.beta.) comprising the following nucleic acid
sequences identified in the sequence listing under SEQ ID NO. 1-56
and 137 or comprising the following nucleic acid sequences
identified in the sequence listing under SEQ ID NO. 57 to 136 each
of the nucleic acids having a DNA- or RNA-type structure.
Inventors: |
Sclingensiepen,
Georg-Fredinand; (Gottingen, DE) ; Brysch,
Wolfgang; (Gottingen, DE) ; Schlingensiepen,
Karl-Hermann; (Bovenden, DE) ; Schlingensiepen,
Reimar; (Gottingen, DE) ; Bogdahn, Ulrich;
(Wurzburg, DE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
Biognostik Gesellschaft Fur
Biomolekulare Diagnostik mbH
Gottingen
DE
|
Family ID: |
26133181 |
Appl. No.: |
10/146058 |
Filed: |
May 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10146058 |
May 16, 2002 |
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08535249 |
Oct 30, 1995 |
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6455689 |
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08535249 |
Oct 30, 1995 |
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PCT/EP94/01362 |
Apr 29, 1994 |
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Current U.S.
Class: |
514/44A ;
536/23.5; 536/25.34 |
Current CPC
Class: |
C12N 2310/315 20130101;
C12N 15/1136 20130101; A61P 35/00 20180101; A61P 37/02 20180101;
A61P 7/00 20180101; A61P 37/04 20180101; A61P 9/00 20180101 |
Class at
Publication: |
514/44 ;
536/23.5; 536/25.34 |
International
Class: |
A61K 048/00; C07H
021/04; C07H 021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1993 |
EP |
93107089.0 |
May 13, 1993 |
EP |
93107849.7 |
Claims
1. Antisense-oligonucleotides or effective derivatives thereof
hybridizing with an area of a gene coding for transforming growth
factor-.beta. (TGF-.beta.) comprising the following nucleic acid
sequences identified in the sequence listing under SEQ ID NO. 1-56
and 137 or comprising the following nucleic acid sequences
identified in the sequence listing under SEQ ID NO. 57 to 136 each
of the nucleic acids having a DNA- or RNA-type structure.
2. Antisense-oligonucleotides according to claim 1 wherein said
nucleic acid hybridizes with an area of a gene coding for
transforming growth factor-.beta..sub.1, -.beta..sub.2 and/or
-.beta..sub.3.
3. Antisense-oligonucleotides according to claim 1 and/or 2 wherein
said nucleic acid is hybridizing with areas of a gene region coding
for TGF-.beta. and/or areas of a gene region coding and non-coding
for TGF-.beta..
4. Antisense-oligonucleotide according to any one of the claims 1-3
wherein the antisense-oligonucleotide is a phosphorothioate
oligodeoxynucleotide.
5. Antisense-oligonucleotide according to any one of the claims 1
to 4 obtainable by solid phase synthesis using phosphite triester
chemistry by growing the nucleotide chain in 3'-5' direction in
that the respective nucleotide is coupled to the first nucleotide
which is covalently attached to the solid phase comprising the
steps of cleaving 5'DMT protecting group of the previous
nucleotide, adding the respective nucleotide for chain propagation,
modifying phosphite groups subsequently cap unreacted 5'-hydroxyl
groups and cleaving the oligonucleotide from the solid support,
followed by working up the synthesis product.
6. Antisense-oligonucleotide according to any on of the claims 1 to
5 wherein the oligonucleotides of the SEQ ID NO. 1 to 56 and 137
are antisense-oligodeoxy-nucleotides of TGF-.beta..sub.1 and
oligonucleotides of the SEQ ID NO. 57 to 136 are
phosphorothioate-antisense-oligo-deoxynuc- leotide of
TGF-.beta..sub.2.
7. Oligonucleotide as nonsense-control-nucleotide with an identical
amount of GC as the antisense-oligonucleotide according to any one
of the claims 1 to 6.
8. Pharmaceutical composition comprising an antisense-nucleic acid
according to any one of the claims 1 to 6.
9. Use of antisense-oligonucleotides according to any one of the
claims 1 to 6 for the manufacturing of a pharmaceutical composition
of claim 8 for the treatment of tumors in which expression of
TGF-.beta. is of relevance for pathogenicity and/or inhibition of
pathological angiogenesis.
10. Use according to claim 9 for manufacturing pharmaceutical
composition for the treatment of the immunosuppressive effect of
TGF-.beta., augmentation of the proliferation of cytotoxic
lymphocytes, for the treatment of endogenous hyper expression of
TGF-.beta., for treatment of breast tumors, for treatment of
neuro-fibroma, malignant glioma inculding glioblastoma for the
treatment and prophylaxis of skin carcinogenesis as well as
treatment of esophageal and gastric carcinomas.
11. Method of treating tumors in which the expression of TGF-.beta.
is of relevance for pathogenicity by inhibiting TGF-.beta. and
thereby reducing an immuno suppression and/or inhibition of
pathological angiogenesis by administration of an effective dose of
an antisense-TGF-.beta.-oligonucle- otides according to any one of
the claims 1 to 6.
12. Method according to claim 11 wherein the reduction of immuno
suppression is accompanied by an augmented proliferation of
cytotoxic lymphocytes in comparison with the status before
administration of the antisense-TGF-.beta.-oligonucleotides and
thereupon starting cytotoxic activity decreases the numbers of
tumor cells.
Description
[0001] The present invention is related to
antisense-oligonucleotides or effective derivatives thereof
hybridizing with an area of a gene coding for transforming growth
factor-.beta. (TGF-.beta.), oligonucleotides as nonsense control
nucleotides, a pharmaceutical composition comprising at least one
anti-sense-oligonucleotide or effective derivatives thereof
hybridizing with an area of a gene coding for TGF-.beta. as well as
a use of antisense-oligonucleotides for the manufacturing of a
pharmaceutical composition for the treatment of tumors and/or the
treatment of the immunosuppressive effect of TGF-.beta..
