U.S. patent application number 13/821669 was filed with the patent office on 2013-11-21 for sdf-1 binding nucleic acids and the use thereof in cancer treatment.
This patent application is currently assigned to NOXXON PHARMA AG. The applicant listed for this patent is Klaus Buchner, Nicole Dinse, Dirk Eulberg, Florian Jarosch, Sven Klussmann, Christian Maasch, Werner Purschke, Dirk Zboralski. Invention is credited to Klaus Buchner, Nicole Dinse, Dirk Eulberg, Florian Jarosch, Sven Klussmann, Christian Maasch, Werner Purschke, Dirk Zboralski.
Application Number | 20130310442 13/821669 |
Document ID | / |
Family ID | 44653255 |
Filed Date | 2013-11-21 |
United States Patent
Application |
20130310442 |
Kind Code |
A1 |
Purschke; Werner ; et
al. |
November 21, 2013 |
SDF-1 Binding Nucleic Acids and the use Thereof in Cancer
Treatment
Abstract
The present invention is related to a nucleic acid molecule
capable of binding to SDF-1, preferably capable of inhibiting
SDF-1, whereby the nucleic acid molecule is for use in a method for
the treatment and/or prevention of a disease or disorder, for use
in a method for the treatment of a subject suffering from a disease
or disorder or being at risk of developing a disease or disorder as
an adjunct therapy, or for use as a medicament for the treatment
and/or prevention of a disease or disorder, whereby the disease or
disorder is cancer.
Inventors: |
Purschke; Werner; (Berlin,
DE) ; Jarosch; Florian; (Berlin, DE) ;
Eulberg; Dirk; (Berlin, DE) ; Klussmann; Sven;
(Berlin, DE) ; Buchner; Klaus; (Berlin, DE)
; Maasch; Christian; (Berlin, DE) ; Dinse;
Nicole; (Berlin, DE) ; Zboralski; Dirk;
(Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purschke; Werner
Jarosch; Florian
Eulberg; Dirk
Klussmann; Sven
Buchner; Klaus
Maasch; Christian
Dinse; Nicole
Zboralski; Dirk |
Berlin
Berlin
Berlin
Berlin
Berlin
Berlin
Berlin
Berlin |
|
DE
DE
DE
DE
DE
DE
DE
DE |
|
|
Assignee: |
NOXXON PHARMA AG
Berlin
DE
|
Family ID: |
44653255 |
Appl. No.: |
13/821669 |
Filed: |
September 9, 2011 |
PCT Filed: |
September 9, 2011 |
PCT NO: |
PCT/EP2011/004554 |
371 Date: |
August 5, 2013 |
Current U.S.
Class: |
514/44R ;
536/23.1 |
Current CPC
Class: |
C12N 15/115 20130101;
A61K 31/7076 20130101; C12N 2320/31 20130101; A61K 31/69 20130101;
A61P 35/02 20180101; A61K 31/7105 20130101; C12N 2320/30 20130101;
A61K 45/06 20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61K
35/12 20130101; C12N 2310/351 20130101; A61K 31/7088 20130101; A61K
31/713 20130101; C12N 2310/16 20130101; C12N 2310/30 20130101; A61K
31/7088 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/44.R ;
536/23.1 |
International
Class: |
A61K 31/7105 20060101
A61K031/7105; A61K 31/69 20060101 A61K031/69; A61K 35/12 20060101
A61K035/12 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 9, 2010 |
EP |
10009379.0 |
Claims
1-10. (canceled)
107. A nucleic acid molecule capable of binding to SDF-1,
preferably capable of inhibiting SDF-1, whereby the nucleic acid
molecule is for use in a method for the treatment and/or prevention
of a disease or disorder, for use in a method for the treatment of
a subject suffering from a disease or disorder or being at risk of
developing a disease or disorder as an adjunct therapy, or for use
as a medicament for the treatment and/or prevention of a disease or
disorder, whereby the disease or disorder is cancer.
108. The nucleic acid molecule according to claim 107, hereby the
cancer is a cancer selected from the group of hematological cancer
and solid tumors, whereby preferably the hematological cancer is
selected from the group comprising leukemia and myeloma and the
solid tumors are selected from the group comprising glioblastoma,
colorectal cancer, breast cancer, lymphoma, prostate cancer,
pancreatic cancer, renal cancer, ovarian cancer and lung
cancer.
109. The nucleic acid molecule according to claim 107 or 108,
whereby the adjunct therapy sensitizes the subject, wherein the
sensitized subject is more responsive to a therapy for the
treatment and/or prevention of the disease or disorder.
110. The nucleic acid according, to claim 109, whereby the therapy
for the treatment and/or prevention of the disease or disorder
comprises the administration of a further pharmaceutically active
agent and/or irradiating the subject and/or surgery and/or cellular
therapy.
111. The nucleic acid molecule according to claim 110, whereby the
further pharmaceutically active agent is selected from the group
comprising of an antibody, an alkylating agent, an anti-metabolite,
a plant alkaloid, a plant terpenoid, a topoisomerase inhibitor,
Leucovorin, Methotrexate, Tamoxifen, Sorafenib, Lenalidomide,
Bortezomib, Dexamethasone, Fluorouracil, and Prednisone.
112. The nucleic acid molecule according to claim 107, whereby the
nucleic acid molecule is capable of blocking the interaction
between SDF-1 and an SDF-1 receptor, whereby the SDF-1 receptor is
selected from the group comprising CXCR4 and CXCR7.
113. The nucleic acid molecule according to claim 107, whereby the
treatment or prevention of the disease or disorder is caused by the
nucleic acid molecule inhibiting the interaction between SDF-1 and
an SDF-1 receptor.
114. The nucleic acid according to claim 107, comprising in
5'.fwdarw.3' direction a first terminal stretch of nucleotides, a
central stretch of nucleotides, and a second terminal stretch of
nucleotides, or a second terminal stretch of nucleotides, the
central stretch of nucleotides, and a first terminal stretch of
nucleotides, whereby the nucleic acid molecule is selected from the
group comprising an SDF-1 binding nucleic acid molecule of type B,
an SDF-1 binding nucleic acid molecule of type C, an SDF-1 binding
nucleic acid molecule of type A and an SDF-1 binding nucleic acid
molecule of type D; whereby the central stretch of nucleotides of
the SDF-1 binding nucleic acid molecule of type B comprises the
nucleotide sequence: 5' GUGUGAUCUAGAUGUADWGGCUGWUCCUAGUYAGG 3' (SEQ
ID NO:52), and whereby the first terminal stretch of nucleotides of
the SDF-1 binding nucleic acid molecule of type B comprises a
nucleotide sequence of 5' X.sub.1X.sub.2SVNS 3' and the second
terminal stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type B comprises a nucleotide sequence of 5'
BVBSX.sub.3X.sub.4 3', whereby X.sub.1 is either absent or is A,
X.sub.2 is G, X.sub.3 is C and X.sub.4 is either absent or is U; or
X.sub.1 is absent, X.sub.2 is either absent or is G, X.sub.3 is
either absent or is C and X.sub.4 is absent; whereby the central
stretch of nucleotides of the SDF-1 binding nucleic acid molecule
of type C comprises the following nucleotide sequence:
GGUYAGGGCUHRXAAGUCGG (SEQ ID NO:108), whereby XA is either absent
or is A, and whereby the first terminal stretch of nucleotides of
the SDF-1 binding nucleic acid molecule of type C comprises a
nucleotide sequence of 5' RKSBUSNVGR 3' (SEQ ID NO:138) and the
second stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type C comprises a nucleotide sequence of 5' YYNRCASSMY
3' (SEQ. ID. NO:139); or whereby the first terminal stretch of
nucleotides of the SDF-1 binding nucleic acid molecule of type C
comprises a nucleotide sequence of 5' XSSSSV 3' and the second
terminal stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type C comprises a nucleotide sequence of 5 BSSSXS 3',
whereby XS is either absent or is S, or whereby the first terminal
stretch of nucleotides of the SDF-1 binding nucleic acid molecule
of type C comprises a nucleotide sequence of 5' CGUGCGCUUGAGAUAGG
3' (SEQ ID NO:220) and the second terminal stretch of nucleotides
of the SDF-1 binding nucleic acid molecule of type C comprises a
nucleotide sequence of 5' CUGAUUCUCACG 3' (SEQ ID NO:221); or the
first terminal stretch of nucleotides of the SDF-1 binding nucleic
acid molecule of type C' comprises a nucleotide sequence of 5'
UGAGAUAGG 3' and the second terminal stretch of nucleotides of the
SDF-1 binding nucleic acid molecule of type C comprises a
nucleotide sequence of 5' CUGAUUCUCA 3' (SEQ ID NO:222); or the
first terminal stretch of nucleotides of the SDF-1 binding nucleic
acid molecule of type C comprises a nucleotide sequence of 5'
GAGAUAGG 3' and the second terminal stretch of nucleotides of the
SDF-1 binding nucleic acid molecule of type C comprises a
nucleotide sequence of 5' CUGAUUCUC 3'; whereby the central stretch
of nucleotides of SDF-1 binding nucleic acid molecule of type A,
comprises a nucleotide sequence of 5' AAAGYRACAHGUMAAXAUGAAAGGUARC
3' (SEQ ID NO:74), whereby XA is either absent or is A, and whereby
the first terminal stretch of nucleotides SDF-1 binding nucleic
acid molecule of type A comprises a nucleotide sequence of 5'
X.sub.1X.sub.2NNBV 3' and the second terminal stretch of
nucleotides SDF-1 binding nucleic acid molecule of type A comprises
a nucleotide sequence of 5' BNBNX.sub.3X.sub.4 3' whereby X.sub.1
is either absent or R, X.sub.2 is S, X.sub.3 is S and X.sub.4 is
either absent or Y, or X.sub.1 is absent, X.sub.2 is either absent
or S, X.sub.3 is either absent or S and X.sub.4 is absent; and
whereby the SDF-1 binding nucleic acid molecule of type D comprises
a nucleotide sequence according to any one of SEQ ID NO:142 to SEQ
ID NO: 144.
115. The nucleic acid molecule according: to claim 114, whereby the
central stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type B comprises the following nucleotide sequence: 5'
GUGUGAUCUAGAUGUADUGGCUGAUCCUAGUCAGG 3' (SEQ ID NO:53).
116. The nucleic acid molecule according to claim 113 or 114,
whereby the first terminal stretch of nucleotides of the SDF-1
binding nucleic acid molecule of type B comprises a nucleotide
sequence of 5' X.sub.1X.sub.2SSBS 3' and the second terminal
stretch of nucleotides of the SDF-1 binding nucleic acid molecule
of type B comprises a nucleotide sequence of 5' BVSSX.sub.3X.sub.4
3', whereby X.sub.1 is absent, X.sub.2 is either absent or G,
X.sub.3 is either absent or C, and X.sub.4 is absent, preferably
the first terminal stretch of nucleotides comprises a nucleotide
sequence of 5' GCGUG 3' and the second terminal stretch of
nucleotides comprises a nucleotide sequence of 5' UACGC 3'.
117. The nucleic acid molecule according, to claim 116, whereby the
SDF-1 binding nucleic acid molecule of type B comprises a
nucleotide sequence according to any one of SEQ ID NO:5 to SEQ ID
NO:20 and SEQ ID NO:22 to SEQ ID NO:28, preferably any one of SEQ
ID NO:5 to SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:22 and SEQ ID
NO:28, more preferably any one of SEQ ID NO:22 and SEQ ID
NO:28.
118. The nucleic acid molecule according to claim 114, whereby the
central stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type C comprises a nucleotide sequence of 5'
GGUYAGGGCUHRAAGUCGG 3' (SEQ ID NO:109), 5' GGUYAGGGCUHRAGUCGG 3'
(SEQ ID NO:110) or 5' GGUUAGGGCUHGAAGUCGG 3' (SEQ ID NO:111),
preferably 5' GGUUAGGGCUHGAAGUCGG 3' (SEQ ID NO:111).
119. The nucleic acid molecule according to claim 114 or 118,
whereby the first terminal stretch of nucleotides of the SDF-1
binding nucleic acid molecule of type C comprises a nucleotide
sequence of 5' RKSBUGSVGR 3' (SEQ ID NO:140) and the second
terminal stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type C comprises a nucleotide sequence of 5' YCNRCASSMY
3' (SEQ ID NO:141).
120. The nucleic acid molecule according to claim 114 or 118,
whereby the first terminal stretch of nucleotides of the SDF-1
binding nucleic acid molecule of type C comprises a nucleotide
sequence of 5' SGGSR 3' and the second terminal stretch of
nucleotides of the SDF-1 binding nucleic acid molecule of type C
comprises a nucleotide sequence of 5' YSCCS 3'.
121. The nucleic acid molecule according to claim 120, whereby the
SDF-1 binding nucleic acid molecule of type C comprises a
nucleotide sequence according to any one of SEQ ID NO:95 to SEQ ID
NO:107, SEQ ID NO: 112 to SEQ ID NO:137, SEQ ID NO:223 and SEQ ID
NO:224, preferably any one of SEQ NO:120, SEQ NO:128, SEQ ID
NO:129, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:223 and SEQ ID
NO:224.
122. The nucleic acid molecule according to claim 114, whereby the
central stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type A comprises a nucleotide sequence of 5'
AAAGYRACAHGUMAAUGAAAGGUARC 3' (SEQ ID NO:75), or 5'
AAAGYRACAHGUMAAAUGAAAGGUARC 3' (SEQ ID NO:76), or 5'
AAAGYAACAHGUCAAUGAAAGGUARC 3' (SEQ ID NO:77), preferably the
central stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type A comprises a nucleotide sequence of 5'
AAAGYAACAHGUCAAUGAAAGGUARC 3' (SEQ ID NO: 77).
123. The nucleic acid molecule according to claim 114 or 122,
whereby the first terminal stretch of nucleotides of the SDF-1
binding nucleic acid molecule of type A comprises a nucleotide
sequence of 5' X.sub.2BBBS 3' and the second terminal stretch of
nucleotides of the SDF-1 binding nucleic acid molecule of type A
comprises a nucleotide sequence of 5' SBBVX.sub.3 3', whereby
X.sub.2 is either absent or is S and X.sub.3 is either absent or is
S; preferably the first terminal stretch of nucleotides of the
SDF-1 binding nucleic acid molecule of type A comprises a
nucleotide sequence of 5' CUGUG 3' and the second terminal stretch
of nucleotides of the SDF-1 binding nucleic acid molecule of type A
comprises a nucleotide sequence of 5' CGCAG 3'; or the first
terminal stretch of nucleotides of the SDF-1 binding nucleic acid
molecule of type A comprises a nucleotide sequence of 5' GCGUG 3'
and the second terminal stretch of nucleotides of the SDF-1 binding
nucleic acid molecule of type A comprises a nucleotide sequence of
5' CGCGC 3'.
124. The nucleic acid molecule according to claim 123, whereby the
SDF-1 binding nucleic acid molecule of type A comprises a
nucleotide sequence according to any one of SEQ ID NO:60 to SEQ ID
NO:73, SEQ ID NO:78 to SEQ ID NO:82, SEQ ID NO:84 to SEQ ID NO:87,
SEQ 11D NO:89 to SEQ ID NO:94, and SEQ ID NO:145, preferably any
one of SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:78, SEQ
ID NO:84, and SEQ ID NO:146, more preferably any one of SEQ ID
NO:84 and SEQ ID NO:146.
125. The nucleic acid molecule according to claim 107, whereby the
nucleic acid molecule comprises a modification, whereby the
modification is preferably a high molecular weight moiety and/or
whereby the modification preferably allows to modify the
characteristics of the nucleic acid molecule in terms of residence
time in the animal or human body, preferably the human body.
126. The nucleic acid molecule according to claim 107, whereby the
nucleic acid molecule is an L-nucleic acid molecule.
127. A pharmaceutical composition comprising as a first
pharmaceutically active agent the nucleic acid molecule according
to claim 107 and optionally a further constituent, whereby the
further constituent is selected from the group comprising a
pharmaceutically acceptable excipient, a pharmaceutically
acceptable carrier and a further pharmaceutically active agent, and
whereby the pharmaceutical composition is for use in a method for
the treatment and/or prevention of a disease or disorder, or for
use in a method for the treatment of a subject suffering from a
disease or disorder or being at risk of developing a disease or a
disorder as an adjunct therapy, or for the treatment and/or
prevention of a disease or disorder, whereby the disease or
disorder is cancer.
128. The pharmaceutical composition according to claim 127, whereby
the adjunct therapy sensitizes the subject, wherein the sensitized
subject is more responsive to a therapy for the treatment and/or
prevention of the disease or disorder.
129. The pharmaceutical composition according to claim 128, whereby
the therapy for the treatment and/or prevention of the diseases or
disorder comprises the administration of a further pharmaceutically
active agent and/or irradiating the subject and/or surgery and/or
cellular therapy.
130. The pharmaceutical composition according to any one of claims
127 to 129, whereby the further pharmaceutically active agent is a
pharmaceutically active agent selected from the group comprising an
antibody, an alkylating agent, an anti-metabolite, a plant
alkaloid, preferably vincristine, a plant terpenoid, a
topoisomerase inhibitor, Leucovorin, Methotrexate, Tamoxifen,
Sorafenib, Lenalidomide, Bortezomib, Dexamethasone, Flurouracil,
and Prednisone.
131. The pharmaceutical composition according to claim 130, whereby
the cancer is a cancer selected from the group of hematological
cancer and solid tumors, whereby preferably the hematological
cancer is selected from the group of leukemia and myeloma and the
solid tumors are selected from the group comprising glioblastoma,
colorectal cancer, breast cancer, lymphoma, prostate cancer,
pancreatic cancer, renal cancer, ovarian cancer and lung
cancer.
132. A medicament comprising one or several dosage units of at
least a first pharmaceutically active agent, wherein the first
pharmaceutically active agent is a nucleic acid molecule capable of
binding to SDF-1 as defined in claim 107, whereby the medicament is
for use in a method for the treatment and/or prevention of a
disease or disorder, or for use in a method for the treatment of a
subject suffering from a disease or disorder or being at risk of
developing a disease or a disorder as an adjunct therapy, or for
the treatment and/or prevention of a disease or disorder, whereby
the disease or disorder is cancer.
133. The medicament according to claim 132, whereby the adjunct
therapy sensitizes the subject, wherein the sensitized subject is
more responsive to a therapy for the treatment and/or prevention of
the disease or disorder.
134. The medicament according to claim 133, whereby the therapy for
the treatment and/or prevention of the diseases or disorder
comprises the administration of a further pharmaceutically active
agent and/or irradiating the subject and/or surgery and/or cellular
therapy.
135. The medicament according to any of claims 132 to 134, wherein
the medicament comprises a further pharmaceutically active agent,
preferably one or several dosage units of a further
pharmaceutically active agent, whereby the further pharmaceutically
active agent is selected from the group comprising an antibody, an
alkylating agent, an anti-metabolite, a plant alkaloid, preferably
vincristine, a plant terpenoid, a topoisomerase inhibitor,
Leucovorin, Methotrexate, Tamoxifen, Sorafenib, Lenalidomide,
Bortezomib, Dexamethasone, Fluorouracil, and Prednisone.
136. The medicament according to claim 135, wherein the cancer is a
cancer selected from the group of hematological cancer and solid
tumors, whereby preferably the hematological cancer is selected
from the group comprising leukemia and myeloma and the solid tumors
are selected from the group comprising glioblastoma, colorectal
cancer, breast cancer, lymphoma, prostate cancer, pancreatic
cancer, renal cancer, ovarian cancer and lung cancer.
137. A method for the treatment of as subject suffering from or
being at risk of developing cancer, whereby the method comprises a)
administering to the subject a pharmaceutically effective amount of
a nucleic acid molecule capable of binding, to SDF-1 as defined in
claim 107.
138. The method according to claim 137, whereby the method further
comprises b) irradiating the subject and/or surgery and/or cellular
therapy and/or administering a pharmaceutically effective amount of
a further pharmaceutically active agent to the subject, whereby the
further pharmaceutically active agent is a pharmaceutically active
agent selected from the group comprising an antibody, an alkylating
agent, an anti-metabolite, a plant alkaloid, preferably
vincristine, a plant terpenoid, a topoisomerase inhibitor,
Leucovorin, Methotrexate, Tamoxifen, Sorafenib, Lenalidomide,
Bortezomib, Dexamethasone, Flurouracil, and Prednisone.
139. The method according to claim 138, wherein the
pharmaceutically effective amount of a nucleic acid molecule
capable of binding to SDF-1 is administered as an adjunct therapy
or part of an adjunct therapy.
140. The method according to claim 139, whereby the adjunct therapy
sensitizes the subject, wherein the sensitized subject is more
responsive to a therapy for the treatment and/or prevention of the
disease or disorder.
141. The method according to claim 140, whereby the therapy for the
treatment and/or prevention of the disease or disorder comprises
the administration of a further pharmaceutically active agent
and/or irradiating the subject and/or surgery and/or cellular
therapy as performed in step b).
142. The method according to any one of claims 137 to 141, whereby
the cancer is a cancer selected from the group of hematological
cancer and solid tumors, whereby preferably the hematological
cancer is selected from the group comprising leukemia and myeloma
and the solid tumors are selected from the group comprising
glioblastoma, colorectal cancer, breast cancer, lymphoma, prostate
cancer, pancreatic cancer, renal cancer, ovarian cancer and lung
cancer.
Description
[0001] The present invention is related to nucleic acid molecules
binding to the CXC chemokine stromal cell-derived factor-1 (SDF-1),
methods for the treatment of cancer, and their use in the
manufacture of a medicament.
[0002] Stromal-cell derived factor-1 (abbr.: SDF-1; synonyms,
CXCL12; PBSF [pre-B-cell growth-stimulating factor]; TPAR-1 [TPA
repressed gene 1]; SCYB12; TLSF [thymic lymphoma cell stimulating
factor]; hIRH [human intercrine reduced in hepatomas]) is an
angiogenic CXC chemokine that does not contain the ELR motif
typical of the IL-8-like chemokines (Salcedo, Wasserman et al.
1999; Salcedo and Oppenheim 2003) but binds and activates the
G-protein coupled receptor CXCR4. As a result of alternative
splicing, there are two forms of SDF-1, SDF-1.alpha. (68 amino
acids, SEQ ID NO: 1) and SDF-113 (SEQ ID NO: 2), which, compared to
SDF-1.alpha. carries five additional amino acids at the C-terminus
(Shirozu, Nakano et al. 1995).
[0003] The amino acid sequence conservation between SDF-1 from
different species is remarkable: human SDF-1.alpha. (SEQ. ID. 1)
and murine SDF-1.alpha. (SEQ ID NO: 3) are virtually identical.
There is only a single conservative change of V to I at position 18
(Shirozu, Nakano et al. 1995).
[0004] Since the SDF-1 receptor CXCR4 is widely expressed on
leukocytes, mature dendritic cells, endothelial cells, brain cells,
and megakaryocytes, the activities of SDF-1 are pleiotropic. This
chemokine, more than any other identified thus far, exhibits the
widest range of biological functions. The most significant
functional effects of SDF-1 are: [0005] Homing and attachment of
epithelial cells to neovascular sites in the choroid portion of the
retina; [0006] SDF-1 is required to maintain stem cells and
progenitor cells, e.g. hematopoietic progenitor (usually CD34+)
cells in the bone marrow of the adult; [0007] SDF-1 supports
proliferation of pre-B cells and augments the growth of bone marrow
B cell progenitors and it induces specific migration of pre- and
pro-B cells, while not acting as a significant chemoattractant for
mature B cells; [0008] SDF-1 is one of the most efficacious T cell
chemoattractants; and [0009] SDF-1 and its receptor CXCR4 are
essential for embryonic development.
[0010] Altered expression levels of SDF-1 or its receptor CXCR4 or
altered responses towards those molecules are said to be associated
with many human diseases, such as retinopathy (Brooks, Caballero et
al. 2004; Butler, Guthrie et al. 2005; Meleth, Agron et al. 2005);
cancer of breast (Muller, Homey et al. 2001; Cabioglu, Sahin et al.
2005), ovaries (Scotton, Wilson et al. 2002), pancreas (Koshiba,
Hosotani et al. 2000), thyroid (Hwang, Chung et al. 2003)
andnasopharynx (Wang, Wu et al. 2005); glioma (Zhou, Larsen et al.
2002); neuroblastoma (Geminder, Sagi-Assif et al. 2001); B cell
chronic lymphocytic leukemia (Burger, Tsukada et al. 2000); WHIM
syndrome (WHIM is an abbreviation for Warts, Hypogammaglobulinemia,
Infections, Myelokathexis syndrome) (Gulino, Moratto et al. 2004;
Balabanian, Lagane et al. 2005b; Kawai, Choi et al. 2005);
immunologic deficiency syndromes (Arya, Ginsberg et al. 1999;
Marechal, Arenzana-Seisdedos et al. 1999; Soriano, Martinez et al.
2002); pathologic neovascularization (Salvucci, Yao et al. 2002;
Yamaguchi, Kusano et al. 2003; Grunewald, Avraham et al. 2006);
inflammation (Murdoch 2000; Fedyk, Jones et al. 2001; Wang, Guan et
al. 2001); multiple sclerosis (Krumbholz, Theil et al. 2006);
rheumatoid arthritis/osteoarthritis (Buckley, Amft et al. 2000;
Kanbe, Takagishi et al. 2002; Grassi, Cristino et al. 2004).
[0011] Tumors (including solid and hematological neoplasias and
malignancies) are not just masses of cancer cells: infiltration of
tumors with immune-cells is a characteristic of cancer. Many human
cancers have a complex chemokine network that influences the extent
and phenotype of this infiltrate, as well as tumor growth,
survival, migration, and angiogenesis. Most solid tumors contain
many non-malignant stromal cells. Indeed, stromal cells sometimes
outnumber cancer cells. The predominant stromal cells that are
found in cancers are macrophages, lymphocytes, endothelial cells
and fibroblasts.
[0012] Cells from different cancer types have different profiles of
chemokine-receptor expression, but the SDF-1 receptor CXCR4 is most
commonly found in tumor cells of mouse and man: tumor cells from at
least 23 different types of human cancers of epithelial,
mesenchymal, and haematopoietic origin express CXCR4 (Balkwill
2004) with SDF-1 being the only known ligand for CXCR4. Apart from
the bone marrow and secondary lymphoid tissue, where it is
constitutively expressed, SDF-1 is found in primary tumor sites in
lymphoma (Corcione, Ottonello et al. 2000) and brain tumors of both
neuronal and astrocytic lineage. Furthermore, it is present at high
levels in ovarian (Scotton, Wilson et al. 2002) and pancreatic
cancer (Koshiba, Hosotani et al. 2000) as well as at sites of
metastasis in breast (Muller, Homey et al. 2001) and thyroid cancer
(Hwang, Chung et al. 2003), neuroblastoma and haematological
malignancies (Geminder, Sagi-Assif et al. 2001).
[0013] Besides CXCR4 another SDF-1 receptor was identified:
RDC1/CXCR7 (Balabanian, Lagane et al. 2005a, Burns, Summers et al.
2006). In vitro and in vivo studies with prostate cancer cell lines
suggest that alterations in CXCR7/RDC1 expression are associated
with enhanced adhesive and invasive activities in addition to a
survival advantage. In vitro and in vivo studies have shown that
both receptors for SDF-1, namely CXCR4 and the CXCR7 promote tumor
growth, metastatic potential and resistance to (chemotherapy
induced) apoptosis in a number of tumors, e.g breast cancer,
glioblastomas, ovarian cancer, neuroblastoma, lung cancer
colorectal and prostate cancer (Burns et al, 2006; Li et al, 2008;
Scotton et al, 2002; Yang et al, 2008; Zagzag et al, 2008).
[0014] CXCR4 and CXCR7 expression thus seems to be a general
characteristic of several tumours.
[0015] The problem underlying the present invention is to provide a
means which specifically interacts with SDF-1, whereby the means
are suitable for the prevention and/or treatment of and/or
cancer.
[0016] Another problem underlying the present invention is to
provide a means which supports the therapy of cancer, whereby such
therapy of cancer typically makes use of chemotherapy and/or
radiation.
[0017] A further problem underlying the present invention is to
provide a means which is suitable for use an adjunct therapy in the
treatment of cancer.
[0018] A still further problem underlying the present invention is
to provide a means which is capable of chemosensitizing patient
suffering cancer and/or chemosensitizing cells forming or being
part of a cancer.
[0019] These and other problems underlying the present invention
are solved by the subject matter of the attached independent
claims. Preferred embodiments may be taken from the dependent
claims.
[0020] More specifically, the problem underlying the present
invention is solved in a first aspect which is also the first
embodiment of the first aspect, by a nucleic acid molecule capable
of binding to SDF-1, preferably capable of inhibiting SDF-1,
whereby the nucleic acid molecule is for use in a method for the
treatment and/or prevention of a disease or disorder, for use in a
method for the treatment of a subject suffering from a disease or
disorder or being at risk of developing a disease or disorder as an
adjunct therapy, or for use as a medicament for the treatment
and/or prevention of a disease or disorder, whereby the disease or
disorder is cancer.
[0021] In a second embodiment of the first aspect which is also an
embodiment of the first embodiment of the first aspect, the cancer
is a cancer selected from the group of hematological cancer,
whereby preferably the hematological cancer is selected from the
group comprising leukemia and myeloma.
[0022] In a third embodiment of the first aspect which is also an
embodiment of the second embodiment of the first aspect, leukemia
is selected from the group comprising chronic lymphoid leukemia and
acute myeloid leukemia.
[0023] In a fourth embodiment of the first aspect which is also an
embodiment of the second embodiment of the first aspect, myeloma is
multiple myeloma.
[0024] In a fifth embodiment of the first aspect which is also an
embodiment of the first embodiment of the first aspect, the cancer
is a cancer selected from the group of solid tumors, whereby
preferably the solid tumors are selected from the group comprising
glioblastoma, colorectal cancer, breast cancer, lymphoma, prostate
cancer, pancreatic cancer, renal cancer, ovarian cancer and lung
cancer.
[0025] In a sixth embodiment of the first aspect which is also an
embodiment of the first, the second, the third, the fourth and the
fifth embodiment of the first aspect, the adjunct therapy
sensitizes the subject, wherein the sensitized subject is more
responsive to a therapy for the treatment and/or prevention of the
disease or disorder.
[0026] In a seventh embodiment of the first aspect which is also an
embodiment of the sixth embodiment of the first aspect, the therapy
for the treatment and/or prevention of the diseases or disorder
comprises the administration of a further pharmaceutically active
agent and/or irradiating the subject and/or surgery and/or cellular
therapy.
[0027] In an eighth embodiment of the first aspect which is also an
embodiment of the seventh embodiment of the first aspect, the
further pharmaceutically active agent is selected from the group
comprising of an antibody, an alkylating agent, an anti-metabolite,
a plant alkaloid, a plant terpenoid, a topoisomerase inhibitor,
Leucovorin, Methotrexate, Tamoxifen, Sorafenib, Lenalidomide,
Bortezomib, Dexamethasone, Fluorouracil, and Prednisone.
[0028] In a ninth embodiment of the first aspect which is also an
embodiment of the eighth embodiment of the first aspect, the
antibody is selected from the group comprising Rituximab,
Ofatumumab, Cetuximab, Ibritumomab-Tiuxetan, Tositumomab,
Trastuzumab, Bevacizumab, and Alemtuzumab.
[0029] In a tenth embodiment of the first aspect which is also an
embodiment of the eighth embodiment of the first aspect, the
alkylating agent is selected from the group comprising cisplatin,
carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,
chlorambucil, doxorubicin, lioposomal doxorubicin, bendamustine,
temozolomide and Melphalan.
[0030] In an eleventh embodiment of the first aspect which is also
an embodiment of the eighth embodiment of the first aspect, the
anti-metabolite is selected from the group comprising
purineazathioprine, mercaptopurine, fludarabine, pentostatin, and
cladribine.
[0031] In a twelfth embodiment of the first aspect which is also an
embodiment of the eighth embodiment of the first aspect, the plant
terpenoid is selected from the group comprising a taxane more
preferably selected from the group comprising Docetaxel,
Paclitaxel, podophyllotoxin and epothilone.
[0032] In a thirteenth embodiment of the first aspect which is also
an embodiment of the eighth embodiment of the first aspect, the
topoisomerase inhibitor is selected from the group comprising
camptothecin, irinotecan, and mitoxantrone.
[0033] In a fourteenth embodiment of the first aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth, the tenth,
the eleventh, the twelfth and the thirteenth embodiment of the
first aspect, the nucleic acid molecule is capable of blocking the
interaction between SDF-1 and an SDF-1 receptor, whereby the SDF-1
receptor is selected from the group comprising CXCR4 and CXCR7.
[0034] In a fifteenth embodiment of the first aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth, the tenth,
the eleventh, the twelfth, the thirteenth and the fourteenth
embodiment of the first aspect, the treatment or prevention of the
disease or disorder is caused by the nucleic acid molecule
inhibiting the interaction between SDF-1 and an SDF-1 receptor.
[0035] In a sixteenth embodiment of the first aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth, the tenth,
the eleventh, the twelfth, the thirteenth, the fourteenth and the
fifteenth embodiment of the first aspect, the nucleic acid molecule
is selected from the group comprising an SDF-1 binding nucleic acid
molecule of type B, an SDF-1 binding nucleic acid molecule of type
C, an SDF-1 binding nucleic acid molecule of type A and an SDF-1
binding nucleic acid molecule of type D.
[0036] In a seventeenth embodiment of the first aspect which is
also an embodiment of the sixteenth embodiment of the first aspect,
the SDF-1 binding nucleic acid molecule of type B comprises a
central stretch of nucleotides, whereby the central stretch of
nucleotides comprises the following nucleotide sequence:
TABLE-US-00001 (SEQ ID NO: 52) 5'
GUGUGAUCUAGAUGUADWGGCUGWUCCUAGUYAGG 3'.
[0037] In an eighteenth embodiment of the first aspect which is
also an embodiment of the seventeenth embodiment of the first
aspect, the central stretch of nucleotides comprises the following
nucleotide sequence:
TABLE-US-00002 (SEQ ID NO: 53) 5'
GUGUGAUCUAGAUGUADUGGCUGAUCCUAGUCAGG 3'.
[0038] In a nineteenth embodiment of the first aspect which is also
an embodiment of the seventeenth and the eighteenth embodiment of
the first aspect, the SDF-1 binding nucleic acid molecule of type B
comprises in 5'->3' direction a first terminal stretch of
nucleotides, the central stretch of nucleotides, and a second
terminal stretch of nucleotides.
[0039] In a twentieth embodiment of the first aspect which is also
an embodiment of the seventeenth and the eighteenth embodiment of
the first aspect, the SDF-1 binding nucleic acid molecule of type B
comprises in 5'->3' direction a second terminal stretch of
nucleotides, the central stretch of nucleotides, and a first
terminal stretch of nucleotides.
[0040] In a twenty-first embodiment of the first aspect which is
also an embodiment of the nineteenth and the twentieth embodiment
of the first aspect, the first terminal stretch of nucleotides
comprises a nucleotide sequence of 5' X.sub.1X.sub.2SVNS 3' and the
second terminal stretch of nucleotides comprises a nucleotide
sequence of 5' BVBSX.sub.3X.sub.4 3', whereby
X.sub.1 is either absent or is A, X.sub.2 is G, X.sub.3 is C and
X.sub.4 is either absent or is U; or X.sub.1 is absent, X.sub.2 is
either absent or is G, X.sub.3 is either absent or is C and X.sub.4
is absent.
[0041] In a twenty-second embodiment of the first aspect which is
also an embodiment of the nineteenth, the twentieth and the
twenty-first, preferably the twenty-first, embodiment of the first
aspect, the first terminal stretch of nucleotides comprises a
nucleotide sequence of 5' X.sub.1X.sub.2CRWG 3' and the second
terminal stretch of nucleotides comprises a nucleotide sequence of
5' KRYSX.sub.3X.sub.4 3',
whereby X.sub.1 is either absent or A, X.sub.2 is G, X.sub.3 is C
and X.sub.4 is either absent or U.
