U.S. patent application number 15/119401 was filed with the patent office on 2017-02-23 for methods and pharmaceutical compositions for the treatment of diseases mediated by the nrp-1/obr complex signaling pathway.
The applicant listed for this patent is Assistance Publique-Hopitaux de Paris (APHP), Centre National de la Recherche Scientifique (CNRS), Commissariat a l'Energie Atomique et aux Energies Alternatives, Fondation Imagine, INSERM (Institut National de la Sante et de la Recherche Medicale), Universite de Bourgogne, Universite Grenoble Alpes, Universite Paris Descartes. Invention is credited to Zakia Belaid-Choucair, Claude Cochet, Odile Filhol-Cochet, Carmen Garrido-Fleury, Olivier Hermine, Renaud Seigneuric.
Application Number | 20170051062 15/119401 |
Document ID | / |
Family ID | 50231095 |
Filed Date | 2017-02-23 |
United States Patent
Application |
20170051062 |
Kind Code |
A1 |
Belaid-Choucair; Zakia ; et
al. |
February 23, 2017 |
Methods and Pharmaceutical Compositions for the Treatment of
Diseases Mediated by the NRP-1/OBR Complex Signaling Pathway
Abstract
The present invention relates to methods and pharmaceutical
compositions for the treatment of diseases mediated by the
NRP-1/OBR complex signaling pathway. In particular, the present
invention relates to a method for treating a disease selected from
the group consisting of cancers, obesity and obesity related
diseases, anorexia, autoimmune diseases and infectious diseases in
a subject in need thereof comprising administering the subject with
a therapeutically effective amount of an antagonist of the
NRP-1/OBR signaling pathway.
Inventors: |
Belaid-Choucair; Zakia;
(Paris, FR) ; Hermine; Olivier; (Paris, FR)
; Garrido-Fleury; Carmen; (Dijon Cedex, FR) ;
Cochet; Claude; (Grenoble Cedex 9, FR) ;
Filhol-Cochet; Odile; (Grenoble Cedex 9, FR) ;
Seigneuric; Renaud; (Dijon Cedex, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (Institut National de la Sante et de la Recherche
Medicale)
Universite Paris Descartes
Fondation Imagine
Assistance Publique-Hopitaux de Paris (APHP)
Centre National de la Recherche Scientifique (CNRS)
Universite Grenoble Alpes
Commissariat a l'Energie Atomique et aux Energies Alternatives
Universite de Bourgogne |
Paris
Paris
Paris
Paris
Paris
Saint Martin d'Heres
Paris
Dijon |
|
FR
FR
FR
FR
FR
FR
FR
FR |
|
|
Family ID: |
50231095 |
Appl. No.: |
15/119401 |
Filed: |
February 18, 2015 |
PCT Filed: |
February 18, 2015 |
PCT NO: |
PCT/EP2015/053355 |
371 Date: |
August 17, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/14 20130101;
A61K 39/39558 20130101; A61K 31/7105 20130101; Y02A 50/30 20180101;
Y02A 50/481 20180101; A61P 37/06 20180101; C12N 2310/531 20130101;
G01N 2333/71 20130101; Y02A 50/409 20180101; C12N 15/1138 20130101;
G01N 2500/02 20130101; A61P 43/00 20180101; C12N 2320/31 20130101;
A61P 13/12 20180101; Y02A 50/423 20180101; A61P 3/04 20180101; A61P
7/00 20180101; A61K 31/713 20130101; A61K 38/17 20130101; A61P
13/02 20180101; C07K 16/2863 20130101; A61K 45/06 20130101; C07K
2317/76 20130101; G01N 2333/70503 20130101; A61K 31/4745 20130101;
A61P 31/00 20180101; A61P 1/14 20180101; Y02A 50/411 20180101; G01N
33/566 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; G01N 33/566 20060101
G01N033/566; A61K 31/7105 20060101 A61K031/7105; A61K 31/713
20060101 A61K031/713; A61K 45/06 20060101 A61K045/06; C12N 15/113
20060101 C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 2014 |
EP |
14305220.7 |
Claims
1. A method for treating a disease selected from the group
consisting of cancers, obesity and obesity related diseases,
cachexia, anorexia, ureteral obstructive kidney disease, autoimmune
diseases and infectious diseases in a subject in need thereof
comprising administering to the subject with a therapeutically
effective amount of an antagonist of the NRP-1/OBR signaling
pathway.
2. The method of claim 1 wherein the antagonist of the NRP-1/OBR
signaling pathway is selected from the group consisting of
antibodies, small organic molecules, polypeptides and aptamers.
3. The method of claim 1 wherein the antagonist of the NRP-1/OBR
signaling pathway is a CK2 inhibitor.