[0002] The transforming growth factor-.beta. (TGF-.beta.) is a
factor which is, for example, secreted by human glioma cells. Human
gliomas such as glioblastoma are human tumors for which at present
no satisfactory therapy exists. The TGF-.beta. supports in an
autocrine manner the growing of the respective tumor cells. The
factor shows immunosuppressive effects and reduces (the
proliferation of such cytotoxic T-lymphocytes which otherwise would
be able to destroy the glioma cells.
[0003] The supression of immune responsiveness has been well
documented in patients with malignant gliomas. These patients
express a variety of immunological deficiencies including cutaneous
energy, depressed antibody production, diminished numbers of
circulating T-cells (Brooks, W. H., Netsky, M. G., Horwitz, D. A.,
Normansell, D. E. Cell mediated immunity in patients with primary
brain tumors, J. Exp. Med., 136: 1931-1947, 1972 and Roszman, T.,
Elliott, L., Brooks, W Modulation of T-cell function by gliomas,
Immunol. Today 12: 370-374, 1991). More recent studies indicate
that these impairments may result from malfunctions in
physiological pathways required for normal T-cell activation and
from quantitative and qualitative defects in T-cell subsets.
[0004] In Proceedings of the 82nd Annual meeting of the American
Association for Cancer Research, Houston Tex., USA, May 15-18,
1991, Proc AM ASSOC CANCER RES ANNU MEET 32 (O), 1991,, 427 is
disclosed that factor-.beta.-antisense-oligonucleotides inhibit a
human melanoma cell line under serum-enriched and stimulate under
serum-free culture conditions. The results established indicate
different roles of cellular TGF-.beta..sub.1 in the growth
regulation of HTZ-19-cells depending on the amount of serum present
in the culture medium. In addition this may indicate the biological
potential and possible draw-backs of exogenously administered
TGF-.beta.-antisense.
[0005] J. EXP. MED. 174 (4), 1991, 925-930, Hatzfield J. et al,
"Release of early human hematopoietic progenitors from quiescence
by antisense transforming growth factor .beta.-1 or Rb
oligonucleotides" discloses release of early human hematopietic
progenitors from quiescence by antisense transforming growth factor
.beta.1 or Rb oligonucleotides. Rb antisense TGF-.beta. negatively
regulates the cycling status of early hematopoietic progenitors
through interaction with the Rb gene product.
[0006] Proceedings of the National Academy of Sciences of USA, Vo.
88, February 1991, Washington US, pages 1516-1520, Potts, J. et
al., "Epithelial-mesenchymal transformation of embryonic cardiac
antisense oligodeoxynucleotide to transforming growth factor beta
3'" discloses that epithelial-mesenchymal transformation of
embryonic cardiac endothelial cells is inhibited by a modified
antisense oligodeoxy-nucleotide to transforming growth factor
.beta.3. The transformation depends on the activity of a
transforming growth factor .beta. (TGF-.beta.) molecule produced by
the heart. Modified antisense oligodeoxynucleotides generated to
non-conserved regions of TGF-.beta.1, -2, -3 and -4 were prepared
in order to examine the possible roles of these members in this
transformation. As a result it has been shown that a specific
member of the TGF-.beta. family (TGF-.beta.3) is essential for the
epithelial-mesenchymal transformation.
[0007] WO-A 92/17206 discloses a composition for use in the
treatment of wounds to inhibit scar tissue formation during healing
comprising an effective activity-inhibitor amount of a growth
factor neutralising agent or agents specific against only fibrotic
growth factors together with a pharmaceutically acceptable carrier.
The method of preparation of said composition and method of
administering the composition to a host suffering from tissue
wounding is also disclosed.
[0008] WO-A 90/09180 discloses methods useful in autologous bone
marrow transplantation and cancer therapy. Bone marrow cells from a
patient having cancer are treated with selected antisense
oligonucleotides in order to deplete the bone marrow of malignant
cells prior to infusion back into the bone marrow donor.
[0009] It is an object of the present invention to provide a method
for the treatment of cancer cells which are correlated with an
immunosuppression. Another object of the present invention is to
provide an effective agent which inhibits the growth of tumor cells
which are related to an immunosuppression.
[0010] According to the invention antisense-oligonucleotides or
effective derivatives thereof which hybridizes with an area of gene
region coding for transforming growth factor-.beta. (TGF-.beta.)
comprising the following nucleic acid sequences identified in the
sequence listing under SEQ ID NO. 1-56 and 137 or comprising the
following nucleic acid sequences identified in the sequence listing
under SEQ ID NO. 57 to 136 each of the nucleic acids having a DNA-
or RNA-type structure are able to solve the problems addressed
above. Preferably, the antisense-oligonucleotides hybridize with an
area of a gene region coding for growth factor-.beta..sub.1,
-.beta..sub.2 and/or .beta..sub.3. The antisense-oligonucleotide is
either able to hybridize with areas of a gene region coding for
TGF-.beta. and/or areas of a gene region coding and non coding for
TGF-.beta.. For example, some nucleotides of the
antisense-oligonucleotide sequence hybridizing with an area of a
gene region coding for transforming growth factor-.beta. is
hybridizing with an area which does not code for the transforming
growth factor whereas, the other part of the respective sequence
does hybridize with a gene region coding for TGF-.beta.. Of course,
it is also in the scope of the present invention that the
antisense-oligonucleotide hybridizes with an area of a gene region
just coding for growth factor-.beta.. It is also understood by the
skilled person that fragments having subsequences of the
antisense-oligonucleotide works according to the invention so long
as production of TGF-.beta. is reduced or inhibited.
[0011] In a preferred embodiment of the present invention the
antisense-oligonucleotide or effective derivative thereof is a
phosphorothioate-oligodeoxynucleotide.