[0042] In a twenty-third embodiment of the first aspect which is
also an embodiment of the nineteenth, the twentieth, the
twenty-first and the twenty-second, preferably the twenty-first or
the twenty-second embodiment of the first aspect, the first
terminal stretch of nucleotides comprises a nucleotide sequence of
5' X.sub.1X.sub.2CGUG 3' and the second terminal stretch of
nucleotides comprises a nucleotide sequence of 5'
UACGX.sub.3X.sub.4 3',
whereby X.sub.1 is either absent or A, X.sub.2 is G, X.sub.3 is C,
and X.sub.4 is either absent or U, preferably the first terminal
stretch of nucleotides comprises a nucleotide sequence of 5' AGCGUG
3' and the second terminal stretch of nucleotides comprises a
nucleotide sequence of 5' UACGCU 3'.
[0043] In a twenty-fourth embodiment of the first aspect which is
also an embodiment of the nineteenth, the twentieth and the
twenty-first, preferably the twenty-first embodiment of the first
aspect, the first terminal stretch of nucleotides comprises a
nucleotide sequence of 5' X.sub.1X.sub.2SSBS 3' and the second
terminal stretch of nucleotides comprises a nucleotide sequence of
5' BVSSX.sub.3X.sub.4 3',
whereby X.sub.1 is absent, X.sub.2 is either absent or G, X.sub.3
is either absent or C, and X.sub.4 is absent, preferably the first
terminal stretch of nucleotides comprises a nucleotide sequence of
5' GCGUG 3' and the second terminal stretch of nucleotides
comprises a nucleotide sequence of 5' UACGC 3'.
[0044] In a twenty-fifth embodiment of the first aspect which is
also an embodiment of the sixteenth, the seventeenth, the
eighteenth, the nineteenth, the twentieth, the twenty-first, the
twenty-second, the twenty-third and the twenty-fourth embodiment of
the first aspect, the SDF-1 binding nucleic acid molecule of type B
comprises a nucleotide sequence according to any one of SEQ ID NO:
5 to SEQ ID NO: 20 and SEQ ID NO: 22 to SEQ ID NO: 28,
preferably any one of SEQ ID NO: 5 to SEQ ID NO: 7, SEQ ID NO: 16,
SEQ ID NO: 22 and SEQ ID NO: 28, more preferably any one of SEQ ID
NO: 22 and SEQ ID NO: 28.
[0045] In a twenty-sixth embodiment of the first aspect which is
also an embodiment of the sixteenth embodiment of the first aspect,
the SDF-1 binding nucleic acid molecule of type C comprises a
central stretch of nucleotides, whereby the central stretch of
nucleotides comprises a nucleotide sequence of
GGUYAGGGCUHRX.sub.AAGUCGG (SEQ ID NO: 108),
whereby X.sub.A is either absent or is A.
[0046] In a twenty-seventh embodiment of the first aspect which is
also an embodiment of the twenty-sixth embodiment of the first
aspect, the central stretch of nucleotides comprises a nucleotide
sequence of 5' GGUYAGGGCUHRAAGUCGG 3' (SEQ ID NO: 109), 5'
GGUYAGGGCUHRAGUCGG 3' (SEQ ID NO: 110) or 5' GGUUAGGGCUHGAAGUCGG 3'
(SEQ ID NO: 111), preferably 5' GGUUAGGGCUHGAAGUCGG 3' (SEQ ID NO:
111).
[0047] In a twenty-eighth embodiment of the first aspect which is
also an embodiment of the twenty-sixth and the twenty-seventh
embodiment of the first aspect, the SDF-1 binding nucleic acid
molecule of type C comprises in 5'->3' direction a first
terminal stretch of nucleotides, the central stretch of
nucleotides, and a second terminal stretch of nucleotides.
[0048] In a twenty-ninth embodiment of the first aspect which is
also an embodiment of the twenty-sixth and the twenty-seventh
embodiment of the first aspect, the SDF-1 binding nucleic acid
molecule of type C comprises in 5'->3' direction a second
terminal stretch of nucleotides, the central stretch of
nucleotides, and a first terminal stretch of nucleotides.
[0049] In a thirtieth embodiment of the first aspect which is also
an embodiment of the twenty-eighth and the twenty-ninth embodiment
of the first aspect, the first terminal stretch of nucleotides
comprises a nucleotide sequence of 5' RKSBUSNVGR 3' (SEQ ID NO:
138) and the second stretch of nucleotides comprises a nucleotide
sequence of 5' YYNRCASSMY 3' (SEQ ID NO: 139),
preferably the first terminal stretch of nucleotides comprises a
nucleotide sequence of 5' RKSBUGSVGR 3' (SEQ ID NO: 140) and the
second terminal stretch of nucleotides comprises a nucleotide
sequence of 5' YCNRCASSMY 3' (SEQ ID NO: 141).
[0050] In a thirty-first embodiment of the first aspect which is
also an embodiment of the twenty-eighth and the twenty-ninth
embodiment of the first aspect, the first terminal stretch of
nucleotides comprises a nucleotide sequence of 5' X.sub.SSSSV 3'
and the second terminal stretch of nucleotides comprises a
nucleotide sequence of 5' BSSSX.sub.S 3', whereby X.sub.S is either
absent or is S,
preferably the first terminal stretch of nucleotides comprises a
nucleotide sequence of 5' SGGSR 3' and the second terminal stretch
of nucleotides comprises a nucleotide sequence of 5' YSCCS 3'.
[0051] In a thirty-second embodiment of the first aspect which is
also an embodiment of the twenty-eighth and the twenty-ninth
embodiment of the first aspect, [0052] a) the first terminal
stretch of nucleotides comprises a nucleotide sequence of 5' GCCGG
3' and the second terminal stretch of nucleotides comprises a
nucleotide sequence of 5' CCGGC 3'; or [0053] b) the first terminal
stretch of nucleotides comprises a nucleotide sequence of 5'
CGUGCGCUUGAGAUAGG 3' (SEQ ID NO: 220) and the second terminal
stretch of nucleotides comprises a nucleotide sequence of 5'
CUGAUUCUCACG 3' (SEQ ID NO: 221); or [0054] c) the first terminal
stretch of nucleotides comprises a nucleotide sequence of 5'
UGAGAUAGG 3' and the second terminal stretch of nucleotides
comprises a nucleotide sequence of 5' CUGAUUCUCA 3' (SEQ ID NO:
222); or [0055] d) the first terminal stretch of nucleotides
comprises a nucleotide sequence of 5' GAGAUAGG 3' and the second
terminal stretch of nucleotides comprises a nucleotide sequence of
5' CUGAUUCUC 3'.
[0056] In a thirty-third embodiment of the first aspect which is
also an embodiment of the twenty-sixth, the twenty-seventh, the
twenty-eighth, the twenty-ninth, the thirtieth, the thirty-first
and the thirty-second embodiment of the first aspect, the type C
SDF-1 binding nucleic acid molecule comprises a nucleotide sequence
according to any one of SEQ ID NO: 95 to SEQ ID NO: 107, SEQ ID NO:
112 to SEQ ID NO: 137, SEQ ID NO: 223 and SEQ ID NO: 224,
preferably any one of SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO:
129, SEQ ID NO:134, SEQ ID NO: 135, SEQ ID NO: 223 and SEQ ID NO:
224.
[0057] In a thirty-fourth embodiment of the first aspect which is
also an embodiment of the sixteenth embodiment of the first aspect,
the SDF-1 binding nucleic acid molecule of type A comprises a
central stretch of nucleotides, whereby the central stretch of
nucleotides comprises a nucleotide sequence of 5'
AAAGYRACAHGUMAAX.sub.AUGAAAGGUARC 3' (SEQ ID NO: 74),
whereby X.sub.A is either absent or is A.
[0058] In a thirty-fifth embodiment of the first aspect which is
also an embodiment of the thirty-fourth embodiment of the first
aspect, the central stretch of nucleotides comprises a nucleotide
sequence of
5'AAAGYRACAHGUMAAUGAAAGGUARC 3' (SEQ ID NO: 75), or
5' AAAGYRACAHGUMAAAUGAAAGGUARC 3' (SEQ ID NO: 76), or
[0059] 5' AAAGYAACAHGUCAAUGAAAGGUARC 3'(SEQ ID NO: 77), preferably
the central stretch of nucleotides comprises a nucleotide sequence
of 5' AAAGYAACAHGUCAAUGAAAGGUARC 3' (SEQ ID NO: 77).
[0060] In a thirty-sixth embodiment of the first aspect which is
also an embodiment of the thirty-fourth and thirty-fifth embodiment
of the first aspect, the SDF-1 binding nucleic acid molecule of
type A comprises in 5'->3' direction a first terminal stretch of
nucleotides, the central stretch of nucleotides, and a second
terminal stretch of nucleotides.
[0061] In a thirty-seventh embodiment of the first aspect which is
also an embodiment of the thirty-fourth and the thirty-fifth
embodiment of the first aspect, the SDF-1 binding nucleic acid
molecule of type A comprises in 5'->3' direction a second
terminal stretch of nucleotides, the central stretch of
nucleotides, and a first terminal stretch of nucleotides.
[0062] In a thirty-eighth embodiment of the first aspect which is
also an embodiment of the thirty-sixth and the thirty-seventh
embodiment of the first aspect, the first terminal stretch of
nucleotides comprises a nucleotide sequence of 5'
X.sub.1X.sub.2NNBV 3' and the second terminal stretch of
nucleotides comprises a nucleotide sequence of 5'
BNBNX.sub.3X.sub.4 3'
whereby X.sub.1 is either absent or R, X.sub.2 is 5, X.sub.3 is S
and X.sub.4 is either absent or Y; or X.sub.1 is absent, X.sub.2 is
either absent or S, X.sub.3 is either absent or S and X.sub.4 is
absent.
[0063] In a thirty-ninth embodiment of the first aspect which is
also an embodiment of the thirty-sixth, the thirty-seventh and the
thirty-eighth, preferably the thirty-eighth embodiment of the first
aspect, the first terminal stretch of nucleotides comprises a
nucleotide sequence of 5' RSHRYR 3' and the second terminal stretch
of nucleotides comprises a nucleotide sequence of 5' YRYDSY 3',
preferably the first terminal stretch of nucleotides comprises a
nucleotide sequence of 5' GCUGUG 3' and the second terminal stretch
of nucleotides comprises a nucleotide sequence of 5' CGCAGC 3'.
[0064] In a fortieth embodiment of the first aspect which is also
an embodiment of the thirty-sixth, the thirty-seventh and the
thirty-eighth, preferably the thirty-eighth embodiment of the first
aspect, the first terminal stretch of nucleotides comprises a
nucleotide sequence of 5' X.sub.2BBBS 3' and the second terminal
stretch of nucleotides comprises a nucleotide sequence of 5'
SBBVX.sub.3 3',
whereby X.sub.2 is either absent or is S and X.sub.3 is either
absent or is S; preferably the first terminal stretch of
nucleotides comprises a nucleotide sequence of 5' CUGUG 3' and the
second terminal stretch of nucleotides comprises a nucleotide
sequence of 5' CGCAG 3'; or the first terminal stretch of
nucleotides comprises a nucleotide sequence of 5' GCGUG 3' and the
second terminal stretch of nucleotides comprises a nucleotide
sequence of 5' CGCGC 3'.
[0065] In a forty-first embodiment of the first aspect which is
also an embodiment of the thirty-fourth, the thirty-fifth, the
thirty-sixth, the thirty-seventh, the thirty-eighth, the
thirty-ninth and the fortieth embodiment of the first aspect, the
SDF-1 binding nucleic acid molecule of type A comprises a
nucleotide sequence according to any one of SEQ ID NO: 60 to SEQ ID
NO: 73, SEQ ID NO: 78 to SEQ ID NO: 82, SEQ ID NO: 84 to SEQ ID NO:
87, SEQ ID NO: 89 to SEQ ID NO: 94, and SEQ ID NO: 145,
preferably any one of SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66,
SEQ ID NO: 78, SEQ ID NO: 84, and SEQ ID NO: 146, more preferably
any one of SEQ ID NO: 84 and SEQ ID NO: 146.
[0066] In a forty-second embodiment of the first aspect which is
also an embodiment of the sixteenth embodiment of the first aspect,
the SDF-1 binding nucleic acid molecule of type D comprises a
nucleotide sequence according to any one of SEQ ID NO: 142 to SEQ
ID NO: 144.
[0067] In a forty-third embodiment of the first aspect which is
also an embodiment of the first, the second, the third, the fourth,
the fifth, the sixth, the seventh, the eighth, the ninth, the
tenth, the eleventh, the twelfth, the thirteenth, the fourteenth,
the fifteenth, the sixteenth, the seventeenth, the eighteenth, the
nineteenth, the twentieth, the twenty-first, the twenty-second, the
twenty-third, the twenty-fourth, the twenty-fifth, the
twenty-sixth, the twenty-seventh, the twenty-eighth, the
twenty-ninth, the thirtieth, the thirty-first, the thirty-second,
the thirty-third, the thirty-fourth, the thirty-fifth, the
thirty-sixth, the thirty-seventh, the thirty-eighth, the
thirty-ninth, the fortieth, the forty-first and the forty-second
embodiment of the first aspect, the SDF-1 is human SDF-1, whereby
preferably the human SDF-1 is human SDF-1 alpha or human SDF-1
beta, more preferably the human SDF-1 is human SDF-1 alpha.
[0068] In a forty-fourth embodiment of the first aspect which is
also an embodiment of the first, the second, the third, the fourth,
the fifth, the sixth, the seventh, the eighth, the ninth, the
tenth, the eleventh, the twelfth, the thirteenth, the fourteenth,
the fifteenth, the sixteenth, the seventeenth, the eighteenth, the
nineteenth, the twentieth, the twenty-first, the twenty-second, the
twenty-third, the twenty-fourth, the twenty-fifth, the
twenty-sixth, the twenty-seventh, the twenty-eighth, the
twenty-ninth, the thirtieth, the thirty-first, the thirty-second,
the thirty-third, the thirty-fourth, the thirty-fifth, the
thirty-sixth, the thirty-seventh, the thirty-eighth, the
thirty-ninth, the fortieth, the forty-first, the forty-second and
the forty-third embodiment of the first aspect, the nucleic acid
molecule comprises a modification, whereby the modification is
preferably a high molecular weight moiety and/or whereby the
modification preferably allows to modify the characteristics of the
nucleic acid molecule in terms of residence time in the animal or
human body, preferably the human body.
[0069] In a forty-fifth embodiment of the first aspect which is
also an embodiment of the forty-fourth embodiment of the first
aspect, the modification is selected from the group comprising a
HES moiety, a PEG moiety, biodegradable modifications and
combinations thereof.
[0070] In a forty-sixth embodiment of the first aspect which is
also an embodiment of the forty-fifth embodiment of the first
aspect, the modification is a PEG moiety consisting of a straight
or branched PEG, whereby preferably the molecular weight of the
straight or branched PEG is from about 20,000 to 120,000 Da, more
preferably from about 30,000 to 80,000 Da and most preferably about
40,000 Da.
[0071] In a forty-seventh embodiment of the first aspect which is
also an embodiment of the forty-fifth embodiment of the first
aspect, the modification is a HES moiety, whereby preferably the
molecular weight of the HES moiety is from about 10,000 to 200,000
Da, more preferably from about 30,000 to 170.000 Da and most
preferably about 150,000 Da.
[0072] In a forty-eighth embodiment of the first aspect which is
also an embodiment of the forty-fourth, the forty-fifth, the
forty-sixth and the forty-seventh embodiment of the first aspect,
the modification is attached to the nucleic acid molecule via a
linker, wherein preferably the linker is a biostable or
biodegradable linker.
[0073] In a forty-ninth embodiment of the first aspect which is
also an embodiment of the forty-fourth, the forty-fifth, the
forty-sixth, the forty-seventh and the forty-eighth embodiment of
the first aspect, the modification is attached to the nucleic acid
molecule at the 5'-terminal nucleotide of the nucleic acid molecule
and/or the 3'-terminal nucleotide of the nucleic acid molecule
and/or to a nucleotide of the nucleic acid molecule between the
5'-terminal nucleotide of the nucleic acid molecule and the
3'-terminal nucleotide of the nucleic acid molecule
[0074] In a fiftieth embodiment of the first aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth, the tenth,
the eleventh, the twelfth, the thirteenth, the fourteenth, the
fifteenth, the sixteenth, the seventeenth, the eighteenth, the
nineteenth, the twentieth, the twenty-first, the twenty-second, the
twenty-third, the twenty-fourth, the twenty-fifth, the
twenty-sixth, the twenty-seventh, the twenty-eighth, the
twenty-ninth, the thirtieth, the thirty-first, the thirty-second,
the thirty-third, the thirty-fourth, the thirty-fifth, the
thirty-sixth, the thirty-seventh, the thirty-eighth, the
thirty-ninth, the fortieth, the forty-first, the forty-second, the
forty-third, the forty-fourth, the forty-fifth, the forty-sixth,
the forty-seventh, the forty-eighth and the forty-ninth embodiment
of the first aspect, the nucleotides of the nucleic acid molecule
or the nucleotides forming the nucleic acid molecule are
L-nucleotides.
[0075] In a fifty-first embodiment of the first aspect which is
also an embodiment of the first, the second, the third, the fourth,
the fifth, the sixth, the seventh, the eighth, the ninth, the
tenth, the eleventh, the twelfth, the thirteenth, the fourteenth,
the fifteenth, the sixteenth, the seventeenth, the eighteenth, the
nineteenth, the twentieth, the twenty-first, the twenty-second, the
twenty-third, the twenty-fourth, the twenty-fifth, the
twenty-sixth, the twenty-seventh, the twenty-eighth, the
twenty-ninth, the thirtieth, the thirty-first, the thirty-second,
the thirty-third, the thirty-fourth, the thirty-fifth, the
thirty-sixth, the thirty-seventh, the thirty-eighth, the
thirty-ninth, the fortieth, the forty-first, the forty-second, the
forty-third, the forty-fourth, the forty-fifth, the forty-sixth,
the forty-seventh, the forty-eighth, the forty-ninth and the
fiftieth embodiment of the first aspect, the nucleic acid molecule
is an L-nucleic acid molecule.
[0076] The problem underlying the present invention is solved in a
second aspect which is also the first embodiment of the second
aspect, by a pharmaceutical composition comprising as a first
pharmaceutically active agent the nucleic acid molecule according
to any one of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth, the tenth,
the eleventh, the twelfth, the thirteenth, the fourteenth, the
fifteenth, the sixteenth, the seventeenth, the eighteenth, the
nineteenth, the twentieth, the twenty-first, the twenty-second, the
twenty-third, the twenty-fourth, the twenty-fifth, the
twenty-sixth, the twenty-seventh, the twenty-eighth, the
twenty-ninth, the thirtieth, the thirty-first, the thirty-second,
the thirty-third, the thirty-fourth, the thirty-fifth, the
thirty-sixth, the thirty-seventh, the thirty-eighth, the
thirty-ninth, the fortieth, the forty-first, the forty-second, the
forty-third, the forty-fourth, the forty-fifth, the forty-sixth,
the forty-seventh, the forty-eighth, the forty-ninth, the fiftieth
and the fifty-first embodiment of the first aspect and optionally a
further constituent, whereby the further constituent is selected
from the group comprising a pharmaceutically acceptable excipient,
a pharmaceutically acceptable carrier and a further
pharmaceutically active agent, and whereby the pharmaceutical
composition is for use in a method for the treatment and/or
prevention of a disease or disorder, or for use in a method for the
treatment of a subject suffering from a disease or disorder or
being at risk of developing a disease or a disorder as an adjunct
therapy, or for the treatment and/or prevention of a disease or
disorder, whereby the disease or disorder is cancer.
[0077] In a second embodiment of the second aspect which is also an
embodiment of the first embodiment of the second aspect, the
adjunct therapy sensitizes the subject, wherein the sensitized
subject is more responsive to a therapy for the treatment and/or
prevention of the disease or disorder.
[0078] In a third embodiment of the second aspect which is also an
embodiment of the second embodiment of the second aspect, the
therapy for the treatment and/or prevention of the diseases or
disorder comprises the administration of a further pharmaceutically
active agent and/or irradiating the subject and/or surgery and/or
cellular therapy.
[0079] In a fourth embodiment of the second aspect which is also an
embodiment of the first, the second and the third embodiment of the
second aspect, the further pharmaceutically active agent is a
pharmaceutically active agent selected from the group comprising an
antibody, an alkylating agent, an anti-metabolite, a plant
alkaloid, preferably vincristine, a plant terpenoid, a
topoisomerase inhibitor, Leucovorin, Methotrexate, Tamoxifen,
Sorafenib, Lenalidomide, Bortezomib, Dexamethasone, Fluorouracil,
and Prednisone.
[0080] In a fifth embodiment of the second aspect which is also an
embodiment of the fourth embodiment of the second aspect, the
antibody is selected from the group comprising Rituximab,
Ofatumumab, Cetuximab, Ibritumomab-Tiuxetan, Tositumomab,
Trastuzumab, Bevacizumab, and Alemtuzumab.
[0081] In a sixth embodiment of the second aspect which is also an
embodiment of the fourth embodiment of the second aspect, the
alkylating agent is selected from the group comprising cisplatin,
carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide,
chlorambucil, doxorubicin, lioposomal doxorubicin, bendamustine,
temozolomide and Melphalan.
[0082] In a seventh embodiment of the second aspect which is also
an embodiment of the fourth embodiment of the second aspect, the
anti-metabolite is selected from the group comprising
purineazathioprine, mercaptopurine, fludarabine, pentostatin, and
cladribine.
[0083] In an eighth embodiment of the second aspect which is also
an embodiment of the fourth embodiment of the second aspect, the
plant terpenoid is selected from the group comprising a taxane more
preferably selected from the group comprising Docetaxel,
Paclitaxel, podophyllotoxin and epothilone.
[0084] In a ninth embodiment of the second aspect which is also an
embodiment of the fourth embodiment of the second aspect, the
topoisomerase inhibitor is selected from the group comprising
camptothecin, irinotecan, and mitoxantrone.
[0085] In a tenth embodiment of the second aspect which is also an
embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth and the ninth embodiment
of the second aspect, the cancer is a cancer selected from the
group of hematological cancer, whereby preferably the hematological
cancer is selected from the group of leukemia and myeloma.
[0086] In an eleventh embodiment of the second aspect which is also
an embodiment of the tenth embodiment of the second aspect,
leukemia is selected from the group comprising chronic lymphoid
leukemia and acute myeloid leukemia.
[0087] In a twelfth embodiment of the second aspect which is also
an embodiment of the tenth embodiment of the second aspect, myeloma
is multiple myeloma.
[0088] In a thirteenth embodiment of the second aspect which is
also an embodiment of the first, the second, the third, the fourth,
the fifth, the sixth, the seventh, the eighth and the ninth
embodiment of the second aspect, the cancer is a cancer selected
from the group of solid tumors, whereby preferably the solid tumors
are selected from the group comprising glioblastoma, colorectal
cancer, breast cancer, lymphoma, prostate cancer, pancreatic
cancer, renal cancer, ovarian cancer and lung cancer.
[0089] The problem underlying the present invention is solved in a
third aspect which is also the first embodiment of the third
aspect, by a medicament comprising one or several dosage units of
at least a first pharmaceutically active agent, wherein the first
pharmaceutically active agent is a nucleic acid molecule capable of
binding to SDF-1 as defined in any one of the first, the second,
the third, the fourth, the fifth, the sixth, the seventh, the
eighth, the ninth, the tenth, the eleventh, the twelfth, the
thirteenth, the fourteenth, the fifteenth, the sixteenth, the
seventeenth, the eighteenth, the nineteenth, the twentieth, the
twenty-first, the twenty-second, the twenty-third, the
twenty-fourth, the twenty-fifth, the twenty-sixth, the
twenty-seventh, the twenty-eighth, the twenty-ninth, the thirtieth,
the thirty-first, the thirty-second, the thirty-third, the
thirty-fourth, the thirty-fifth, the thirty-sixth, the
thirty-seventh, the thirty-eighth, the thirty-ninth, the fortieth,
the forty-first, the forty-second, the forty-third, the
forty-fourth, the forty-fifth, the forty-sixth, the forty-seventh,
the forty-eighth, the forty-ninth, the fiftieth and the fifty-first
embodiment of the first aspect, whereby the medicament is for use
in a method for the treatment and/or prevention of a disease or
disorder, or for use in a method for the treatment of a subject
suffering from a disease or disorder or being at risk of developing
a disease or a disorder as an adjunct therapy, or for the treatment
and/or prevention of a disease or disorder, whereby the disease or
disorder is cancer.
[0090] In a second embodiment of the third aspect which is also an
embodiment of the first embodiment of the third aspect, the adjunct
therapy sensitizes the subject, wherein the sensitized subject is
more responsive to a therapy for the treatment and/or prevention of
the disease or disorder.
[0091] In a third embodiment of the third aspect which is also an
embodiment of the second embodiment of the third aspect, the
therapy for the treatment and/or prevention of the diseases or
disorder comprises the administration of a further pharmaceutically
active agent and/or irradiating the subject and/or surgery and/or
cellular therapy.
[0092] In a fourth embodiment of the third aspect which is also an
embodiment of the first, the second and the third, preferably the
first embodiment of the third aspect, the medicament comprises a
further pharmaceutically active agent, preferably one or several
dosage units of a further pharmaceutically active agent, whereby
the further pharmaceutically active agent is selected from the
group comprising an antibody, an alkylating agent, an
anti-metabolite, a plant alkaloid, preferably vincristine, a plant
terpenoid, a topoisomerase inhibitor, Leucovorin, Methotrexate,
Tamoxifen, Sorafenib, Lenalidomide, Bortezomib, Dexamethasone and
Fluorouracil.
[0093] In a fifth embodiment of the third aspect which is also an
embodiment of the third embodiment of the third aspect, the
medicament comprises the further pharmaceutically active agent,
preferably one or several dosage units of the further
pharmaceutically active agent, whereby the further pharmaceutically
active agent is selected from the group comprising an antibody, an
alkylating agent, an anti-metabolite, a plant alkaloid, preferably
vincristine, a plant terpenoid, a topoisomerase inhibitor,
Leucovorin, Methotrexate, Tamoxifen, Sorafenib, Lenalidomide,
Bortezomib, Dexamethasone Fluorouracil, and Prednisone.
[0094] In a sixth embodiment of the third aspect which is also an
embodiment of the fourth and the fifth embodiment of the third
aspect, the antibody is selected from the group comprising
Rituximab, Ofatumumab, Cetuximab, Ibritumomab-Tiuxetan,
Tositumomab, Trastuzumab, Bevacizumab, and Alemtuzumab.
[0095] In a seventh embodiment of the third aspect which is also an
embodiment of the fourth and the fifth embodiment of the third
aspect, the alkylating agent is selected from the group comprising
cisplatin, carboplatin, oxaliplatin, mechlorethamine,
cyclophosphamide, chlorambucil, doxorubicin, lioposomal
doxorubicin, bendamustine, temozolomide and Melphalan.
[0096] In an eighth embodiment of the third aspect which is also an
embodiment of the fourth and the fifth embodiment of the third
aspect, the anti-metabolite is selected from the group comprising
purineazathioprine, mercaptopurine fludarabine, pentostatin, and
cladribine.
[0097] In a ninth embodiment of the third aspect which is also an
embodiment of the fourth and the fifth embodiment of the third
aspect, the plant terpenoid is selected from the group of a taxane,
more preferably selected from the group comprising Docetaxel,
Paclitaxel, podophyllotoxin and epothilone.
[0098] In a tenth embodiment of the third aspect which is also an
embodiment of the fourth and the fifth embodiment of the third
aspect, the topoisomerase inhibitor is selected from the group
comprising camptothecin, irinotecan and mitoxantrone.
[0099] In an eleventh embodiment of the third aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth and the tenth
embodiment of the third aspect, wherein the cancer is a cancer
selected from the group of hematological cancer, whereby preferably
the hematological cancer is selected from the group comprising
leukemia and myeloma.
[0100] In a twelfth embodiment of the third aspect which is also an
embodiment of the eleventh embodiment of the third aspect, leukemia
is selected from the group comprising chronic lymphoid leukemia and
acute myeloid leukemia.
[0101] In a thirteenth embodiment of the third aspect which is also
an embodiment of the eleventh embodiment of the third aspect,
myeloma is multiple myeloma.
[0102] In a fourteenth embodiment of the third aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth and the tenth
embodiment of the third aspect, the cancer is a cancer selected
from the group of solid tumors, whereby preferably the solid tumors
are selected from the group comprising glioblastoma, colorectal
cancer, breast cancer, lymphoma, prostate cancer, pancreatic
cancer, renal cancer, ovarian cancer and lung cancer.
[0103] The problem underlying the present invention is solved in a
fourth aspect which is also the first embodiment of the fourth
aspect, by use of a nucleic acid molecule as defined in any one of
the first, the second, the third, the fourth, the fifth, the sixth,
the seventh, the eighth, the ninth, the tenth, the eleventh, the
twelfth, the thirteenth, the fourteenth, the fifteenth, the
sixteenth, the seventeenth, the eighteenth, the nineteenth, the
twentieth, the twenty-first, the twenty-second, the twenty-third,
the twenty-fourth, the twenty-fifth, the twenty-sixth, the
twenty-seventh, the twenty-eighth, the twenty-ninth, the thirtieth,
the thirty-first, the thirty-second, the thirty-third, the
thirty-fourth, the thirty-fifth, the thirty-sixth, the
thirty-seventh, the thirty-eighth, the thirty-ninth, the fortieth,
the forty-first, the forty-second, the forty-third, the
forty-fourth, the forty-fifth, the forty-sixth, the forty-seventh,
the forty-eighth, the forty-ninth, the fiftieth and the fifty-first
embodiment of the first aspect, for the manufacture of a medicament
for the treatment and/or prevention of a disease or disorder or for
use in a method for the treatment of a subject suffering from a
disease or disorder or being at risk of developing a disease or a
disorder as an adjunct therapy, whereby the disease or disorder is
cancer.
[0104] In a second embodiment of the fourth aspect which is also an
embodiment of the first embodiment of the fourth aspect, the
adjunct therapy sensitizes the subject, wherein the sensitized
subject is more responsive to a therapy for the treatment and/or
prevention of the disease or disorder.
[0105] In a third embodiment of the fourth aspect which is also an
embodiment of the second embodiment of the fourth aspect, the
therapy for the treatment and/or prevention of the diseases or
disorder comprises the administration of a further pharmaceutically
active agent and/or irradiating the subject and/or surgery and/or
cellular therapy.
[0106] In a fourth embodiment of the fourth aspect which is also an
embodiment of the first, the second and the third, preferably the
first embodiment of the fourth aspect, the medicament is used in
combination with a further pharmaceutically active agent, whereby
the further pharmaceutically active agent is a pharmaceutically
active agent selected from the group comprising an antibody, an
alkylating agent, an anti-metabolite, a plant alkaloid, preferably
vincristine, a plant terpenoid, a topoisomerase inhibitor,
Leucovorin, Methotrexate, Tamoxifen, Sorafenib, Lenalidomide,
Bortezomib, Dexamethasone, Fluorouracil, and Prednisone.
[0107] In a fifth embodiment of the fourth aspect which is also an
embodiment of the third embodiment of the fourth aspect, the
further pharmaceutically active agent is a pharmaceutically active
agent selected from the group comprising an antibody, an alkylating
agent, an anti-metabolite, a plant alkaloid, preferably
vincristine, a plant terpenoid, a topoisomerase inhibitor,
Leucovorin, Methotrexate, Tamoxifen, Sorafenib, Lenalidomide,
Bortezomib, Dexamethasone, Fluorouracil, and Prednisone.
[0108] In a sixth embodiment of the fourth aspect which is also an
embodiment of the fourth and the fifth embodiment of the fourth
aspect, the antibody is selected from the group comprising
Rituximab, Cetuximab, Ibritumomab-Tiuxetan, Tositumomab,
Trastuzumab, Bevacizumab, and Alemtuzumab.
[0109] In a seventh embodiment of the fourth aspect which is also
an embodiment of the fourth and the fifth embodiment of the fourth
aspect, the alkylating agent is selected from the group comprising
cisplatin, carboplatin, oxaliplatin, mechlorethamine,
cyclophosphamide, chlorambucil, doxorubicin, lioposomal
doxorubicin, bendamustine, temozolomide and Melphalan.
[0110] In an eighth embodiment of the fourth aspect which is also
an embodiment of the fourth and the fifth embodiment of the fourth
aspect, the anti-metabolite is selected from the group comprising
purineazathioprine, mercaptopurine fludarabine, pentostatin, and
cladribine.
[0111] In a ninth embodiment of the fourth aspect which is also an
embodiment of the fourth and the fifth embodiment of the fourth
aspect, the plant terpenoid is selected from the group comprising a
taxane, more preferably selected from the group of Docetaxel,
Paclitaxel, podophyllotoxin and epothilone.
[0112] In a tenth embodiment of the fourth aspect which is also an
embodiment of the fourth and the fifth embodiment of the fourth
aspect, the topoisomerase inhibitor is selected from the group
comprising camptothecin, irinotecan, and mitoxantrone.
[0113] In an eleventh embodiment of the fourth aspect which is also
an embodiment of the fourth and the fifth embodiment of the fourth
aspect, the cancer is a cancer selected from the group of
hematological cancer, whereby preferably the hematological cancer
is selected from the group comprising leukemia and myeloma.
[0114] In a twelfth embodiment of the fourth aspect which is also
an embodiment of the eleventh embodiment of the fourth aspect,
leukemia is selected from the group comprising chronic lymphoid
leukemia and acute myeloid leukemia.
[0115] In a thirteenth embodiment of the fourth aspect which is
also an embodiment of the eleventh embodiment of the fourth aspect,
myeloma is multiple myeloma.
[0116] In a fourteenth embodiment of the fourth aspect which is
also an embodiment of the first, the second, the third, the fourth,
the fifth, the sixth, the seventh, the eighth, the ninth and the
tenth embodiment of the fourth aspect, the cancer is a cancer
selected from the group of solid tumors, whereby preferably the
solid tumors are selected from the group comprising glioblastoma,
colorectal cancer, breast cancer, lymphoma, prostate cancer,
pancreatic cancer, renal cancer, ovarian cancer and lung
cancer.
[0117] The problem underlying the present invention is solved in a
fifth aspect which is also the first embodiment of the fifth
aspect, by a method for the treatment of a subject suffering from
or being at risk of developing cancer, whereby the method comprises
[0118] a step a) of administering to the subject a pharmaceutically
effective amount of a nucleic acid molecule capable of binding to
SDF-1 as defined in any one of the first, the second, the third,
the fourth, the fifth, the sixth, the seventh, the eighth, the
ninth, the tenth, the eleventh, the twelfth, the thirteenth, the
fourteenth, the fifteenth, the sixteenth, the seventeenth, the
eighteenth, the nineteenth, the twentieth, the twenty first, the
twenty second, the twenty third, the twenty fourth, the twenty
fifth, the twenty-sixth, the twenty seventh, the twenty-eighth, the
twenty-ninth, the thirtieth, the thirty-first, the thirty-second,
the thirty-third, the thirty-fourth, the thirty-fifth, the
thirty-sixth, the thirty-seventh, the thirty-eighth, the
thirty-ninth, the fortieth, the forty-first, the forty-second, the
forty-third, the forty-fourth, the forty-fifth, the forty-sixth,
the forty-seventh, the forty-eighth, the forty-ninth, the fiftieth
and the fifty-first embodiment of the first aspect.