4. The method of claim 3 wherein the CK2 inhibitor is an allosteric
CK2 inhibitor.
5. The method of claim 1 wherein the antagonist of the NRP-1/OBR
signaling pathway is an antibody having an ability to block
interaction between NRP-1 and OBR or an ability to block
interaction between one or both of NRP-1 and Leptin.
6. The method of claim 5 wherein the antibody is directed to an
extracellular domain of NRP-1 or OBR or a domain of Leptin.
7. The method of claim 1 wherein the antagonist of the NRP-1/OBR
signaling pathway is combined with an anti-VEGF agent.
8. A method for the treatment of cancer in a subject in need
thereof comprising administering to the subject a therapeutically
effective amount of an anti-VEGF agent and a therapeutically
effective amount of a CK2 inhibitor.
9. A method for preventing or reducing an adaptive-evasive response
induced by a prolonged exposure to anti-VEGF treatment in a subject
having a cancer comprising administering to the subject a
therapeutically effective amount of an antagonist of the
NRP-1/OBR/Leptin signaling pathway.
10. The method of claim 9 wherein the antagonist of the
NRP-1/OBR/Leptin signaling pathway is a CK2 inhibitor.
11. A method for screening a plurality of test substances useful
for prevention or treatment of a disease selected from the group
consisting of cancers, obesity and obesity related diseases,
cachexia, anorexia, ureteral obstructive kidney disease, autoimmune
diseases and infectious diseases comprising (a) testing each of the
plurality of test substances for its ability to inhibit the
NRP-1/OBR/Leptin signaling pathway and (b) and positively selecting
test substances capable of inhibiting said NRP-1/OBR/Leptin
signaling pathway.
12. The method of claim 11 wherein step (a) comprises determining
whether each of the plurality of test substances i) inhibits
formation of a complex between NRP-1 and OBR or Leptin, ii)
inhibits phosphorylation of OBR and NRP1 induced by CK2, iii)
inhibits nuclear translocation of NRP-1/OBR complexes and iv)
inhibits expression of genes which are under control of the
NRP-1/OBR/Leptin signaling pathway.
13. The method of claim 11 wherein step a) comprises: a1)
contacting a test substance with a mixture of a first NRP-1
polypeptide or a substantially homologous or substantially similar
amino acid sequence thereof and (2) a second OBR polypeptide or a
substantially homologous or substantially similar amino acid
sequence thereof, and a2) determining the ability of said test
substance to modulate binding between said NRP-1 polypeptide or
said substantially homologous or substantially similar amino acid
sequence thereof and said second OBR polypeptide or said
substantially homologous or substantially similar amino acid
sequence thereof.
14. The method of claim 11 wherein step a) comprises: a1)
contacting a test substance with a mixture of a first NRP-1
polypeptide or a substantially homologous or substantially similar
amino acid sequence thereof and (2) a second Leptin polypeptide or
a substantially homologous or substantially similar amino acid
sequence thereof, and a2) determining the ability of said test
substance to modulate binding between said NRP-1 polypeptide and
said second Leptin polypeptide.
15. The method of claim 11 wherein step a) comprises: a1)
contacting a test substance with a mixture of a first NRP-1 or OBR
polypeptide or a substantially homologous or substantially similar
amino acid sequence thereof and (2) a second CK2 alpha polypeptide
or a substantially homologous or substantially similar amino acid
sequence thereof, and a2) determining the ability of said test
substance to modulate binding between said first NRP-1 or OBR
polypeptide or said substantially homologous or substantially
similar amino acid sequence thereof and said second CK2.alpha.
polypeptide or said substantially homologous or substantially
similar amino acid sequence thereof.
16. The method of claim 11 wherein step a) comprises: a1)
contacting a test substance with a mixture of a first NRP-1 or
Leptin polypeptide or a substantially homologous or substantially
similar amino acid sequence thereof and (2) a second CK2 alpha
polypeptide or a substantially homologous or substantially similar
amino acid sequence thereof, and a2) determining the ability of
said test substance to modulate binding between said NRP-1 or
Leptin polypeptide or said substantially homologous or
substantially similar amino acid sequence thereof and said second
CK2 alpha polypeptide or said substantially homologous or
substantially similar amino acid sequence thereof.
17. The method of claim 12, wherein step (a) comprises determining
whether each of the plurality of test substances inhibits
interaction between extracellular domains of NRP1 and OBR and a
domain of Leptin that interacts with NRP-1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and pharmaceutical
compositions for the treatment of diseases mediated by the
NRP-1/OBR complex signaling pathway.