[0012] According to the invention the antisense-oligonucleotides
are obtainable by solid phase synthesis using phosphite triester
chemistry by growing the nucleotide chain in 3'-5' direction in
that the respective nucleotide is coupled to the first nucleotide
which is covalently attached to the solid phase comprising the
steps of
[0013] cleaving 5'DMT protecting group of the previous
nucleotide,
[0014] adding the respective nucleotide for chain propagation,
[0015] modifying the phosphite group subsequently cap unreacted
5'-hydroxyl groups and
[0016] cleaving the oligonucleotide from the solid support,
[0017] followed by working up the synthesis product.
[0018] The chemical structures of oligodeoxy-ribonucleotides are
given in FIG. 1 as well as the respective structures of antisense
oligo-ribonucleotides are given in FIG. 2. The oligonucleotide
chain is to be understood as a detail out of a longer nucleotide
chain.
[0019] In FIG. 1 lit. B means an organic base such as adenine (A),
guanin (G), cytosin (C) and thymin (T) which are coupled via
N9(A,G) or N1(D,T) to the desoxyribose. The sequence of the bases
is the reverse complement of the genetic target sequence
(mRNA-sequence). The modifications used are
[0020] 1. Oligodeoxy-ribonucleotides where all R.sup.1 are
substituted by
1 1.1 R.sup.1 = O 1.2 R.sup.1 = S 1.3 R.sup.1 = F 1.4 R.sup.1 =
CH.sub.3 1.5 R.sup.1 = OEt
[0021] 2. Oligodeoxy-ribonucleotides where R.sup.1 is varied at the
internucleotide phosphates within one oligonucleotide 1
[0022] where B=deoxy-ribonucleotide dA, dC, dG or dT depending on
gene sequence
[0023] p=internucleotide phosphate
[0024] n=an oligodeoxy-ribonucleotide stretch of length 6-20
bases
2 2.1 R.sup.1a = S; R.sup.1b = O 2.2 R.sup.1a = CH.sub.3; R.sup.1b
= O 2.3 R.sup.1a = S; R.sup.1b = CH.sub.3 2.4 R.sup.1a = CH.sub.3;
R.sup.1b = S
[0025] 3. Oligodeoxy-ribonucleotides where R.sup.1 is alternated at
the internucleotide phosphates within one oligonucleotide 2
[0026] where B=deoxy-ribonucleotide dA, dC, dG or dT depending on
gene sequence
[0027] p=internucleotide phosphate
[0028] n=an oligodeoxy-ribodincleotide stretch of length 4-12
dinucleotides
3 3.2 R.sup.1a = S; R.sup.1b = O 3.2 R.sup.1a = CH.sub.3; R.sup.1b
= O 3.3 R.sup.1a = S; R.sup.1b = CH.sub.3
[0029] 4. Any of the compounds 1.1-1.5; 2.1-2.4; 3.1-3.3 coupled at
R.sup.2 with the following compounds which are covalently coupled
to increase cellular uptake
4 4.1 cholesterol 4.2 poly(L)lysine 4.3 transferrin
[0030] 5. Any of the compounds 1.1-1.5; 2.1-2.4; 3.1-3.3 coupled at
R.sup.3 with the following compounds which are covalently coupled
to increase cellular uptake
5 5.1 cholesterol 5.2 poly(L)lysine 5.3 transferrin
[0031] In the case of the RNA-oligonucleotides (FIG. 2) are the
basis (adenin (A), guanin (G), cytosin (C), uracil (U)) coupled via
N9 (A,G) or N1 (C,U) to the ribose. The sequence of the basis is
the reverse complement of the genetic target sequence
(mRNA-sequence) . The modifications in the oligonucleotide sequence
used are as follows
[0032] 6. Oligo-ribonucleotides where all R.sup.1 are substituted
by
6 6.1 R.sup.1 = O 6.2 R.sup.1 = S 6.3 R.sup.1 = F 6.4 R.sup.1 =
CH.sub.3 6.5 R.sup.1 = OEt
[0033] 7. Oligo-ribonucleotides where R.sup.1 is varied at the
internucleotide phosphates within one oligonucleotide 3
[0034] where B=ribonucleotide dA, dC, dG or dT depending on gene
sequence
[0035] p=internucleotide phosphate
[0036] n=an oligo-ribonucleotide stretch of length 4-20 bases
7 7.1 R.sup.1a = S; R.sup.1b = O 7.2 R.sup.1a = CH.sub.3; R.sup.1b
= O 7.3 R.sup.1a = S; R.sup.1b = CH.sub.3 7.4 R.sup.1a = CH.sub.3;
R.sup.1b = S
[0037] 8. Oligo-ribonucleotides where R.sup.1 is alternated at the
internucleotide phosphates within one oligonucleotide 4
[0038] where B=ribonucleotide dA, dC, dG or dT depending on gene
sequence
[0039] p=internucleotide phosphate
[0040] n=an oligo-ribodinucleotide stretch of length 4-12
dinucleotides
8 8.2 R.sup.1a = S; R.sup.1b = O 8.2 R.sup.1a = CH.sub.3; R.sup.1b
= O 8.3 R.sup.1a = S; R.sup.1b = CH.sub.3
[0041] 9. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3 coupled at
R.sup.2 with the following compounds which are covalently coupled
to increase cellular uptake
9 9.1 cholesterol 9.2 poly(L)lysine 9.3 transferrin
[0042] 10. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3 coupled
at R.sup.3 the following compounds are covalently coupled to
increase cellular uptake
10 10.1 cholesterol 10.2 poly(L)lysine 10.3 transferrin
[0043] 11. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3; 9.1-9.3;
10.1-10.3 where all R.sup.4 are substituted by
11 11.1 R.sup.4 = O 11.2 R.sup.4 = F 11.3 R.sup.4 = CH.sub.3
[0044] Modifications of the antisense-oligonucleotides are
advantageous since they are not as fast destroyed by endogeneous
factors when applied as this is valid for naturally occurring
nucleotide sequences. However, it is understood by the skilled
person that also naturally occurring nucleotides having the
disclosed sequence can be used according to the invention. In a
very preferred embodiment the modification is a phosphorothioat
modification.