[0119] In a second embodiment of the fifth aspect which is also an
embodiment of the first embodiment of the fifth aspect, the method
comprises [0120] a step b) of irradiating the subject and/or
surgery and/or cellular therapy and/or administering a
pharmaceutically effective amount of a further pharmaceutically
active agent to the subject, whereby the further pharmaceutically
active agent is a pharmaceutically active agent selected from the
group comprising an antibody, an alkylating agent, an
anti-metabolite, a plant alkaloid, preferably vincristine, a plant
terpenoid, a topoisomerase inhibitor, Leucovorin, Methotrexate,
Tamoxifen, Sorafenib, Lenalidomide, Bortezomib, Dexamethasone,
Fluorouracil, and Prednisone.
[0121] In a third embodiment of the fifth aspect which is also an
embodiment of the second embodiment of the fifth aspect, the
pharmaceutically effective amount of a nucleic acid molecule
capable of binding to SDF-1 as defined in any one of the first, the
second, the third, the fourth, the fifth, the sixth, the seventh,
the eighth, the ninth, the tenth, the eleventh, the twelfth, the
thirteenth, the fourteenth, the fifteenth, the sixteenth, the
seventeenth, the eighteenth, the nineteenth, the twentieth, the
twenty first, the twenty second, the twenty third, the twenty
fourth, the twenty fifth, the twenty-sixth, the twenty seventh, the
twenty-eighth, the twenty-ninth, the thirtieth, the thirty-first,
the thirty-second, the thirty-third, the thirty-fourth, the
thirty-fifth, the thirty-sixth, the thirty-seventh, the
thirty-eighth, the thirty-ninth, the fortieth, the forty-first, the
forty-second, the forty-third, the forty-fourth, the forty-fifth,
the forty-sixth, the forty-seventh, the forty-eighth, the
forty-ninth, the fiftieth and the fifty-first embodiment of the
first aspect is administered as an adjunct therapy or part of an
adjunct therapy.
[0122] In a fourth embodiment of the fifth aspect which is also an
embodiment of the third embodiment of the fifth aspect, the adjunct
therapy sensitizes the subject, wherein the sensitized subject is
more responsive to a therapy for the treatment and/or prevention of
the disease or disorder.
[0123] In a fifth embodiment of the fifth aspect which is also an
embodiment of the fourth embodiment of the fifth aspect, the
therapy for the treatment and/or prevention of the disease or
disorder comprises the administration of a further pharmaceutically
active agent and/or irradiating the subject and/or surgery and/or
cellular therapy as performed in step b).
[0124] In a sixth embodiment of the fifth aspect which is also an
embodiment of the second, the third, the fourth and the fifth
embodiment of the fifth aspect, the antibody is selected from the
group comprising Rituximab, Cetuximab, Ibritumomab-Tiuxetan,
Tositumomab, Trastuzumab, Bevacizumab, and Alemtuzumab.
[0125] In a seventh embodiment of the fifth aspect which is also an
embodiment of the second, the third, the fourth and the fifth
embodiment of the fifth aspect, the alkylating agent is selected
from the group comprising cisplatin, carboplatin, oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, doxorubicin,
lioposomal doxorubicin, bendamustine, temozolomide and
Melphalan.
[0126] In an eighth embodiment of the fifth aspect which is also an
embodiment of the second, the third, the fourth and the fifth
embodiment of the fifth aspect, the anti-metabolite is selected
from the group comprising purineazathioprine, mercaptopurine,
fludarabine, pentostatin, and cladribine.
[0127] In a ninth embodiment of the fifth aspect which is also an
embodiment of the second, the third, the fourth and the fifth
embodiment of the fifth aspect, the plant terpenoid is selected
from the group comprising taxanes, more preferably selected from
the group of Docetaxel, Paclitaxel, podophyllotoxin and
epothilone.
[0128] In a tenth embodiment of the fifth aspect which is also an
embodiment of the second, the third, the fourth and the fifth
embodiment of the fifth aspect, the topoisomerase inhibitor is
selected from the group comprising camptothecin, irinotecan, and
mitoxantrone.
[0129] In an eleventh embodiment of the fifth aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth and the tenth
embodiment of the fifth aspect, the cancer is a cancer selected
from the group of hematological cancer, whereby preferably the
hematological cancer is selected from the group comprising leukemia
and myeloma.
[0130] In a twelfth embodiment of the fifth aspect which is also an
embodiment of the eleventh embodiment of the fifth aspect, leukemia
is selected from the group comprising chronic lymphoid leukemia and
acute myeloid leukemia.
[0131] In a thirteenth embodiment of the fifth aspect which is also
an embodiment of the eleventh and the twelfth embodiment of the
fifth aspect, myeloma is multiple myeloma.
[0132] In a fourteenth embodiment of the fifth aspect which is also
an embodiment of the first, the second, the third, the fourth, the
fifth, the sixth, the seventh, the eighth, the ninth and the tenth
embodiment of the fifth aspect, the cancer is a cancer selected
from the group of solid tumors, whereby preferably the solid tumors
are selected from the group comprising glioblastoma, colorectal
cancer, breast cancer, lymphoma, prostate cancer, pancreatic
cancer, renal cancer, ovarian cancer and lung cancer.
[0133] While not wishing to be bound by any theory, the present
inventors have found that the nucleic acid molecules according to
the present invention inhibit the binding of SDF-1 to its SDF-1
receptors and thus, either directly or indirectly, are used for the
treatment of cancer. Furthermore, the instant inventors have found
that the nucleic acid molecules according to the present invention
are suitable to block the interaction of SDF-1 with the SDF-1
receptors CXCR4 and CXCR7, respectively. Insofar, the SDF-1 binding
nucleic acid molecule according to the present invention can also
be viewed as antagonists of CXCR4 and CXCR7, respectively.
[0134] As to the various diseases, conditions and disorders which
may be treated or prevented by using the nucleic acid molecules
according to the present invention or compositions, preferably
pharmaceutical compositions comprising the same, it has to be
acknowledged that such diseases, conditions and disorders are those
which are described herein, including and in particular those
described and set forth in the introductory part of the instant
application. Insofar, the respective passages form an integral part
of the present disclosure teaching the suitability of the nucleic
acid molecules for the prevention and treatment, respectively, for
said diseases, conditions, and disorders.
[0135] As used herein the term SDF-1 refers to any SDF-1 including,
but not limited to, mammalian SDF-1. Preferably, the mammalian
SDF-1 is selected from the group comprising mice, rat, rabbit,
hamster, monkey and human SDF-1. More preferably the SDF-1 is human
SDF-1 also referred to as SDF-1.alpha. (SEQ ID NO: 1) and/or human
SDF-1.beta. (SEQ ID NO: 2), most preferably human SDF-1 also
referred to as SDF-1.alpha. (SEQ ID NO: 1)
[0136] SDF-1 acts through two different receptors, the receptors
CXCR4 and RDC1/CXCR7 (Balabanian, Lagane et al. 2005a, Burns,
Summers et al. 2006) (see the introductory part of the instant
application). Elevated expression of CXCR4 and CXCR7 was shown for
several cancer types as described herein.
[0137] Because SDF-1 acts through two different receptors, a
treatment of an SDF-1 related disease or disorder by a compound
specific for one out of the two SDF-1 receptors CXCR4 and CXCR7:
[0138] a) should be less effective due to the two different SDF-1
receptors expressed on cells, preferably cancer cells; [0139] b) is
limited to a distinct population of cells, preferably to a distinct
population of cancer cells, due to the individual SDF-1 receptors
expressed on the cells.
[0140] Cancer is a term for malignant neoplasms, a great and
heterogeneous group of diseases in which cells display uncontrolled
growth, invasion and often metastasizes, wherein the cancer cells
spread to other locations in the body, to regional lymph nodes or
distant body sites like brain, bone, liver, or other organs. These
three malignant properties of cancer differentiate malignant tumors
from benign tumors, whereby, as used hererin, the term cancer shall
also encompass malignant tumors which in turn are also referred to
herein as tumors. Malignant tumors fall into two categories based
on their origin: Hematological and solid tumors. Hematological
tumors are cancer types affecting blood, bone marrow, and lymph
nodes. Solid tumors are formed by an abnormal growth of body tissue
cells other than blood, bone marrow or lymphatic cells.
[0141] Preferred forms of cancer are the following ones:
Adrenocortical Carcinoma
[0142] AIDS-Related Cancers such as Kaposi Sarcoma and Lymphoma
Anal Cancer
Appendix Cancer
Atypical Teratoid/Rhabdoid Tumor
Basal Cell Carcinoma
Bile Duct Cancer, Extrahepatic
Bladder Cancer
Bone Cancer
Osteosarcoma
Malignant Fibrous Histiocytoma
Brain Stem Glioma
[0143] Brain Tumor such as Astrocytomas, Brain and Spinal Cord
Tumors, Brain Stem Glioma, Childhood, Central Nervous System
Atypical Teratoid/Rhabdoid Tumor, Central Nervous System Embryonal
Tumors, Craniopharyngioma, Ependymoblastoma, Ependymoma,
Medulloblastoma, Medulloepithelioma, Pineal Parenchymal Tumors of
Intermediate Differentiation, Supratentorial Primitive
Neuroectodermal Tumors and Pineoblastoma
Breast Cancer
Bronchial Tumors
Carcinoid Tumor
Carcinoma of Unknown Primary
[0144] Cancer of Central Nervous System such as Atypical
Teratoid/Rhabdoid Tumor and
Lymphoma
Cervical Cancer
Childhood Cancers
Chordoma
Chronic Myeloproliferative Disorders
Colon Cancer
Colorectal Cancer
Craniopharyngioma
Cutaneous T-Cell Lymphoma
Embryonal Tumors
Endometrial Cancer
Ependymoblastoma
Ependymoma,
Esophageal Cancer
Esthesioneuroblastoma
Ewing Sarcoma Family of Tumors
Extracranial Germ Cell Tumor
Extragonadal Germ Cell Tumor
Extrahepatic Bile Duct Cancer
[0145] Eye Cancer such as Intraocular Melanoma and
Retinoblastoma
Fibrous Histiocytoma of Bone
Osteosarcoma
Gallbladder Cancer
Gastric (Stomach) Cancer
Gastrointestinal Carcinoid Tumor
Gastrointestinal Stromal Tumors (GIST)
[0146] Germ Cell Tumor (extracranial, extragonadal or ovarian)
Gestational Trophoblastic Tumor
Glioma
Hairy Cell Leukemia
Head and Neck Cancer
Heart Cancer
Hepatocellular (Liver) Cancer
Histiocytosis
Hypopharyngeal Cancer
Intraocular Melanoma
Islet Cell Tumors (Endocrine Pancreas)
Kaposi Sarcoma
Kidney Cancer
Langerhans Cell Histiocytosis
Laryngeal Cancer
[0147] Leukemia such Acute Lymphoblastic Leukemia (abbr. ALL),
Acute Myeloid Leukemia (abbr. AML), Chronic Lymphocytic Leukemia
(abbr. CLL), Chronic Myelogenous Leukemia (abbr. CML) and Hairy
Cell Leukemia
Lip and Oral Cavity Cancer
Liver Cancer (Primary)
Lobular Carcinoma In Situ (LCIS)
Lung Cancer
[0148] Lymphoma such as AIDS-Related Lymphoma, Burkitt, Mycosis
Fungoides and Sezary Syndrome, Hodgkin, Non-Hodgkin and leukemia of
Primary Central Nervous System (abbr. CNS)
Macroglobulinemia
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma
Medulloblastoma
Medulloepithelioma
Melanoma
Merkel Cell Carcinoma
Mesothelioma
[0149] Metastatic Squamous Neck Cancer with Occult Primary
Midline Tract Carcinoma Involving NUT Gene
Mouth Cancer
Multiple Endocrine Neoplasia Syndromes
Multiple Myeloma
Mycosis Fungoides
Myelodysplastic Syndromes
Myelodysplastic/Myeloproliferative Neoplasms
Myeloproliferative Disorders
Nasal Cavity and Paranasal Sinus Cancer
Nasopharyngeal Cancer
Neuroblastoma
Non-Small Cell Lung Cancer
Oral Cancer
Oral Cavity Cancer
Oropharyngeal Cancer
Osteosarcoma and Malignant Fibrous Histiocytoma of Bone
Ovarian Cancer
Pancreatic Cancer
Papillomatosis
Paraganglioma
Paranasal Sinus and Nasal Cavity Cancer
Parathyroid Cancer
Penile Cancer
Pharyngeal Cancer
Pheochromocytoma
Pineal Parenchymal Tumors of Intermediate Differentiation
Pineoblastoma and Supratentorial Primitive Neuroectodermal
Tumors
Pituitary Tumor
Plasma Cell Neoplasm/Multiple Myeloma
Pleuropulmonary Blastoma, Childhood
[0150] Primary Central Nervous System (abbr CNS) Lymphoma
Prostate Cancer
Rectal Cancer
Renal Cell (Kidney) Cancer
Renal Pelvis and Ureter, Transitional Cell Cancer
Retinoblastoma
Rhabdomyosarcoma
Salivary Gland Cancer
[0151] Sarcoma such as Ewing Sarcoma Family of Tumors, Kaposi
Sarcoma, Soft Tissue Sarcoma, Uterine Sarcoma Skin Cancer such
Melanoma, Merkel Cell Carcinoma and Nonmelanoma
Small Cell Lung Cancer
Small Intestine Cancer
Soft Tissue Sarcoma
Squamous Cell Carcinoma
Squamous Neck Cancer Stomach (Gastric) Cancer
Supratentorial Primitive Neuroectodermal Tumors
T-Cell Lymphoma
Testicular Cancer
Throat Cancer
Thymoma and Thymic Carcinoma
Thyroid Cancer
Transitional Cell Cancer of the Renal Pelvis and Ureter
Trophoblastic Tumor, Gestational
Ureter and Renal Pelvis, Transitional Cell Cancer
Urethral Cancer
Uterine Cancer, Endometrial
Uterine Sarcoma
Vaginal Cancer
Vulvar Cancer
[0152] Waldenstrom Macroglobulinemia
Wilms Tumor
[0153] The SDF-1-CXCR4 axis has been shown to play a role in stem
cell mobilization including cancer stem cells, vasculogenesis,
tumor growth and metastasis. The SDF-1 receptor CXCR4 is expressed
in a variety of cancers and hematological malignancies in vivo as
is CXCR7 (Maksym, Tarnowski et al., 2009; Wang, Shiosawa et al.,
2008; Miao, Lucker et al., 2007). The growth and invasion signal
for tumor cells is SDF-1, in particular if the cells express the
receptors for SDF-1 (Batchelor et al., 2007; Zhu et al., 2009; Xu
et al., 2009; Kozin et al., 2010).
[0154] CXCR4 as well as SDF-1 are induced by hypoxia (Ceradini et
al. 2004). Together with VEGF they represent a potent synergistic
axis that initiates and maintains angiogenic/vascologenic pathways
(Kryczek et al. 2005). The role in vasculogenesis is supported by
evidence that SDF-1 attracts CXCR4 expressing endothelial
progenitor cells from the circulation (Sengupta et al. 2005).
SDF-1-CXCR4 mediated recruitment of bone marrow derived cells that
support vascularization may also be the reason for recurrence of
glioblastoma after irradiation therapy (Kioi et al., 2010). As
demonstrated by Kioi et al. in an intracranial glioblastoma
multiforme (abbr. GBM) mouse xenograft model the treatment of GBM
patients with high dosis of radiation is less effective due to
irradiation induced recruitment of bone-marrow derived cells (abbr.
BMDCs). The blockade of the interaction of SDF-1 and its receptor
CXCR4 by the CXCR4 antagonist AMD3100 prevented the influx of BMDCs
in the irradiated tumor (Kioi et al., 2010). In 2010 Tseng et al.
presented data with an ENU induced glioblastoma rat model, a model
that closely mimics human GBM, that besides CXCR4 also CXCR7 is
involved in irradiation induced recruitment of BMDCs. In this study
the CXCR7 antagonist CCX2206 prevented the influx of BMDCs in the
irradiated tumor (Tseng et al., 2010). In accordance thereof and
because the nucleic acid molecules according to the present
invention are able to block the interaction of both SDF-1 and CXCR4
and SDF-1 and CXCR7 the effect on survival after irradiation is
expected to be better than shown for the use of one of the CXCR4
and CXCR7 antagonist alone.
[0155] In addition, SDF-1 induces VEGF secretion, while VEGF
increases CXCR4 expression (Salcedo et al. 1999) and angiogenesis
signals. Therefore inhibition of the SDF-1-CXCR4 axis may reduce or
prevent tumor growth by inhibition of angiogenesis/vasculogenesis
either with monotherapy or particularly in combination with other
antivascular agents such as VEGF inhibitors.
[0156] Furthermore it is suggested that `homing` of CXCR4
expressing cancer cells to SDF-1-expressing organs directs
metastatic cells preferentially to the liver, bone marrow, lung and
lymph nodes (Alsayed et al. 2007; Burger & Peled 2009) and
therefore the SDF-1-CXCR4 axis plays a role in metastasis.
[0157] Hence, the inhibition of the SDF-1-CXCR4 axis and the
SDF-1-CXCR7 axis with only one compound such as the SDF-1 binding
nucleic acid molecule according to the present invention should be
effective in treating cancer and/or tumors, in particular a wide
range of both haematological and solid tumors either as monotherapy
or in combination with other treatments such as, but not limited
to, drug therapy, cellular therapy, irradiation and surgery.
Moreover, in comparison to a compound that binds and inhibits one
out of the two SDF-1 receptors CXCR4 and CXCR7, the inhibition of
the SDF-1-CXCR4 axis and the SDF-1-CXCR7 axis with only one
compound such as the SDF-1 binding nucleic acid molecule according
to the present invention should be more effective in treating
cancer and/or tumors, in particular a wide range of both
haematological and solid tumors either as monotherapy or in
combination with other treatments such as but not limited to drug
therapy, cellular therapy, irradiation and surgery.
[0158] It is within the present invention that drug therapy
comprises the treatment and/or prevention of a disease or disorder
by a drug, preferably a pharmaceutically active agent, more
preferably a pharmaceutically active agent as defined herein.
[0159] As preferably used herein, in cell therapy also referred to
as cellular therapy, processed tissue from the organs, embryos, or
fetuses of animals such as sheep or cows is injected into a subject
suffering from or being at risk of developing a disease or
disorder, whereby preferably the disease or disorder is cancer and
cell therapy a form of cancer treatment.
[0160] In theory, non-hematological cancers can be cured if
entirely removed by surgery. When the cancer has metastasized to
other sites in the body prior to surgery, complete surgical
excision is usually impossible. Examples of surgical procedures or
surgery for cancer include mastectomy for breast cancer,
prostatectomy for prostate cancer, and lung cancer surgery for
non-small cell lung cancer. The goal of the surgery can be either
the removal of only the tumor, or of the entire organ. Surgery is
often combined with other cancer treatments or therapies, such as
chemotherapy and radiation. Cancer surgery may be used to achieve
one or more goals. Such goals may include, but are not limited to,
cancer prevention, diagnosis, staging, primary treatment, debulking
and relieving symptoms or side effects.
[0161] Radiotherapy (also referred to X-ray therapy or irradiation)
is the use of ionizing radiation to kill cancer cells. Radiotherapy
is used in the medical art to treat almost every type of solid
tumor. Irradiation is also used to treat leukemia and lymphoma.
Radiotherapy injures or destroys cells in the area being treated by
damaging their genetic material, making it impossible for these
cells to continue to grow and divide. The effects of radiotherapy
are localized and confined to the region being treated. Radiation
dose to each site depends on a number of factors, including the
radiosensitivity of each cancer type and whether there are tissues
and organs nearby that may be damaged by radiation. The goal of
radiotherapy is to damage as many cancer cells as possible, while
limiting harm to nearby healthy tissue.
[0162] Additionally, an SDF-1 binding nucleic molecule according to
the present invention is preferred if the physiological effect of
the SDF-1-CXCR4 axis and/or SDF-1-CXCR7 axis is related to higher
plasma levels of SDF-1. For instance, particular therapeutic agents
such as paclitaxel and bevacizumab produce an elevation of plasma
SDF-1 levels which can have a negative effect on tumor therapy by
releasing more bone marrow derived endothelial progenitor cells or
by stimulating growth, invasiveness or metastasis (Shaked, Henke et
al., 2008; Xu, Duda et al., 2009). In this case the co-application
of an SDF-1 binding nucleic acid will ameliorate the effects of
elevated plasma SDF-1 levels.
[0163] Moreover, the inhibition of the SDF-1-CXCR4 axis and/or
SDF-1-CXCR7 axis by an SDF-1 binding nucleic molecule according to
the present invention will enhance the anti-tumor effects of other
therapeutic agents by disrupting the adhesive stromal interactions
with leukemia and other cancer cells that confer survival and drug
resistance to these therapies (Jin et al. 2008; Nervi et al. 2009).
Such use of SDF-1 binding nucleic molecule is known as a process
known as chemosensitization.
[0164] The sensitization of tumor cells to chemotherapy or
radiotherapy is known as `chemosensitization` or
`radiosensitization`, respectively. Such `chemosensitization` or
`radiosensitization`, preferably by the nucleic acid molecules
according to the present invention, sensitizes the subject
suffering from a disease or disorder, whereby the sensitized
subject is more responsive to a therapy for the treatment and/or
prevention of the disease or disorder, whereby preferably the
disease or the disorder is cancer. Such treatment used together
with a primary treatment, preferably a cancer treatment, is an
adjunct therapy according to the present invention and also
referred to as adjunctive therapy. The purpose of such adjunct
therapy is to assist a primary treatment, preferably a primary
cancer treatment. Hence, the inhibition of the SDF-1-CXCR4 axis
and/or SDF-1-CXCR7 axis will be particularly effective in treating
a wide range of both haematological and solid tumors either as
monotherapy or in combination with other treatments such as but not
limited to drug therapy, cellular therapy, irradiation and
surgery.
[0165] By these means and in view of the outlined involvement of
SDF-1 and SDF-1 receptors--such as CXCR4 and CXCR7-, the SDF-1
binding and the interaction between SDF-1 and SDF-1 receptor
inhibiting nucleic acid molecules according to the present
invention can help to attenuate such diseases, whereby inhibition
of SDF-1 by the SDF-1 binding nucleic acid molecules according to
the present invention leads to chemosensitization of malignant
cells to be treated by chemotherapy, reduction or inhibition of
growth and invasiveness, inhibition of angiogenesis/vasculogenesis,
inhibition of metastasis and/or inhibition of elevated plasma SDF-1
levels derived from the response of the host to chemotherapy.
[0166] Moreover, the present invention is based on the surprising
finding that it is possible to generate nucleic acid molecules
binding specifically and with high affinity to SDF-1, thereby
inhibiting and antagonizing the effects of SDF-1, in particular the
effects of SDF-1 on its receptors such as CXCR4 and CXCR7.
[0167] An antagonists to SDF-1 is a molecule that binds to
SDF-1--such as the SDF-1 binding nucleic acid molecules according
to the present invention--and inhibits the function of SDF-1,
preferably in an in vitro assay or in an in vivo model as described
in the Examples.
[0168] It is within the present invention that the nucleic acid
according to the present invention is a nucleic acid molecule.
Insofar the terms nucleic acid and nucleic acid molecule are used
herein in a synonymous manner if not indicated to the contrary.
Moreover, such nucleic acids are preferably also referred to herein
as the nucleic acid molecules according to the present invention,
the nucleic acids according to the present invention, the inventive
nucleic acids or the inventive nucleic acid molecules.
[0169] The features of the nucleic acid according to the present
invention as described herein can be realised in any aspect of the
present invention where the nucleic acid is used, either alone or
in any combination.
[0170] As outlined in more detail herein, the present inventors
have identified a number of different SDF-1 binding nucleic acid
molecules, whereby the nucleic acid molecules can be characterised
in terms of stretches of nucleotides which are also referred to
herein as Boxes (see Example 1). As experimentally shown in
examples 5 to 11 the inventors could surprisingly demonstrate in
several systems that SDF-1 binding nucleic acid molecules are
suitable for the treatment of cancer and actually capable of
treating cancer.
[0171] The different types of SDF-1 binding nucleic acid molecules
comprise three different stretches of nucleotides: the first
terminal stretch of nucleotides, the central stretch of nucleotides
and second terminal stretch of nucleotides. In general, SDF-1
binding nucleic acid molecules of the present invention comprise at
their 5'-end and the 3'-end the terminal stretches of nucleotides:
the first terminal stretch of nucleotides and the second terminal
stretch of nucleotides (also referred to as 5'-terminal stretch of
nucleotides and 3'-terminal stretch of nucleotides). The first
terminal stretch of nucleotides and the second terminal stretch of
nucleotides can, in principle due to their base complementarity,
hybridize to each other, whereby upon hybridization a
double-stranded structure is formed. However, such hybridization is
not necessarily realized in the molecule under physiological and/or
non-physiological conditions. The three stretches of nucleotides of
SDF-1 binding nucleic acid molecules--the first terminal stretch of
nucleotides, the central stretch of nucleotides and second terminal
stretch of nucleotides--are arranged to each other in
5'.fwdarw.3'-direction: the first terminal stretch of
nucleotides--the central stretch of nucleotides--the second
terminal stretch of nucleotides. However, alternatively, the second
terminal stretch of nucleotides, the central stretch of nucleotides
and the terminal first stretch of nucleotides are arranged to each
other in 5'.fwdarw.3'-direction.
[0172] The differences in the sequences of the defined boxes or
stretches between the different SDF-1 binding nucleic acid
molecules influence the binding affinity to SDF-1. Based on binding
analysis of the different SDF-1 binding nucleic acid molecules of
the present invention the central stretch and the nucleotides
forming the same are individually and more preferably in their
entirety essential for binding to human SDF-1.
[0173] The terms `stretch` and `stretch of nucleotide` are used
herein in a synonymous manner if not indicated to the contrary.
[0174] In a preferred embodiment the nucleic acid according to the
present invention is a single nucleic acid molecule. In a further
embodiment, the single nucleic acid molecule is present as a
multitude of the single nucleic acid molecule or as a multitude of
the single nucleic acid molecule species.
[0175] It will be acknowledged by the ones skilled in the art that
the nucleic acid molecule in accordance with the invention
preferably consists of nucleotides which are covalently linked to
each other, preferably through phosphodiester links or
linkages.
[0176] It is within the present invention that the nucleic acids
according to the present invention comprise two or more stretches
or part(s) thereof can, in principle, hybridise with each other.
Upon such hybridisation a double-stranded structure is formed. It
will be acknowledged by the ones skilled in the art that such
hybridisation may or may not occur, particularly under in vitro
and/or in vivo conditions. Also, in case of such hybridisation, it
is not necessarily the case that the hybridisation occurs over the
entire length of the two stretches where, at least based on the
rules for base pairing, such hybridisation and thus formation of a
double-stranded structure may, in principle, occur. As preferably
used herein, a double-stranded structure is a part of a nucleic
acid molecule or a structure formed by two or more separate strands
or two spatially separated stretches of a single strand of a
nucleic acid molecule, whereby at least one, preferably two or more
base pairs exist which are base pairing preferably in accordance
with the Watson-Crick base pairing rules. It will also be
acknowledged by the one skilled in the art that other base pairing
such as Hoogsten base pairing may exist in or form such
double-stranded structure. It is also to be acknowledged that the
feature that two stretches hybridize preferably indicates that such
hybridization is assumed to happen due to base complementarity of
the two stretches.
[0177] In a preferred embodiment the term arrangement as used
herein, means the order or sequence of structural or functional
features or elements described herein in connection with the
nucleic acids disclosed herein.
[0178] It will be acknowledged by the person skilled in the art
that the nucleic acids according to the present invention are
capable of binding to SDF-1. Without wishing to be bound by any
theory, the present inventors assume that the SDF-1 binding results
from a combination of three-dimensional structural traits or
elements of the claimed nucleic acid molecule, which are caused by
orientation and folding patterns of the primary sequence of
nucleotides forming such traits or elements, whereby preferably
such traits or elements are the first terminal stretch of
nucleotides, the central stretch of nucleotides and the second
terminal stretch of nucleotides of SDF-1 binding nucleic acid
molecules. It is evident that the individual trait or element may
be formed by various different individual sequences the degree of
variation of which may vary depending on the three-dimensional
structure such element or trait has to form. The overall binding
characteristic of the claimed nucleic acid results from the
interplay of the various elements and traits, respectively, which
ultimately results in the interaction of the claimed nucleic acid
with its target, i.e. SDF-1. Again without being wished to be bound
by any theory, the central stretch of nucleotides that is
characteristic for SDF-1 binding nucleic acids seems to be
important for mediating the binding of the claimed nucleic acid
molecules with SDF-1. Accordingly, the nucleic acids according to
the present invention are suitable for the interaction with SDF-1.
Also, it will be acknowledged by the person skilled in the art that
the nucleic acids according to the present invention are
antagonists to SDF-1. Because of this the nucleic acids according
to the present invention are suitable for the treatment and
prevention, respectively, of any disease or condition which is
associated with or caused by SDF-1. Such diseases and conditions
may be taken from the prior art which establishes that SDF-1 is
involved or associated with said diseases and conditions,
respectively, and which is incorporated herein by reference
providing the scientific rationale for the therapeutic use of the
nucleic acids according to the invention.
[0179] The nucleic acids according to the present invention shall
also comprise nucleic acids which are essentially homologous to the
particular sequences disclosed herein. The term substantially
homologous shall be understood such as the homology is at least
75%, preferably 85%, more preferably 90% and most preferably more
that 95%, 96%, 97%, 98% or 99%.
[0180] The actual percentage of homologous nucleotides present in
the nucleic acid according to the present invention will depend on
the total number of nucleotides present in the nucleic acid. The
percent modification can be based upon the total number of
nucleotides present in the nucleic acid.
[0181] The homology between two nucleic acid molecules can be
determined as known to the person skilled in the art. More
specifically, a sequence comparison algorithm may be used for
calculating the percent sequence homology for the test sequence(s)
relative to the reference sequence, based on the designated program
parameters. The test sequence is preferably the sequence or nucleic
acid molecule which is said to be homologous or to be tested
whether it is homologous, and if so, to what extent, to a different
nucleic acid molecule, whereby such different nucleic acid molecule
is also referred to as the reference sequence. In an embodiment,
the reference sequence is a nucleic acid molecule as described
herein, preferably a nucleic acid molecule having a sequence
according to any one of SEQ ID NO: 5 to SEQ ID NO: 225, more
preferably a nucleic acid molecule having a sequence according to
any one of SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 120, SEQ ID NO:
128, SEQ ID NO: 129, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:
223, SEQ ID NO: 224, SEQ ID NO: 84, SEQ ID NO: 146, SEQ ID NO: 142,
SEQ ID NO: 143, and SEQ ID NO: 144. Optimal alignment of sequences
for comparison can be conducted, e.g., by the local homology
algorithm of Smith & Waterman (Smith & Waterman, 1981) by
the homology alignment algorithm of Needleman & Wunsch
(Needleman & Wunsch, 1970) by the search for similarity method
of Pearson & Lipman (Pearson & Lipman, 1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
visual inspection.
[0182] One example of an algorithm that is suitable for determining
percent sequence identity is the algorithm used in the basic local
alignment search tool (hereinafter "BLAST"), see, e.g. Altschul et
al (Altschul et al. 1990 and Altschul et al, 1997). Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information (hereinafter "NCBI").
The default parameters used in determining sequence identity using
the software available from NCBI, e.g., BLASTN (for nucleotide
sequences) and BLASTP (for amino acid sequences) are described in
McGinnis et al (McGinnis et al, 2004).
[0183] The nucleic acids according to the present invention shall
also comprise nucleic acids which have a certain degree of identity
relative to the nucleic acids disclosed herein and defined by their
nucleotide sequence. More preferably, the instant invention also
comprises those nucleic acid molecules which have an identity of at
least 75%, preferably 85%, more preferably 90% and most preferably
more than 95%, 96%, 97%, 98% or 99% relative to the nucleic acids
disclosed herein and defined by their nucleotide sequence or a part
thereof.
[0184] The term inventive nucleic acid or nucleic acid according to
the present invention shall also comprise those nucleic acids
comprising the nucleic acids sequences disclosed herein or part
thereof, such as, e.g., a metabolite or derivative of the nucleic
acid according to the present invention, preferably to the extent
that the nucleic acids or said parts are involved in the or capable
of binding to SDF-1. Such a nucleic acid may be derived from the
ones disclosed herein, e.g., by truncation. Truncation may be
related to either or both of the ends of the nucleic acids as
disclosed herein. Also, truncation may be related to the inner
sequence of nucleotides, i.e. it may be related to the
nucleotide(s) between the 5' and the 3' terminal nucleotide,
respectively. Moreover, truncation shall comprise the deletion of
as little as a single nucleotide from the sequence of the nucleic
acids disclosed herein. Truncation may also be related to more than
one stretch of the inventive nucleic acid(s), whereby the stretch
can be as little as one nucleotide long. The binding of a nucleic
acid according to the present invention can be determined by the
ones skilled in the art using routine experiments or by using or
adopting a method as described herein, preferably as described
herein in the example part.
[0185] The nucleic acids according to the present invention may be
either D-nucleic acids or L-nucleic acids. Preferably, the
inventive nucleic acids are L-nucleic acids. In addition it is
possible that one or several parts of the nucleic acid are present
as D-nucleic acids or at least one or several parts of the nucleic
acids are L-nucleic acids. The term "part" of the nucleic acids
shall mean as little as one nucleotide. Such nucleic acids are
generally referred to herein as D- and L-nucleic acids,
respectively. Therefore, in a particularly preferred embodiment,
the nucleic acids according to the present invention consist of
L-nucleotides and comprise at least one D-nucleotide. Such
D-nucleotide is preferably attached to a part different from the
stretches defining the nucleic acids according to the present
invention, preferably those parts thereof, where an interaction
with other parts of the nucleic acid is involved. Preferably, such
D-nucleotide is attached at a terminus of any of the stretches and
of any nucleic acid according to the present invention,
respectively. In a further preferred embodiment, such D-nucleotides
may act as a spacer or a linker, preferably attaching modifications
such as PEG and HES to the nucleic acids according to the present
invention.
[0186] It is also within the present invention that each and any of
the nucleic acid molecules described herein in their entirety in
terms of their nucleic acid sequence(s) are limited to the
particular nucleotide sequence(s). In other words, the terms
"comprising" or "comprise(s)" shall be interpreted in such
embodiment in the meaning of containing or consisting of:
[0187] It is also within the present invention that the nucleic
acids according to the present invention are part of a longer
nucleic acid whereby this longer nucleic acid comprises several
parts whereby at least one such part is a nucleic acid, or a part
thereof, according to the present invention. The other part(s) of
these longer nucleic acids can be either one or several D-nucleic
acid(s) or L-nucleic acid(s). Any combination may be used in
connection with the present invention. These other part(s) of the
longer nucleic acid can exhibit a function which is different from
binding, preferably from binding to SDF-1. One possible function is
to allow interaction with other molecules, whereby such other
molecules preferably are different from SDF-1, such as, e.g., for
immobilization, cross-linking, detection or amplification. In a
further embodiment of the present invention the nucleic acids
according to the invention comprise, as individual or combined
moieties, several of the nucleic acids of the present invention.
Such nucleic acid comprising several of the nucleic acids of the
present invention is also encompassed by the term longer nucleic
acid.
[0188] L-nucleic acids as used herein are nucleic acids consisting
of L-nucleotides, preferably consisting completely of
L-nucleotides.
[0189] D-nucleic acids as used herein are nucleic acids consisting
of D-nucleotides, preferably consisting completely of
D-nucleotides.
[0190] The terms nucleic acid and nucleic acid molecule are used
herein in an interchangeable manner if not explicitly indicated to
the contrary.
[0191] Also, if not indicated to the contrary, any nucleotide
sequence is set forth herein in 5'.fwdarw.3' direction.