BACKGROUND OF THE INVENTION
[0002] Neuropilin-1 (NRP-1) a trans-membrane receptor that plays a
central role in neuronal development (Fujisawa et al., 1995) has
been subsequently shown to be also involved in blood vessel
development as a co-receptor for two different types of ligands,
the semaphorin (SEMA) family of axon guidance modulators and the
vascular endothelial growth factor (VEGF) family of angiogenesis
stimulators, respectively. NRP-1 needs to form complexes with
receptors belonging to the plexin family, which serve as the
signal-transducing element for the axonal repulsion and collapse of
neuronal growth cones after SEMA binding to the NRP-1/plexin
complex (He and Tessier-Lavigne, 1997). NRP-1 also interacts as
well with VEGF receptors (VEGFR) forming a complex, which can be
activated by VEGF-A165 for normal developmental angiogenesis (Soker
et al., 1998). In addition, it has been shown that NRP-1 plays a
critical role in the regulation of the immune system by modulating
interactions between dendritic and T cells in the periphery and
between thymocytes and thymic epithelial cells in the thymus
(Tordjman et al, 2002) (Lepelletier et al., 2007). Amongst these
normal physiologic functions NRP-1 is also involved in
physiopathology. For example, NRP-1 is a receptor for the HTLV-1
virus (Ghez et al., 2006). In addition, data are accumulating
showing that NRP-1 is involved in oncogenesis (Soker et al., 2001;
Ellis et al, 2006), but its role remains controversial. SEMA
inhibit cell growth and decrease cell survival by inducing PTEN
activity, an inhibitor of the PI3kinase pathway (Cantly et al.,
1999), whereas VEGF plays an opposite role by competing with SEMA
for binding to NRP-1 and by providing signaling for cell
proliferation and survival through NRP-1/VEGFR complex (Bachelder
et al., 2003; Narazaki and Tosato, 2006). In some cancer cells,
including breast cancer cells, the expression of NRP-1 increases
cell proliferation and invasiveness through mechanisms that do not
necessarily involved neither SEMA nor VEGF suggesting that
alternative ligands and/or receptors may use NRP-1 as a co-receptor
(Kigel et al., 2008).
[0003] Leptin is a small non glycosylated protein expressed not
only by the benign primary source adipocytes but by cancer cells
also (Clin Cancer Research 2004). OBR is the receptor of leptin.
Leptin has been shown to regulate gene expression. More than 64
genes were identified including those for growth, cell cycle
regulators, extracellular matrix proteins and gene associated with
metastasis (J of Endocrinol 2008; EBM 2008). Obesity is considered
a risk for many cancers. Serum leptin levels are often elevated in
obese people. Leptin acts as a mitogenic agent in many tissues;
therefore, it may act to promote cancer cell growth. In fact,
leptin was shown to act as a growth factor for prostate cancer
cells in vitro, to induce increased migration of prostate cancer
cells and expression of growth factors such as vascular endothelial
growth factor (VEGF), transforming growth factor-beta 1
(TGF-.beta.I), and basic fibroblast growth factor (bFGF), and to
enhance prostate cancer growth. Leptin has been shown recently to
promote T helper 1 (Thl)-cell differentiation and to modulate the
onset and progression of autoimmune responses in several animal
models of disease. If leptin's role is fundamental in Thl-mediated
autoimmune diseases or inflammatory diseases, such as inflammatory
bowel syndrome, then a therapeutic effect can be anticipated by
blocking peripheral leptin action. Leptin has also been shown to be
involved in the pathogenesis of rheumatoid arthritis and in the
development of experimental autoimmune encephalomyelitis (EAE), a
mouse model for multiple sclerosis. Consequently, inhibitors of the
leptin signaling pathway are highly desirable for therapeutic
purposes.
SUMMARY OF THE INVENTION
[0004] The present invention relates to methods and pharmaceutical
compositions for the treatment of diseases mediated by the
NRP-1/OBR complex signaling pathway. In particular, the present
invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION
[0005] Several studies have demonstrated Neuropilin-1 (NRP-1)
implication in tumor progression independently of its known
co-receptors and cognate ligands. On the basis of growing
connections between breast cancer and obesity, and Leptin and its
receptor OBR involved in this process the inventors postulated that
NRP-1 is a new target of treatment of breast cancer and by
extension other cancer in which OBR and Leptin are involved. In
addition inhibition of VEGF interaction (for example with Avastin)
with its cognate receptor and NRP-1 may favor increase signaling
with Leptin with NRP-1/OBR receptor complex resulting in an
increase of metastasis and shortening overall survival.