[0045] The synthesis of the oligodeoxy-nucleotide of the invention
is described as an example in a greater detail as follows.
[0046] Oligodeoxy-nucleotides were synthesized by stepwise 5'
addition of protected nucleosides using phosphite triester
chemistry. The nucleotide A was introduced as
5'-dimethoxy-trityl-deoxyadenosine(N.sup.4-benzoyl)-N-
,N'-diisopropyl-2-cyano-ethyl phosphoramidite (0.1 M); C was
introduced by a
5'-dimethoxytrityl-deoxycytidine(N.sup.4-benzoyl)-N,N'-diisopropyl-2-cy-
anoethyl phosphoramidite; G was introduced as
5'-dimethoxy-trityl-deoxygua-
nosine(N.sup.8-isobutyryl)-N,N'-diisopropyl-2-cyanoethyl
phosphoramidite and the T was introduced as
5'-dimethodytrityl-deoxythymidine-N,N'-diisop- ropyl-2-cyanoethyl
phosphoramidite. The nucleosides were preferably applied in 0.1 M
concentration dissolved in acetonitrile.
[0047] Synthesis was performed on controlled pore glass particles
of approximately 150 .mu.m diameter (pore diameter 500 .ANG.) to
which the most 3' nucleoside is covalently attached via a
long-chain alkylamin linker (average loading 30 .mu.mol/g solid
support).
[0048] The solid support was loaded into a cylindrical synthesis
column, capped on both ends with filters which permit adequate flow
of reagents but hold back the solid synthesis support. Reagents
were delivered and withdrawn from the synthesis column using
positive pressure of inert gas. The nucleotides were added to the
growing oligonucleotide chain in 3'->5 direction. Each
nucleotide was coupled using one round of the following synthesis
cycle:
[0049] cleave 5'DMT (dimethoxytrityl) protecting group of the
previous nucleotide with 3-chloroacetic acid in di chloromethane
followed by washing the column with anhydrous acetonitrile. Then
simultaneously one of the bases in form of their protected
derivative depending on the sequence was added plus tetrazole in
acetonitrile. After reaction the reaction mixture has been
withdrawn and the phosphite was oxidized with a mixture of sulfur
(S.sub.8) in carbon disulfid/pyridine/triethylamine. After the
oxidation reaction the mixture was withdrawn and the column was
washed with acetonitrile. The unreacted 5'-hydroxyl groups were
capped with simultaneous addition of 1-methylimidazole and acetic
anhydryide/lutidine/tetrahydrofuran. Thereafter, the synthesis
column was washed with acetonitrile and the next cycle was
started.
[0050] The work up procedure and purification of the synthesis
products occured as follows.
[0051] After the addition of the last nucleotide the
deoxynucleotides were cleaved from the solid support by incubation
in ammonia solution. Exoxyclic base protecting groups were removed
by further incubation in ammonia. Then the ammonia was evaporated
under vacuum. Full-length synthesis products still bearing the
5'DMT protecting group were separated from shorter failure
contaminants using reverse phase high performance liquid
chromatography on silica C.sub.18 stationary phase. Eluents from
the product peak were collected, dried under vacuum and the 5'-DMT
protecting group cleaved by incubation in acetic acid which was
evaporated thereafter under vacuum. The synthesis products were
solubilized in the deionized water and extracted three times with
diethylether. Then the products were dried in vacuo. Another
HPLC-AX chromatography was performed and the eluents from the
product peak were dialysed against excess of Trisbuffer as well as
a second dialysis against deionized water. The final products were
lyophilized and stored dry.
[0052] The antisense-oligonucleotides of the invention can be used
as pharmaceutical composition or medicament. This medicament can be
used for treating tumors in which the expression of TGF-.beta. is
of relevance for pathogenicity by inhibiting the tranforming growth
factor-.beta. and thereby reducing an immunosuppression and/or
inhibiting pathological angiogenesis The reduction of
immunosuppression caused by the administration of an effective dose
of an antisense TGF-.beta.-oligonucleotides may be accompanied by
an augmentated proliferation of cycto-toxic lymphocytes in
comparison with the status before administration of the medicament.
Thereupon, the lymphocytes are starting their cytotoxic activity
decreasing the numbers of tumor cells.
[0053] The medicament of the present invention is further useful
for the treatment of endogeneous hyperexpression of TGF-.beta., for
treatment of rest tumors, for treatment of neurofibroma, malignant
glioma including glioblastoma and for the treatment and prophylaxis
of skin carcinogenesis as well as treatment of esophageal and
gastric carcinomas.
[0054] The effect of TGF-.beta..sub.2-specific
antisense-oligonucleotides on human T cell proliferation and
cytotoxicity upon stimulation with autologous cultured glioma cells
was investigated. It was demonstrated that TGF-.beta..sub.2-derived
phosphorothioat-derivatives S-ODN's may specifically inhibit
protein expression of TGF-.beta. in glioma cells. In addition,
TGF-.beta..sub.2-specific S-ODN's revers--to a significant
amount--immunosuppressive effects of TGF-.beta. upon T-cell
proliferation and cytotoxicity.
[0055] It has been shown that T-cell response in human brain tumor
patients is clearly reduced and that tumor infiltrating lymphocytes
have only marginal impact upon tumor progression of individual
patients (Palma, L., Di Lorenzo, N., Guidett, B. Lymphocytes
infiltrates in primary glioblastomas and recidivous gliomas, J.
Neurosurg., 49: 854-861, 1978 and Ridley, A., Cavanagh, J. B.