[0192] As preferably used herein any position of a nucleotide is
determined or referred to relative to the 5' end of a sequence, a
stretch or a substretch. Accordingly, a second nucleotide is the
second nucleotide counted from the 5' end of the sequence, stretch
and substretch, respectively. Also, in accordance therewith, a
penultimate nucleotide is the second nucleotide counted from the 3'
end of a sequence, stretch and substretch, respectively.
[0193] Irrespective of whether the inventive nucleic acid consists
of D-nucleotides, L-nucleotides or a combination of both with the
combination being e.g. a random combination or a defined sequence
of stretches consisting of at least one L-nucleotide and at least
one D-nucleic acid, the nucleic acid may consist of
desoxyribonucleotide(s), ribonucleotide(s) or combinations
thereof.
[0194] Designing the inventive nucleic acids as L-nucleic acid is
advantageous for several reasons. L-nucleic acids are enantiomers
of naturally occurring nucleic acids. D-nucleic acids, however, are
not very stable in aqueous solutions and particularly in biological
systems or biological samples due to the widespread presence of
nucleases. Naturally occurring nucleases, particularly nucleases
from animal cells are not capable of degrading L-nucleic acids.
Because of this the biological half-life of the L-nucleic acid is
significantly increased in such a system, including the animal and
human body. Due to the lacking degradability of L-nucleic acid no
nuclease degradation products are generated and thus no side
effects arising therefrom observed. This aspect delimits the
L-nucleic acid of factually all other compounds which are used in
the therapy of diseases and/or disorders involving the presence of
SDF-1. L-nucleic acids which specifically bind to a target molecule
through a mechanism different from Watson Crick base pairing, or
aptamers which consists partially or completely of L-nucleotides,
particularly with those parts of the aptamer being involved in the
binding of the aptamer to the target molecule, are also called
spiegelmers. Aptamers and spiegelmers as such are known to a person
skilled in the art and are, among others, described in `The Aptamer
Handbook` (eds. Klussmann, 2006).
[0195] It is also within the present invention that the inventive
nucleic acids, regardless whether they are present as D-nucleic
acids, L-nucleic acids or D,L-nucleic acids or whether they are DNA
or RNA, may be present as single stranded or double stranded
nucleic acids. Typically, the inventive nucleic acids are single
stranded nucleic acids which exhibit defined secondary structures
due to the primary sequence and may thus also form tertiary
structures. The inventive nucleic acids, however, may also be
double stranded in the meaning that two strands which are
complementary or partially complementary to each other are
hybridised to each other.
[0196] The inventive nucleic acids may be modified. Such
modifications may be related to the single nucleotide of the
nucleic acid and are well known in the art. Examples for such
modification are described by, among others, Venkatesan et al.
(Venkatesan, Kim et al. 2003) and Kusser (Kusser 2000). Such
modification can be a H atom, a F atom or O--CH.sub.3 group or
NH.sub.2-group at the 2' position of the individual nucleotide of
which the nucleic acid consists. Also, the nucleic acid according
to the present invention can comprises at least one LNA nucleotide.
In an embodiment the nucleic acid according to the present
invention consists of LNA nucleotides.
[0197] In an embodiment, the nucleic acids according to the present
invention may be a multipartite nucleic acid. A multipartite
nucleic acid as used herein, is a nucleic acid which consists of at
least two separate nucleic acid strands. These at least two nucleic
acid strands form a functional unit whereby the functional unit is
a ligand to a target molecule. The at least two nucleic acid
strands may be derived from any of the inventive nucleic acids by
either cleaving the nucleic acid molecule to generate two strands
or by synthesising one nucleic acid corresponding to a first part
of the inventive, i.e. overall nucleic acid and another nucleic
acid corresponding to the second part of the overall nucleic acid.
It is to be acknowledged that both the cleavage and the synthesis
may be applied to generate a multipartite nucleic acid where there
are more than two strands as exemplified above. In other words, the
at least two separate nucleic acid strands are typically different
from two strands being complementary and hybridising to each other
although a certain extent of complementarity between said at least
two separate nucleic acid strands may exist and whereby such
complementarity may result in the hybridisation of said separate
strands.
[0198] Finally it is also within the present invention that a fully
closed, i.e. circular structure for the nucleic acids according to
the present invention is realized, i.e. that the nucleic acids
according to the present invention are closed in an embodiment,
preferably through a covalent linkage, whereby more preferably such
covalent linkage is made between the 5' end and the 3' end of the
nucleic acid sequences as disclosed herein or any derivative
thereof.
[0199] A possibility to determine the binding constants of the
nucleic acid molecules according to the present invention is the
use of the methods as described in example 3 and 4 which confirms
the above finding that the nucleic acids according to the present
invention exhibit a favourable K.sub.D value range. An appropriate
measure in order to express the intensity of the binding between
the individual nucleic acid molecule and the target which is in the
present case SDF-1 is the so-called K.sub.D value which as such as
well the method for its determination are known to the one skilled
in the art.
[0200] Preferably, the K.sub.D value shown by the nucleic acids
according to the present invention is below 1 .mu.M. A K.sub.D
value of about 1 .mu.M is said to be characteristic for a
non-specific binding of a nucleic acid to a target. As will be
acknowledged by the ones skilled in the art, the K.sub.D value of a
group of compounds such as the nucleic acids according to the
present invention is within a certain range. The above-mentioned
K.sub.D of about 1 .mu.M is a preferred upper limit for the K.sub.D
value. The lower limit for the K.sub.D of target binding nucleic
acids can be as little as about 10 picomolar or can be higher. It
is within the present invention that the K.sub.D values of
individual nucleic acids binding to SDF-1 is preferably within this
range. Preferred ranges can be defined by choosing any first number
within this range and any second number within this range.
Preferred upper K.sub.D values are 250 nM and 100 nM, preferred
lower K.sub.D values are 50 nM, 10 nM, 1 nM, 100 pM and 10 pM. The
more preferred upper K.sub.D value is 2.5 nM, the more preferred
lower K.sub.D value is 100 pM.
[0201] In addition to the binding properties of the nucleic acid
molecules according to the present invention, the nucleic acid
molecules according to the present invention inhibit the function
of the respective target molecule which is in the present case
SDF-1. The inhibition of the function of SDF-1--for instance the
stimulation of the respective receptors as described previously--is
achieved by binding of nucleic acid molecules according to the
present invention to SDF-1 and forming a complex of a nucleic acid
molecule according to the present invention and MCP-1 and SDF-1.
Such complex of a nucleic acid molecule and SDF-1 cannot stimulate
the receptors that normally are stimulated by SDF-1. Accordingly,
the inhibition of receptor function by nucleic acid molecules
according to the present invention is independent from the
respective receptor that can be stimulated by SDF-1 but results
from preventing the stimulation of the receptor by MCP-1 and SDF-1
by the nucleic acid molecules according to the present
invention.
[0202] A possibility to determine the inhibitory constant of the
nucleic acid molecules according to the present invention is the
use of the methods as described in example 5 and 6 (for the CXCR4
and CXCR7, respectively) which confirms the above finding that the
nucleic acids according to the present invention exhibit a
favourable inhibitory constant which allows the use of said nucleic
acids in a therapeutic treatment scheme. An appropriate measure in
order to express the intensity of the inhibitory effect of the
individual nucleic acid molecule on interaction of the target which
is in the present case SDF-1 and the respective receptor, is the
so-called half maximal inhibitory concentration (abbr. IC.sub.50)
which as such as well the method for its determination are known to
the one skilled in the art.
[0203] Preferably, the IC.sub.50 value shown by the nucleic acid
molecules according to the present invention is below 1 .mu.M. An
IC.sub.50 value of about 1 .mu.M is said to be characteristic for a
non-specific inhibition of target functions by a nucleic acid
molecule. As will be acknowledged by the ones skilled in the art,
the IC.sub.50 value of a group of compounds such as the nucleic
acid molecules according to the present invention is within a
certain range. The above-mentioned IC.sub.50 of about 1 .mu.M is a
preferred upper limit for the IC.sub.50 value. The lower limit for
the IC.sub.50 of target binding nucleic acid molecules can be as
little as about 10 picomolar or can be higher. It is within the
present invention that the IC.sub.50 values of individual nucleic
acids binding to SDF-1 is preferably within this range. Preferred
ranges can be defined by choosing any first number within this
range and any second number within this range. Preferred upper
IC.sub.50 values are 250 nM and 100 nM, preferred lower IC.sub.50
values are 50 nM, 10 nM, 1 nM, 100 pM and 10 pM. The more preferred
upper IC.sub.50 value is 2.5 nM, the more preferred lower IC.sub.50
value is 100 pM.
[0204] The nucleic acid molecules according to the present
invention may have any length provided that they are still able to
bind to the target molecule. It will be acknowledged in the art
that there are preferred lengths of the nucleic acids according to
the present inventions. Typically, the length is between 15 and 120
nucleotides. It will be acknowledged by the ones skilled in the art
that any integer between 15 and 120 is a possible length for the
nucleic acids according to the present invention. More preferred
ranges for the length of the nucleic acids according to the present
invention are lengths of about 20 to 100 nucleotides, about 20 to
80 nucleotides, about 20 to 60 nucleotides, about 20 to 50
nucleotides and about 29 to 450 nucleotides.
[0205] It is within the present invention that the nucleic acids
disclosed herein comprise a moiety which preferably is a high
molecular weight moiety and/or which preferably allows to modify
the characteristics of the nucleic acid in terms of, among others,
residence time in the animal body, preferably the human body. A
particularly preferred embodiment of such modification is
PEGylation and HESylation of the nucleic acids according to the
present invention. As used herein PEG stands for poly(ethylene
glycole) and HES for hydroxyethly starch. PEGylation as preferably
used herein is the modification of a nucleic acid according to the
present invention whereby such modification consists of a PEG
moiety which is attached to a nucleic acid according to the present
invention. HESylation as preferably used herein is the modification
of a nucleic acid according to the present invention whereby such
modification consists of a HES moiety which is attached to a
nucleic acid according to the present invention. These
modifications as well as the process of modifying a nucleic acid
using such modifications, is described in European patent
application EP 1 306 382, the disclosure of which is herewith
incorporated in its entirety by reference.
[0206] In the case of PEG being such high molecular weight moiety
the molecular weight is preferably about 20,000 to about 120,000
Da, more preferably from about 30,000 to about 80,000 Da and most
preferably about 40,000 Da. In the case of HES being such high
molecular weight moiety the molecular weight is preferably from
about 50 to about 1000 kDa, more preferably from about 100 to about
700 kDa and most preferably from 200 to 500 kDa. HES exhibits a
molar substitution of 0.1 to 1.5, more preferably of 1 to 1.5 and
exhibits a substitution sample expressed as the C2/C6 ratio of
approximately 0.1 to 15, preferably of approximately 3 to 10. The
process of HES modification is, e.g., described in German patent
application DE 1 2004 006 249.8 the disclosure of which is herewith
incorporated in its entirety by reference.
[0207] The modification can, in principle, be made to the nucleic
acid molecules of the present invention at any position thereof.
Preferably such modification is made either to the 5'-terminal
nucleotide, the 3'-terminal nucleotide and/or any nucleotide
between the 5' nucleotide and the 3' nucleotide of the nucleic acid
molecule.
[0208] The modification and preferably the PEG and/or HES moiety
can be attached to the nucleic acid molecule of the present
invention either directly or indirectly, preferably through a
linker. It is also within the present invention that the nucleic
acid molecule according to the present invention comprises one or
more modifications, preferably one or more PEG and/or HES moiety.
In an embodiment the individual linker molecule attaches more than
one PEG moiety or HES moiety to a nucleic acid molecule according
to the present invention. The linker used in connection with the
present invention can itself be either linear or branched. This
kind of linkers are known to the ones skilled in the art and are
further described in patent applications WO2005/074993 and
WO2003/035665.
[0209] In a preferred embodiment the linker is a biodegradable
linker. The biodegradable linker allows to modify the
characteristics of the nucleic acid according to the present
invention in terms of, among other, residence time in an animal
body, preferably in a human body, due to release of the
modification from the nucleic acid according to the present
invention. Usage of a biodegradable linker may allow a better
control of the residence time of the nucleic acid according to the
present invention. A preferred embodiment of such biodegradable
linker is a biodegradable linker as described in, but not limited
to, international patent applications WO2006/052790, WO2008/034122,
WO2004/092191 and WO2005/099768.
[0210] It is within the present invention that the modification or
modification group is a biodegradable modification, whereby the
biodegradable modification can be attached to the nucleic acid
molecule of the present invention either directly or indirectly,
preferably through a linker. The biodegradable modification allows
to modify the characteristics of the nucleic acid according to the
present invention in terms of, among other, residence time in an
animal body, preferably in a human body, due to release or
degradation of the modification from the nucleic acid according to
the present invention. Usage of biodegradable modification may
allow a better control of the residence time of the nucleic acid
according to the present invention. A preferred embodiment of such
biodegradable modification is biodegradable as described in, but
not restricted to, international patent applications WO2002/065963,
WO2003/070823, WO2004/113394 and WO2000/41647, preferably in
WO2000/41647, page 18, line 4 to 24.
[0211] Beside the modifications as described above, other
modifications can be used to modify the characteristics of the
nucleic acids according to the present invention, whereby such
other modifications may be selected from the group of proteins,
lipids such as cholesterol and sugar chains such as amylase,
dextran etc.
[0212] Without wishing to be bound by any theory, it seems that by
modifying the nucleic acids according to the present invention with
high molecular weight moiety such as a polymer and more
particularly one or several of the polymers disclosed herein, which
are preferably physiologically acceptable, the excretion kinetic is
changed. More particularly, it seems that due to the increased
molecular weight of such modified inventive nucleic acids and due
to the nucleic acids of the invention not being subject to
metabolism particularly when in the L form, excretion from an
animal body, preferably from a mammalian body and more preferably
from a human body is decreased. As excretion typically occurs via
the kidneys, the present inventors assume that the glomerular
filtration rate of the thus modified nucleic acids is significantly
reduced compared to the nucleic acids not having this kind of high
molecular weight modification which results in an increase in the
residence time in the animal body. In connection therewith it is
particularly noteworthy that, despite such high molecular weight
modification the specificity of the nucleic acids according to the
present invention is not affected in a detrimental manner. Insofar,
the nucleic acids according to the present invention have among
others, the surprising characteristic--which normally cannot be
expected from pharmaceutically active compounds--such that a
pharmaceutical formulation providing for a sustained release is not
necessarily required to provide for a sustained release of the
nucleic acids according to the present invention. Rather the
nucleic acids according to the present invention in their modified
form comprising a high molecular weight moiety, can as such already
be used as a sustained release-formulation as they act, due to
their modification, already as if they were released from a
sustained-release formulation. Insofar, the modification(s) of the
nucleic acid molecules according to the present invention as
disclosed herein and the thus modified nucleic acid molecules
according to the present invention and any composition comprising
the same may provide for a distinct, preferably controlled
pharmacokinetics and biodistribution thereof. This also includes
residence time in circulation and distribution to tissues. Such
modifications are further described in the patent application
WO2003/035665.
[0213] However, it is also within the present invention that the
nucleic acids according to the present invention do not comprise
any modification and particularly no high molecular weight
modification such as PEGylation or HESylation. Such embodiment is
particularly preferred when the nucleic acid according to the
present invention shows preferential distribution to any target
organ or tissue in the body or when a fast clearance of the nucleic
acid according to the present invention from the body after
administration is desired. Nucleic acids according to the present
invention as disclosed herein with a preferential distribution
profile to any target organ or tissue in the body would allow
establishment of effective local concentrations in the target
tissue while keeping systemic concentration of the nucleic acids
low. This would allow the use of low doses which is not only
beneficial from an economic point of view, but also reduces
unnecessary exposure of other tissues to the nucleic acid agent,
thus reducing the potential risk of side effects. Fast clearance of
the nucleic acids according to the present invention from the body
after administration might be desired, among others, in case of in
vivo imaging or specific therapeutic dosing requirements using the
nucleic acids according to the present invention or medicaments
comprising the same.
[0214] The nucleic acids according to the present invention, and/or
the antagonists according to the present invention may be used for
the generation or manufacture of a medicament. Such medicament or a
pharmaceutical composition according to the present invention
contains at least one of the inventive nucleic acids selected from
the group of SDF-1 binding nucleic acids, optionally together with
further pharmaceutically active compounds, whereby the inventive
nucleic acid preferably acts as pharmaceutically active compound
itself. Such medicaments comprise in preferred embodiments at least
a pharmaceutically acceptable carrier. Such carrier may be, e.g.,
water, buffer, PBS, glucose solution, preferably a 5% glucose salt
balanced solution, starch, sugar, gelatine or any other acceptable
carrier substance. Such carriers are generally known to the one
skilled in the art. It will be acknowledged by the person skilled
in the art that any embodiments, use and aspects of or related to
the medicament of the present invention is also applicable to the
pharmaceutical composition of the present invention and vice
versa.
[0215] The indication, diseases and disorders for the treatment
and/or prevention of which the nucleic acids, the pharmaceutical
compositions and medicaments in accordance with or prepared in
accordance with the present invention result from the involvement,
either direct or indirect, of SDF-1 in the respective pathogenetic
mechanism.
[0216] Of course, because the SDF-1 binding nucleic acids according
to the present invention interact with or bind to human or murine
SDF-1, a skilled person will generally understand that the SDF-1
binding nucleic acids according to the present invention can easily
be used for the treatment, prevention and/or diagnosis of any
disease as described herein of humans and animals. In connection
therewith, it is to be acknowledged that the nucleic acid molecules
according to the present invention can be used for the treatment
and prevention of any of the diseases, disorder or condition
described herein, irrespective of the mode of action underlying
such disease, disorder and condition.
[0217] In the following the rational for the use of the nucleic
acid molecules according to the present invention in connection
with the various diseases, disorders and conditions is provided,
thus rendering the claimed therapeutic, preventive and diagnostic
applicability of the nucleic acid molecules according to the
present invention plausible. In order to avoid any unnecessary
repetition, it should be acknowledged that due to the involvement
of the SDF-1-SDF-1 receptor axis as outlined in connection
therewith said axis may be addressed by the nucleic acid molecules
according to the present invention such that the claimed
therapeutic, preventive and diagnostic effect is achieved. It
should furthermore be acknowledged that the particularities of the
diseases, disorders and conditions, of the patients and any detail
of the treatment regimen described in connection therewith, may be
subject to preferred embodiments of the instant application.
[0218] For haematological malignancies, in particular, there is
considerable evidence that leukemia cells may be protected from
conventional therapies (chemotherapy combined with various targeted
agents such as specific antibodies or kinase inhibitors) within
particular tissue microenvironments, referred to as niches. Such
niches are found particularly in the bone marrow where they can
harbour malignant cells that are then able to expand and produce a
relapse following the initial therapy (Burger and Kipps, 2002;
Burger and Burkle, 2007; Meads et al., 2008; Burger, Ghia et al.,
2009). This preservation of malignant cells during chemotherapy is
thought to be largely due to direct contact between the malignant
cells and stromal cells (Lagneaux, Delforge et al. 1998; Kurtova,
Balakrishnan et al., 2009; Damiano, Cress et al., 1999) however in
the complexity of this microenvironment there are multiple cellular
and molecular signals that may lead to resistance of the malignant
cells to chemotherapy. Despite this complexity it is clear that
stromal cells produce the chemokine SDF-1 and that both normal and
malignant cells that express CXCR4 migrate to and are held in such
niches. That this molecular pathway is key for this interaction is
demonstrated by the fact that specific inhibition of this
interaction is sufficient to release both normal and malignant
cells from the niches (Broxmeyer, Orschell et al., 2005; Devine,
Flomenberg et al., 2004; Azab, Runnels et al., 2009). In addition
to weakening the interaction with the niches it has been shown for
numerous hematological malignancies that disruption of the
SDF-1-CXCR4 axis results in increasing the vulnerability of the
cells to other therapies--so called `chemosensitization`. This
chemosensitization has been described for multiple myeloma (Azab,
Runnels et al., 2009) and various acute and chronic leukemias
(Dillmann, Veldwijk et al., 2009; Lagneaux, Delforge et al.
1998).
[0219] Therefore use of SDF-1 binding nucleic acids according to
the present invention to disrupt cross talk between malignant cells
and their milieu to sensitize them to other therapies is an
attractive strategy for the treatment of haematological
malignacies. Examples of therapies that can be enhanced by
combination with SDF-1 binding nucleic acids according to the
present invention include the following but not limited to
Fludarabine, Cyclophosphamide, Rituxan, Chlorambucil,
Lenalidomide,.Bortezomib,.Dexamethasone,.Melphalan, Imatinib or
Nilotinib.
[0220] The foregoing description emphasized the role of bone marrow
stromal cells and bone marrow niches in the protection of malignant
cells from the effects of chemotherapy or other targeted therapies
for haematological malignancies. However there is evidence for
similar interactions occurring locally within solid tumors as a
large proportion of the cells in solid tumors are not cancer cells
but rather stromal, immune or vascular cells derived from the host
that interact intimately with the tumor cells. Many different types
of solid tumors express CXCR4 (Engl, Relja et al., 2006; Muller,
Homey et al., 2001; Koshiba, Hosotani et al., 2000, Ehtesham,
Stevenson, et al., 2008; Zeelenberg, Ruuls-Van Stalle et al., 2003;
Sauer, Seidler et al., 2005; Su, Zhang et al., 2005) and/or CXCR7
(Burns, Summers et al. 2006; Miao et al., 2007; Wang et al., 2008;
Zheng, Li et al., 2010) receptors either constitutively or in
response to hypoxia or various treatments. Malignant cells may use
this signaling pathway for survival and migration by activation of
Akt and Erk. SDF-1 can be produced by the malignant cells
themselves or by the stromal cells within the tumor. Once again in
this complex environment the exact mechanism by which tumour cells
grow and escape from chemotherapy or other therapeutic approaches
are not clearly defined. However it is clear that the SDF-1-CXCR4
axis and the SDF-1-CXCR7 play an important role. For example
inhibition of CXCR4 sensitizes glioma cell lines to in vitro
chemotherapy (Redjal et al., 2006) and high expression of CXCR4 is
predictive of poor outcome in breast cancer (Holm, Abreo et al.,
2008; Mizell, Smith et al., 2009) and gastro-intestinal cancers
(Schimanski et al., 2008). Therefore the use of SDF-1 binding
nucleic acids according to the present invention to inhibit the
action of SDF-1 on either CXCR4 or CXCR7 receptors in a wide
variety of solid tumors will enhance current therapy by making the
cells more vulnerable to the therapy either by direct action or by
blocking interactions with other cells in the tumor.
[0221] In addition to the above aspects CXCR4 also conveys signals
that are thought to be critical for recruitment and retention of
pro-angiogenic and immunosuppressive bone marrow-derived cells
(BMDCs). This pathway may therefore also be used for
VEGF-independent angiogenesis. As a consequence, blocking the
SDF1-CXCR4 axis to sensitize tumors to anti-VEGF therapy or
radiation has emerged as an attractive strategy treatment for solid
cancers.
[0222] However, there is a concern that CXCR4 blockade may not be
sufficient to block the effects of SDF-1, which may also bind to
CXCR7 on cancer or stromal cells. For example, CXCR7 has been
recently reported to be expressed in brain tumor cells and mediate
anti-apoptotic effects, and has also been shown to regulate the
invasion, angiogenesis and tumor growth of human hepatocellular
carcinomas. In such cases the action of SDF-1 binding nucleic acids
to block the action of SDF-1 on both the CXCR7 and CXCR4 receptors
in a single agent would provide a particular efficacy compared to
specific receptor blockers.
[0223] The medicament according to the present invention may be
used in combination with a further medicament or a further
pharmaceutically active agent, whereby the further medicament or
the further pharmaceutically active agent damages, destroys and/or
labels (the) cancer cells. If the nucleic acid molecule according
to the present invention is used with a further medicament or a
further pharmaceutically active agent, the therapy which is based
on the nucleic acid molecule is preferably an adjunct therapy to
the therapy making use of or being based on the further medicament
or further pharmaceutically active agent. Such further medicament
or further pharmaceutically active agent are preferably selected
from but not restricted to the group comprising [0224] a)
antibodies such as Rituximab (target: CD20), Cetuximab (target:
epidermal growth factor receptor), Ibritumomab-Tiuxetan (target:
CD20), Tositumomab (target: CD20), Trastuzumab (target: HER2/neu),
Bevacizumab (target: VEGF), Alemtuzumab (target: CD52); [0225] b)
alkylating agents such as cisplatin, carboplatin, oxaliplatin,
mechlorethamine, cyclophosphamide, chlorambucil, Doxorubicin,
liposomal Doxorubicin, bendamustine, Melphalan, temozolomide [0226]
c) anti-metabolites such as purineazathioprine, mercaptopurine,
fludarabine, pentostatin, cladribine; [0227] d) plant alkaloids
such vinca alkaloids, plant terpenoids such as taxanes, preferably
Docetaxel, Paclitaxel, podophyllotoxin, epothilone; [0228] e)
topoisomerase inhibitors such as camptothecins, irinitecan,
mitoxantrone; [0229] f) and other such as Leucovorin, Methotrexate,
Tamoxifen, Sorafenib, Lenalidomide, Bortezomib, Dexamethasone,
Fluorouracil and Prendnisone.
[0230] Other agents that can be used as further pharmaceutically
active agent in the treatment of cancer are well known in the art
and include, but are not limited toimmunsuppressive drugs,
cytokines and cytostatic drugs (for reference: "Allgemeine und
Spezielle Pharmakologie und Toxikologie 2011", editor: Thomas
Karow; Pulheim, Germany). Such agents well known in the art are
used in the treatment of cancer according to the current standard
of care for the particular cancer patient population.
[0231] It will be acknowledged that the above specified further
pharmaceutically active agents can be used in connection with each
any aspect of the present invention which makes use of such further
pharmaceutically active agent.
[0232] The further medicament or pharmaceutically active agent has
or may provide the function of a chemotherapy. Alternatively or
additionally to chemotherapy radiotherapy can be used.
[0233] The medicament according to the present invention, in
combination with or without the further medicament or further
pharmaceutically active agent, and with or without radiotherapy,
can be used for the treatment and/or prevention of cancer,
preferably [0234] a) hematological cancer, whereby more preferably
the hematological cancer is selected from the group of leukemia,
and myeloma. [0235] b) solid tumors, whereby more solid tumors are
selected from the group of glioblastoma, colorectal cancer, breast
cancer, lymphoma, prostate cancer, pancreatic cancer, lung cancer,
renal cancer, andovarian cancer
[0236] Preferably breast cancer is selected from the group of
advanced HER2-negative breast cancer.
[0237] Preferably leukemia is selected from the group of chronic
lymphoid leukemia and acute myeloid leukemia.
[0238] Preferably myeloma is selected from the group of multiple
myeloma.
[0239] The preferred further medicament or a further
pharmaceutically active agent for the treatment of Glioblastoma is
radiotherapy or chemotherapy with temozolomide or therapy with
bevacizumab. The preferred further medicament or a further
pharmaceutically active agent for the treatment of colorectal
cancer is selected from the group comprising
fluorouracil,.Leucovorin, Oxaliplatin, Irinotecan and
bevacizumab.
[0240] The preferred further medicament or a further
pharmaceutically active agent for the treatment of advanced
HER2-negative breast cancer is selected from the group of
Doxorubicin,. Paclitaxel,
Docetaxel,.Methotrexate,.Fluorouracil,.Bevacizumab,.Tamoxifen, and
aromatase inhibitors.
[0241] The preferred further medicament or a further
pharmaceutically active agent for the treatment of chronic lymphoid
leukemia is.selected from the group comprising
fludarabine,.cyclophosphamide,.rituximab, Chlorambucil,
alemtuzumab, vincristine, pentostatin, mitoxantrone, doxorubicin,
cladribine, and bendamustine.
[0242] The preferred further medicament or a further
pharmaceutically active agent for the treatment of multiple myeloma
is selected from the group comprising
Lenalidomide,.Bortezomib,.Dexamethasone,.Melphalan,
Cyclophosphamide, liposomal doxorubicin, and prednisone.
[0243] In one embodiment of the medicament of the present
invention, such medicament is for use in combination with other
treatments for any of the diseases disclosed herein, particularly
those for which the medicament of the present invention is to be
used.
[0244] "Combination therapy" (or "co-therapy") includes the
administration of a medicament of the invention and at least a
second or further agent as part of a specific treatment regimen
intended to provide the beneficial effect from the co-action of
these therapeutic agents, i.e. the medicament of the present
invention and said second or further agent. The beneficial effect
of the combination includes, but is not limited to, pharmacokinetic
or pharmacodynamic co-action resulting from the combination of
therapeutic agents. Administration of these therapeutic agents in
combination typically is carried out over a defined time period
(usually minutes, hours, days or weeks depending upon the
combination selected).
[0245] "Combination therapy" may be, but generally is not, intended
to encompass the administration of two or more of these therapeutic
agents as part of separate monotherapy regimens. "Combination
therapy" is intended to embrace administration of these therapeutic
agents in a sequential manner, that is, wherein each therapeutic
agent is administered at a different time, as well as
administration of these therapeutic agents, or at least two of the
therapeutic agents, in a substantially simultaneous manner.
Substantially simultaneous administration can be accomplished, for
example, by administering to a subject a single capsule having a
fixed ratio of each therapeutic agent or in multiple, single
capsules for each of the therapeutic agents.
[0246] Sequential or substantially simultaneous administration of
each therapeutic agent can be effected by any appropriate route
including, but not limited to, topical routes, oral routes,
intravenous routes, intramuscular routes, and direct absorption
through mucous membrane tissues. The therapeutic agents can be
administered by the same route or by different routes. For example,
a first therapeutic agent of the combination selected may be
administered by injection while the other therapeutic agents of the
combination may be administered topically.
[0247] Alternatively, for example, all therapeutic agents may be
administered topically or all therapeutic agents may be
administered by injection. The sequence in which the therapeutic
agents are administered is not narrowly critical unless noted
otherwise. "Combination therapy" also can embrace the
administration of the therapeutic agents as described above in
further combination with other biologically active ingredients.
Where the combination therapy further comprises a non-drug
treatment, the non-drug treatment may be conducted at any suitable
time so long as a beneficial effect from the co-action of the
combination of the therapeutic agents and non-drug treatment is
achieved. For example, in appropriate cases, the beneficial effect
is still achieved when the non-drug treatment is temporally removed
from the administration of the therapeutic agents, perhaps by days
or even weeks.
[0248] As outlined in general terms above, the medicament according
to the present invention can be administered, in principle, in any
form known to the ones skilled in the art. A preferred route of
administration is systemic administration, more preferably by
parenteral administration, preferably by injuction. Alternatively,
the medicament may be administered locally. Other routes of
administration comprise intramuscular, intraperitoneal, and
subcutaneous, per orum, intranasal, intratracheal or pulmonary with
preference given to the route of administration that is the least
invasive, while ensuring efficiancy.
[0249] Parenteral administration is generally used for
subcutaneous, intramuscular or intravenous injections and
infusions. Additionally, one approach for parenteral administration
employs the implantation of a slow-release or sustained-released
systems, which assures that a constant level of dosage is
maintained, that are well known to the ordinary skill in the
art.
[0250] Furthermore, preferred medicaments of the present invention
can be administered in intranasal form via topical use of suitable
intranasal vehicles, inhalants, or via transdermal routes, using
those forms of transdermal skin patches well known to those of
ordinary skill in that art. To be administered in the form of a
transdermal delivery system, the dosage administration will, of
course, be continuous rather than intermittent throughout the
dosage regimen. Other preferred topical preparations include
creams, ointments, lotions, aerosol sprays and gels.
[0251] Subjects that will respond favorably to the method of the
invention include medical and veterinary subjects generally,
including human beings and human patients. Among other subjects for
whom the methods and means of the invention are useful are cats,
dogs, large animals, avians such as chickens, and the like.
[0252] The medicament of the present invention will generally
comprise an effective amount of the active component(s) of the
therapy, including, but not limited to, a nucleic acid molecule of
the present invention, dissolved or dispersed in a pharmaceutically
acceptable medium. Pharmaceutically acceptable media or carriers
include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art.
Supplementary active ingredients can also be incorporated into the
medicament of the present invention.
[0253] In a further aspect the present invention is related to a
pharmaceutical composition. Such pharmaceutical composition
comprises at least one of the nucleic acids according to the
present invention and preferably a pharmaceutically acceptable
binder. Such binder can be any binder used and/or known in the art.
More particularly such binder is any binder as discussed in
connection with the manufacture of the medicament disclosed herein.
In a further embodiment, the pharmaceutical composition comprises a
further pharmaceutically active agent.
[0254] The preparation of a medicament and a pharmaceutical
composition will be known to those of skill in the art in light of
the present disclosure. Typically, such compositions may be
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection; as tablets or other solids for oral
administration; as time release capsules; or in any other form
currently used, including eye drops, creams, lotions, salves,
inhalants and the like. The use of sterile formulations, such as
saline-based washes, by surgeons, physicians or health care workers
to treat a particular area in the operating field may also be
particularly useful. Compositions may also be delivered via
microdevice, microparticle or sponge.
[0255] Upon formulation, a medicament will be administered in a
manner compatible with the dosage formulation, and in such amount
as is pharmacologically effective. The formulations are easily
administered in a variety of dosage forms, such as the type of
injectable solutions described above, but drug release capsules and
the like can also be employed.
[0256] The medicament of the invention can also be administered in
oral dosage forms as timed release and sustained release tablets or
capsules, pills, powders, granules, elixirs, tinctures,
suspensions, syrups and emulsions. Suppositories are advantageously
prepared from fatty emulsions or suspensions.
[0257] The pharmaceutical composition or medicament may be
sterilized and/or contain adjuvants, such as preserving,
stabilizing, wetting or emulsifying agents, solution promoters,
salts for regulating the osmotic pressure and/or buffers. In
addition, they may also contain other therapeutically valuable
substances. The compositions are prepared according to conventional
mixing, granulating, or coating methods, and typically contain
about 0.1% to 75%, preferably about 1% to 50%, of the active
ingredient.
[0258] Liquid, particularly injectable compositions can, for
example, be prepared by dissolving, dispersing, etc. The active
compound is dissolved in or mixed with a pharmaceutically pure
solvent such as, for example, water, saline, aqueous dextrose,
glycerol, ethanol, and the like, to thereby form the injectable
solution or suspension. Additionally, solid forms suitable for
dissolving in liquid prior to injection can be formulated.
[0259] The medicaments and nucleic acid molecules, respectively, of
the present invention can also be administered in the form of
liposome delivery systems, such as small unilamellar vesicles,
large unilamellar vesicles and multilamellar vesicles. Liposomes
can be formed from a variety of phospholipids, containing
cholesterol, stearylamine or phosphatidylcholines. In some
embodiments, a film of lipid components is hydrated with an aqueous
solution of drug to a form lipid layer encapsulating the drug, what
is well known to the ordinary skill in the art. For example, the
nucleic acid molecules described herein can be provided as a
complex with a lipophilic compound or non-immunogenic, high
molecular weight compound constructed using methods known in the
art. Additionally, liposomes may bear such nucleic acid molecules
on their surface for targeting and carrying cytotoxic agents
internally to mediate cell killing. An example of nucleic-acid
associated complexes is provided in U.S. Pat. No. 6,011,020.
[0260] The medicaments and nucleic acid molecules, respectively, of
the present invention may also be coupled with soluble polymers as
targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropyl-methacrylamide-phenol,
polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine
substituted with palmitoyl residues. Furthermore, the medicaments
and nucleic acid molecules, respectively, of the present invention
may be coupled to a class of biodegradable polymers useful in
achieving controlled release of a drag, for example, polylactic
acid, polyepsilon capro lactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates
and cross-linked or amphipathic block copolymers of hydrogels.