[0006] By using a well described MDA-MB231 (NRP-1 positive and OBR
positive) and T47D (OBR low and NRP-1 negative) breast cancer cell
lines model transduced either by shNRP-1 or cDNA encoding for NRP-1
the inventors have shown that: [0007] 1) In vitro Leptin decreased
cell proliferation and increased migration and these effects were
dependent on NRP-1 expression. In vivo studies on xenograft models
either by overexpressing NRP-1 in T47D or by silencing NRP-1 in
MDA-MB231 have also shown a correlation between NRP-1 expression
and lymph node infiltration and this infiltration increased with
tumor treatment with leptin [0008] 2) NRP-1 forms a complex with
OBR [0009] 3) the NRP-1/OBR complex formation is leptin dependent
[0010] 4) the NRP-1/OBR complex translocates to the nucleus [0011]
5) the NRP-1/OBR complex formation and nuclear translocation are
dependent on NRP-1 and OBR phosphorylation by the Serine/threonine
Protein-kinase CK2 (CK2). This was confirmed by the inhibition of
CK2 by 3 different chemical compounds (TBB, DRB and CX4945) and by
RNA silencing that prevented not only NRP-1 and OBR phosphorylation
but also the formation and the nuclear translocation of the
NRP-1/OBR complex. [0012] 6) As other known NRP-1 ligands (VEGF,
Sema3A, TGF.beta. and PDGF), NRP-1 does bind directly to leptin and
induces OBR oligomerization upon leptin binding to OBR. The
oligomerization results in OBR signaling increase. This last result
show that leptin can a be targeted as a true NRP-1 ligand to
prevent its binding as it was the case for VEGF and SEMA to block
metastasis induced by NRP-1/OBR complex.
[0013] Accordingly, the characterization of the NRP-1/OBR complex
and its nuclear translocation shed a light on an eventual function
of NRP-1 as transcription factor or activator since the inventors
detected NRP-1/OBR complex in the nucleus and NRP-1 Chip-Seq
analysis of sample generated from MDA-MB-231 led to identify genes
implicated in metabolism, in immune cell response and breast cancer
metastasis and with enriched sequences containing RNA polymerase II
(Pol2) and transcription factor binding sites. This was confirmed
by the detection of NRP-1 and Pol2 interaction.
[0014] The new complex NRP-1/OBR, its phosphorylation by CK2, its
identification as nuclear receptor and the association of NRP-1
with RNA polymerase II thus open a wide field of investigations for
the understanding of the metabolism and metabolism-associated
disorders, mainly obesity and anorexia, leading thus to new
therapeutic strategies such as the use of CK2 inhibitors.
[0015] Accordingly a first aspect of the invention relates to a
method for treating a disease selected from the group consisting of
cancers, obesity and obesity related diseases, cachexia, anorexia,
ureteral obstructive kidney disease, autoimmune diseases and
infectious diseases in a subject in need thereof comprising
administering the subject with a therapeutically effective amount
of an antagonist of the NRP-1/OBR signaling pathway.
[0016] As used herein, the terms "treatment," "treat," and
"treating" refer to reversing, alleviating, inhibiting the progress
of a disease or disorder as described herein, or delaying,
eliminating or reducing the incidence or onset of a disorder or
disease as described herein, as compared to that which would occur
in the absence of the measure taken. The terms "prophylaxis" or
"prophylactic use" and "prophylactic treatment" as used herein,
refer to any medical or public health procedure whose purpose is to
prevent a disease. As used herein, the terms "prevent",
"prevention" and "preventing" refer to the reduction in the risk of
acquiring or developing a given condition, or the reduction or
inhibition of the recurrence or said condition in a subject who is
not ill, but who has been or may be near a subject with the
disease.
[0017] The term "obesity" refers to a condition characterized by an
excess of body fat. The operational definition of obesity is based
on the Body Mass Index (BMI), which is calculated as body weight
per height in meter squared (kg/m.sup.2). Obesity refers to a
condition whereby an otherwise healthy subject has a BMI greater
than or equal to 30 kg/m.sup.2, or a condition whereby a subject
with at least one co-morbidity has a BMI greater than or equal to
27 kg/m.sup.2. An "obese subject" is an otherwise healthy subject
with a BMI greater than or equal to 30 kg/m.sup.2 or a subject with
at least one co-morbidity with a BMI greater than or equal 27
kg/m.sup.2. A "subject at risk of obesity" is an otherwise healthy
subject with a BMI of 25 kg/m.sup.2 to less than 30 kg/m.sup.2 or a
subject with at least one co-morbidity with a BMI of 25 kg/m.sup.2
to less than 27 kg/m.sup.2. The increased risks associated with
obesity may occur at a lower BMI in people of Asian descent. In
Asian and Asian-Pacific countries, including Japan, "obesity"
refers to a condition whereby a subject with at least one
obesity-induced or obesity-related co-morbidity that requires
weight reduction or that would be improved by weight reduction, has
a BMI greater than or equal to 25 kg/m.sup.2. An "obese subject" in
these countries refers to a subject with at least one
obesity-induced or obesity-related co-morbidity that requires
weight reduction or that would be improved by weight reduction,
with a BMI greater than or equal to 25 kg/m.sup.2. In these
countries, a "subject at risk of obesity" is a person with a BMI of
greater than 23 kg/m.sup.2 to less than 25 kg/m.sup.2.