Lymphocytes infiltration in gliomas, Evidence of possible host
resistance. Brain, 4: 117-124, 1971). Isolated tumor infiltrating
lymphocytes from brain tumors are functionally incompetent, these
immunosuppressive effects have been attributed to TGF-.beta..sub.2
in vitro and in vivo (Bodmer, S., Stromer, K., Frei, K., Siepl,
Ch., de Tribolet, N., Heid, I. , Fontana, A. , Immunosuppression
and transforming growth factor-.beta..sub.2 in glioblastoma, J.
Immunol., 143: 3222-3229, 1989; Couldwell, W. T., Dore-Duffy, P.,
Apuzzo, M. L. J., Antel, J. P. Malignant glioma modulation of
immune function, relative contribution of different soluble
factors, J. Neuroimmunol., 33: 89-96, 1991; Kuppner, M. C., Hamou,
M. F., Sawamura, Y., Bodner, S., de Tribolet, N., Inhibition of
lymphocyte function by glioblastoma derived transforming growth
factor .beta..sub.2, J. Neurosurg., 71: 211-217, 1989; Maxwell, M.,
Galanopoulos, T., Neville-Golden, J., Antoniades, H. N., Effect of
the expression of transforming growth factor-.beta..sub.2 in
primary human glioblastomas on immunsuppression and loss of immune
surveillance, J. Neurosurg., 76: 799-804, 1992; Palladino, M. A.,
Morris, R. E., Fletscher Starnes, H., Levinson, A. D., The
transforming growth factor betas, A new family of immunoregulatory
molecules, Ann. N.Y. Acad. Sci., 59: 181 to 187, 1990; Roszman, T.,
Elliott, L., Brooks, W., Modulation of T-cell function by gliomas,
Immunol Today 12: 370-374, 1991).
[0056] FIG. 3: IGF-.beta. western blot analysis of serum free
glioma culture cell lysates. Lanes 2 (HTZ-153), 3 (HTZ-209), and 4
(HTZ-243) indicate blots of respective cell lysates with
TGF-.beta..sub.2 specific antibody. Lane 1 represents a TGF-.beta.
positive control employing 50 ng pure TGF-.beta..sub.2.
TGF-.beta..sub.2-antisense treated cells are displayed in lanes A.
Untreated control cells are depicted in lanes B. Cells were treated
with antisense oligonucleotides for 48 hrs (1 .mu.M final
concentration).
[0057] FIG. 4: IGF-.beta..sub.1-mRNA expression in glioma cells.
Each lane contained 20 .mu.g of cytoplasmatic RNA from tumors A
(HTZ-153), B (HTZ-209), C (HTZ-243) that hybridized to a
.sup.32P-labeled TGF-.beta..sub.1 oligonucleotide probe. To verify
equal amounts of RNA, the blot was stained with methylene blue
prior to hybridization (lanes A', B', C').
[0058] FIG. 5: TGF-.beta..sub.2-mRNA expression in glioma cells.
Each lane contained 20 .mu.g of cytoplasmatic RNA from tumors A
(HTZ-153), B (HTZ-209), C (HTZ-243) that hybridized to a
.sup.32P-labeled TGF-.beta..sub.2 oligonucleotide probe. To verify
equal amounts of RNA, the blot was stained with methylene blue
prior to hybridization (A', B', C').
[0059] FIG. 6: TGF-.beta..sub.2-mRNA expression in glioma cells
after TGF-.beta..sub.2-S-ODN treatment. Cytoplasmatic RNA of
untreaated glioma cells A (HTZ-153) , B (HTZ-209) and C (HTZ-243)
or glioma cells A', B' and C' treated for 48 hours with 1 .mu.M
(f.c.) TGF-.beta..sub.2-specific S-ODN's under serum-enriched
culture conditions, was isolated and processed for Northern blot
analysis. Each lane contained 20 .mu.g of cytoplasmatic RNA
hybridized to a .sup.32 P-labeled TGF-.beta..sub.2 oligonucleotide
probe.
[0060] FIG. 7: Effect of TGF-.beta..sub.2-specific S-ODN's and
TGF-.beta. neutralizing antibody on cytotoxicity of PBMC's against
autologous cultured glioma cells (target/effector 1:10). After 6
days culture of PBMC's with IL-1.alpha. and II-2 the cells were
collected, washed, irradiated (30 Gy) and added in target/effector
ratios of 1:10, 1:5, 1:1 to autologous glioma cells. Glioma targets
were pretreated with either TGF-.beta. specific S-ODN's or
TGF-.beta. antibody. Cytotoxicity was assessed employing a modified
microcytotoxicity assay. Data are means of triplicate samples,
error bars represents SE. Data points reflect individual controls,
where tumor targets were treated with medium alone (control).
TGF-.beta. antibody (100 .mu.g/ml) , or S-ODN's (1 .mu.M resp. 5
.mu.M) as references for cytotoxicity effects. Thereby, effects
upon target cells of antibody or S-ODN's alone could be
excluded.
[0061] FIG. 8: Dose-dependent effects of TGF-.beta..sub.2-specific
and nonsense S-ODN's on proliferation of lymphocytes, glioma cells
and lymphocytes cocultured with autologous glioma cells (MLTC). A:
HTZ-153, B: (HTZ-209, C: HTZ-243. PBMC'x were preactivated for 6
days with IL-1.alpha. and IL-2 and incubated for additional 6 days
with autologous irradiated (60 Gy) and TGF-.beta..sub.2-(No.6) and
nonsense (no. 5) S-ODSN-treated glioma cells (MLTC).
Simultaneously, part of preactivated PBMC's (lymphocytes) and
glioma cells (tumor) were incubated with TGF-.beta..sub.2 specific
(Ly: No. 2, Tu: No. 4) and nonsense) S-ODN's (Ly: No. 1, Tu: No. 3)
for 3 days, to evaluate putative direct effects of S-ODN's upon
effector- or target cells alone. Proliferation of lymphocytes and
glioma cells was assessed employing a .sup.3H Tdr incorporation
assay. Data are means of triplicate samples, error bars represent
SE.