[0261] If desired, the pharmaceutical composition and medicament,
respectively, to be administered may also contain minor amounts of
non-toxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and other substances such as for
example, sodium acetate, and triethanolamine oleate.
[0262] The dosage regimen utilizing the nucleic acid molecules and
medicaments, respectively, of the present invention is selected in
accordance with a variety of factors including type, species, age,
weight, sex and medical condition of the patient; the severity of
the condition to be treated; the route of administration; the renal
and hepatic function of the patient; and the particular aptamer or
salt thereof employed. An ordinarily skilled physician or
veterinarian can readily determine and prescribe the effective
amount of the drug required to prevent, counter or arrest the
progress of the condition.
[0263] Effective plasma levels of the nucleic acid according to the
present invention preferably range from 500 fM to 200 .mu.M,
preferably from 1 nM to 20 .mu.M, more preferably from 5 nM to 20
.mu.M, most preferably 50 nM to 20 .mu.M in the treatment of any of
the diseases disclosed herein.
[0264] The nucleic acid molecules and medicaments, respectively, of
the present invention may preferably be administered in a single
daily dose, every second or third day, weekly, every second week,
in a single monthly dose or every third month.
[0265] It is within the present invention that the medicament as
described herein constitutes the pharmaceutical composition
disclosed herein.
[0266] In a further aspect the present invention is related to a
method for the treatment of a subject who is in need of such
treatment, whereby the method comprises the administration of a
pharmaceutically active amount of at least one of the nucleic acids
according to the present invention. In an embodiment, the subject
suffers from a disease or is at risk to develop such disease,
whereby the disease is any of those disclosed herein, particularly
any of those diseases disclosed in connection with the use of any
of the nucleic acids according to the present invention for the
manufacture of a medicament.
[0267] As preferably used herein, the term treatment comprises in a
preferred embodiment additionally or alternatively prevention
and/or follow-up.
[0268] As preferably used herein, the terms disease and disorder
shall be used in an interchangeable manner, if not indicated to the
contrary.
[0269] As used herein, the term comprise is preferably not intended
to limit the subject matter followed or described by such term.
However, in an alternative embodiment the term comprises shall be
understood in the meaning of containing and thus as limiting the
subject matter followed or described by such term.
[0270] The various SEQ ID NOs:, the chemical nature of the nucleic
acid molecules according to the present invention and the target
molecules SDF-1 as used herein, the actual sequence thereof and the
internal reference number is summarized in the following table. It
has to be noticed that the nucleic acids were characterized on the
aptamer, i.e. D-nucleic acid level (D-RNA) with the biotinylated
human D-SDF-1 (SEQ ID NO: 4) or on the Spiegelmer level, i.e.
L-nucleic acid (L-RNA) with the natural configuration of SDF-1, the
L-SDF-1 (human SDF-1 .alpha., SEQ ID NO: 1). The different nucleic
acids share one internal reference name but one SEQ ID Nos: for the
D-RNA (Aptamer) molecule and one SEQ ID Nos: for the L-RNA
(Spiegelmer) molecule, respectively.
TABLE-US-00003 TABLE 1 SEQ ID NO: RNA/Peptide Sequence Internal
Reference 1 L-peptide
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR human/monkey/cat
SDF- QVCIDPKLKWIQEYLEKALNK 1.alpha. human/monkey/cat SDF-1 2
L-peptide KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR
human/monkey/cat SDF- QVCIDPKLKWIQEYLEKALNKRFKM 1.beta. 3 L-peptide
KPVSLSYRCPCRFFESHIARANVKHLKILNTPNCALQIVARLKNNNR murine SDF-1.alpha.
QVCIDPKLKWIQEYLEKALNK murine SDF-1 4 D-peptide
KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR biotinylated hu
D-SDF- QVCIDPKLKWIQEYLEKALNKRFK-Biotin 1 5 L-RNA
AGCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUACGCU 193-C2-001 6 L-RNA
AGCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGCU 193-G2-001 7 L-RNA
AGCGUGGUGUGAUCUAGAUGUAAUGGCUGAUCCUAGUCAGGUGCGCU 193-F2-001 8 L-RNA
GCGAGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUGCGC 193-G1-002 9 L-RNA
GCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUGCGC 193-D2-002 10 L-RNA
GCAUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUGCCC 193-A1-002 11 L-RNA
GCGUGGUGUGAUCUAGAUGUAAUGGCUGAUCCUAGUCAGGGACGC 193-D3-002 12 L-RNA
GCGUGGUGUGAUCUAGAUGUAGAGGCUGAUCCUAGUCAGGUACGC 193-B3-002 13 L-RNA
GCGUGGUGUGAUCUAGAUGUAAAGGCUGAUCCUAGUCAGGUACGC 193-H3-002 14 L-RNA
GUGGUGUGAUCUAGAUGUAGUGGCUGUUCCUAGUCAGGUAUGC 193-E3-002 15 L-RNA
GCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUUAGGUACGC 193-D1-002 16 L-RNA
GCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUACGC 193-C2-002 17 L-RNA
CGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUACG 193-C2-003 18 L-RNA
GUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUAC 193-C2-004 19 L-RNA
UGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUA 193-C2-005 20 L-RNA
GGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGU 193-C2-006 21 L-RNA
GUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGG 193-C2-007 22 L-RNA
GCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGC 193-G2-012 23 L-RNA
GCGCGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGCGCGC 193-G2-013 24 L-RNA
GCGCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGCGC 193-G2-014 25 L-RNA
GGGCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGCCC 193-G2-015 26 L-RNA
GGCCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGGCC 193-G2-016 27 L-RNA
GCCCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGGGC 193-G2-017 28 L-RNA
5'-40 kDa-PEG- 193-G2-012-5'-PEG,
GCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGC NOX-A12 29 D-RNA
AGCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUACGCU 193-C2-001 30 D-RNA
AGCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGCU 193-G2-001 31 D-RNA
AGCGUGGUGUGAUCUAGAUGUAAUGGCUGAUCCUAGUCAGGUGCGCU 193-F2-001 32 D-RNA
GCGAGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUGCGC 193-G1-002 33 D-RNA
GCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUGCGC 193-D2-002 34 D-RNA
GCAUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUGCCC 193-A1-002 35 D-RNA
GCGUGGUGUGAUCUAGAUGUAAUGGCUGAUCCUAGUCAGGGACGC 193-D3-002 36 D-RNA
GCGUGGUGUGAUCUAGAUGUAGAGGCUGAUCCUAGUCAGGUACGC 193-B3-002 37 D-RNA
GCGUGGUGUGAUCUAGAUGUAAAGGCUGAUCCUAGUCAGGUACGC 193-H3-002 38 D-RNA
GUGGUGUGAUCUAGAUGUAGUGGCUGUUCCUAGUCAGGUAUGC 193-E3-002 39 D-RNA
GCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUUAGGUACGC 193-D1-002 40 D-RNA
GCGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUACGC 193-C2-002 41 D-RNA
CGUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUACG 193-C2-003 42 D-RNA
GUGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUAC 193-C2-004 43 D-RNA
UGGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGUA 193-C2-005 44 D-RNA
GGUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGGU 193-C2-006 45 D-RNA
GUGUGAUCUAGAUGUAGUGGCUGAUCCUAGUCAGG 193-C2-007 46 D-RNA
GCGUGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGUACGC 193-G2-012 47 D-RNA
GCGCGGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGCGCGC 193-G2-013 48 D-RNA
GCGCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGCGC 193-G2-014 49 D-RNA
GGGCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGCCC 193-G2-015 50 D-RNA
GGCCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGGCC 193-G2-016 51 D-RNA
GCCCGUGUGAUCUAGAUGUAUUGGCUGAUCCUAGUCAGGGGGC 193-G2-017 52 L-RNA
GUGUGAUCUAGAUGUADWGGCUGWUCCUAGUYAGG Type B Formula-1 53 L-RNA
GUGUGAUCUAGAUGUADUGGCUGAUCCUAGUCAGG Type B Formula-2 54 L-RNA
AAAGUAACACGUAAAAUGAAAGGUAAC 55 L-RNA AAAGCAACAUGUCAAUGAAAGGUAGC 56
L-RNA GGUUAGGGCUAAAGUCGG 57 L-RNA GGUUAGGGCUAGAAGUCGG 58 L-RNA
GGUUAGGGCUCGAAGUCGG 59 L-RNA GGUUAGGGCUUGAAGUCGG 60 L-RNA
GCUGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCAGC 192-A10-001 61 L-RNA
GCUGUGAAAGUAACAUGUCAAUGAAAGGUAACCACAGC 192-G10 62 L-RNA
GCUGUGAAAGUAACACGUCAAUGAAAGGUAACCGCAGC 192-F10 63 L-RNA
GCUGUGAAAGUAACACGUCAAUGAAAGGUAACCACAGC 192-B11 64 L-RNA
GCUGUAAAAGUAACAUGUCAAUGAAAGGUAACUACAGC 192-C9 65 L-RNA
GCUGUAAAAGUAACAAGUCAAUGAAAGGUAACUACAGC 192-E10 66 L-RNA
GCUGUGAAAGUAACAAGUCAAUGAAAGGUAACCACAGC 192-C10 67 L-RNA
GCAGUGAAAGUAACAUGUCAAUGAAAGGUAACCACAGC 192-D11 68 L-RNA
GCUGUGAAAGUAACAUGUCAAUGAAAGGUAACCACUGC 192-G11 69 L-RNA
GCUAUGAAAGUAACAUGUCAAUGAAAGGUAACCAUAGC 192-H11 70 L-RNA
GCUGCGAAAGCGACAUGUCAAUGAAAGGUAGCCGCAGC 192-D10 71 L-RNA
GCUGUGAAAGCAACAUGUCAAUGAAAGGUAGCCACAGC 192-E9 72 L-RNA
GCUGUGAAAGUAACAUGUCAAUGAAAGGUAGCCGCAGC 192-H9 73 L-RNA
AGCGUGAAAGUAACACGUAAAAUGAAAGGUAACCACGCU 191-A6 74 L-RNA
AAAGYRACAHGUMAAX.sub.AUGAAAGGUARC; X.sub.A = A or absent Type A
Formula-1 75 L-RNA AAAGYRACAHGUMAAUGAAAGGUARC Type A Formula-2 76
L-RNA AAAGYRACAHGUMAAAUGAAAGGUARC Type A Formula-3 77 L-RNA
AAAGYAACAHGUCAAUGAAAGGUARC Type A Formula-4 78 L-RNA
CUGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCAG 192-A10-002 79 L-RNA
UGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCA 192-A10-003 80 L-RNA
GUGAAAGCAACAUGUCAAUGAAAGGUAGCCGC 192-A10-004 81 L-RNA
UGAAAGCAACAUGUCAAUGAAAGGUAGCCG 192-A10-005 82 L-RNA
GAAAGCAACAUGUCAAUGAAAGGUAGCC 192-A10-006 83 L-RNA
AAAGCAACAUGUCAAUGAAAGGUAGC 192-A10-007 84 L-RNA
GCGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCGC 192-A10-008 85 L-RNA
GCGCGAAAGCAACAUGUCAAUGAAAGGUAGCCGCGC 192-A10-015 86 L-RNA
GCGGAAAGCAACAUGUCAAUGAAAGGUAGCCCGC 192-A10-014 87 L-RNA
CGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCG 192-A10-016 88 L-RNA
GCGCAAAGCAACAUGUCAAUGAAAGGUAGCGUGC 192-A10-017 89 L-RNA
GUGCAAAGCAACAUGUCAAUGAAAGGUAGCGCGC 192-A10-018 90 L-RNA
CGCGAAAGCAACAUGUCAAUGAAAGGUAGCCGUG 192-A10-019 91 L-RNA
GGGCAAAGCAACAUGUCAAUGAAAGGUAGCGCCC 192-A10-020 92 L-RNA
GGCCAAAGCAACAUGUCAAUGAAAGGUAGCGGCC 192-A10-021 93 L-RNA
GCCCAAAGCAACAUGUCAAUGAAAGGUAGCGGGC 192-A10-022 94 L-RNA
CCCCAAAGCAACAUGUCAAUGAAAGGUAGCGGGG 192-A10-023 95 L-RNA
GUGCUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAGCAC 197-B2 96 L-RNA
AGCGUGGCGAGGUUAGGGCUAGAAGUCGGUCGACACGCU 191-D5-001 97 L-RNA
GUGUUGCGGAGGUUAGGGCUAGAAGUCGGUCAGCAGCAC 197-H1 98 L-RNA
CGUGCGGCCUAAGAGGUUAGGGCUUAAAGUCGGUCUUUGGCCA 190-D3 ACACG 99 L-RNA
CGUGCGCUUGAGAUAGGGGUUAGGGCUUAAAGUCGGCUGAUUC 190-A3-001 UCACG 100
L-RNA CGUGAUUGGUGAGGGGUUAGGGCUUGAAGUCGGCCUUGUCCAG 190-A2 UCACG 101
L-RNA AGCGUGAAGGGGUUAGGGCUCGAAGUCGGCUGACACGCU 191-A5 102 L-RNA
GUGCUGCGGGGGUUAGGGCUCGAAGUCGGCCCGCAGCAC 197-H3 103 L-RNA
GUGUUCCCGGGGUUAGGGCUUGAAGUCGGCCGGCAGCAC 197-B1 104 L-RNA
GUGUUGCAGGGGUUAGGGCUUGAAGUCGGCCUGCAGCAC 197-E3 105 L-RNA
GUGCUGCGGGGGUUAGGGCUCAAAGUCGGCCUGCAGCAC 197-H2 106 L-RNA
GUGCUGCCGGGGUUAGGGCUAA-AGUCGGCCGACAGCAC 197-D1 107 L-RNA
GUGCUGUGGGGGUCAGGGCUAGAAGUCGGCCUGCAGCAC 197-D2 108 L-RNA
GGUYAGGGCUHRX.sub.AAGUCGG; X.sub.A = A or absent Type C Formula-1
109 L-RNA GGUYAGGGCUHRAAGUCGG Type C Formula-2 110 L-RNA
GGUYAGGGCUHRAGUCGG Type C Formula-3 111 L-RNA GGUUAGGGCUHGAAGUCGG
Type C Formula-4 112 L-RNA UGAGAUAGGGGUUAGGGCUUAAAGUCGGCUGAUUCUCA
190-A3-003 113 L-RNA GAGAUAGGGGUUAGGGCUUAAAGUCGGCUGAUUCUC
190-A3-004 114 L-RNA GGGGUUAGGGCUUAAAGUCGGCUGAUUCU 190-A3-007 115
L-RNA GCGUGGCGAGGUUAGGGCUAGAAGUCGGUCGACACGC 191-D5-002
116 L-RNA CGUGGCGAGGUUAGGGCUAGAAGUCGGUCGACACG 191-D5-003 117 L-RNA
CGGGCGAGGUUAGGGCUAGAAGUCGGUCGACCG 191-D5-004 118 L-RNA
CGGGCGAGGUUAGGGCUAGAAGUCGGUCGCCCG 191-D5-005 119 L-RNA
CGGCGAGGUUAGGGCUAGAAGUCGGUCGCCG 191-D5-006 120 L-RNA
CGGGAGGUUAGGGCUAGAAGUCGGUCCCG 191-D5-007 121 L-RNA
GGGAGGUUAGGGCUAGAAGUCGGUCCC 191-D5-010 122 L-RNA
CCGCGGUUAGGGCUAGAAGUCGGGCGG 191-D5-017 123 L-RNA
CCCGGGUUAGGGCUAGAAGUCGGCGGG 191-D5-029 124 L-RNA
GGCGGGUUAGGGCUAGAAGUCGGCGCC 191-D5-024 125 L-RNA
CCCGCGGUUAGGGCUAGAAGUCGGGCGGG 191-D5-017-29a 126 L-RNA
GCCGCGGUUAGGGCUAGAAGUCGGGCGGC 191-D5-017-29b 127 L-RNA
CCCCGGGUUAGGGCUAGAAGUCGGCGGGG 191-D5-019-29a 128 L-RNA
CGGCGGGUUAGGGCUAGAAGUCGGCGCCG 191-D5-024-29a 129 L-RNA
GGGCGGGUUAGGGCUAGAAGUCGGCGCCC 191-D5-024-29b 130 L-RNA
UGCUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAGCA 197-B2-001 131 L-RNA
GCUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAGC 197-B2-002 132 L-RNA
CUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAG 197-B2-003 133 L-RNA
UGCGGGGGUUAGGGCUAGAAGUCGGCCUGCA 197-B2-004 134 L-RNA
GCGGGGGUUAGGGCUAGAAGUCGGCCUGC 197-B2-005 135 L-RNA
GCCGGGGUUAGGGCUAGAAGUCGGCCGGC 197-B2-006 136 L-RNA
GGCCGGGGUUAGGGCUAGAAGUCGGCCGGCC 197-B2-006-31a 137 L-RNA
CGCCGGGGUUAGGGCUAGAAGUCGGCCGGCG 197-B2-006-31b 138 L-RNA RKSBUSNVGR
Type C Formula-5-5' 139 L-RNA YYNRCASSMY Type C Formula-5-3' 140
L-RNA RKSBUGSVGR Type C Formula-6-5' 141 L-RNA YCNRCASSMY Type C
Formula-6-3' 142 L-RNA
CGUGGUCCGUUGUGUCAGGUCUAUUCGCCCCGGUGCAGGGCAUCCGCG 194-A2-001 143
L-RNA GCAGUGUGACGCGGACGUGAUAGGACAGAGCUGAUCCCGCUCAGGUGAG 196-B12-003
144 L-RNA CAACAGCAGUGUGACGCGGACGUGAUAGGACAGAGCUGAUCCCGCUCAG
196-B12-004 145 L-RNA 5'-40
kDa-PEG-GCGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCGC 192-A10-008-5'-PEG 146
D-RNA GCUGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCAGC 192-A10-001 147 D-RNA
GCUGUGAAAGUAACAUGUCAAUGAAAGGUAACCACAGC 192-G10 148 D-RNA
GCUGUGAAAGUAACACGUCAAUGAAAGGUAACCGCAGC 192-F10 149 D-RNA
GCUGUGAAAGUAACACGUCAAUGAAAGGUAACCACAGC 192-B11 150 D-RNA
GCUGUAAAAGUAACAUGUCAAUGAAAGGUAACUACAGC 192-C9 151 D-RNA
GCUGUAAAAGUAACAAGUCAAUGAAAGGUAACUACAGC 192-E10 152 D-RNA
GCUGUGAAAGUAACAAGUCAAUGAAAGGUAACCACAGC 192-C10 153 D-RNA
GCAGUGAAAGUAACAUGUCAAUGAAAGGUAACCACAGC 192-D11 154 D-RNA
GCUGUGAAAGUAACAUGUCAAUGAAAGGUAACCACUGC 192-G11 155 D-RNA
GCUAUGAAAGUAACAUGUCAAUGAAAGGUAACCAUAGC 192-H11 156 D-RNA
GCUGCGAAAGCGACAUGUCAAUGAAAGGUAGCCGCAGC 192-D10 157 D-RNA
GCUGUGAAAGCAACAUGUCAAUGAAAGGUAGCCACAGC 192-E9 158 D-RNA
GCUGUGAAAGUAACAUGUCAAUGAAAGGUAGCCGCAGC 192-H9 159 D-RNA
AGCGUGAAAGUAACACGUAAAAUGAAAGGUAACCACGCU 191-A6 160 D-RNA
CUGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCAG 192-A10-002 161 D-RNA
UGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCA 192-A10-003 162 D-RNA
GUGAAAGCAACAUGUCAAUGAAAGGUAGCCGC 192-A10-004 163 D-RNA
UGAAAGCAACAUGUCAAUGAAAGGUAGCCG 192-A10-005 164 D-RNA
GAAAGCAACAUGUCAAUGAAAGGUAGCC 192-A10-006 165 D-RNA
AAAGCAACAUGUCAAUGAAAGGUAGC 192-A10-007 166 D-RNA
GCGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCGC 192-A10-008 167 D-RNA
GCGCGAAAGCAACAUGUCAAUGAAAGGUAGCCGCGC 192-A10-015 168 D-RNA
GCGGAAAGCAACAUGUCAAUGAAAGGUAGCCCGC 192-A10-014 169 D-RNA
CGUGAAAGCAACAUGUCAAUGAAAGGUAGCCGCG 192-A10-016 170 D-RNA
GCGCAAAGCAACAUGUCAAUGAAAGGUAGCGUGC 192-A10-017 171 D-RNA
GUGCAAAGCAACAUGUCAAUGAAAGGUAGCGCGC 192-A10-018 172 D-RNA
CGCGAAAGCAACAUGUCAAUGAAAGGUAGCCGUG 192-A10-019 173 D-RNA
GGGCAAAGCAACAUGUCAAUGAAAGGUAGCGCCC 192-A10-020 174 D-RNA
GGCCAAAGCAACAUGUCAAUGAAAGGUAGCGGCC 192-A10-021 175 D-RNA
GCCCAAAGCAACAUGUCAAUGAAAGGUAGCGGGC 192-A10-022 176 D-RNA
CCCCAAAGCAACAUGUCAAUGAAAGGUAGCGGGG 192-A10-023 177 D-RNA
GUGCUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAGCAC 197-B2 178 D-RNA
AGCGUGGCGAGGUUAGGGCUAGAAGUCGGUCGACACGCU 191-D5-001 179 D-RNA
GUGUUGCGGAGGUUAGGGCUAGAAGUCGGUCAGCAGCAC 197-H1 180 D-RNA
CGUGCGGCCUAAGAGGUUAGGGCUUAAAGUCGGUCUUUGGCCA 190-D3 ACACG 181 D-RNA
CGUGCGCUUGAGAUAGGGGUUAGGGCUUAAAGUCGGCUGAUUC 190-A3-001 UCACG 182
D-RNA CGUGAUUGGUGAGGGGUUAGGGCUUGAAGUCGGCCUUGUCCAG 190-A2 UCACG 183
D-RNA AGCGUGAAGGGGUUAGGGCUCGAAGUCGGCUGACACGCU 191-A5 184 D-RNA
GUGCUGCGGGGGUUAGGGCUCGAAGUCGGCCCGCAGCAC 197-H3 185 D-RNA
GUGUUCCCGGGGUUAGGGCUUGAAGUCGGCCGGCAGCAC 197-B1 186 D-RNA
GUGUUGCAGGGGUUAGGGCUUGAAGUCGGCCUGCAGCAC 197-E3 187 D-RNA
GUGCUGCGGGGGUUAGGGCUCAAAGUCGGCCUGCAGCAC 197-H2 188 D-RNA
GUGCUGCCGGGGUUAGGGCUAA-AGUCGGCCGACAGCAC 197-D1 189 D-RNA
GUGCUGUGGGGGUCAGGGCUAGAAGUCGGCCUGCAGCAC 197-D2 190 D-RNA
UGAGAUAGGGGUUAGGGCUUAAAGUCGGCUGAUUCUCA 190-A3-003 191 D-RNA
GAGAUAGGGGUUAGGGCUUAAAGUCGGCUGAUUCUC 190-A3-004 192 D-RNA
GGGGUUAGGGCUUAAAGUCGGCUGAUUCU 190-A3-007 193 D-RNA
GCGUGGCGAGGUUAGGGCUAGAAGUCGGUCGACACGC 191-D5-002 194 D-RNA
CGUGGCGAGGUUAGGGCUAGAAGUCGGUCGACACG 191-D5-003 195 D-RNA
CGGGCGAGGUUAGGGCUAGAAGUCGGUCGACCG 191-D5-004 196 D-RNA
CGGGCGAGGUUAGGGCUAGAAGUCGGUCGCCCG 191-D5-005 197 D-RNA
CGGCGAGGUUAGGGCUAGAAGUCGGUCGCCG 191-D5-006 198 D-RNA
CGGGAGGUUAGGGCUAGAAGUCGGUCCCG 191-D5-007 199 D-RNA
GGGAGGUUAGGGCUAGAAGUCGGUCCC 191-D5-010 200 D-RNA
CCGCGGUUAGGGCUAGAAGUCGGGCGG 191-D5-017 201 D-RNA
CCCGGGUUAGGGCUAGAAGUCGGCGGG 191-D5-029 202 D-RNA
GGCGGGUUAGGGCUAGAAGUCGGCGCC 191-D5-024 203 D-RNA
CCCGCGGUUAGGGCUAGAAGUCGGGCGGG 191-D5-017-29a 204 D-RNA
GCCGCGGUUAGGGCUAGAAGUCGGGCGGC 191-D5-017-29b 205 D-RNA
CCCCGGGUUAGGGCUAGAAGUCGGCGGGG 191-D5-019-29a 206 D-RNA
CGGCGGGUUAGGGCUAGAAGUCGGCGCCG 191-D5-024-29a 207 D-RNA
GGGCGGGUUAGGGCUAGAAGUCGGCGCCC 191-D5-024-29b 208 D-RNA
UGCUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAGCA 197-B2-001 209 D-RNA
GCUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAGC 197-B2-002 210 D-RNA
CUGCGGGGGUUAGGGCUAGAAGUCGGCCUGCAG 197-B2-003 211 D-RNA
UGCGGGGGUUAGGGCUAGAAGUCGGCCUGCA 197-B2-004 212 D-RNA
GCGGGGGUUAGGGCUAGAAGUCGGCCUGC 197-B2-005 213 D-RNA
GCCGGGGUUAGGGCUAGAAGUCGGCCGGC 197-B2-006 214 D-RNA
GGCCGGGGUUAGGGCUAGAAGUCGGCCGGCC 197-B2-006-31a 215 D-RNA
CGCCGGGGUUAGGGCUAGAAGUCGGCCGGCG 197-B2-006-31b 216 D-RNA
CGUGGUCCGUUGUGUCAGGUCUAUUCGCCCCGGUGCAGGGCAUCCGCG 194-A2-001 217
D-RNA GCAGUGUGACGCGGACGUGAUAGGACAGAGCUGAUCCCGCUCAGGUGAG 196-B12-003
218 D-RNA CAACAGCAGUGUGACGCGGACGUGAUAGGACAGAGCUGAUCCCGCUCAG
196-B12-004 219 L-RNA 5'-40 kDa-PEG- Control Spiegelmer
UAAGGAAACUCGGUCUGAUGCGGUAGCGCUGUGCAGAGCU 220 L-RNA
CGUGCGCUUGAGAUAGG 221 L-RNA CUGAUUCUCACG 222 L-RNA CUGAUUCUCA 223
L-RNA 5'-40 kDa-PEG-GCCGGGGUUAGGGCUAGAAGUCGGCCGGC 197-B2-006-5'-PEG
224 L-RNA 5'-40 kDa-PEG-CGGGAGGUUAGGGCUAGAAGUCGGUCCCG
191-D5-007-5'PEG 225 L-RNA 5'-40 kDa-PEG- revNOX-A12
CGCAUGGACUGAUCCUAGUCGGUUAUGUAGAUCUAGUGUGGUGC G
[0271] The present invention is further illustrated by the figures,
examples and the sequence listing from which further features,
embodiments and advantages may be taken, wherein
[0272] FIG. 1 shows an alignment of sequences of SDF-1 binding
nucleic acid molecules of "type A"
[0273] FIG. 2A+B show derivatives of SDF-1 binding nucleic acid
molecule 192-A10-001 (SDF-1 binding nucleic acid molecules of "type
A");
[0274] FIG. 3 shows an alignment of sequences of SDF-1 binding
nucleic acid molecules of "type B";
[0275] FIG. 4A+B show derivatives of SDF-1 binding nucleic acid
molecules 193-C2-001 and 193-G2-001 (SDF-1 binding nucleic acid
molecules of type B);
[0276] FIG. 5 shows an alignment of sequences of SDF-1 binding
nucleic acid molecules of "type C";
[0277] FIG. 6 shows derivatives of SDF-1 binding nucleic acid
molecule 190-A3-001 (SDF-1 binding nucleic acid molecules of "type
C");
[0278] FIGS. 7A+B show derivatives of SDF-1 binding nucleic acid
moleculs 190-D5-001 (SDF-1 binding nucleic acid molecules of "type
C");
[0279] FIG. 8 shows derivatives of SDF-1 binding nucleic acid
molecule 197-B2 (SDF-1 binding nucleic acid molecule of "type
C");
[0280] FIG. 9 shows further SDF-1 binding nucleic acid molecules
molecules which are, in addition to other SDF-1 binding nucleic
acid molecules, also referred to as SDF-1 binding nucleic acid
molecules of "type D";
[0281] FIG. 10 shows the efficacy of SDF-1 binding Spiegelmers
193-G2-012-5'-PEG (also referred to as NOX-A12), 197-B2-006-5'-PEG,
191-D5-007-5'-PEG and 191-A10-008-5'-PEG in a chemotaxis assay with
the human T cell leukemia cell line Jurkat whereby cells were
allowed to migrate towards 0.3 nM human SDF-1 preincubated at
37.degree. C. with various amounts of Spiegelmers
193-G2-012-5'-PEG, 197-B2-006-5'-PEG, 191-D5-007-5'-PEG and
191-A10-008-5'-PEG, represented as percentage of control over
concentration of Spiegelmers 193-G2-012-5'-PEG, 197-B2-006-5'-PEG,
191-D5-007-5'-PEG and 191-A10-008-5'-PEG;
[0282] FIG. 11A shows the efficacy of SDF-1 binding Spiegelmer
NOX-A12 in a chemotaxis assay with the human pre-B ALL cell line
Nalm-6 whereby cells were allowed to migrate towards 0.3 nM human
SDF-1 preincubated at 37.degree. C. with various amounts of
Spiegelmer NOX-A12 represented as percentage of control over
concentration of Spiegelmer NOX-A12;
[0283] FIG. 11B shows the efficacy of SDF-1 binding Spiegelmer
NOX-A12 in a chemotaxis assay with the human leukemic monocyte
lymphoma cell line U937 whereby cells were allowed to migrate
towards 3 nM human SDF-1 preincubated at 37.degree. C. with various
amounts of Spiegelmer NOX-A12 represented as percentage of control
over concentration of Spiegelmer NOX-A12;
[0284] FIG. 12 shows the efficacy of SDF-1 binding Spiegelmer
NOX-A12 in a chemotaxis assay with the human pre-B cell leukemia
cell line BV-173 whereby cells were allowed to migrate towards 3 nM
human SDF-1 preincubated at 37.degree. C. with various amounts of
Spiegelmer NOX-A12 represented as percentage of control over
concentration of Spiegelmer NOX-A12;
[0285] FIG. 13 shows the efficacy of SDF-1 binding Spiegelmer
NOX-A12 in a complementation assay with CHO cells stably expressing
CXCR7 and .beta.-arrestin both fused to a fragment of
.beta.-galactosidase whereby CXCR7 of the cells were activated
towards 10 nM human SDF-1 preincubated at 37.degree. C. with
various amounts of Spiegelmer NOX-A12 represented as percentage of
control over concentration of Spiegelmer NOX-A12;
[0286] FIG. 14 shows the inhibition of SDF-1 induced sprouting by
human SDF-1 binding Spiegelmer 193-G2-012-5'-PEG (also referred to
as NOX-A12) and by PEGylated Control Spiegelmer in aortic ring
sprouting assay, whereby rings from rat aorta were embedded in
collagen matrix and incubated for 6 days with SDF-1 with or without
Spiegelmers (a: control; b: 10 nM SDF-1; c: 10 nM SDF-1+1 .mu.M
human SDF-1 binding Spiegelmer 193-G2-012-5'-PEG; d: 10 nM SDF-1+1
.mu.M PEGylated Control Spiegelmer);
[0287] FIG. 15 shows the inhibition of SDF-1 induced sprouting by
human SDF-1 binding Spiegelmer 193-G2-012-5'-PEG (also referred to
as NOX-A12) and by PEGylated Control Spiegelmer in aortic ring
sprouting assay whereby sprouting indices are shown as mean+/-SD
for 5 rings per condition (*: the value for SDF-1 is significantly
different from control (Mann-Whitney-test; p=0.009); **: the value
for SDF-1+human SDF-1 binding Spiegelmer 193-G2-012-5'-PEG is
significantly different from that for SDF-1 (Mann-Whitney-test;
p=0.028)
[0288] FIG. 16 shows the efficacy of human SDF-1 binding Spiegelmer
NOX-A12 to sensitize RPMI-8226 MM cells to F-ara-A (Fludarabine),
whereby confluent murine BM stromal MS-5 cells secreting SDF-1 were
incubated with human SDF-1 binding Spiegelmer NOX-A12 or the
non-functional revNOX-A 12 and subsequently cocultured with
RPMI-8226 MM cells; cells were treated with 1 .mu.M F-ara-A for 40
hours and cell viability was measured by Flow Cytometry using
ViaCount Reagent; Error bars indicate SD, N=5, * p=0.0134, ***
p=0.0003 (two-tailed, unpaired t-test);
[0289] FIG. 17 shows the efficacy of human SDF-1 binding Spiegelmer
NOX-A12 to inhibit the proliferation of Jurkat cells in coculture
with stromal MS-5 cells, whereby murine stromal MS-5 cells
secreting SDF-1 were incubated with increasing concentrations of
human SDF-1 binding Spiegelmer NOX-A12; Jurkat cells were added to
the confluent MS-5 cell layer and cell counts were measured after
40 hours by Flow Cytometry using ViaCount Reagent. Error bars
indicate SD, N=4, *** p=0.0008 (two-tailed, unpaired t-test);
[0290] FIG. 18A+B show the efficacy of human SDF-1 binding
Spiegelmer NOX-A12 to reverse SDF-1 dose-dependent adhesion of
Jurkat cells to fibronectin, whereby Jurkat cells were incubated
with SDF-1 alone (A), with SDF-1 and increasing concentrations of
human SDF-1 binding Spiegelmer NOX-A12 or with SDF-1 and increasing
concentrations of control Spiegelmer revNOX-A12 (B) for 30 minutes
and seeded on fibronectin coated plates for 15 minutes; cells were
subsequently washed off with media and attached cells were
quantified using Cell Titer Glo Reagent; error bars indicate
SD.
EXAMPLE 1
Nucleic Acids that Bind Human SDF-1
[0291] In the following the terms `nucleic acid` and `nucleic acid
molecule` are used herein in a synonymous manner if not indicated
to the contrary. Moreover, the terms `stretch` and `stretch of
nucleotide` are used herein in a synonymous manner if not indicated
to the contrary.
[0292] L-nucleic acid molecules that bind to human SDF-1 and the
respective nucleotide sequences are depicted in FIGS. 1 to 9. The
nucleic acids were characterized on the aptamer, i.e. D-nucleic
acid level using competitive or direct pull-down binding assays
with biotinylated human D-SDF-1 (protocol, see Example 3).
Spiegelmers were tested with the natural configuration of SDF-1
(L-SDF-1) by surface plasmon resonance measurement using a Biacore
2000 instrument (protocol, see Example 5) and a cell culture in
vitro chemotaxis assay (protocol, see Example 4).
[0293] The SDF-1 binding nucleic acid molecules exhibit different
sequence motifs, three main types are defined in FIGS. 1, 2A and 2B
(Type A), FIGS. 3, 4A and 4B (Type B), FIGS. 5, 4, 7A, 7B and 8
(Type C). The nucleic acid molecules exhibit different sequence
motifs. For definition of nucleotide sequence motifs, the IUPAC
abbreviations for ambiguous nucleotides is used:
TABLE-US-00004 S strong G or C; W weak A or U; R purine G or A; Y
pyrimidine C or U; K keto G or U; M imino A or C; B not A C or U or
G; D not C A or G or U; H not G A or C or U; V not U A or C or G; N
all A or G or C or U
[0294] If not indicated to the contrary, any nucleic acid sequence
or sequence of stretches and boxes, respectively, is indicated in
the 5'.fwdarw.3' direction.