[0018] The method of the invention is particularly suitable for the
prophylactic treatment of obesity related disorders.
[0019] The term "obesity-related diseases" encompasses disorders
that are associated with, caused by, or result from obesity.
Examples of obesity-related disorders include overeating and
bulimia, diabetes, hypertension, elevated plasma insulin
concentrations and insulin resistance, dyslipidemia,
hyperlipidemia, breast, prostate, endometrial and colon cancer,
heart disease, cardiovascular disorders, abnormal heart rhythms and
arrhythmias, myocardial infarction, congestive heart failure,
coronary heart disease, angina pectoris, cerebral infarction,
cerebral thrombosis and transient ischemic attack, and
osteoarthritis. Other examples include pathological conditions
showing reduced metabolic activity or a decrease in resting energy
expenditure as a percentage of total fat-free mass. Further
examples of obesity-related disorders include metabolic syndrome,
also known as syndrome X, insulin resistance syndrome, type II
diabetes, impaired fasting glucose, impaired glucose tolerance,
inflammation, such as systemic inflammation of the vasculature,
atherosclerosis, hypercholesterolemia, hyperuricaemia, as well as
secondary outcomes of obesity such as left ventricular hypertrophy.
Obesity-related disorders also include the liver abnormalities
associated with obesity such as non-alcoholic fatty liver disease
(NAFLD) a rising cause of cirrhosis associated to obesity and
metabolic syndrome. Indeed, NAFLD can present as simple steatosis
or evolve towards inflammation and steatohepatitis (NASH), with a
20% risk of cirrhosis after 20 years. "Dyslipidemia" is a major
risk factor for coronary heart disease (CHD). Low plasma levels of
high density lipoprotein (HDL) cholesterol with either normal or
elevated levels of low density (LDL) cholesterol is a significant
risk factor for developing atherosclerosis and associated coronary
artery disease in humans. Dyslipidemia is often associated with
obesity.
[0020] As used herein, the term "cancer" has its general meaning in
the art and includes, but is not limited to, solid tumors and blood
borne tumors. The term cancer includes diseases of the skin,
tissues, organs, bone, cartilage, blood and vessels. The term
"cancer" further encompasses both primary and metastatic cancers.
Examples of cancers that may treated by methods and compositions of
the invention include, but are not limited to, cancer cells from
the bladder, blood, bone, bone marrow, brain, breast, colon,
esophagus, gastrointestinal, gum, head, kidney, liver, lung,
nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,
or uterus. In addition, the cancer may specifically be of the
following histological type, though it is not limited to these:
neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant
and spindle cell carcinoma; small cell carcinoma; papillary
carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma;
basal cell carcinoma; pilomatrix carcinoma; transitional cell
carcinoma; papillary transitional cell carcinoma; adenocarcinoma;
gastrinoma, malignant; cholangiocarcinoma; hepatocellular
carcinoma; combined hepatocellular carcinoma and
cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic
carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,
familial polyposis coli; solid carcinoma; carcinoid tumor,
malignant; branchiolo-alveolar adenocarcinoma; papillary
adenocarcinoma; chromophobe carcinoma; acidophil carcinoma;
oxyphilic adenocarcinoma; basophil carcinoma; clear cell
adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;
papillary and follicular adenocarcinoma; nonencapsulating
sclerosing carcinoma; adrenal cortical carcinoma; endometroid
carcinoma; skin appendage carcinoma; apocrine adenocarcinoma;
sebaceous adenocarcinoma; ceruminous; adenocarcinoma;
mucoepidermoid carcinoma; cystadenocarcinoma; papillary
cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous
cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell
carcinoma; infiltrating duct carcinoma; medullary carcinoma;
lobular carcinoma; inflammatory carcinoma; paget's disease,
mammary; acinar cell carcinoma; adenosquamous carcinoma;
adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian
stromal tumor, malignant; thecoma, malignant; granulosa cell tumor,
malignant; and roblastoma, malignant; Sertoli cell carcinoma;
leydig cell tumor, malignant; lipid cell tumor, malignant;
paraganglioma, malignant; extra-mammary paraganglioma, malignant;
pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic
melanoma; superficial spreading melanoma; malign melanoma in giant
pigmented nevus; epithelioid cell melanoma; blue nevus, malignant;
sarcoma; fibrosarcoma; fibrous histiocytoma, malignant;
myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;
embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal
sarcoma; mixed tumor, malignant; mullerian mixed tumor;
nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma,
malignant; brenner tumor, malignant; phyllodes tumor, malignant;
synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal
carcinoma; teratoma, malignant; struma ovarii, malignant;
choriocarcinoma; mesonephroma, malignant; hemangio sarcoma;
hemangioendothelioma, malignant; kaposi's sarcoma;
hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;
juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma,
malignant; mesenchymal chondrosarcoma; giant cell tumor of bone;
ewing's sarcoma; odontogenic tumor, malignant; ameloblastic
odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma;
pinealoma, malignant; chordoma; glioma, malignant; ependymoma;
astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma;
astroblastoma; glioblastoma; oligodendroglioma;
oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;
ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory
neurogenic tumor; meningioma, malignant; neurofibrosarcoma;
neurilemmoma, malignant; granular cell tumor, malignant; malignant
lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;
malignant lymphoma, small lymphocytic; malignant lymphoma, large
cell, diffuse; malignant lymphoma, follicular; mycosis fungoides;
other specified non-Hodgkin's lymphomas; malignant histiocytosis;
multiple myeloma; mast cell sarcoma; immunoproliferative small
intestinal disease; leukemia; lymphoid leukemia; plasma cell
leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid
leukemia; basophilic leukemia; eosinophilic leukemia; monocytic
leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid
sarcoma; and hairy cell leukemia.
[0021] "Autoimmune diseases" in the context of the present
invention, relates to diseases arising from an overactive immune
response of the body against substances and tissues normally
present in the body. In other words, the body actually attacks its
own cells or components. The immune system mistakes some part of
the body as a pathogen and attacks it. This may be restricted to
certain organs (e.g. in thyroiditis) or involve a particular tissue
in different places (e.g. Goodpasture's disease which may affect
the basement membrane in both the lung and the kidney). The
treatment of autoimmune and/or inflammatory diseases is typically
with immunosuppression--medication which decreases the immune
response. Example of autoimmune and/or inflammatory disease include
but are not limited to sarcoidosis, Ankylosing Spondylitis, Crohns
Disease (one of two types of idiopathic inflammatory bowel disease
"IBD"), Dermatomyositis, Diabetes mellitus type 1, Goodpasture's
syndrome, Graves' disease, Guillain-Barre syndrome (GBS),
Hashimoto's disease, Hidradenitis suppurativa, Idiopathic
thrombocytopenic purpura, systemic Lupus erythematosus, Mixed
Connective Tissue Disease, Multiple Sclerosis Myasthenia gravis,
Myositis, Narcolepsy, Pemphigus vulgaris, Pernicious anaemia,
Psoriasis, Psoriatic Arthritis, Polymyositis, Primary biliary
cirrhosis, Relapsing polychondritis, Rheumatoid arthritis, Systemic
sclerosis, Temporal arteritis (also known as "giant cell
arteritis"), Ulcerative Colitis (one of two types of idiopathic
inflammatory bowel disease "IBD"), Vasculitis, Wegener's
granulomatosis.
[0022] Typically infectious diseases include but are not limited to
chronic infectious diseases and more preferably viral infections,
e.g. Herpes (HSV), HIV, Hepatitis B, Hepatitis C, etc.,
intracellular bacterial infections, e.g. tuberculosis,
salmonellosis, listeriosis, etc., and parasite infections, e.g.
malaria, leishmaniasis, schistosomiasis.
[0023] As used herein, the term "NRP-1" has its general meaning in
the art and refers to Neuropilin-1. Neuropilins are 120 to 130 kDa
non-tyrosine kinase receptors. There are multiple NRP-1 and NRP-2
splice variants and soluble isoforms. The basic structure of
neuropilins comprises five domains: three extracellular domains
(a1a2, b1b2 and c), a transmembrane domain, and a cytoplasmic
domain. The a1a2 domain is homologous to complement components C1r
and C1s (CUB), which generally contains four cysteine residues that
form two disculfid bridges. The b1b2 domain is homologous to
coagulation factors V and VIII. The central portion of the c domain
is designated as MAM due to its homology to meprin, AS and receptor
tyrosine phosphotase .mu. proteins. The a1a2 and b1b2 domains are
responsible for ligand binding, whereas the c domain is critical
for homodimerization or heterodimerization.
[0024] As used herein the term "OBR" has its general meaning in the
art and refers to the leptin receptor also known as LEPR and CD295.