[0062] The invention is further explained by the following
non-limiting examples.
EXAMPLE 1
[0063] Characterization of tumor cells (autologous target cells)
Tumor cells of 3 patients with high grade malignant gliomas
(HTZ-153 and HTZ 209, glioblastomas, HTZ-243, malignant
astrocytoma, Gr.III-WHO) and their resp. autologous lymphocytes
were studied. Standard tumor cell cultures were established in
Dulbecco's Minimal Essential Medium containing 20% fetal calf serum
(FCS, Seromed, Berlin, Germany), 1 .mu.M L-glutamine, MEM vitamin
solution and nonessential amino acids (GIBCO, Paisley, Scotland, U.
K.) (Bogdahn, U., Fleischer, B., Rupniak, H. T. R., Ali-Osman, F.
T-cell mediated cytotoxicity in human glioma Biology of Brain
Tumor, Martinus Nijhoff Publishers, Boston, 70: 501-507, 1986).
Other target cells included K562 (an NK-sensitive erythromyeloid
leukemic cell line, American Type Culture Collection, Rockville,
Md., USA) . Tumor cell cultures were characterized by
immunocytochemistry employing the PAP-method (Bourne, J. A.,
Handbook of immunoperoxidase staining methods, DAKO Corporation,
Carpinteria Calif., USA, 1983) in Labtek tissue culture slides
(Miles Laboratories Inc., Naperville, Ill., USA) with the following
mono- or polyclonal antibodies to: GFAP, Cytokeratin,
Neurofilament, Desmin, Vimentin, NSE, HLA, DrO, W6/32 (Class I
Antigen), .beta..sub.2-Microglobulin, Fibronectin, Laminin, Ki 67
(Dakopatts, Glostrup, Denmark) and anti-TGF-.beta. (R & D
Systems, Inc., Minneapolis, Minn., USA). TGF-.beta. specific
immunocytochemistry was performed after 48 hours incubation of
glioma culture slides with 1 .mu.M final concentration (f.c.)
TGF-.beta..sub.2-specific S-ODN's and 1 .mu.M (f.c.) nonsense
S-ODN's treated controls.
EXAMPLE 2
[0064] Characterization of Lymphocytes (Effector Cells)
[0065] Peripheral blood mononuclear cells from all glioma patients
were isolated from heparinized venous blood at the day of surgery,
employing Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient
centrifugation and cryopreserved in liquid nitrogen under standard
conditions (Bogdahn, U., Fleischer, B., Rupniak, H. T. R.,
Ali-Osman, F. T-cell mediated cytotoxicity in human glioma Biology
of Brain Tumor, Martinus Nijhoff Publishers, Boston, 70: 501-507,
1986). Lymphocytes were cultured in RPMI 1640 (Flow Laboratories
Inc., Scotland, U.K.) with 10% human pooled AB-serum (Flow
Laboratories Inc. McLean, Va., USA) and 2 mM L-glutamine. Native
and activated (see below) peripheral blood mononuclear cells were
characterized by immunocytochemistry employing alkaline phosphatase
and monoclonal anti-alkaline phosphatase complexes (APAAP-method,
Dakopatts GmbH, Hamburg, Germany) (Cordell, J. L., Falini, B.,
Erber, W. N., et al., Immunoenzymatic labeling of monoclonal
antibodies using immune complexes of alkaline phosphatase and
monoclonal anti-alkaline phosphatase (APAAP complexes), J.
Histochem. Cytochem., 32: 219-229, 1984) with monoclonal antibodies
to the following antigens: CD3, CD4, CD8, CD16, CD25, HLA DR
(Becton Dickinson, Mountain View, Calif. USA).
EXAMPLE 3
[0066] LAK-cell Generation
[0067] As the proliferative and cytotoxic response of peripheral
blood mononuclear cells from glioma patients is suppressed, cells
(2.times.10.sup.6 cells/mi) were preactivated in vitro for 6 days
with interleukin-1.alpha. (10 U/ml). R & D Systems, Inc.,
Minneapolis, Minn., USA) and interleukin-2 (100 U/ml), BIOTEST AG
Frankfurt/M. Germany) in 48 flat bottom tissue culture plates
(2.times.10.sup.6 cells/ml) (Costar, Cambridge, Mass., USA).
EXAMPLE 4
[0068] Proliferation Assay
[0069] In mixed lymphocyte-tumor cell cultures (MLTC)
15.times.10.sup.3 lethally irradiated (60 Gy, .sup.64Co-source)
tumor cells served as stimulators, and were cocultivated with
25.times.10.sup.3 preactivated mononuclear cells (LAK-cells, see
above) for 6 days in 96-well-flat bottom tissue culture plates
(NUNC, Copenhagen, Denmark) . In MLTC-experiments, the same culture
medium conditions were employed as during preactivation. In
antisense experiments, TGF-.beta..sub.2-specific phosphorothioate
oligodeoxynucleotides (S-ODN's) and nonsense oligodeoxynucleotides
(see below) were added to the cultures 12 hours before MLTC assay.
Anti-TGF-.beta.neutralizing antibodies (R & D Systems, Inc.
Minneapolis, Minn., USA) were added to the culture 2 hours before
MLTC.
EXAMPLE 5
[0070] Cytotoxicity Assay
[0071] Cytotoxicity experiments were performed with a modified
microcytotoxicity assay (Bogdahn, U. , Fleischer, B., Rupniak, H.