SDF-1 Binding Nucleic Acid Molecules of Type A
[0295] As depicted in FIG. 1 all sequences of SDF-1 binding nucleic
acid moleculess of type A comprise one central stretch of
nucleotides which is flanked by the first (5'-) terminal and the
second (3'-) terminal stretch of nucleotides (also referred to as
first terminal stretch of nucleotides and second stretch of
nucleotides) whereby both stretches can hybridize to each other.
However, such hybridization is not necessarily given in the
molecule.
[0296] In the following the terms `SDF-1 binding nucleic acid
molecules of type A` and `Type A SDF-1 binding nucleic acids` or
Type A SDF-1 binding nucleic acid molecules` are used herein in a
synonymous manner if not indicated to the contrary.
[0297] The sequences of the defined boxes or stretches of
nucleotides may be different between the SDF-1 binding nucleic
acids of type A which influences the binding affinity to SDF-1.
Based on binding analysis of the different SDF-1 binding nucleic
acids summarized as Type A SDF-1 binding nucleic acids, the central
stretch of nucleotides and its nucleotide sequences as described in
the following are individually and more preferably in their
entirety essential for binding to SDF-1.
[0298] The central stretch of nucleotides of all identified
sequences of Type A SDF-1 binding nucleic acids share the
sequence
##STR00001##
(Type A Formula-1, SEQ ID NO: 74), whereby X.sub.A is either absent
or is `A`. If `A` is absent, the sequence of the central nucleotide
sequence can be summarized as Type A Formula-2
##STR00002##
SEQ ID NO: 75. Type A SDF-1 binding nucleic acid 191-A6 (central
nucleotide sequence:
##STR00003##
SEQ ID NO: 54) carrying the additional nucleotide `A` within the
central nucleotide sequence and still binding to SDF-1 let conclude
an alternative central nucleotide sequence
##STR00004##
Type A Formula-3, SEQ ID NO: 76). Exemplarily for all the other
nucleic acids of Type A SDF-1 binding nucleic acids, the Type A
SDF-1 binding nucleic acid 192-A10-001 was characterized for its
binding affinity to human SDF-1. The equilibrium binding constant
K.sub.D was determined using the pull-down binding assay
(K.sub.D=1.5 nM) and by surface plasmon resonance measurement
(K.sub.D=1.0 nM). The IC.sub.50 (inhibitory concentration 50%) of
0.12 nM for 192-A10-001 was measured using a cell culture in vitro
chemotaxis assay. Consequently, all Type A SDF-1 binding nucleic
acids as depicted in FIG. 1 were analyzed in a competitive
pull-down binding assay vs. 192-A10-001. The Type A SDF-1 binding
nucleic acids 192-B11 and 192-C10 showed equal binding affinities
as 192-A10-001 in these competition experiments. Weaker binding
affinity was determined for Type A SDF-1 binding nucleic acids
192-G10, 192-F10, 192-C9, 192-E10, 192-D11, 192-G11, 192-H11 and
191-A6. The Type A SDF-1 binding nucleic acids 192-D10, 192-E9 and
192-H9 have much weaker binding affinity than 192-A10-001.
[0299] As mentioned above, the Type A SDF-1 binding nucleic acid
192-B11 and 192-C10 exhibit equal binding affinity to SDF-1 as
192-A10-001. However, they show slight differences in the
nucleotide sequence of the central stretch of nucleotides.
Therefore the consensus sequence of the three molecules binding to
SDF-1 with almost the same high affinity can be summarized by the
nucleotide sequence
##STR00005##
(Type A Formula-4, SEQ ID NO: 77)) whereby the nucleotide sequence
of the central stretch of nucleotides of 192-A10-001 (nucleotide
sequence:
##STR00006##
SEQ ID NO: 84) represents the nucleotide sequence with the best
binding affinity of Type A SDF-1 binding nucleic acids.
[0300] Five or six out of the six nucleotides of the 5'-terminal
stretch (also referred to as first terminal stretch) of Type A
SDF-1 binding nucleic acids may hybridize to the respective five or
six nucleotides out of the six nucleotides of the 3'-terminal
stretch (also referred to as second terminal stretch) to form a
terminal helix. Although these nucleotides are variable at several
positions, the different nucleotides allow for hybridization of
five or six out of the six nucleotides of the 5'- and 3'-terminal
stretches each. The 5'-terminal and 3'-terminal stretches of Type A
SDF-1 binding nucleic acids as shown in FIG. 1 can be summarized in
a generic formula for the 5'-terminal stretch (`RSHRYR`, Type A
Formula-5-5') and for the 3'-terminal stretch (`YRYDSY`, Type A
Formula-5-3'). Truncated derivatives of Type A SDF-1 binding
nucleic acid 192-A10-001 were analyzed in a competitive pull-down
binding assay vs. the original molecule 192-A10-001 and 192-A10-008
(FIGS. 2A and 2B). These experiments showed that a reduction of the
six terminal nucleotides (5' end: GCUGUG; 3' end: CGCAGC) of
192-A10-001 to five nucleotides (5' end: CUGUG; 3' end: CGCAG) of
the derivative 192-A10-002 could be done without reduction of
binding affinity. However, the truncation to four terminal
nucleotides (5' end: UGUG; 3' end: CGCA; 192-A10-003) or less
(192-A10-004/-005/-006/-007) led to reduced binding affinity to
SDF-1 (FIG. 2A). The determined 5'-terminal and 3'-terminal
stretches with a length of five and four nucleotides of the
derivatives of Type A SDF-1 binding nucleic acid 192-A10-001 as
shown in FIGS. 2A and 2B can be described in a generic formula for
the 5'-terminal stretch (`X.sub.2BBBS`, Type A Formula-6-5') and of
the 3'-terminal stretch (`SBBVX.sub.3`; Type A Formula-6-3'),
whereby X.sub.2 is either absent or is `S` and X.sub.3 is either
absent or is `S`.
[0301] The nucleotide sequence of the 5'- and 3'-terminal stretches
has an influence on the binding affinity of Type A SDF-1 binding
nucleic acids. This is not only shown by the nucleic acids 192-F10
and 192-E10, but also by derivatives of 192-A10-001 (FIG. 2B). The
central stretch of 192-F10 and 192-E10 are identical to 192-B11 and
192-C10, but comprise slight differences at the 3'-end of
5'-terminal stretch and at the 5'-end of 3'-terminal stretch
resulting in reduced binding affinity.
[0302] The substitution of 5'- and 3'-terminal nucleotides `CUGUG`
and `CGCAG` of Type A SDF-1 binding nucleic acid 192-A10-002 by
`GCGCG` and `CGCGC` (192-A10-015) resulted in a reduced binding
affinity whereas substitutions by `GCGUG` and `CGCGC` (192-A10-008)
resulted in same binding affinity as shown for 192-A10-002 (FIG.
2B). Additionally, nine derivatives of Type A SDF-1 binding nucleic
acid 192-A10-001
(192-A10-014/-015/-016/-017/-018/-019/-020/-021/-022/-023) bearing
four 5'- and 3'-terminal nucleotides respectively were tested as
aptamers for their binding affinity vs. 192-A10-001 or its
derivative 192-A10-008 (both have the identical binding affinity to
SDF-1). All molecules showed weaker, much weaker or very much
weaker binding affinity to SDF-1 as 192-A10-001 (six nucleotides
forming a terminal helix) or as 192-A10-008 with five terminal
nucleotides, respectively (FIG. 2B). Consequently, the sequence and
the number of nucleotides of the 5'- and 3'-terminal stretches are
essential for an effective binding to SDF-1. As shown for Type A
SDF-1 binding nucleic acids 192-A10-002 and 192-A10-08 the
preferred combination of 5'- and 3'-terminal stretches are `CUGUG`
and `CGCAG` (5'- and 3'-terminal stretches of Type A SDF-1 binding
nucleic acid 192-A10-002) and `GCGUG` and `CGCGC` (5'- and
3'-terminal stretches of Type A SDF-1 binding nucleic acid
192-A10-008).
[0303] However, combining the 5'- and 3'-terminal stretches of all
tested Type A SDF-1 binding nucleic acids the generic formula for
the 5'-terminal stretch of Type A SDF-1 binding nucleic acids is
`X.sub.1X.sub.2NNBV` (Type A Formula-7-5') and the generic formula
for the 3'-terminal stretch of Type A SDF-1 binding nucleic acids
is `BNBNX.sub.3X.sub.4` (Type A Formula-7-3'), whereas
X.sub.1 is `R` or absent, X.sub.2 is `S`, X.sub.3 is `S` and
X.sub.4 is `Y` or absent; or X.sub.1 is absent, X.sub.2 is `S` or
absent, X.sub.3 is `S` or absent and X.sub.4 is absent.
[0304] In order to prolong the Spiegelmer's plasma residence time
in vivo, Spiegelmers 192-A10-008 was covalently coupled to a 40 kDa
polyethylene glycol (PEG) moiety at the 5'-end as described in
chapter 2. The PEG-moiety has no influence on Spiegelmers potency
to inhibit SDF-1 induced chemotaxis.
SDF-1 Binding Nucleic Acid Molecules of Type B
[0305] As depicted in FIG. 3 all sequences of SDF-1 binding nucleic
acids of type B comprise one central stretch of nucleotides which
is flanked by 5'- and 3'-terminal stretches (also referred to as
first and second terminal stretch of nucleotides) that can
hybridize to each other. However, such hybridization is not
necessarily given in the molecule.
[0306] In the following the terms `SDF-1 binding nucleic acid
molecules of type B` and `Type B SDF-1 binding nucleic acids` or
Type B SDF-1 binding nucleic acid molecules` are used herein in a
synonymous manner if not indicated to the contrary.
[0307] The sequences of the defined boxes or stretches may be
different between the SDF-1 binding nucleic acids which influences
the binding affinity to SDF-1. Based on binding analysis of the
different SDF-1 binding nucleic acids, the central stretch of
nucleotides and its nucleotide sequences as described in the
following are individually and more preferably in their entirety
essential for binding to SDF-1.
[0308] The central stretch of nucleotides of all identified
sequences of SDF-1 binding nucleic acids 193-C2-001, 193-G2-001,
193-F2-001, 193-G1-002, 193-D2-002, 193-A1-002, 193-D3-002,
193-B3-002, 193-H3-002, 193-E3-002 and 193-D1-002 share the
sequence
##STR00007##
(Type B Formula-1, SEQ ID NO: 52). The SDF-1 binding nucleic acids
193-G2-001, 193-C2-001 and 193-F2-001 that differ in one position
of the central stretch of nucleotides (consenus sequence of central
stretch of nucleotides:
##STR00008##
(Type B Formula-2, SEQ ID NO: 53) were analyzed in a competitive
pull-down binding assay vs. the SDF-1 binding nucleic acid
192-A10-001 (K.sub.D of 1.5 nM determined in a pull-down binding
assay, IC.sub.50 of 0.12 nM). Each of the SDF-1 binding nucleic
acids 193-G2-001, 193-C2-001 and 193-F2 showed superior binding to
human SDF-1 in comparison to SDF-1 binding nucleic acid 192-A10-001
whereby the binding affinity of 193-G2-001 is as good as 193-C2-001
and 193-F2-001 (FIG. 3). The data suggests that the difference in
the nucleotide sequence of the central stretch of nucleotides of
SDF-1 binding nucleic acids 193-G2-001, 193-C2-001 and 193-F2-001
has no influence on the binding affinity to SDF-1. The SDF-1
binding nucleic acids 193-G1-002, 193-D2-002, 193-A1-002,
193-D3-002, 193-B3-002, 193-H3-002, 193-E3-002 and 193-D1-002
showed reduced binding to human SDF-1 in comparison to SDF-1
binding nucleic acid 193-G2-001. SDF-1 binding nucleic acid
193-G2-001 was characterized for its binding affinity to human
SDF-1. The equilibrium binding constant K.sub.D was determined
using the pull-down binding assay (K.sub.D=0.3 nM). The IC.sub.50
(inhibitory concentration 50%) of 0.08 nM for 193-G2-001 was
measured using a cell culture in vitro chemotaxis assay.
[0309] Four, five or six nucleotides out of the six nucleotides of
the 5'-terminal stretch of SDF-1 binding nucleic acids may
hybridize to the respective four, five or six out of the six
nucleotides of the 3'-terminal stretch of SDF-1 binding nucleic
acids to form a terminal helix. Although the nucleotides are
variable at several positions, the different nucleotides allow the
hybridization for four, five or six nucleotides out of the six
nucleotides of the 5'- and 3'-terminal stretches each. The
5'-terminal and 3'-terminal stretches of SDF-1 binding nucleic
acids as shown in FIG. 3 can be summarized in a generic formula for
the 5'-terminal stretch (`X.sub.1X.sub.2GCRWG` whereas X.sub.1 is
`A` or absent, X.sub.2 is `G`) and of the 3'-terminal stretch
(`KRYSCX.sub.3X.sub.4` whereas X.sub.3 is `G`, X.sub.4 is `U` or
absent). SDF-1 binding nucleic acids 193-G1-002, 193-D2-002,
193-A1-002 and 193-D3-002 have weaker binding affinities to SDF-1
although they share the identical central stretch of nucleotides
with 193-C2-001, 193-G2-001 and 193-F2-001 (FIG. 3).
The-unfavorable binding properties of SDF-1 binding nucleic acids
193-G1-002, 193-D2-002, 193-A1-002 and 193-D3-002 may be due to the
number of nucleotides and sequence of the 5'- and 3'-terminal
stretches.
[0310] Truncated derivatives of the SDF-1 binding nucleic acids
193-G2-001 and 193-C2-001 were analyzed in a competitive pull-down
binding assay vs. 193-G2-001 and 193-G2-012, respectively (FIGS. 4A
and 4B). These experiments showed that a reduction of the six
terminal nucleotides (5' end: AGCGUG; 3' end: UACGCU) of SDF-1
binding nucleic acids 193-G2-001 and 193-C2-001 to five nucleotides
(5' end: GCGUG; 3' end: UACGC) lead to molecules with similar
binding affinity (193-C2-002 and 193-G2-012). The equilibrium
dissociation constant K.sub.D was determined using the pull-down
binding assay (K.sub.D=0.3 nM). A truncation to four (5' end: CGUG;
3' end: UACG; 193-C2-003) or less nucleotides (193-C2-004,
193-C2-005, 193-C2-006, 193-C2-007) resulted in a reduced binding
affinity to SDF-1 which was measured by using the competition
pull-down binding assay (FIG. 4A). The nucleotide sequence of the
five terminal nucleotides at the 5'- and 3'-end, respectively, has
an influence on the binding affinity of SDF-1 binding nucleic
acids. The substitution of 5'- and 3'-terminal nucleotides `GCGUG`
and `UACGC` (193-C2-002, 193-G2-12) by `GCGCG` and `CGCGC`
(193-G2-013) resulted in a reduced binding affinity. Additionally,
the four different derivatives of SDF-1 binding nucleic acid
193-G2-001 with a terminal helix with a length of four base-pairing
nucleotides (193-G2-014/-015/-016/-017) were tested. All of them
showed reduced binding affinity to SDF-1 (FIG. 4B). Therefore the
sequence and the length of the 5'- and 3'-terminal nucleotides are
essential for an effective binding to SDF-1. The 5'-terminal and
3'-terminal stretches with a length of five and four nucleotides of
the derivatives of SDF-1 binding nucleic acids 193-C2-003 and
193-G2-012 as shown in FIGS. 4A and 4B can be described in a
generic formula for the 5'-terminal stretch (`X.sub.1X.sub.2SSBS`),
whereby X.sub.1 is absent, X.sub.2 is either absent or is `G`, and
of the 3'-terminal stretch (`BVSSX.sub.3X.sub.4`), and whereby
X.sub.3 is either absent or is `C` and X.sub.4 is absent. As shown
for SDF-1 binding nucleic acids 193-G2-001 and 193-C2-01 and their
derivatives 193-G2-012 and 193-C2-002 the preferred combination of
5'- and 3'-terminal stretches are `X.sub.1X.sub.2GCGUG`
(5'-terminal stretch) and `UACGCX.sub.3X.sub.4` (3'-terminal
stretch), whereas X.sub.1 is either `A` or absent, X.sub.2 is `G`
and X.sub.3 is `C` and `X.sub.4 is `U` or absent.
[0311] However, combining the 5'- and 3'-terminal stretches of all
tested SDF-1 binding nucleic acids the generic formula for the
5'-terminal stretch of SDF-1 binding nucleic acids is
`X.sub.1X.sub.2SVNS` and the generic formula for the 3'-terminal
stretch SDF-1 binding nucleic acids is `BVBSX.sub.3X.sub.4`,
whereas
X.sub.1 is `A` or absent, X.sub.2 is `G`, X.sub.3 is `C` and
X.sub.4 is `U` or absent; or X.sub.1 is absent, X.sub.2 is `G` or
absent, X.sub.3 is `C` or absent and X.sub.4 is absent.
[0312] In order to prolong the Spiegelmer's plasma residence time
in vivo, Spiegelmers 193-G2-012 was covalently coupled to a 40 kDa
polyethylene glycol (PEG) moiety at the 5'-end as described in
chapter 2 (PEGylated-nucleic acid molecule: 193-G2-012-5'-PEG also
referred to as NOX-A12). The PEGylated Spiegelmer NOX-A12 was
analyzed in cell culture in an in vitro chemotaxis-assay and an
inhibition of SDF-1 induced chemotaxis was determined (IC.sub.50 of
0.2 nM). The PEGylated Spiegelmer NOX-A12 was analyzed by Biacore
measurement and a binding constant (K.sub.D) of 0.2 nM was
determined.
SDF-1 Binding Nucleic Acid Molecules of Type C
[0313] As depicted in FIG. 12 all sequences of SDF-1 binding
nucleic acids of type C comprise one central stretch of nucleotides
which is flanked by 5'- and 3'-terminal stretches (also referred to
as first terminal stretch and second terminal stretch of
nucleotides) that can hybridize to each other. However, such
hybridization is not necessarily given in the molecule.
[0314] In the following the terms `SDF-1 binding nucleic acid
molecules of type C` and `Type C SDF-1 binding nucleic acids` or
Type C SDF-1 binding nucleic acid molecules` are used herein in a
synonymous manner if not indicated to the contrary.
[0315] The sequences of the defined boxes or stretches may be
different between the SDF-1 binding nucleic acids of Type C which
influences the binding affinity to SDF-1. Based on binding analysis
of the different SDF-1 binding nucleic acids summarized as Type C
SDF-1 binding nucleic acids, the central stretch of nucleotides and
its nucleotide sequence as described in the following are
individually and more preferably in their entirety essential for
binding to SDF-1.
[0316] The central stretch of nucleotides of all identified
sequences of Type C SDF-1 binding nucleic acids share the
sequence
##STR00009##
(Type C Formula-1, SEQ ID NO: 108), whereby X.sub.A is either
absent or is `A`. With the exception of Type C SDF-1 binding
nucleic acid 197-D1 the central stretch of nucleotides of all
identified sequences of Type C SDF-1 binding nucleic acids share
the nucleotide sequence
##STR00010##
(Type C Formula-2, SEQ ID NO: 109). Type C SDF-1 binding nucleic
acid 197-D1 (central stretch of nucleotides:
##STR00011##
(SEQ ID NO: 56) missing one nucleotide `A` within the central
stretch of nucleotides and still binding to SDF-1 let conclude an
alternative central stretch of nucleotides
##STR00012##
Type C Formula-3, SEQ ID NO: 110). Initially, all Type C SDF-1
binding nucleic acids as depicted in FIG. 5 were analyzed in a
competitive pull-down binding assay vs. Type A SDF-1 binding
nucleic acid 192-A10-001 (K.sub.D=1.5 nM determined by pull-down
assay and by surface plasmon resonance measurements; IC.sub.50=0.12
nM). The Type C SDF-1 binding nucleic acids 191-D5-001, 197-B2,
190-A3-001, 197-H1, 197-H3 and 197-E3 showed weaker binding
affinities than 192-A10-001 in competition experiments. Much weaker
binding affinity was determined for 191-A5, 197-B1, 197-D1, 197-H2
and 197-D2 (FIG. 5). The molecules or derivatives thereof were
further characterized by further competitive pull-down binding
assays, plasmon resonance measurements and an in vitro chemotaxis
assay. The Type C SDF-1 binding nucleic acid 191-D5-001 was
characterized for its binding affinity to human SDF-1 whereas the
equilibrium binding constant K.sub.D was determined by surface
plasmon resonance measurement (K.sub.D=0.8 nM). The IC.sub.50
(inhibitory concentration 50%) of 0.2 nM for 191-D5-001 was
measured using a cell-culture in vitro chemotaxis assay. The
binding affinity of Type C SDF-1 binding nucleic acid 197-B2 for
human SDF-1 was determined by surface plasmon resonance measurement
(K.sub.D=0.9 nM), its IC.sub.50 (inhibitory concentration 50%) of
0.2 nM was analyzed in a cell-culture in vitro chemotaxis assay.
These data indicates that Type C SDF-1 binding nucleic acids
191-D5-001 and 197-B2 have the similar binding affinity to SDF-1
(FIGS. 5 and 8).
[0317] Type C SDF-1 binding nucleic acid 190-A3-001 comprises a
5'-terminal stretch of 17 nucleotides (`CGUGCGCUUGAGAUAGG`, SEQ ID
NO: 220) and a 3'-terminal stretch of 12 nucleotides
(`CUGAUUCUCACG`, SEQ ID NO: 221) whereby on the one hand the four
nucleotides at the 5'-end of the 5'-terminal stretch and the four
nucleotides at the 3'-end of the 3'-terminal stretch may hybridize
to each other to form a terminal helix. Alternatively the
nucleotides `UGAGA` in the 5'-terminal stretch may hybridize to the
nucleotides `UCUCA` in the 3'-terminal stretch to form a terminal
helix. A reduction to nine nucleotides of the 5'-terminal stretch
(`UGAGAUAGG`) and to ten (`CUGAUUCUCA`, SEQ ID NO: 222) nucleotides
of the 3'-terminal stretch (`CUGAUUCUC`) of molecule 190-A3-001
does not have an influence on the binding affinity to SDF-1
(190-A3-003; FIG. 13). A reduction to eight nucleotides of the
5'-terminal stretch (`GAGAUAGG`) and to nine nucleotides of the
3'-terminal stretch (`CUGAUUCUC`) of molecule 190-A3-001 does not
have an influence on the binding affinity to SDF-1 (190-A3-004;
FIG. 6). The equilibrium binding constant K.sub.D of 190-A3-004 was
determined using the pull-down binding assay (K.sub.D=4.6 nM) and
by surface plasmon resonance measurement (K.sub.D=4.7 nM). The
IC.sub.50 (inhibitory concentration 50%) of 0.1 nM for 190-A3-004
was measured using a cell-culture in vitro chemotaxis assay.
However, the truncation to two nucleotides at the 5'-terminal
stretch leads to a very strong reduction of binding affinity
(190-A3-007; FIG. 6).
[0318] The Type C SDF-1 binding nucleic acids 191-D5-001, 197-B2
and 197-H1 (central stretch of nucleotides:
##STR00013##
SEQ ID 57, 197-H3/191-A5 (central stretch of nucleotides:
##STR00014##
SEQ ID NO: 58 and 197-E3/197-B1 (central stretch of
nucleotides:
##STR00015##
SEQ ID NO: 59 share an almost identical central stretch of
nucleotides (Type C formula-4; nucleotide sequence:
##STR00016##
SEQ ID NO: 111). 191-D5-001, 197-B2 and 197-H1 do not share a
similar 5'- and 3'-terminal stretch (197-H3 and 197-E3 have the
identical 5'- and 3'-terminal stretch as 197-B2). However, the
respective ten (197-B2, 197-E3, 197-H3) or nine out of the ten
(191-D5-001, 197-H1) nucleotides of the 5'-terminal stretch may
hybridize to the respective ten (197-B2, 197-E3, 197-H3) or nine
out of the ten (191-D5-001, 197-H1) nucleotides of the 3'-terminal
stretch (FIG. 5). Thus, the 5'-terminal stretch of Type C SDF-1
binding nucleic acids 197-B2, 191-D5-001, 197-H1, 197-E3 and 197-H3
as mentioned above plus 191-A5, 197-B1, 197-H2, 197-D1 and 197-D2
comprise a common generic nucleotide sequence of `RKSBUSNVGR` (Type
C Formula-5-5', SEQ ID NO: 138). The 3'-terminal stretch of Type C
SDF-1 binding nucleic acids 197-B2, 191-D5-001, 197-H1, 197-E3, and
197-H3 as mentioned above plus 191-A5, 197-B1, 197-H2, 197-D1 and
197-D2 comprise a common generic nucleotide sequence of
`YYNRCASSMY` (Type C Formula-5-3', SEQ ID NO: 139), whereby the 5'
and the 3'-terminal stretches of Type C SDF-1 binding nucleic acids
197-B2, 191-D5-001, 197-H1, 197-E3 and 197-H3 are preferred. These
preferred 5'- and 3'-terminal stretches of Type C SDF-1 binding
nucleic acids 197-B2, 191-D5-001, 197-H1, 197-E3 and 197-H3 can be
summarized in the generic formula `RKSBUGSVGR` (Type C
Formula-6-5'; 5'-terminal stretch, SEQ ID NO: 140) and `YCNRCASSMY`
(Type C Formula-6-3'; 3'-terminal stretch, SEQ ID NO: 141).
[0319] Truncated derivatives of Type C SDF-1 binding nucleic acid
191-D5-001 were constructed and tested in a competitive pull-down
binding assay vs. the original molecule 191-D5-001 (FIG. 7A, FIG.
7B). At first the length of the 5'- and 3'-terminal stretches were
shortened from ten nucleotides (191-D5-001) each to seven
nucleotides each (191-D5-004) as depicted in FIG. 14A whereby nine
out of the ten (191-D5-001) or six out of the seven nucleotides
(191-D5-004) of the 5'-terminal stretch and of the 3'-terminal
stretch, respectively can hybridize to each other. The reduction to
seven nucleotides of the 5'- and 3'-terminal stretch respectively
(whereas six out of the seven nucleotides can hybridize to each
other) led to reduced binding affinity to SDF-1 (191-D5-004). The
terminal stretches of Type C SDF-1 binding nucleic acid 191-D5-004
were modified whereby the non-pairing nucleotide `A` within the
3'-terminal stretch of 191-D5-004 was substituted by a `C`
(191-D5-005). This modification led to an improvement of binding.
This derivative, Type C SDF-1 binding nucleic acid 191-D5-005,
showed similar binding to SDF-1 as 191-D5-001. Further truncation
of the 5'- and 3'-terminal stretch to five nucleotides respectively
led to a molecule with a length of total 29 nucleotides
(191-D5-007). Because of the similarities of 191-D5-001 and of the
Type C SDF-1 binding nucleic acids 197-B2, 191-D5-001, 197-H1,
191-A5, 197-H3, 197-B1, 197-E3, 197-D1, 197-H2 and 197-D2 and
because of the data shown for 191-D5-007 it may assume that the 5'-
and 3'-terminal stretch can in principle be truncated down to five
nucleotides whereby the nucleotide sequence `CGGGA` for 5'-terminal
stretch and `UCCCG` for the 3'-terminal stretch was successfully
tested (Type C SDF-1 binding nucleic acid 191-D5-007). Type C SDF-1
binding nucleic acid 191-D5-007 surprisingly binds somewhat better
to SDF-1 than 191-D5-001 (determined on aptamer level using the
competition binding assay). The equilibrium binding constant
K.sub.D of 191-D5-007 was determined using the pull-down binding
assay (K.sub.D=2.2 nM) and by surface plasmon resonance measurement
(K.sub.D=0.8 nM). The IC.sub.50 (inhibitory concentration 50%) of
0.1 nM for 191-D5-007 was measured using a cell-culture in vitro
chemotaxis assay. Further truncation of both terminal stretches to
four nucleotides (191-D5-010, FIG. 7A).
[0320] Further derivatives of Type C SDF-1 binding nucleic acid
191-D5-001 (191-D5-017/-024/-029) bearing 5'- and 3'-terminal
stretches of respectively four nucleotides also showed reduced
binding affinity to SDF-1 in the competition pull-down binding
assay vs. 191-D5-007 (FIG. 7B). Alternative 5'- and 3'-terminal
stretches with a length of respectively five nucleotides were
additionally tested, too (191-D5-017-29a, 191-D5-017-29b,
191-D5-019-29a, 191-D5-024-29a, 191-D5-024-29b). The generic
formula of these derivatives for the 5'-terminal stretch is
`X.sub.SSSSV` (Type C Formula-7-5') and for the 3'-stretch is
`BSSSX.sub.S` Type C Formula-7-3'), whereby X.sub.S is absent or
`S`. Two out of the five tested variants showed identical binding
affinity to SDF-1 as 191-D5-007 (191-D5-024-29a, 191-D5-024-29b;
FIG. 7B). The sequences of the 5'-terminal and 3'-terminal
stretches of 191-D5-001-derivatives that show the best binding
affinity to SDF-1 and comprise a 5'-terminal and 3'-terminal
stretch of five nucleotides respectively (191-D5-007,
191-D5-024-29a, 191-D5-024-29b) can be summarized in a generic
formula (5'-terminal stretch: `SGGSR`, Type C Formula-8-5';
3'-terminal stretch: `YSCCS`, Type C Formula-8-3').
[0321] Truncated derivatives of Type C SDF-1 binding nucleic acid
197-B2 were analyzed in a competitive pull-down binding assay vs.
the original molecule 197-B2 and 191-D5-007 (FIG. 7). Using the
competitive pull-down binding assay vs. 191-D5-007 it was shown
that 197-B2 has the same binding affinity to SDF-1 as 191-D5-007.
The 5'- and 3'-terminal stretches were shortened without loss of
binding affinity from ten nucleotides (197-B2) each to five
nucleotides each (197-B2-005) whereby the nucleotides of the
5'-terminal stretch and of the 3'-terminal stretch can completely
hybridize to each other. If the 5'-terminal (`GCGGG`) and
3'-terminal (`CCUGC`) stretch of 197-B2-005 was substituted by
`GCCGG` (5'-terminal stretch) and by `CCGGC` (3'-terminal stretch)
of 197-B2-006, the binding affinity to SDF-1 fully persisted.
Because 197-B2 and 191-D5-001 (and their derivatives) share the
identical core nucleotide sequence and several derivatives of
191-D5 with 5'- and 3'-terminal stretches with a length of
respectively four nucleotides were tested, a further truncation of
the 5'- and 3'-terminal stretch was omitted. Two further
derivatives were designed that comprise six nucleotides at the 5'-
and 3'-end (5'- and 3'-terminal stretches) respectively. The
binding affinity to SDF-1 of both molecules (197-B2-006-31a and
197-B2-006-31b) is the same as shown for 191-D5-007 and 197-B2-006
(FIG. 15). The sequences of the 5'-terminal and 3'-terminal
stretches of 197-B2 derivatives that show the best binding affinity
to SDF-1 and comprise a 5'-terminal and 3'-terminal stretch of five
nucleotides respectively can be summarized in a generic formula
(5'-terminal stretch: `GCSGG`, Type C Formula-9-5'; 3'-terminal
stretch: `CCKGC`, Type C Formula-9-3').
[0322] Combining the preferred 5'- and 3'-stretches of truncated
derivatives of Type C SDF-1 binding nucleic acids 191-D5-001
(5'-terminal stretch: `SGGSR`, Type C Formula-8-5'; 3'-terminal
stretch: `YSCCS`, Type C Formula-8-3') and 197-B2 (5'-terminal
stretch: `GCSGG`, Type C Formula-9-5'; 3'-terminal stretch:
`CCKGC`, Type C Formula-9-3') the common preferred generic formula
for the 5'-terminal and the 3'-terminal stretch is `SSSSR`
(5'-terminal stretch, Type C Formula-10-5') and `YSBSS`
(3'-terminal stretch: Type C Formula-10-3').
[0323] In order to prolong the Spiegelmer's plasma residence time
in vivo, Spiegelmers 197-B2-006 and 191-D5-007 were covalently
coupled to a 40 kDa polyethylene glycol (PEG) moiety at their
5'-ends as described in chapter 2. The PEGylated Spiegelmers
197-B2-006 and 191-D5-007 were analyzed in cell culture in an in
vitro chemotaxis. The PEG-moiety has no influence on Spiegelmers
potency to inhibit SDF-1 induced chemotaxis.
SDF-1 Binding Nucleic Acid Molecules of Type D
[0324] Additionally, further three SDF-1 binding nucleic acids that
do not share the SDF-1 binding motifs of `Type A`, `Type B` and
`Type C` were identified and are referred to herein as "type D".
There were analyzed as aptamers using the pull-down binding assay
(FIG. 9).
[0325] It is to be understood that any of the sequences shown in
FIGS. 1 through 9 are nucleic acid molecules according to the
present invention, including those truncated forms thereof but also
including those extended forms thereof under the proviso, however,
that the thus truncated and extended, respectively, nucleic acid
molecules are still capable of binding to the target.
EXAMPLE 2
Synthesis and Derivatization of Aptamers and Spiegelmers
Small Scale Synthesis
[0326] Aptamers and Spiegelmers were produced by solid-phase
synthesis with an ABI 394 synthesizer (Applied Biosystems, Foster
City, Calif., USA) using 2'TBDMS RNA phosphoramidite chemistry
(Damha and Ogilvie, 1993). rA(N-Bz)-, rC(Ac)-, rG(N-ibu)-, and
rU-phosphoramidites in the D- and L-configuration were purchased
from ChemGenes, Wilmington, Mass. Aptamers and Spiegelmers were
purified by gel electrophoresis.
Large Scale Synthesis Plus Modification
[0327] The Spiegelmers were produced by solid-phase synthesis with
an AktaPilot100 synthesizer (Amersham Biosciences; General Electric
Healthcare, Freiburg) using 2'TBDMS RNA phosphoramidite chemistry
(Damha and Ogilvie, 1993). L-rA(N-Bz)-, L-rC(Ac)-, L-rG(N-ibu)-,
and L-rU-phosphoramidites were purchased from ChemGenes
(Wilmington, Mass., USA). The 5'-amino-modifier was purchased from
American International Chemicals Inc. (Framingham, Mass., USA).
Synthesis of the Spiegelmers was started on L-riboG; L-riboC,
L-riboA, L-riboU respectively modified CPG pore size 1000 .ANG.
(Link Technology, Glasgow, UK). For coupling (15 min per cycle),
0.3 M benzylthiotetrazole (American International Chemicals Inc.,
Framingham, Mass., USA) in acetonitrile, and 3.5 equivalents of the
respective 0.2 M phosphoramidite solution in acetonitrile was used.
An oxidation-capping cycle was used. Further standard solvents and
reagents for oligonucleotide synthesis were purchased from Biosolve
(Valkenswaard, NL). The Spiegelmers were synthesized DMT-ON; after
deprotection, it was purified via preparative RP-HPLC (Wincott F.
et al., 1995) using Source15RPC medium (Amersham). The 5'DMT-group
was removed with 80% acetic acid (90 min at RT). Subsequently,
aqueous 2 M NaOAc solution was added and the Spiegelmer was
desalted by tangential-flow filtration using a 5 K regenerated
cellulose membrane (Millipore, Bedford, Mass.).
Pegylation
[0328] In order to prolong the Spiegelmer's plasma residence time
in vivo, the Spiegelmers were covalently coupled to a 40 kDa
polyethylene glycol (PEG) moiety at the 5'-end.
[0329] For PEGylation (for technical details of the method for
PEGylation see European patent application EP 1 306 382), the
purified 5'-amino modified Spiegelmerd were dissolved in a mixture
of H.sub.2O (2.5 ml), DMF (5 ml), and buffer A (5 ml; prepared by
mixing citric acid.H.sub.2O [7 g], boric acid [3.54 g], phosphoric
acid [2.26 ml], and 1 M NaOH [343 ml] and adding water to a final
volume of 1 l; pH=8.4 was adjusted with 1 M HCl).