The human transmembrane receptor has at least four different
isoforms with different C-terminus cytoplasmatic domains (Barr et
al., 1999, J. Biol. Chem. 274 (30): 21416-21424). The full form of
ObR (ObR1) is 1,165 amino acids long and contains extracellular,
transmembrane and intracellular domains. The extracellular domain
binds ligand, whereas the intracellular tail recruits and activates
signaling substrates.
[0025] As used herein the term "NRP-1/OBR complex" refers to the
complex resulting from the heterodimerization between NRP-1 and OBR
as described in the EXAMPLE. The formation of the complex is
mediated by the binding of leptin to OBR and NRP-1. The NRP-1/OBR
complex formation and nuclear translocation are dependent on NRP-1
and OBR phosphorylation by the Serine/threonine Protein-kinase CK2.
As described in the EXAMPLE, the NRP-1/OBR complex translocates to
the nucleus and interacts with RNA polymerase II (RNApol2) so as to
trigger the expression of various genes. In particular, in cancer
cells, the complex triggers the expression of genes implicated in
cancer metastasis. All the effects mediated by the formation of the
NRP-1/OBR complex are referred as the "NRP-1/OBR/Leptin signaling
pathway".
[0026] The term "antagonist of the NRP-1/OBR signaling pathway"
means any compound that attenuates signal transduction mediated by
the formation of the NRP-1/OBR complex as described in the EXAMPLE.
In particular the antagonist of the NRP-1/OBR signaling pathway is
a compound that inhibits, reduces, abolishes or otherwise reduces
the formation of said complex. In other terms the antagonist of the
NRP-1/OBR signaling pathway is a compound that inhibits, reduces,
abolishes or otherwise reduces the signaling pathway triggered by
the formation of the complex. Such inhibition may result where: (i)
the antagonist of the NRP-1/OBR signaling pathway of the invention
binds to NRP-1 or OBR without triggering signal transduction, to
reduce or block the formation of the NRP-1/OBR complex; (ii) the
antagonist of the NRP-1/OBR signaling pathway inhibits the
stability of the NRP-1/OBR complex by impeding the phosphorylation
mediated by the Protein-kinase CK2; or (iii) the antagonist of the
NRP-1/OBR signaling pathway binds to, or otherwise inhibits the
activity of, a molecule that is part of a regulatory chain that,
when not inhibited, has the result of stimulating or otherwise
facilitating the signal transduction mediated by the NRP-1/OBR
complex.
[0027] Typically, the antagonist of the NRP-1/OBR signaling pathway
includes but is not limited to an antibody, a small organic
molecule, a polypeptide and an aptamer.
[0028] In some embodiments, the antagonist of the NRP-1/OBR
signaling pathway is a small organic molecule.
[0029] The term "small organic molecule" refers to a molecule of a
size comparable to those organic molecules generally used in
pharmaceuticals. The term excludes biological macromolecules (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, more preferably up to 2000 Da,
and most preferably up to about 1000 Da.
[0030] In a particular embodiment, the antagonist of the NRP-1/OBR
signaling pathway is a CK2 inhibitor.
[0031] CK2 inhibitors are commonly classified into three
categories: (1) inhibitors that target the regulatory subunit of
CK2 (e.g., genetically selected peptide aptamers); (2) inhibitors
of the catalytic activity of CK2 (e.g., quinobene, TBB, DMAT, IQA);
and (3) disruptors of CK2 holoenzymes, which are often molecules
binding to the CK2 subunit interface and inhibit the high affinity
interaction of its subunits. The CK2 inhibitors of each class can
be any type of molecule, such as, small molecules, functional
nucleic acids, or peptide mimetics, etc. Typically, CK2 inhibitors
consist of a diverse array of chemicals, including flavonoids (e.g.
apigenin), derivatives of hydro xyantraquinones/xantenones (e.g.,
emodin), derivatives of hydroxycoumarines (e.g., DBC), derivatives
of tetrabromotriazole/imidazole (e.g., DRB, TBB, DMAT, TBCA, TBBz),
and derivatives of indoloquinazolines (e.g., IQA). More
particularly, many ATP-competitive inhibitors of CK2 have been
reported in the literature, including
5,6-dichloro-l-P-D-ribofuranosylbenzimidazole (D B),
6-methyl-1,3,8-trihydroxyanthraquinone (emodin),
2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT),
4,5,6,7-tetrabromobenzotriazole (TBB), resorufin,
4,4',5,5',6,6'-Hexahydroxydiphenic acid 2,6,2',6'-dilactone
(ellagic acid),
[5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA),
and 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one
(quercetin). See, e.g., Zhu et al., 2009, Mol. Cell. Biochem. 333:
159-67; Lopez-Ramos et al., 2010, Faseb J. 24: 3171-85; and Cozza
et al, 2010, Med. Res. Rev. 30: 419-62.