T. R., Ali Osman, F. T-cell mediated cytotoxicity in human glioma
Biology of Brain Tumor, Martinus Nijhoff Publishers, Boston, 70:
501-507, 1986). Briefly, 1.5.times.10.sup.3 target cells were
seeded into 96-well flat bottom tissue culture plates. Twelve hours
after plating, TGF-.beta..sub.2-specific S-ODN's and nonsense
oligodeoxynucleotides (anti-sense-controls) were added to the
culture. Anti-TGF-.beta. neutralizing antibodies and normal rabbit
serum (antibody-controls, R & D Systems, Inc. Minneapolis,
Minn., USA) were added to the culture 22 hours after plating.
Various ratios (target/effector ratio of 1:1, 1:5, 1:10 of
preactivated effector cells (LAK-cells) were irradiated (30 Gy) ,
and added to respective targets 24 hours after plating for 3 days
under standard culture conditions (RPMI 1640 culture medium
containing 10% pooled AB-serum and 2 .mu.M L-Glutamine). No
cytotokines were added to the culture during cytotoxicity
experiments. An incubation period of 3 days was selected, as
statistical evaluation of data turned out to be optimal at this
time point. Killing of target cells was demonstrated by
incorporation of Trypan blue dye (data not presented). Target cell
proliferation in LAK-cell treated targets) was assessed with a
standard .sup.3H-Thymidine incorporation assay
(6-.sup.3H-Thymidine, 1 .mu.Ci/well, spec. Activity 27 Ci/mmol).
Liquid scintillation counting of .sup.3H-thymidine incorporation
was performed after 18 hours of incubation of cells. The specific
cytotoxicity was calculated as:
(cpm.sub.(control)-cpm.sub.(probe)/cpm.sub.(control)).times.100%.
EXAMPLE 6
[0072] Northern and Western Blot Analysis
[0073] Cytoplasmatic RNA was prepared by lysing glioma cells
treated with 1 .mu.M (f.c.) TGF-.beta..sub.2-specific S-ODN's for
48 hours and untreated controls in buffer containing 0.5% NP-40
(Sambrook, J., Fritsch, E. F., Maniatis, T. Molecular cloning. A
laboratory manual, 2nd Edition, Cold Spring Harbor Laboratory
Press. 1989). For Northern hybridization aliquots of 20 .mu.g
denaturated RNA were separated by electrophoresis on 1%
agarose-formaldehyd gel. The quality and quantity of immobilized
RNA was verified by methylene-blue staining of the Hybond-N
membranes (Amersham/Buchler, Braunschweig, Germany) after transfer.
Blots were hybridized overnight with specific TGF-.beta..sub.1- or
TGF-.beta..sub.2-synthetic oligonucleotide probes (40-mer, Oncogen
Science, Seattle, USA) , 5' labeled with (gamma-.sup.32P) -ATP
employing T4 polynucleotide kinase (Pharmacia, Freiburg, Germany)
and exposed to X-ray film.
[0074] For Western blotting, TGF-.beta.-S-ODN treated (48 hours, 1
.mu.M f. c.) resp. untreated glioma cells were grown in medium
containing 10% FCS washed and further cultured in defined serum
free medium for 24 hours. The cells were lysed employing a lysis
buffer containing NP-40. 30 .mu.g of total cellular protein were
loaded onto each lane of a 12% polyacrylamide-SDS gel. Fractionated
proteins were then electroblotted to a nitrocellulose membrane for
20 minutes at 0.8 mA/cm.sup.2 as described (Towbin, H., Staehelin,
T., Gordon, J. Electrophoretic transfer of proteins from PAGE to
nitrocellulose sheets: procedure and some applications, Proc. Natl.
Acad. Sci., USA, 76: 4350-4354, 1979). Filters were probed with a
polyclonal antibody of TGF-.beta..sub.2 (R & D Systems Inc.
Minneapolis, USA) 50 .mu.g of TGF-.beta. served as control.
EXAMPLE 7
[0075] Phosphorothioate Modified Antisense Oligodeocynucleotides
(S-ODN's)
[0076] TGF-.beta..sub.2-specific antisense oligodeoxynucleotides
(antisense direction of TGF-.beta..sub.2 mRNA primer sequene
oligonucleotide seequence: CAGCACACAGTACT) and randomized nonsense
sequence with the same GC-content as the specific S-ODN's (nonsense
oligonucleotide sequence: GTCCCTATACGAAC) were synthesized on an
Applied Biosystems model 380 B DNA Synthesizer (Schlingensiepen, K.
-H., Brysch, W. Phosphorothioate oligomers. Inhibitors of oncogene
expression in tumor cells and tools for gene function analysis in :
Erikson, R., Izant., J. (Eds.) Gene regulation by antisense nucleic
acids. Raven Press New York 1992). S-ODN's were removed from the
solid support with 33% ammonnia. Oligonucleotides still bearing the
5' trityl protecting group were purified by reverse phase HPLC,
with an Aquapore RP-300, C8-column( Brownlee). Solvents: A--0.1 M
TEAA pH 7, B-Acetonitrile. Gradient 3--35% B over 30 Min. linear.
Trityl bearing fraction of oligonucleotides, corresponding to the
full-length product were detritylated in 80% acetic acid/ETOH for
20 Min. extracted twice with diethyl-ether, desalted on a Sephadex
G 25 (Pharmacia) column, ethanol precipitated (2.times.) and
finally diluted in 0.1 M Tris/HCL pH 7.6. S-ODN's were judged from
polyacrylamid-gel-electrophoresis to be more than 85% full-length
material.
EXAMPLE 8
[0077] Characterization of Tumor Cells
[0078] All glioma cell cultures expressed GFAP, TGF-.beta.,
vimentin, and HLA-Class I antigens, as well as
.beta.-microglobulin, fibro-nectin, and KI 67, inconsistent
expression was found with desmin, HLA-Class II antigen (positive:
HTZ-209) and NSE (positive: HTZ-209, HTZ-243). No expression was
found for cytokeratin, laminin and neurofilaments, indicating the
glial origin of these tumor cells.