[0330] The pH of the Spiegelmer solution was brought to 8.4 with 1
M NaOH. Then, 40 kDa PEG-NHS ester (JenKem Technology USA Inc.,
Allen, Tex.) was added at 37.degree. C. every 30 min in six
portions of 0.25 equivalents until a maximal yield of 75 to 85% was
reached. The pH of the reaction mixture was kept at 8-8.5 with 1 M
NaOH during addition of the PEG-NHS ester.
[0331] The reaction mixture was blended with 4 ml urea solution (8
M), and 4 ml buffer B (0.1 M triethylammonium acetate in H.sub.2O)
and heated to 95.degree. C. for 15 min. The PEGylated Spiegelmer
was then purified by RP-HPLC with Source 15RPC medium (Amersham),
using an acetonitrile gradient (buffer B; buffer C, 0.1 M
triethylammonium acetate in acetonitrile). Excess PEG eluted at 5%
buffer C, PEGylated Spiegelmer at 10-15% buffer C. Product
fractions with a purity of >95% (as assessed by HPLC) were
combined and mixed with 40 ml 3 M NaOAC. The PEGylated Spiegelmer
was desalted by tangential-flow filtration (5 K regenerated
cellulose membrane, Millipore, Bedford Mass.).
EXAMPLE 3
Determination of Binding Constants (Pull-Down Binding Assay)
Direct Pull-Down Binding Assay
[0332] The affinity of aptamers to biotinlayted human D-SDF-1 was
measured in a pull-down binding assay format at 37.degree. C.
Aptamers were 5'-phosphate labeled by T4 polynucleotide kinase
(Invitrogen, Karlsruhe, Germany) using [.gamma.-.sup.32P]-labeled
ATP (Hartmann Analytic, Braunschweig, Germany). The specific
radioactivity of labeled aptamers was 200,000-800,000 cpm/pmol.
Aptamers were incubated after de- and renaturation at 10, 20, 30 or
40 .mu.M concentration at 37.degree. C. in selection buffer (20 mM
Tris-HCl pH 7.4; 137 mM NaCl; 5 mM KCl; 1 mM MgCl.sub.2; 1 mM
CaCl.sub.2; 0.1% [w/vol] Tween-20) together with varying amounts of
biotinlayted human D-SDF-1 for 4-12 hours in order to reach
equilibrium at low concentrations. Selection buffer was
supplemented with 10 .mu.g/ml human serum albumin (Sigma-Aldrich,
Steinheim, Germany), and 10 .mu.g/ml yeast RNA (Ambion, Austin,
USA) in order to prevent adsorption of binding partners with
surfaces of used plasticware or the immobilization matrix. The
concentration range of biotinlayted human D-SDF-1 was set from 8
.mu.M to 100 nM; total reaction volume was 1 ml. Peptide and
peptide-aptamer complexes were immobilized on 1.5 .mu.l
Streptavidin Ultralink Plus particles (Pierce Biotechnology,
Rockford, USA) which had been preequilibrated with selection buffer
and resuspended in a total volume of 6 .mu.l. Particles were kept
in suspension for 30 min at the respective temperature in a
thermomixer. Immobilized radioactivity was quantitated in a
scintillation counter after detaching the supernatant and
appropriate washing. The percentage of binding was plotted against
the concentration of biotinlayted human D-SDF-1 and dissociation
constants were obtained by using software algorithms (GRAFIT;
Erithacus Software; Surrey U.K.) assuming a 1:1 stoichiometry.
Competitive Pull-Down Binding Assay
[0333] In order to compare different D-SDF-1 binding aptamers, a
competitive ranking assay was performed. For this purpose the most
affine aptamer available was radioactively labeled (see above) and
served as reference. After de- and renaturation it was incubated at
37.degree. C. with biotinlayted human D-SDF-1 in 1 ml selection
buffer at conditions that resulted in around 5-10% binding to the
peptide after immobilization and washing on NeutrAvidin agarose or
Streptavidin Ultralink Plus (both from Pierce) without competition.
An excess of de- and renatured non-labeled D-RNA aptamer variants
was added to different concentrations (e.g. 2, 10, and 50 nM) with
the labeled reference aptamer to parallel binding reactions. The
aptamers to be tested competed with the reference aptamer for
target binding, thus decreasing the binding signal in dependence of
their binding characteristics. The aptamer that was found most
active in this assay could then serve as a new reference for
comparative analysis of further aptamer variants.
EXAMPLE 4
Binding Analysis by Surface Plasmon Resonance Measurement
[0334] The Biacore 2000 instrument (Biacore AB, Uppsala, Sweden)
was used to analyze binding of Spiegelmers to human SDF-1.alpha..
When coupling of human SDF-1.alpha. was to be achieved via amine
groups, human SDF-1.alpha. was dialyzed against water for 1-2 h
(Millipore VSWP mixed cellulose esters; pore size, 0.025 .mu.M) to
remove interfering amines. CM4 sensor chips (Biacore AB, Uppsala,
Sweden) were activated before protein coupling by a 35-.mu.l
injection of a 1:1 dilution of 0.4 M NHS and 0.1 M EDC at a flow of
5 .mu.l/min. Human MCP-1 or human SDF-1.alpha. was then injected in
concentrations of 0.1-1.5 .mu.g/ml at a flow of 2 .mu.l/min until
the instrument's response was in the range of 1000-2000 RU
(relative units). Unreacted NHS esters were deactivated by
injection of 35 .mu.l ethanolamine hydrochloride solution (pH 8.5)
at a flow of 5 .mu.l/min. The sensor chip was primed twice with
binding buffer and equilibrated at 10 .mu.l/min for 1-2 hours until
the baseline appeared stable. For all proteins, kinetic parameters
and dissociation constants were evaluated by a series of Spiegelmer
injections at concentrations of 1000, 500, 250, 125, 62.5, 31.25,
and 0 nM in selection buffer (Tris-HCl, 20 mM; NaCl, 137 mM; KCl, 5
mM; CaCl.sub.2, 1 mM; MgCl.sub.2, 1 mM; Tween20, 0.1% [w/v]; pH
7.4). In all experiments, the analysis was performed at 37.degree.
C. using the Kinject command defining an association time of 180
and a dissociation time of 360 seconds at a flow of 10 .mu.l/min.
Data analysis and calculation of dissociation constants (K.sub.D)
was done with the BIAevaluation 3.0 software (BIACORE AB, Uppsala,
Sweden) using the Langmuir 1:1 stoichiometric fitting
algorithm.
EXAMPLE 5
Analysis of the Inhibition of SDF-1-Induced Chemotaxis by
SDF-1-Binding Spiegelmers
[0335] The human T cell leukemia cell line Jurkat, the human
leukemic monocyte lymphoma cell line U937, the human pre-B cell
leukemia cell line BV-173 and human pre-B ALL cell line Nalm-6
express CXCR4. While Jurkat cells do not express CXCR7, the
leukemia lines BV-173 and U-937 were tested positive for CXCR7
expression. All cells used were obtained from the DSMZ
(Braunschweig). All cell lines were cultivated at 37.degree. C. and
5% CO2 in RPMI 1640 medium with Glutamax (Invitrogen, Karlsruhe,
Germany) which contains 10% fetal bovine serum, 100 units/ml
penicillin and 100 .mu.g/ml streptomycin (Invitrogen, Karlsruhe,
Germany). One day before the experiment, cells were seeded in a new
T175 flask with a density of 0.3.times.10.sup.6/ml (Jurkat, U937,
BV-173) or 0.75.times.10.sup.6/ml (Nalm-6), respectively.
[0336] For the experiment, cells were centrifuged (5 min at 300 g),
resuspended, counted and washed once with 15 ml HBH (Hanks balanced
salt solution containing 1 mg/ml bovine serum albumin and 20 mM
HEPES; Invitrogen, Karlsruhe, Germany). Then the cells were
resuspended at 1.33.times.10.sup.6/ml (Jurkat, U937, BV-173) or
2.67.times.10.sup.6/ml (Nalm-6), respectively. Cells were then
allowed to migrate through the porous membranes of the filter
plates for three hours towards a solution containing SDF-1 and
various amounts of Spiegelmer. The stimulation solutions
(SDF-1+various concentrations of Spiegelmer) were made up as
10.times. solutions in a 0.2 ml low profile 96-tube plate. 212
.mu.l HBH were pipetted into the lower compartments of the
transport plate and 23.5 .mu.l of the stimulation solutions were
added. All conditions were made up as triplicates. After 20 to 30
min the filter plate was inserted into the plate containing the
stimulation solutions and 75 .mu.l of a cell suspension with
1.33.times.10.sup.6/ml or 2.67.times.10.sup.6/ml, respectively,
were added to the wells of the filter plate (1.times.10.sup.5 or
2.times.10.sup.5 cells/well). The cells were then allowed to
migrate for 3 h at 37.degree. C. For calibration, 0, 10 and 30
.mu.l of the cell suspension was added to 235, 225 and 205 .mu.l
HBH, respectively, in wells of a separate 96-well plate. After 3
hours incubation, the insert plate was removed and 30 .mu.l
resazurin working solution (440 .mu.M in PBS) were added to the
lower wells and to the wells of the calibration plate. The plates
were then incubated at 37.degree. C. for 2.5 h. After incubation,
100 .mu.l of each well were transferred to a black 96 well
plate.
[0337] For evaluation, fluorescence values were corrected for
background fluorescence (no cells in well). Then the difference
between experimental conditions with and without SDF-1 was
calculated. The value for the sample without Spiegelmer (SDF-1
only) was set 100% and the values for the samples with Spiegelmer
were calculated as percent of this. For a dose-response curve the
percent-values were plotted against Spiegelmer concentration and
the IC.sub.50-value (concentration of Spiegelmer at which 50% of
the activity without Spiegelmer is present) was determined
graphically from the resulting curve.
Results
[0338] Human SDF-1 was found to stimulate migration of Jurkat cells
in a dose dependent manner, with half-maximal stimulation at about
0.3 nM.
[0339] Human SDF-1 was found to stimulate migration of cells of the
human leukemic monocyte lymphoma cell line U937 in a dose dependent
manner, with half-maximal stimulation at about 3 nM.
[0340] Human SDF-1 was found to stimulate migration of cells of the
human pre-B cell leukemia cell line BV-173 in a dose dependent
manner, with half-maximal stimulation at about 3 nM.
[0341] Human SDF-1 was found to stimulate migration of cells of the
human pre-B ALL cell line Nalm-6 in a dose dependent manner, with
half-maximal stimulation at about 0.3 nM.
[0342] When cells were allowed to migrate towards a solution
containing human SDF-1 plus increasing concentrations of SDF-1
binding Spiegelmers, dose-dependent inhibition was observed. The
respective IC50s of the tested Spiegelmers as specified in Example
1 were determined in human T cell leukemia cell line Jurkat cells.
For example, for SDF-1 binding Spiegelmer NOX-A12 (also referred to
as 193-G2-012-5'-PEG) an IC50 of 0.2 nM was determined (FIG. 10).
When an unspecific Control Spiegelmer was used instead of SDF-1
binding Spiegelmers, no inhibitory effect was observed up to 1
.mu.M.
[0343] Inhibition of the SDF-1 induced chemotaxis by SDF-1 binding
spiegelmer NOX-A12 was also observed in three other different
leukemia cell types: the human leukemic monocyte lymphoma cell line
U937 (FIG. 11B), the human pre-B cell leukemia cell line BV-173
(FIG. 12) and the human pre-B ALL cell line Nalm-6 (FIG. 11A).
Furthermore, we have evidence that primary chronic lymphocytic
leukemia cells migrate towards SDF-1 and that SDF-1 dependent
chemotaxis is effectively blocked by NOX-A12.
[0344] The leukemia lines BV-173 and U-937 were tested positive
also for CXCR7 expression. The potency of SDF-binding spiegelmer
NOX-A12 to block interaction of SDF-1 and CXCR7 was determined as
shown in Example 6.
EXAMPLE 6
Inhibition of CXCR7 Activation by SDF-1-Binding Spiegelmer
NOX-A12
[0345] Besides CXCR4, SDF-1 also binds to the chemokine receptor
CXCR7. The inhibitory potential of SDF-1-binding Spiegelmer NOX-A12
towards CXCR7 was tested in a complementation assay with CHO cells
stably expressing CXCR7 and .beta.-arrestin both fused to a
fragment of 13-galactosidase (PathHunter.TM.-.beta.-arrestin assay,
DiscoveRX, CA, USA). Upon SDF-1 binding .beta.-arrestin complexed
with CXCR7 and thus led to complementation and activation of the
.beta.-galactosidase which was measured with a chemiluminescence
substrate.
Method
[0346] PathHunter eXpress CHO-K1 Human CXCR7 .beta.-arrestin cells
were plated for 48 hours in OCC2 Medium and stimulated with 10 nM
SDF-1 and various concentrations of SDF-1-binding Spiegelmer
NOX-A12 for 90 minutes. Following stimulation, signal was detected
using the PathHUnter Detection Kit and the manufacturer's
recommended protocol (DiscoveRX, CA, USA).
Results
[0347] Stimulation of .beta.-galactosidase and hence CXCR7
activation with 10 nM human SDF-1 was efficiently blocked by
SDF-1-binding Spiegelmer NOX-A12 with an IC50 of 5.4 nM (FIG.
13).
EXAMPLE 7
Functional Analysis of Human SDF-1 Binding Spiegelmer
193-G2-012-5'-PEG in an Aortic Ring Sprouting Assay
[0348] To test whether human SDF-1 binding Spiegelmer
193-G2-012-5'-PEG is functional also in a standard angiogenesis
organ culture assay, aortic ring sprouting assays were performed.
This assay, in which the length and abundance of vessel-like
extensions from the explants are evaluated, has become the most
widely used organ culture model for angiogenesis (Auerbach et al.
2003). It has already been shown that SDF-1 induces sprouting in
this type of assay (Salcedo et al. 1999).
[0349] Rat aortae were cut into rings, embedded in a collagen
matrix and incubated with SDF-1 and SDF-1 plus human SDF-1 binding
Spiegelmer 193-G2-012-5'-PEG or SDF plus an non-functional
PEGylated Control Spiegelmer that does not bind SDF-1. After 6 to 7
days, sprouting (i.e. outgrowth of endothelial cells) was analysed
by taking pictures and determining a sprouting index.
Method
[0350] Aortae from male rats were obtained from Bagheri Life
sciences (Berlin, Germany). The aortae were prepared freshly and
transported on ice in MCDB 131-Medium (Invitrogen, Karlsruhe,
Germany) containing 50 units/ml penicillin, 50 .mu.g/ml
streptomycin (both Invitrogen, Karlsruhe, Germany) and 2.5 .mu.g/ml
fungizone (Cambrex, USA).
[0351] For an experiment a single aorta was transferred to a cell
culture dish together with the medium and residual connective
tissue was removed. Then the aorta was cut with a scalpel into
rings of about 1 to 2 mm length. The rings were washed intensively
(at least five times) in Medium199 (Invitrogen, Karlsruhe, Germany)
and then placed in wells of a 24 well plate, containing 450 .mu.l
of collagen solution per well. This collagen solution was prepared
by mixing 9 ml rat tail collagen (3 mg/ml in 0.1% acetic acid;
Sigma, Deisenhofen, Germany) with 1.12 ml 10.times. Medium 199
(Invitrogen, Karlsruhe, Germany), 1.12 ml 10.times. Collagen-buffer
(0.05 N NaOH, 200 mM HEPES, 260 mM NaHCO.sub.3) and 0.6 ml 200 mM
Glutamin. The rings were oriented such that the trimmed edges were
perpendicular to the bottom of the well. The collagen was allowed
to solidify by incubating the plates for at least one hour at
37.degree. C. Thereafter 1 ml MCDB131-medium with additions (SDF-1
and Spiegelmers) was added per well. Rings were then incubated at
37.degree. C. for six to seven days. As control for sprouting the
experiments were additionally done with VEGF (Vascular endothelial
growth factor).
[0352] Sprouting was documented by taking pictures with a digital
camera. In some cases rings were fixed by addition of 1 ml 10%
paraformaldehyde and stored at 2-8.degree. C. for further
documentation. Pictures were analysed with the Scion Image image
processing software. After calibration with the help of a picture
taken from a stage micrometer, a line was drawn in a distance of
0.33 mm from one edge of a ring. A plot histogram along this line
was generated by the software, histograms were printed and peaks
(representing sprouts crossing the line) were counted. This number
was taken as sprouting index. 4 to 5 rings per condition were
evaluated. Statistical analysis was performed with WinSTAT for
Excel.
Results
[0353] It could be demonstrated that SDF-1 induces sprouting and
that this effect could be blocked with human SDF-1 binding
Spiegelmer 193-G2-012-5'-PEG No blockage of SDF-1 induced sprouting
was observed by the non-functional PEGylated Control Spiegelmer
(FIGS. 14 and 15).
EXAMPLE 8
Effect of SDF-1 Binding Spiegelmer NOX-A12 on Chemosensitization of
Leukemia Cells
[0354] There is considerable evidence that leukemia cells may be
protected from conventional chemotherapies by interaction between
their CXCR4 receptors with SDF-1 secreted by stromal cells within
particular tissue microenvironments such as the bone marrow (abbr.
BM) niche. Therefore, targeting the CXCR4-SDF-1 axis by using the
SDF-1 binding Spiegelmer NOX-A 12 is an attractive approach for
disrupting the protective effects of SDF-1-secreting stromal cells
and for sensitizing leukemia cells towards subsequent
chemotherapy.
[0355] In order to mimic the in vivo interaction of the BM
microenvironment with leukemia cells, an in vitro coculture system
with murine BM stromal MS-5 cells and the multiple myeloma (abbr.
MM) cell line RPMI 8226 was established. Aim of the experiment was
to show whether SDF-1 binding Spiegelmer NOX-A12 sensitizes MM
cells in coculture with stromal cells to effects of
chemotherapeutic agents. Stromal MS-5 cells secreting SDF 1 were
incubated with SDF-1 binding Spiegelmer NOX-A12 or the
non-functional revNOX-A12. The MM cell line RPMI-8226 was added to
the confluent stromal cell layer. The cells were then incubated
with the chemotherapeutic agent F ara A (Fludarabine) for 40 hours.
Cell Viability viability was measured.
Method
[0356] The murine stromal cell line MS-5 (ACC 441) was purchased
from the DSMZ, the Multiple Myeloma cell line RPMI8226 (CCL-155)
was purchased from the ATCC. The Multiple Myeloma cell line
RPMI8226 was maintained in RPMI medium 1640 GlutaMAX (Invitrogen)
supplemented with 10% FBS (Biochrom) and penicillin-streptomycin,
the MS-5 cells were cultured in MEM alpha GlutaMAX (Invitrogen)
with 10% FBS and penicillin-streptomycin. For chemosensitization
coculture experiments stromal MS-5 cells were seeded the day before
onto 24-well plates (the inner eight wells) at a concentration of
8.times.104/mL/well in MEM alpha GlutaMAX medium (+10% FBS) and
incubated at 37.degree. C. in 5% CO.sub.2. The confluent stromal
cell layer was washed and 0.5 mL RPMI medium 1640 (+1% FBS) was
added to the wells. SDF-1 binding Spiegelmer NOX-A12 or revNOX-A12
was subsequently added to the wells to a final concentration of 100
nM and incubated for four hours. 3.5.times.105 RPMI8226 cells in
RPMI medium 1640 (+1% FBS) were added to the stromal cell layer.
Four hours later, 1 .mu.M F-ara-A (Sigma Aldrich) was added to the
cells when indicated. After 40 hours of incubation the cells were
collected in 15 mL tubes, first the supernatant was harvested and
then the attached cells were trypsinized including MS-5 cells. The
collected cells were washed twice with PBS (+1% BSA) and
resuspended in 2 mL PBS (+1% BSA). 150 .mu.L of the cell suspension
was transferred in a u-shape 96-well plate and then incubated with
50 .mu.l of ViaCount Reagent (Millipore) for 15 minutes at room
temperature. Cell viability and cell number were determined by Flow
Cytometry using the Guava EasyCyte 6HT/2L (Millipore).
Results
[0357] Cell viability of RPMI-8226 cells cocultured with stromal
MS-5 cells was only slightly affected by SDF-1 binding Spiegelmer
NOX A12. 1 .mu.M F-ara-A showed no significant effect on the
viability of RPMI-8226 cells. However, when NOX-A12 and F-ara-A
were combined, a synergistic decrease of cell viability was
observed (FIG. 16). Thus, SDF-1 binding Spiegelmer NOX-A12 was
shown to sensitize the MM cell line RPMI-8226 towards treatment of
the chemotherapeutic agent F-ara-A when cocultured with the BM
stromal cell line MS 5. The viability of stromal MS-5 cells is
neither affected by F-ara-A nor by NOX-A12 (data not shown). These
results demonstrate a proof of principle in the potential of NOX
A12 in disrupting to disrupt the protective effects of SDF-1
secreted by BM stromal cells.
EXAMPLE 9
Effect of SDF-1 Binding Spiegelmer NOX-A12 on Proliferation of
Leukemia Cells
[0358] Aim of the experiment was to show whether SDF-1 binding
Spiegelmer NOX-A12 has an impact on proliferation of leukemia cells
in coculture with bone marrow (abbr. BM) stromal cells. Murine
stromal MS-5 cells secreting SDF-1 were incubated with SDF-1
binding Spiegelmer NOX-A12 or the non-functional Spiegelmer
revNOX-A12. The leukemic T-cell line Jurkat was added to the
confluent stromal cell layer and incubated for 40 hours at
37.degree. C. and 5% CO2. Cell numbers were quantified by Flow
Cytometry using the Guava EasyCyte and ViaCount Reagent.
Method
[0359] The murine stromal cell line MS-5 (ACC 441) were purchased
from the DSMZ and were cultured in MEM alpha GlutaMAX (Invitrogen)
with 10% FBS and penicillin-streptomycin. For proliferation
coculture experiments stromal MS-5 cells were seeded the day before
onto 24-well plates (the inner eight wells) at a concentration of
8.times.104/mL/well in MEM alpha GlutaMAX medium (+10% FBS) and
incubated at 37.degree. C. in 5% CO2. The confluent stromal cell
layer was washed and 0.5 mL RPMI medium 1640 (+1% FBS) was added to
the wells. SDF-1 binding Spiegelmer NOX-A12 or Spiegelmer revNOX
A12 was subsequently added to the wells to a final concentration of
100 nM and incubated for four hours. 2.times.105 Jurkat cells
(.about.logarithmic growth phase; washed once) in RPMI medium 1640
(+1% FBS) were added to the confluent stromal cell layer and
incubated for 48 hours at 37.degree. C. with 5% CO2. The cells were
then collected in 15 mL tubes, attached cells were trypsinized
including MS-5 cells. The collected cells were washed twice with
PBS (+1% BSA). 150 .mu.L of this cell suspension was transferred in
a u-shape 96-well plate and then incubated with 50 .mu.L ViaCount
Reagent (Millipore) for 15 minutes at room temperature. Cell
viability and cell number were determined by Flow Cytometry using
the Guava EasyCyte 6HT/2L.
Results
[0360] While 1 nM SDF-1 binding Spiegelmer NOX-A12 showed no effect
on the Jurkat cell number after 40 hours of cultivation, the cell
number was reduced up to 20% when stromal MS-5 cells were
preincubated with 10 or 100 nM SDF-1 binding Spiegelmer NOX-A12
(FIG. 17). Thus, SDF-1 secreted by stromal cells apparently
stimulates the proliferation of Jurkat cells. The SDF-1 dependent
induction of proliferation can be blocked by SDF-1 binding
Spiegelmer NOX-A12 leading to the detection of fewer a lower amount
of leukemic cells.
EXAMPLE 10
Effect of SDF-1 Binding Spiegelmer NOX-A12 on Adhesive Properties
of Leukemia Cells
[0361] The interaction of leukemic cells with extracellular matrix
(abbr. ECM) proteins plays a crucial role in leukemia pathogenesis.
Therefore we tested the effect of SDF-1 binding Spiegelmer NOX-A12
on adhesion of leukemia cells on the ECM protein fibronectin.
Stimulation of the Jurkat leukemia T-cell line with SDF-1 led to a
dose-dependent modulation of adhesion on to fibronectin.
Methods
[0362] The T cell leukemia Jurkat (ACC 282) were purchased from the
DSMZ were maintained in RPMI medium 1640 GlutaMAX (Invitrogen)
supplemented with 10% FBS (Biochrom) and penicillin-streptomycin.
For the adhesion experiments 96-well culture plates were incubated
with 10 .mu.g/mL human fibronectin (R&D systems) in PBS for 2
hours at 37.degree. C. The plates were washed twice with 100 .mu.L
PBS and subsequently blocked with PBS-BSA (0.1%) for two hours at
37.degree. C. The wells were then washed with RPMI medium. Jurkat
cells from logarithmic growth phase were washed with RPMI medium
(+0.1% BSA) and incubated with various concentrations of human
SDF-1 (R&D systems) and NOX-A12 for 15 minutes at 37.degree. C.
NOX-A12 and SDF-1 were preincubated for 30 minutes. 1.times.105
stimulated Jurkat cells were seeded to the Fibronectin-coated
96-well plates and incubated for 30 minutes. The plates were then
washed five times with RPMI medium. Attached cells were quantified
by using Cell Titer Glo Reagent (Promega). Therefor, 50 .mu.L RPMI
medium was added to each well, followed by 50 .mu.L of Cell Titer
Glo Reagent. The plates were mixed for two minutes, followed by
incubation at room temperature for 10 minutes. Cell number was
quantified by relative luminescence signal.
Results
[0363] Low to medium concentrations of SDF-1 (1-10 nM) decreased
the adhesion of Jurkat cells to fibronectin, while higher
concentrations (30-300 nM) increased the adhesive properties of
Jurkat cells (FIG. 18A). SDF-1 binding Spiegelmer NOX-A12 was shown
to reverse this effect, the control Spiegelmer revNOX-A12 not (FIG.
18B). Thus SDF-1 binding Spiegelmer NOX-A12 might have an impact on
the disruption of leukemic cell interactions with their protective
ECM environment. Furthermore, this example might explain SDF-1
binding Spiegelmer NOX-A12 dependent detachment and mobilization of
hematopoetic cells from the bone marrow niche.
EXAMPLE 11
Disruption of the Interaction of Multiple Myeloma Cells with the
Bone Marrow Environment In Vivo Thereby Enhancing the Sensitivity
of the Multiple Myeloma Cells to Therapy
[0364] The SDF-1/CXCR4 axis plays a major role in homing and
trafficking of multiple myeloma (abbr. MM) cells to the bone marrow
(abbr. BM). Therefore, de-adhesion of MM cells from the surrounding
BM milieu through SDF-1 inhibition enhances MM sensitivity to
therapeutic agents. Azab et al. published a protocol to test the
CXCR4 inhibitor AMD3100 potency to disrupt the interaction of MM
cells with the BM environment in vivo that affects localization MM
cells [, which in turn enhances the sensitivity of MM cells to
chemotherapy. They reported that the blockade of the SDF-1 receptor
CXCR4 by the CXCR4 specific antagonist led to a disruption of the
interaction of MM cells with the BM environment in vivo, to
enhanced sensitivity of the MM cells to therapy, and as a result to
enhanced tumor reduction induced by bortezomib (Azab et al. 2009).
Based on this protocol (Azab et al. 2009) the SDF-1 binding
Spiegelmer NOX-A12 is tested for its potency to disrupt the
interaction of MM cells with the BM environment in vivo thereby
enhancing the sensitivity of the MM cells to therapy.
[0365] For the MM animal model severe combined immunodeficient
(SCID) mice are used whereby Luc+/GFP+ MM.1S cells
(2.times.10.sup.6/mouse) are injected into the tail vein of SCID
mice. After 3 to 4 weeks, sufficient tumor progression is detected
by bioluminescence imaging (for protocol see Azab et al. 2009).
Mice are randomly divided into 4 groups: group 1, control mice
(received vehicle: 5% glucose); group 2, mice treated every other
day with 20 mg/kg NOX-A12 subcutaneous injection; group 3, mice
treated with intraperitoneal bortezomib injection of 0.5 mg/kg
twice a week; group 4, mice treated with intraperitoneal bortezomib
injection of 0.5 mg/kg twice a week and every other day with 20
mg/kg NOX-A12 subcutaneous injection.
[0366] The localization of the MM tumor cells in the bone marrow is
determined in vivo confocal microscopy using a fluorescence
labelled anti-SDF antibody (for protocol see Azab et al. 2009),
whereby the administration of NOX-A12 leads to MM cell mobilization
from bone marrow to the blood (as determined by ex vivo flow
cytometry; for protocol see Azab et al. 2009) and to a reduction of
tumor growth when administered together with bortezomib (by in vivo
bioluminescence detection; for protocol see Mitsiades et al., 2003;
Mitsiades et al. 2004). The stronger effects on tumor growth by
bortezomib plus NOX-A12 in comparison to a treatment with
bortezomib alone support the data of Example 8 showing positive
effects of NOX-12 on chemosensitization of MM cells.
REFERENCES
[0367] The complete bibliographic data of the documents recited
herein are, if not indicated to the contrary, as follows, whereby
the disclosure of said references is incorporated herein by
reference. [0368] Alsayed Y., Ngo H., et al. (2007) Mechanisms of
regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and homing
in multiple myeloma. Blood 109(7): 2708-17. [0369] Altschul S. F.,
Gish W., et al. (1990) Basic local alignment search tool. J Mol
Biol. 215(3):403-10. [0370] Altschul S. F., Madden T. L., et al.
(1997) Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acids Res. 25(17):3389-402.
[0371] Arya S. K., Ginsberg C. C., et al. (1999) In vitro phenotype
of SDF1 gene mutant that delays the onset of human immunodeficiency
virus disease in vivo. J Hum Virol 2(3): 133-8. [0372] Auerbach R.,
Lewis R., et al. (2003) Angiogenesis assays: a critical overview.
Clin Chem. 49(1):32-40. Review. [0373] Azab A. K., Runnels J. M.,
et al. (2009) CXCR4 inhibitor AMD3100 disrupts the interaction of
multiple myeloma cells with the bone marrow microenvironment and
enhances their sensitivity to therapy. Blood. 113(18):4341-51.
[0374] Batchelor T. T., Sorensen A. G., et al. (2007) AZD2171, a
pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor
vasculature and alleviates edema in glioblastoma patients. Cancer
Cell. 11(1):83-95. [0375] Balabanian K., Lagane B., et al. (2005)
The chemokine SDF-1/CXCL12 binds to and signals through the orphan
receptor RDC1 in T lymphocytes. J Biol Chem 280(42): 35760-35766
[0376] Balabanian, K., Lagane B., et al. (2005) WHIM syndromes with
different genetic anomalies are accounted for by impaired CXCR4
desensitization to CXCL12. Blood 105(6): 2449-57. [0377] Balkwill
F. (2004) Cancer and the chemokine network. Nat Rev Cancer 4(7):
540-50. [0378] Brooks H. L. Jr., Caballero S. Jr., et al. (2004)
Vitreous levels of vascular endothelial growth factor and
stromal-derived factor 1 in patients with diabetic retinopathy and
cystoid macular edema before and after intraocular injection of
triamcinolone. Arch Ophthalmol 122(12): 1801-7. [0379] Broxmeyer H.
E., Orschell C. M., (2005) Rapid mobilization of murine and human
hematopoietic stem and progenitor cells with AMD3100, a CXCR4
antagonist. J Exp Med. 201(8):1307-18. [0380] Buckley C. D., Amft
N., et al. (2000) Persistent induction of the chemokine receptor
CXCR4 by TGF-beta 1 on synovial T cells contributes to their
accumulation within the rheumatoid synovium. J Immunol 165(6):
3423-9. [0381] Burger J. A., Burkle A, et al. (2007) The CXCR4
chemokine receptor in acute and chronic leukaemia: a marrow homing
receptor and potential therapeutic target. Br J Haematol.
137(4):288-96. [0382] Burger J. A., Ghia P., et al. (2009) The
microenvironment in mature B-cell malignancies: a target for new
treatment strategies. Blood. 114(16):3367-75. Review [0383] Burger
J. A., Kipps T. J. et al. (2002) Chemokine receptors and stromal
cells in the homing and homeostasis of chronic lymphocytic leukemia
B cells. Leuk Lymphoma. 43(3):461-6 [0384] Burger J. A. and Peled
A. (2009) CXCR4 antagonists: targeting the microenvironment in
leukemia and other cancers. Leukemia 23(1): 43-52. [0385] Burger J.
A., Tsukada N., et al. (2000) Blood-derived nurse-like cells
protect chronic lymphocytic leukemia B cells from spontaneous
apoptosis through stromal cell-derived factor-1. Blood.
96(8):2655-63. [0386] Burns J. M., Summers B. C., et al. (2006) A
novel chemokine receptor for SDF-1 and I-TAC involved in cell
survival, cell adhesion, and tumor development. J Exp Med 203(9):
2201-2213 [0387] Butler J. M., Guthrie S. M., et al. (2005) SDF-1
is both necessary and sufficient to promote proliferative
retinopathy. J Clin Invest 115(1): 86-93 [0388] Cabioglu, N., Sahin
A., et al. (2005) Chemokine receptor CXCR4 expression in breast
cancer as a potential predictive marker of isolated tumor cells in
bone marrow. Clin Exp Metastasis 22(1): 39-46. [0389] Ceradini D.
J., Kulkarni A. R., et al. (2004) Progenitor cell trafficking is
regulated by hypoxic gradients through HIF-1 induction of SDF-1.
Nat Med. 10(8):858-64. [0390] Corcione A., Ottonello L., et al.
(2000) Stromal cell-derived factor-1 as a chemoattractant for
follicular center lymphoma B cells. J Natl Cancer Inst 92(8):
628-35. [0391] Damha M. J., Ogilvie K. K., et al. (1993)
Oligoribonucleotide synthesis. The silyl-phosphoramidite method.
Methods Mol Biol. 20:81-114. [0392] Damiano J. S., Cress A. E., et
al. (1999) Cell adhesion mediated drug resistance (CAM-DR): role of
integrins and resistance to apoptosis in human myeloma cell lines.
Blood. 93(5):1658-67 [0393] Devine S. M., Flomenberg N., et al.
(2004) Rapid mobilization of CD34+ cells following administration
of the CXCR4 antagonist AMD3100 to patients with multiple myeloma
and non-Hodgkin's lymphoma. J Clin Oncol. 22(6):1095-102. [0394]
Dillmann F., Veldwijk M. R., et al. (2009) Plerixafor inhibits
chemotaxis toward SDF-1 and CXCR4-mediated stroma contact in a
dose-dependent manner resulting in increased susceptibility of
BCR-ABL+ cell to Imatinib and Nilotinib. Leuk Lymphoma. 50(10):16
[0395] Ehtesham M., Stevenson C. B., et al. (2008) Preferential
expression of chemokine receptor CXCR4 by highly malignant human
gliomas and its association with poor patient survival.
Neurosurgery. 63(4):E820 [0396] Engl T., Relja B., et al. (2006)
CXCR4 chemokine receptor mediates prostate tumor cell adhesion
through alpha5 and beta3 integrins. Neoplasia. 8(4):290-301. [0397]
Fedyk E. R., Jones D., et al. (2001) Expression of stromal-derived
factor-1 is decreased by IL-1 and TNF and in dermal wound healing.
J Immunol. 166(9):5749-54. [0398] Geminder H., Sagi-Assif 0., et
al. (2001) A possible role for CXCR4 and its ligand, the CXC
chemokine stromal cell-derived factor-1, in the development of bone
marrow metastases in neuroblastoma. J Immunol 167(8): 4747-57.
[0399] Grassi F., Cristino S., et al. (2004) CXCL12 chemokine
up-regulates bone resorption and MMP-9 release by human
osteoclasts: CXCL12 levels are increased in synovial and bone
tissue of rheumatoid arthritis patients. J Cell Physiol 199(2):
244-51. [0400] Grunwald M., Avraham I., et al. (2006) VEGF-induced
adult neovascularization: recruitment, retention, and role of
accessory cells. Cell 124(1): 175-89. [0401] Gulino, A. V., Moratto
D., et al. (2004) Altered leukocyte response to CXCL12 in patients
with warts hypogammaglobulinemia, infections, myelokathexis (WHIM)
syndrome. Blood 104(2): 444-52. [0402] Holm N. T., Abreo F., et al.
(2009) Elevated chemokine receptor CXCR4 expression in primary
tumors following neoadjuvant chemotherapy predicts poor outcomes
for patients with locally advanced breast cancer (LABC). Breast
Cancer Res Treat. 113(2):293-9. Epub 2008 Feb. 13 [0403] Hwang J.
H., Chung H. K., et al. (2003) CXC chemokine receptor 4 expression
and function in human anaplastic thyroid cancer cells. J Clin
Endocrinol Metab 88(1): 408-16. [0404] Jin L., Tabe Y., et al.
(2008) CXCR4 up-regulation by imatinib induces chronic myelogenous
leukemia (CML) cell migration to bone marrow stroma and promotes
survival of quiescent CML cells. Mol Cancer Ther 7(1): 48-58 [0405]
Kanbe K., Takagishi K., et al. (2002) Stimulation of matrix
metalloprotease 3 release from human chondrocytes by the
interaction of stromal cell-derived factor 1 and CXC chemokine
receptor 4. Arthritis Rheum 46(1): 130-7. [0406] Kawai T., Choi U.,
et al. (2005) Enhanced function with decreased internalization of
carboxy-terminus truncated CXCR4 responsible for WHIM syndrome. Exp
Hematol 33(4): 460-8. [0407] Kioi M., Vogel H., et al. (2010)
Inhibition of vasculogenesis, but not angiogenesis, prevents the
recurrence of glioblastoma after irradiation in mice. J Clin Invest
120(3): 694-705. [0408] Klussmann S. (2006). The Aptamer
Handbook--Functional Oligonucleotides and their Applications.
Edited by S. Klussmann. WILEY-VCH, Weinheim, Germany, ISBN
3-527-31059-2 [0409] Koshiba T., Hosotani R., et al. (2000)
Expression of stromal cell-derived factor 1 and CXCR4 ligand
receptor system in pancreatic cancer: a possible role for tumor
progression. Clin Cancer Res 6(9): 3530-5. [0410] Kozin S. V.,
Kamoun W. S., et al. (2010) Recruitment of myeloid but not
endothelial precursor cells facilitates tumor regrowth after local
irradiation. Cancer Res 70(14): 5679-85. [0411] Krumbholz M., Theil
D., et al. (2006) Chemokines in multiple sclerosis: CXCL12 and
CXCL13 up-regulation is differentially linked to CNS immune cell
recruitment. Brain 129: 200-211. [0412] Kryczek I., Lange A., et
al. (2005) CXCL12 and vascular endothelial growth factor
synergistically induce neoangiogenesis in human ovarian cancers.
Cancer Res 65(2): 465-72. [0413] Kurtova A. V., Balakrishnan K., et
al. (2009) Diverse marrow stromal cells protect CLL cells from
spontaneous and drug-induced apoptosis: development of a reliable
and reproducible system to assess stromal cell adhesion-mediated
drug resistance. Blood. 114(20):4441-50. [0414] Kusser W. (2000)
Chemically modified nucleic acid aptamers for in vitro selections:
evolving evolution. J Biotechnol 74(1): 27-38. [0415] Lagneaux L.,
Delforge A., et al. (1998) Chronic lymphocytic leukemic B cells but
not normal B cells are rescued from apoptosis by contact with
normal bone marrow stromal cells. Blood. 91(7):2387-96. [0416] Li
J. K., Yu L., et al. (2008) Inhibition of CXCR4 activity with
AMD3100 decreases invasion of human colorectal cancer cells in
vitro. World J Gastroenterol 14(15): 2308-2313 [0417] Maksym R. B.,
Tarnowski M., et al. (2009) The role of stromal-derived
factor-1--CXCR7 axis in development and cancer. Eur J Pharmacol.
625(1-3):31-40. Review. [0418] Marechal V., Arenzana-Seisdedos F.,
et al. (1999) Opposite effects of SDF-1 on human immunodeficiency
virus type 1 replication. J Virol 73(5): 3608-15. [0419] McGinnis
S., Madden T. L. et al. (2004) BLAST: at the core of a powerful and
diverse set of sequence analysis tools. Nucleic Acids Res. 32(Web
Server issue):W20-5. [0420] Meads M. B., Hazlehurst L. A., et al.
(2008) The bone marrow microenvironment as a tumor sanctuary and
contributor to drug resistance. Clin Cancer Res. 14(9):2519-26.
Review. [0421] Meleth A. D., Agron E., et al. (2005) Serum
inflammatory markers in diabetic retinopathy. Invest Ophthalmol Vis
Sci 46(11): 4295-301. [0422] Miao, Z., Luker K. E., et al. (2007)
CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is
expressed on tumor-associated vasculature. Proc Natl Acad Sci USA
104(40): 15735-40. [0423] Mitsiades C. S., Mitsiades N. S., et al.
(2003) Fluorescence imaging of multiple myeloma cells in a
clinically relevant SCID/NOD in vivo model: biologic and clinical
implications. Cancer Res. 63(20):6689-96. [0424] Mitsiades C. S.,
Mitsiades N., et al. (2004) Focus on multiple myeloma. Cancer Cell.
6(5):439-44. Review. [0425] Mizell J., Smith M., et al. (2009)
Overexpression of CXCR4 in primary tumor of patients with HER-2
negative breast cancer was predictive of a poor disease-free
survival: a validation study. Ann Surg Oncol. 16(10):2711-6. [0426]
Muller, A., Homey B., et al. (2001) Involvement of chemokine
receptors in breast cancer metastasis. Nature 410(6824): 50-6.
[0427] Murdoch, C. (2000) CXCR4: chemokine receptor extraordinaire.
Immunol Rev 177: 175-84. [0428] Needleman and Wunsch (1970) A
general method applicable to the search for similarities in the
amino acid sequence of two proteins. J Mol Biol. 48(3):443-53.
[0429] Nervi B., Ramirez P., et al. (2009) Chemosensitization of
acute myeloid leukemia (AML) following mobilization by the CXCR4
antagonist AMD3100. Blood. 113(24):6206-14. Epub 2008 Dec. 2 [0430]
Pearson and Lipman (1988) Improved tools for biological sequence
comparison. Proc. Nat'l. Acad. Sci. USA 85: 2444 [0431] Redjal N.,
Chan J. A., et al. (2006) CXCR4 inhibition synergizes with
cytotoxic chemotherapy in gliomas. Clin Cancer Res. 12(22):6765-71.
[0432] Salcedo R., Wasserman K., et al. (1999) Vascular endothelial
growth factor and basic fibroblast growth factor induce expression
of CXCR4 on human endothelial cells: In vivo neovascularization
induced by stromal-derived factor-1 alpha. Am J Pathol 154(4):
1125-1135 [0433] Salcedo, R. and Oppenheim J. J. (2003) Role of
chemokines in angiogenesis: CXCL12/SDF-1 and CXCR4 interaction, a
key regulator of endothelial cell responses. Microcirculation
10(3-4): 359-70. [0434] Salvucci O., Yao L., et al. (2002)
Regulation of endothelial cell branching morphogenesis by
endogenous chemokine stromal-derived factor-1. Blood 99(8):
2703-11. [0435] Saur D., Seidler B., et al. (2005) CXCR4 expression
increases liver and lung metastasis in a mouse model of pancreatic
cancer. Gastroenterology. 129(4):1237-50. [0436] Schimanski C. C.,
Galle P. R., et al. (2008) Chemokine receptor CXCR4-prognostic
factor for gastrointestinal tumors. World J Gastroenterol.
14(30):4721-4. [0437] Scotton C. J., Wilson J. L., et al. (2002)
Multiple actions of the chemokine CXCL12 on epithelial tumor cells
in human ovarian cancer. Cancer Res. 62(20):5930-8 [0438] Sengupta
N., Caballero S., et al. (2005) Preventing stem cell incorporation
into choroidal neovascularization by targeting homing and
attachment factors. Invest Ophthalmol V is Sci. 46(1):343-8. [0439]
Shaked Y., Henke E., et al. (2008) Rapid chemotherapy-induced acute
endothelial progenitor cell mobilization: implications for
antiangiogenic drugs as chemosensitizing agents. Cancer Cell.
14(3):263-73 [0440] Shirozu M., Nakano T., et al. (1995) Structure
and chromosomal localization of the human stromal cell-derived
factor 1 (SDF1) gene. Genomics 28(3): 495-500. [0441] Smith and
Waterman (1981), Adv. Appl. Math. 2: 482 [0442] Soriano A.,
Martinez C., et al. (2002) Plasma stromal cell-derived factor
(SDF)-1 levels, SDF1-3'A genotype, and expression of CXCR4 on T
lymphocytes: their impact on resistance to human immunodeficiency
virus type 1 infection and its progression. J Infect Dis 186(7):
922-31. [0443] Su L., Zhang J, et al. (2005) Differential
expression of CXCR4 is associated with the metastatic potential of
human non-small cell lung cancer cells. Clin Cancer Res.
11(23):8273-80. [0444] Tseng D., Lartey F. et al. (2010) J. K., Yu
L., et al. (2008) (MS108) Inhibition of SDF-1/CXCR7 radiosensitizes
ENU induced glioblastomas in the rat. 56th Annual Meeting Radiation
Research Society, Sep. 25-29, 2010, Grand Wailea Resort Hotel and
Spa, Maui, Hi., USA [0445] Venkatesan N., Kim S. J., et al. (2003)
Novel phosphoramidite building blocks in synthesis and applications
toward modified oligonucleotides. Curr Med Chem 10(19): 1973-91.
[0446] Wang J., Shiozawa Y., et al. (2008) The role of CXCR7/RDC1
as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J Biol
Chem 283(7): 4283-4294. Epub 2007 Dec. 5. [0447] Wang J., Guan E.,
et al. (2001) Role of tyrosine phosphorylation in
ligand-independent sequestration of CXCR4 in human primary
monocytes-macrophages. J Biol Chem 276(52): 49236-43.
[0448] Wang N., Wu Q. L., et al. (2005) Expression of chemokine
receptor CXCR4 in nasopharyngeal carcinoma: pattern of expression
and correlation with clinical outcome. J Transl Med 3: 26. [0449]
Xu L., Duda D. G., et al. (2009) Direct evidence that bevacizumab,
an anti-VEGF antibody, up-regulates SDF1alpha, CXCR4, CXCL6, and
neuropilin 1 in tumors from patients with rectal cancer. Cancer
Res. 69(20):7905-10. [0450] Yamaguchi J., Kusano K. F., et al.
(2003) Stromal cell-derived factor-1 effects on ex vivo expanded
endothelial progenitor cell recruitment for ischemic
neovascularization. Circulation 107(9): 1322-8. [0451] Yang J.,
Zhang B. et al. (2008) Breast cancer metastasis suppressor 1
inhibits SDF-1 alpha-induced migration of non-small cell lung
cancer by decreasing CXCR4 expression. [0452] Cancer Lett
269(1):46-56 [0453] Zagzag D., Esencay M., et al. (2008) Hypoxia-
and vascular endothelial growth factor-induced stromal cell-derived
factor-1 alpha/CXCR4 expression in glioblastomas: one plausible
explanation of Scherer's structures. Am J Pathol 173(2): 545-560
[0454] Zeelenberg I. S., Ruuls-Van Stalle L., et al. (2003) The
chemokine receptor CXCR4 is required for outgrowth of colon
carcinoma micrometastases. Cancer Res. 63(13):3833-9. [0455] Zheng
K., Li H. Y., et al. (2010) Chemokine receptor CXCR7 regulates the
invasion, angiogenesis and tumor growth of human hepatocellular
carcinoma cells. J Exp Clin Cancer Res. 29:31. [0456] Zhou Y.,
Larsen P. H., et al. (2002) CXCR4 is a major chemokine receptor on
glioma cells and mediates their survival. J Biol Chem 277(51):
49481-7. [0457] Zhu A. X., Sahani D. V., et al. (2009) Efficacy,
safety, and potential biomarkers of sunitinib monotherapy in
advanced hepatocellular carcinoma: a phase II study. J Clin Oncol
27(18): 3027-35.
[0458] The features of the present invention disclosed in the
specification, the claims and/or the drawings may both separately
and in any combination thereof be material for realizing the
invention in various forms thereof.
Sequence CWU 1
1
225168PRTHomo sapiensmisc_featureL-peptide 1Lys Pro Val Ser Leu Ser
Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg
Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 30 Asn Cys
Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45
Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50
55 60 Ala Leu Asn Lys 65 272PRTHomo sapiensmisc_featureL-peptide
2Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1
5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr
Pro 20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn
Asn Arg Gln 35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln
Glu Tyr Leu Glu Lys 50 55 60 Ala Leu Asn Lys Arg Phe Lys Met 65 70
368PRTMus musculusmisc_featureL-peptide 3Lys Pro Val Ser Leu Ser
Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15 His Ile Ala Arg
Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 30 Asn Cys
Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45
Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50
55 60 Ala Leu Asn Lys 65 471PRTHomo sapiensmisc_featureD-peptide
4Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1
5 10 15 His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr
Pro 20 25 30 Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn
Asn Arg Gln 35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln
Glu Tyr Leu Glu Lys 50 55 60 Ala Leu Asn Lys Arg Phe Lys 65 70
547DNAartificialsynthetic 5agcguggugu gaucuagaug uaguggcuga
uccuagucag guacgcu 47647DNAartificialsynthetic 6agcguggugu
gaucuagaug uauuggcuga uccuagucag guacgcu
47747DNAartificialsynthetic 7agcguggugu gaucuagaug uaauggcuga
uccuagucag gugcgcu 47845DNAartificialsynthetic 8gcgaggugug
aucuagaugu aguggcugau ccuagucagg ugcgc 45945DNAartificialsynthetic
9gcguggugug aucuagaugu aguggcugau ccuagucagg ugcgc
451045DNAartificialsynthetic 10gcauggugug aucuagaugu aguggcugau
ccuagucagg ugccc 451145DNAartificialsynthetic 11gcguggugug
aucuagaugu aauggcugau ccuagucagg gacgc 451245DNAartificialsynthetic
12gcguggugug aucuagaugu agaggcugau ccuagucagg uacgc
451345DNAartificialsynthetic 13gcguggugug aucuagaugu aaaggcugau
ccuagucagg uacgc 451445DNAartificialsynthetic 14gcguggugug
aucuagaugu aguggcuguu ccuagucagg uaugc 451545DNAartificialsynthetic
15gcguggugug aucuagaugu aguggcugau ccuaguuagg uacgc
451645DNAartificialsynthetic 16gcguggugug aucuagaugu aguggcugau
ccuagucagg uacgc 451743DNAartificialsynthetic 17cgugguguga
ucuagaugua guggcugauc cuagucaggu acg 431841DNAartificialsynthetic
18guggugugau cuagauguag uggcugaucc uagucaggua c
411939DNAartificialsynthetic 19uggugugauc uagauguagu ggcugauccu
agucaggua 392037DNAartificialsynthetic 20ggugugaucu agauguagug
gcugauccua gucaggu 372135DNAartificialsynthetic 21gugugaucua
gauguagugg cugauccuag ucagg 352245DNAartificialsynthetic
22gcguggugug aucuagaugu auuggcugau ccuagucagg uacgc
452345DNAartificialsynthetic 23gcgcggugug aucuagaugu auuggcugau
ccuagucagg cgcgc 452443DNAartificialsynthetic 24gcgcguguga
ucuagaugua uuggcugauc cuagucaggg cgc 432543DNAartificialsynthetic
25gggcguguga ucuagaugua uuggcugauc cuagucaggg ccc
432643DNAartificialsynthetic 26ggccguguga ucuagaugua uuggcugauc
cuagucaggg gcc 432743DNAartificialsynthetic 27gcccguguga ucuagaugua
uuggcugauc cuagucaggg ggc 432845DNAartificialsynthetic 28gcguggugug
aucuagaugu auuggcugau ccuagucagg uacgc 452947DNAartificialsynthetic
29agcguggugu gaucuagaug uaguggcuga uccuagucag guacgcu
473047DNAartificialsynthetic 30agcguggugu gaucuagaug uauuggcuga
uccuagucag guacgcu 473147DNAartificialsynthetic 31agcguggugu
gaucuagaug uaauggcuga uccuagucag gugcgcu
473245DNAartificialsynthetic 32gcgaggugug aucuagaugu aguggcugau
ccuagucagg ugcgc 453345DNAartificialsynthetic 33gcguggugug
aucuagaugu aguggcugau ccuagucagg ugcgc 453445DNAartificialsynthetic
34gcauggugug aucuagaugu aguggcugau ccuagucagg ugccc
453545DNAartificialsynthetic 35gcguggugug aucuagaugu aauggcugau
ccuagucagg gacgc 453645DNAartificialsynthetic 36gcguggugug
aucuagaugu agaggcugau ccuagucagg uacgc 453745DNAartificialsynthetic
37gcguggugug aucuagaugu aaaggcugau ccuagucagg uacgc
453845DNAartificialsynthetic 38gcguggugug aucuagaugu aguggcuguu
ccuagucagg uaugc 453945DNAartificialsynthetic 39gcguggugug
aucuagaugu aguggcugau ccuaguuagg uacgc 454045DNAartificialsynthetic
40gcguggugug aucuagaugu aguggcugau ccuagucagg uacgc
454143DNAartificialsynthetic 41cgugguguga ucuagaugua guggcugauc
cuagucaggu acg 434241DNAartificialsynthetic 42guggugugau cuagauguag
uggcugaucc uagucaggua c 414339DNAartificialsynthetic 43uggugugauc
uagauguagu ggcugauccu agucaggua 394437DNAartificialsynthetic
44ggugugaucu agauguagug gcugauccua gucaggu
374535DNAartificialsynthetic 45gugugaucua gauguagugg cugauccuag
ucagg 354645DNAartificialsynthetic 46gcguggugug aucuagaugu
auuggcugau ccuagucagg uacgc 454745DNAartificialsynthetic
47gcgcggugug aucuagaugu auuggcugau ccuagucagg cgcgc
454843DNAartificialsynthetic 48gcgcguguga ucuagaugua uuggcugauc
cuagucaggg cgc 434943DNAartificialsynthetic 49gggcguguga ucuagaugua
uuggcugauc cuagucaggg ccc 435043DNAartificialsynthetic 50ggccguguga
ucuagaugua uuggcugauc cuagucaggg gcc 435143DNAartificialsynthetic
51gcccguguga ucuagaugua uuggcugauc cuagucaggg ggc
435235DNAartificialsynthetic 52gugugaucua gauguadwgg cugwuccuag
uyagg 355335DNAartificialsynthetic 53gugugaucua gauguadugg
cugauccuag ucagg 355427DNAartificialsynthetic 54aaaguaacac
guaaaaugaa agguaac 275526DNAartificialsynthetic 55aaagcaacau
gucaaugaaa gguagc 265618DNAartificialsynthetic 56gguuagggcu
aaagucgg 185719DNAartificialsynthetic 57gguuagggcu agaagucgg
195819DNAartificialsynthetic 58gguuagggcu cgaagucgg
195919DNAartificialsynthetic 59gguuagggcu ugaagucgg
196038DNAartificialsynthetic 60gcugugaaag caacauguca augaaaggua
gccgcagc 386138DNAartificialsynthetic 61gcugugaaag uaacauguca
augaaaggua accacagc 386238DNAartificialsynthetic 62gcugugaaag
uaacacguca augaaaggua accgcagc 386338DNAartificialsynthetic
63gcugugaaag uaacacguca augaaaggua accacagc
386438DNAartificialsynthetic 64gcuguaaaag uaacauguca augaaaggua
acuacagc 386538DNAartificialsynthetic 65gcuguaaaag uaacaaguca
augaaaggua acuacagc 386638DNAartificialsynthetic 66gcugugaaag
uaacaaguca augaaaggua accacagc 386738DNAartificialsynthetic
67gcagugaaag uaacauguca augaaaggua accacagc
386838DNAartificialsynthetic 68gcugugaaag uaacauguca augaaaggua
accacugc 386938DNAartificialsynthetic 69gcuaugaaag uaacauguca
augaaaggua accauagc 387038DNAartificialsynthetic 70gcugcgaaag
cgacauguca augaaaggua gccgcagc 387138DNAartificialsynthetic
71gcugugaaag caacauguca augaaaggua gccacagc
387238DNAartificialsynthetic 72gcugugaaag uaacauguca augaaaggua
gccgcagc 387339DNAartificialsynthetic 73agcgugaaag uaacacguaa
aaugaaaggu aaccacgcu 397427DNAartificialsynthetic 74aaagyracah
gumaaaugaa agguarc 277526DNAartificialsynthetic 75aaagyracah
gumaaugaaa gguarc 267627DNAartificialsynthetic 76aaagyracah
gumaaaugaa agguarc 277726DNAartificialsynthetic 77aaagyaacah
gucaaugaaa gguarc 267836DNAartificialsynthetic 78cugugaaagc
aacaugucaa ugaaagguag ccgcag 367934DNAartificialsynthetic
79ugugaaagca acaugucaau gaaagguagc cgca
348032DNAartificialsynthetic 80gugaaagcaa caugucaaug aaagguagcc gc
328130DNAartificialsynthetic 81ugaaagcaac augucaauga aagguagccg
308228DNAartificialsynthetic 82gaaagcaaca ugucaaugaa agguagcc
288326DNAartificialsynthetic 83aaagcaacau gucaaugaaa gguagc
268436DNAartificialsynthetic 84gcgugaaagc aacaugucaa ugaaagguag
ccgcgc 368536DNAartificialsynthetic 85gcgcgaaagc aacaugucaa
ugaaagguag ccgcgc 368634DNAartificialsynthetic 86gcggaaagca
acaugucaau gaaagguagc ccgc 348734DNAartificialsynthetic
87cgugaaagca acaugucaau gaaagguagc cgcg
348834DNAartificialsynthetic 88gcgcaaagca acaugucaau gaaagguagc
gugc 348934DNAartificialsynthetic 89gugcaaagca acaugucaau
gaaagguagc gcgc 349034DNAartificialsynthetic 90cgcgaaagca
acaugucaau gaaagguagc cgug 349134DNAartificialsynthetic
91gggcaaagca acaugucaau gaaagguagc gccc
349234DNAartificialsynthetic 92ggccaaagca acaugucaau gaaagguagc
ggcc 349334DNAartificialsynthetic 93gcccaaagca acaugucaau
gaaagguagc gggc 349434DNAartificialsynthetic 94ccccaaagca
acaugucaau gaaagguagc gggg 349539DNAartificialsynthetic
95gugcugcggg gguuagggcu agaagucggc cugcagcac
399639DNAartificialsynthetic 96agcguggcga gguuagggcu agaagucggu
cgacacgcu 399739DNAartificialsynthetic 97guguugcgga gguuagggcu
agaagucggu cagcagcac 399848DNAartificialsynthetic 98cgugcggccu
aagagguuag ggcuuaaagu cggucuuugg ccaacacg
489948DNAartificialsynthetic 99cgugcgcuug agauaggggu uagggcuuaa
agucggcuga uucucacg 4810048DNAartificialsynthetic 100cgugauuggu
gagggguuag ggcuugaagu cggccuuguc cagucacg
4810139DNAartificialsynthetic 101agcgugaagg gguuagggcu cgaagucggc
ugacacgcu 3910239DNAartificialsynthetic 102gugcugcggg gguuagggcu
cgaagucggc ccgcagcac 3910339DNAartificialsynthetic 103guguucccgg
gguuagggcu ugaagucggc cggcagcac 3910439DNAartificialsynthetic
104guguugcagg gguuagggcu ugaagucggc cugcagcac
3910539DNAartificialsynthetic 105gugcugcggg gguuagggcu caaagucggc
cugcagcac 3910638DNAartificialsynthetic 106gugcugccgg gguuagggcu
aaagucggcc gacagcac 3810739DNAartificialsynthetic 107gugcuguggg
ggucagggcu agaagucggc cugcagcac 3910819DNAartificialsynthetic
108gguyagggcu hraagucgg 1910919DNAartificialsynthetic 109gguyagggcu
hraagucgg 1911018DNAartificialsynthetic 110gguyagggcu hragucgg
1811119DNAartificialsynthetic 111gguuagggcu hgaagucgg
1911238DNAartificialsynthetic 112ugagauaggg guuagggcuu aaagucggcu
gauucuca 3811336DNAartificialsynthetic 113gagauagggg uuagggcuua
aagucggcug auucuc 3611429DNAartificialsynthetic 114gggguuaggg
cuuaaagucg gcugauucu 2911537DNAartificialsynthetic 115gcguggcgag
guuagggcua gaagucgguc gacacgc 3711635DNAartificialsynthetic
116cguggcgagg uuagggcuag aagucggucg acacg
3511733DNAartificialsynthetic 117cgggcgaggu uagggcuaga agucggucga
ccg 3311833DNAartificialsynthetic 118cgggcgaggu uagggcuaga
agucggucgc ccg 3311931DNAartificialsynthetic 119cggcgagguu
agggcuagaa gucggucgcc g 3112029DNAartificialsynthetic 120cgggagguua
gggcuagaag ucggucccg 2912127DNAartificialsynthetic 121gggagguuag
ggcuagaagu cgguccc 2712227DNAartificialsynthetic 122ccgcgguuag
ggcuagaagu cgggcgg 2712327DNAartificialsynthetic 123cccggguuag
ggcuagaagu cggcggg 2712427DNAartificialsynthetic 124ggcggguuag
ggcuagaagu cggcgcc 2712529DNAartificialsynthetic 125cccgcgguua
gggcuagaag ucgggcggg 2912629DNAartificialsynthetic 126gccgcgguua
gggcuagaag ucgggcggc 2912729DNAartificialsynthetic 127ccccggguua
gggcuagaag ucggcgggg 2912829DNAartificialsynthetic 128cggcggguua
gggcuagaag ucggcgccg 2912929DNAartificialsynthetic 129gggcggguua
gggcuagaag ucggcgccc 2913037DNAartificialsynthetic 130ugcugcgggg
guuagggcua gaagucggcc ugcagca 3713135DNAartificialsynthetic
131gcugcggggg uuagggcuag aagucggccu gcagc
3513233DNAartificialsynthetic 132cugcgggggu uagggcuaga agucggccug
cag 3313331DNAartificialsynthetic 133ugcggggguu agggcuagaa
gucggccugc a 3113429DNAartificialsynthetic 134gcggggguua gggcuagaag
ucggccugc 2913529DNAartificialsynthetic 135gccgggguua gggcuagaag
ucggccggc 2913631DNAartificialsynthetic 136ggccgggguu agggcuagaa
gucggccggc c 3113731DNAartificialsynthetic 137cgccgggguu agggcuagaa
gucggccggc g 3113810DNAartificialsynthetic 138rksbusnvgr
1013910DNAartificialsynthetic 139yynrcassmy
1014010DNAartificialsynthetic 140rksbugsvgr
1014110DNAartificialsynthetic 141ycnrcassmy
1014248DNAartificialsynthetic 142cgugguccgu ugugucaggu cuauucgccc
cggugcaggg cauccgcg 4814349DNAartificialsynthetic 143gcagugugac
gcggacguga uaggacagag cugaucccgc ucaggugag
4914449DNAartificialsynthetic 144caacagcagu gugacgcgga cgugauagga
cagagcugau cccgcucag 4914536DNAartificialsynthetic 145gcgugaaagc
aacaugucaa ugaaagguag ccgcgc 3614638DNAartificialsynthetic
146gcugugaaag caacauguca augaaaggua gccgcagc
3814738DNAartificialsynthetic 147gcugugaaag uaacauguca augaaaggua
accacagc 3814838DNAartificialsynthetic 148gcugugaaag uaacacguca
augaaaggua accgcagc 3814938DNAartificialsynthetic 149gcugugaaag
uaacacguca augaaaggua accacagc 3815038DNAartificialsynthetic
150gcuguaaaag uaacauguca augaaaggua acuacagc
3815138DNAartificialsynthetic 151gcuguaaaag uaacaaguca augaaaggua
acuacagc 3815238DNAartificialsynthetic 152gcugugaaag uaacaaguca
augaaaggua accacagc 3815338DNAartificialsynthetic 153gcagugaaag
uaacauguca augaaaggua accacagc 3815438DNAartificialsynthetic
154gcugugaaag uaacauguca augaaaggua accacugc
3815538DNAartificialsynthetic 155gcuaugaaag uaacauguca augaaaggua
accauagc 3815638DNAartificialsynthetic 156gcugcgaaag cgacauguca
augaaaggua gccgcagc 3815738DNAartificialsynthetic 157gcugugaaag
caacauguca augaaaggua gccacagc 3815838DNAartificialsynthetic
158gcugugaaag uaacauguca augaaaggua gccgcagc
3815939DNAartificialsynthetic 159agcgugaaag uaacacguaa aaugaaaggu
aaccacgcu 3916036DNAartificialsynthetic 160cugugaaagc aacaugucaa
ugaaagguag ccgcag 3616134DNAartificialsynthetic 161ugugaaagca
acaugucaau gaaagguagc cgca 3416232DNAartificialsynthetic
162gugaaagcaa caugucaaug aaagguagcc gc
3216330DNAartificialsynthetic 163ugaaagcaac augucaauga aagguagccg
3016428DNAartificialsynthetic 164gaaagcaaca ugucaaugaa agguagcc
2816526DNAartificialsynthetic 165aaagcaacau gucaaugaaa gguagc
2616636DNAartificialsynthetic 166gcgugaaagc aacaugucaa ugaaagguag
ccgcgc 3616736DNAartificialsynthetic 167gcgcgaaagc aacaugucaa
ugaaagguag ccgcgc 3616834DNAartificialsynthetic 168gcggaaagca
acaugucaau gaaagguagc ccgc 3416934DNAartificialsynthetic
169cgugaaagca acaugucaau gaaagguagc cgcg
3417034DNAartificialsynthetic 170gcgcaaagca acaugucaau gaaagguagc
gugc 3417134DNAartificialsynthetic 171gugcaaagca acaugucaau
gaaagguagc gcgc 3417234DNAartificialsynthetic 172cgcgaaagca
acaugucaau gaaagguagc cgug 3417334DNAartificialsynthetic
173gggcaaagca acaugucaau gaaagguagc gccc
3417434DNAartificialsynthetic 174ggccaaagca acaugucaau gaaagguagc
ggcc 3417534DNAartificialsynthetic 175gcccaaagca acaugucaau
gaaagguagc gggc 3417634DNAartificialsynthetic 176ccccaaagca
acaugucaau gaaagguagc gggg 3417739DNAartificialsynthetic
177gugcugcggg gguuagggcu agaagucggc cugcagcac
3917839DNAartificialsynthetic 178agcguggcga gguuagggcu agaagucggu
cgacacgcu 3917939DNAartificialsynthetic 179guguugcgga gguuagggcu
agaagucggu cagcagcac 3918048DNAartificialsynthetic 180cgugcggccu
aagagguuag ggcuuaaagu cggucuuugg ccaacacg
4818148DNAartificialsynthetic 181cgugcgcuug agauaggggu uagggcuuaa
agucggcuga uucucacg 4818248DNAartificialsynthetic 182cgugauuggu
gagggguuag ggcuugaagu cggccuuguc cagucacg
4818339DNAartificialsynthetic 183agcgugaagg gguuagggcu cgaagucggc
ugacacgcu 3918439DNAartificialsynthetic 184gugcugcggg gguuagggcu
cgaagucggc ccgcagcac 3918539DNAartificialsynthetic 185guguucccgg
gguuagggcu ugaagucggc cggcagcac 3918639DNAartificialsynthetic
186guguugcagg gguuagggcu ugaagucggc cugcagcac
3918739DNAartificialsynthetic 187gugcugcggg gguuagggcu caaagucggc
cugcagcac 3918838DNAartificialsynthetic 188gugcugccgg gguuagggcu
aaagucggcc gacagcac 3818939DNAartificialsynthetic 189gugcuguggg
ggucagggcu agaagucggc cugcagcac 3919038DNAartificialsynthetic
190ugagauaggg guuagggcuu aaagucggcu gauucuca
3819136DNAartificialsynthetic 191gagauagggg uuagggcuua aagucggcug
auucuc 3619229DNAartificialsynthetic 192gggguuaggg cuuaaagucg
gcugauucu 2919337DNAartificialsynthetic 193gcguggcgag guuagggcua
gaagucgguc gacacgc 3719435DNAartificialsynthetic 194cguggcgagg
uuagggcuag aagucggucg acacg 3519533DNAartificialsynthetic
195cgggcgaggu uagggcuaga agucggucga ccg
3319633DNAartificialsynthetic 196cgggcgaggu uagggcuaga agucggucgc
ccg 3319731DNAartificialsynthetic 197cggcgagguu agggcuagaa
gucggucgcc g 3119829DNAartificialsynthetic 198cgggagguua gggcuagaag
ucggucccg 2919927DNAartificialsynthetic 199gggagguuag ggcuagaagu
cgguccc 2720027DNAartificialsynthetic 200ccgcgguuag ggcuagaagu
cgggcgg 2720127DNAartificialsynthetic 201cccggguuag ggcuagaagu
cggcggg 2720227DNAartificialsynthetic 202ggcggguuag ggcuagaagu
cggcgcc 2720329DNAartificialsynthetic 203cccgcgguua gggcuagaag
ucgggcggg 2920429DNAartificialsynthetic 204gccgcgguua gggcuagaag
ucgggcggc 2920529DNAartificialsynthetic 205ccccggguua gggcuagaag
ucggcgggg 2920629DNAartificialsynthetic 206cggcggguua gggcuagaag
ucggcgccg 2920729DNAartificialsynthetic 207gggcggguua gggcuagaag
ucggcgccc 2920837DNAartificialsynthetic 208ugcugcgggg guuagggcua
gaagucggcc ugcagca 3720935DNAartificialsynthetic 209gcugcggggg
uuagggcuag aagucggccu gcagc 3521033DNAartificialsynthetic
210cugcgggggu uagggcuaga agucggccug cag
3321131DNAartificialsynthetic 211ugcggggguu agggcuagaa gucggccugc a
3121229DNAartificialsynthetic 212gcggggguua gggcuagaag ucggccugc
2921329DNAartificialsynthetic 213gccgggguua gggcuagaag ucggccggc
2921431DNAartificialsynthetic 214ggccgggguu agggcuagaa gucggccggc c
3121531DNAartificialsynthetic 215cgccgggguu agggcuagaa gucggccggc g
3121648DNAartificialsynthetic 216cgugguccgu ugugucaggu cuauucgccc
cggugcaggg cauccgcg 4821749DNAartificialsynthetic 217gcagugugac
gcggacguga uaggacagag cugaucccgc ucaggugag
4921849DNAartificialsynthetic 218caacagcagu gugacgcgga cgugauagga
cagagcugau cccgcucag 4921940DNAartificialsynthetic 219uaaggaaacu
cggucugaug cgguagcgcu gugcagagcu 4022017DNAartificialsynthetic
220cgugcgcuug agauagg 1722112DNAartificialsynthetic 221cugauucuca
cg 1222210DNAartificialsynthetic 222cugauucuca
1022329DNAartificialsynthetic 223gccgggguua gggcuagaag ucggccggc
2922429DNAartificialsynthetic 224cgggagguua gggcuagaag ucggucccg
2922545DNAartificialsynthetic 225cgcauggacu gauccuaguc gguuauguag
aucuagugug gugcg 45
* * * * *