[0079] Western blot analysis of tumor cell lysates revealed that
HTZ-153, HTZ-209 and HTZ-243 cells produced TGF-.beta..sub.2
protein (FIG. 3).
[0080] Northern blot analysis of cytoplasmatic RNA's from all 3
tumros revealed message for TCF-.beta..sub.1 (2.3 kB) and
TGF-.beta..sub.2 (4.1 kB) (FIGS. 5 and 5): message for
TGF-.beta..sub.1 was fairly well represented in all three tumors
(FIG. 4) , however, tumor HTZ-209 displayed a faint
TGF-.beta..sub.2 signal compared to the remaining tumors (FIG.
5).
EXAMPLE 9
[0081] Modulation of TGF-.beta. Expression by Treatment of Glioma
Cells with TGF-.beta..sub.2 Specific S-ODN's
[0082] The effects of TGF-.beta..sub.2-specific S-ODN-treatment
upon TGF-.beta..sub.2 mRNA- and -protein expression in glioma cells
were analysed by Northern blotting. Western Blotting and
immunocytochemistry. Northern blot analysis of glioma cells treated
with TGF-.beta..sub.2-specific S-ODN's (f.c. 1 .mu.M for 48 hours)
yielded inconsistent results: HTZ-153 displayed an increase in
TGF-.beta..sub.2-message, whereas tumors HTZ-209 and HTZ-243 showed
no detectable message following antisense oligodeoxynucleotides
treatment (FIG. 6). Western blot analysis revealed a decreased
TGF-.beta..sub.2-specific signal for all 3 tumors after S-ODN
treatment (FIG. 3).
[0083] Immunostaining of glioma cultures treated with
TGF-.beta..sub.2-specific S-ODN's (f.c. 1 .mu.M for 48 hours)
revealed a decrease of TGF-.beta.-dependant immunoreactivity
compared to nonsense S-ODN-treated and untreated controls for all 3
tumors. Controls with normal mouse serum and human AB-serum were
negative (slides not presented).
EXAMPLE 10
[0084] Characterization of Lymphocytes
[0085] Autologous effector lymphocytes employed in the following
experiments on tumor dependant lymphocyte proliferation and glioma
cytotoxicity were characterized by conventional lymphocyte
differentiation antigens. Data of characterization experiments are
displayed in table 1, cell populations reflect the phenotype of
lymphocyte subsets of native (Day 0) and activated (Day 6) effector
cells, employed in proliferation and cytotoxicity experiments. The
percentage of CD3.sup.+ cells increased during culture time, up to
85%. The same was true for CD4.sup.+ (up to 80%). CD8.sup.+ (up to
18), CD25.sup.+ (up to 60%)-cells, the fraction of CD16.sup.+ cells
increased to a maximum of 50% (HTZ-243) during the first 6 days of
culture.
EXAMPLE 11
[0086] Cytotoxicity Experiments
[0087] Native PBMC's of tumor-patients investigated in our study
expressed low cytotoxic activity to autologous targets, (below 20%
at target/effector ration 1:10. Preliminary experiments disclosed
that preactivation of autologous effector PBMC's was most
effective, when cells were incubated with 10 U/ML IL-1.alpha. adn
100 U/ml IL-2 for 6 days. These LAK-cells were employed in all
further cytotoxicity/proliferation experiments.
[0088] At a target/effector ration of {fraction (1/10)}, LAK cells
achieved a cytotoxic activity of up to 25% in the autologous target
systems (FIG. 7). Preincubation of tumor cells with neutralizing
TGF-S antibodies (f.c. 100 .mu.g/ml) resulted in a cytotoxicity of
30% -50% (5-30% increase above the untreated controls) (FIG. 7).
When tumor cells were pre-incubated with TGF-.beta..sub.2-specific
antisense S-ODN's cytotoxicity increase in a dose dependent fashion
to a maximum of 79% (5 .mu.M S-ODN's, 25-60% increase above
untreated controls) and 67% (1 .mu.M S-ODNs, 15-45% increase above
untreated autologous lymphocytes. All three effector cell
populations expressed high NK-activity as detected by cytotoxicity
assay against K 562 cell line, ranging from 60% to 75%.
EXAMPLE 12
[0089] Proliferation Experiments
[0090] Lymphocyte proliferation upon stimulation with autologous
tumor cells (MLTC) treated with TGF-.beta..sub.2-specific S-ODNs
was increased in tumors HTZ-153 (FIG. 8a) and HTZ-209 (FIG. 8b),
however, no effect was observed in HTZ-243 cells (FIG. 8c) Nonsense
S-ODN's at a final concentration (f.c.) of 1 .mu.M did not alter
lymphocyte proliferation (FIG. 8). Effects of
TGF-.beta..sub.2-specific S-ODN's were observed in a doese
dependant fashion from 0.1 .mu.M up to 1 .mu.M, higher
concentrations (5 .mu.M) displayed non-specific toxicity towards
PBMC's and tumor cells (FIG. 8): the proliferation of PBMC's in
S-ODN treated MLTC's and tumor cells (FIG. 8): the proliferation of
PBMC's in S-ODN treated MLTC's was persistently lower for
oligonucleotide concentrations above 1 .mu.M. High concentrations
of neutralizing TGF-.beta. antibody (100 .mu.g/ml) did not enhance
lymphocyte proliferation. TGF-.beta..sub.2-specific antisense
S-ODN's had an inhibitory effect upon proliferation of either
cultured lymphocyte populations (marginal effect) or autologous
target cells (FIG. 8) achieving a maximum of 75% at a S-ODN's
concentration of 5 .mu.M (f.c.). Less profound inhibitory effects
were observed with randomized control nonsense S-ODN's (average
20%, up to 40% at 5 .mu.M f.c.).
Sequence CWU 1
1
* * * * *