[0032] CK2 inhibitors as described herein also include, but are not
limited to, the compounds of any of the formulae described in
International Patent Application Nos. PCT/US2007/077464,
PCT/US2008/074820, and PCT7US2009/035609, and U.S. Provisional
Application Ser. No. 61/170,468 (filed 17 Apr. 2009), 61/242,227
(filed 14 Sep. 2009), 61,180,090 (filed 20 May 2009), 61/218,318
(filed 18 Jun. 2009), 61/179,996 (filed 20 May 2009), 61/218,214
(filed 14 Jun. 2009), 61/41,806 (11 Sep. 2009), 61/180,099 (filed
20 May 2009), 61/218,347 (filed 18 Jun. 2009), 61/237,227 (filed 26
Aug. 2009), 61/243,107 (filed 16 Sep. 2009) and 61/243,104 (filed
16 Sep. 2009), the contents of each of which are incorporated
herein by reference in their entirety. CK2 inhibitors can be
synthesized by methods known in the art, including methods
disclosed in International Patent Application Nos.
PCT/US2007/077464, PCT/US2008/074820, and PCT/US2009/035609.
[0033] In some embodiments, the CK2 inhibitors is selected from the
group consisting of:
##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006## ##STR00007##
[0034] In some embodiments, the CK2 inhibitor is CX-4945:
##STR00008##
[0035] CX-4945 is a first-in-class potent, selective and orally
available ATP-competitive inhibitor of CK2 with favorable drug
properties (Siddiqui-Jain A, Bliesath J, Macalino D, Omori M, Huser
N, Streiner N, Ho C B, Anderes K, Proffitt C, O'Brien S E, Lim J K,
Von Hoff D D, Ryckman D M, Rice W G, Drygin D. CK2 inhibitor
CX-4945 suppresses DNA repair response triggered by DNA-targeted
anticancer drugs and augments efficacy: mechanistic rationale for
drug combination therapy. Mol Cancer Ther. 2012 April;
11(4):994-1005. doi: 10.1158/1535-7163.MCT-11-0613. Epub 2012 Jan.
20.).
[0036] In some embodiments, the CK2 inhibitor is a compound
(Compound 1 or Compound 2) having the formula:
##STR00009##
[0037] or a pharmaceutically acceptable salt or ester thereof.
[0038] In some embodiments, the CK2 inhibitor a compound described
in WO 2011/013002 and in particular is selected from the group
consisting of:
##STR00010##
[0039] and pharmaceutically acceptable salts or esters thereof.
[0040] In some embodiments, the CK2 inhibitor is a compound as
described in: [0041] Maksym O. Chekanov, Olga V. Ostrynska, Sergii
S. Tarnayskyi, Anatoliy R. Synyugin, Nadiia V. Briukhovetska,
Volodymyr G. Bdzhola, Alexander E. Pashenko, Andrey A. Fokin,
Sergiy M. Yarmoluk Design, synthesis and biological evaluation of
2-aminopyrimidinones and their 6-aza-analogs as a new class of CK2
inhibitors Journal of Enzyme Inhibition and Medicinal Chemistry
[0042] Maksym O. Chekanov, Olga V. Ostrynska, Anatoliy R. Synyugin,
Volodymyr G. Bdzhola, Sergiy M. Yarmoluk Design, synthesis and
evaluation of 2-phenylisothiazolidin-3-one-1,1-dioxides as a new
class of human protein kinase CK2 inhibitors Journal of Enzyme
Inhibition and Medicinal Chemistry Posted online on 11 Apr. 2013.
[0043] Giorgio Cozza, Lorenzo A Pinna, Stefano Moro Protein kinase
CK2 inhibitors: a patent review Expert Opinion on Therapeutic
Patents September 2012, Vol. 22, No. 9, Pages 1081-1097.
[0044] In some embodiments, the CK2 inhibitor is an allosteric CK2
inhibitor, i.e. a compound which does not compete with ATP but
still inhibits CK2 by modifying the conformation of a CK2 subunit
(e.g. CK2 alpha) in manner that the enzyme is inactive. Examples of
allosteric CK2 inhibitors include but are not limited to
Azonaphthalene derivatives (compound M4) as described in Moucadel
V, Prudent R, Sautel C F, Teillet F, Barette C, Lafanechere L,
Receveur-Brechot V, Cochet C. Antitumoral activity of allosteric
inhibitors of protein kinase CK2. Oncotarget. 2011 December;
2(12):997-1010. Another example includes D3.1 as described in the
EXAMPLE.
[0045] In some embodiments, the allosteric CK2 inhibitor is D3.1
which has the general formula of:
##STR00011##
[0046] In some embodiments, the allosteric CK2 inhibitor is M4
which has the general formula of:
##STR00012##
[0047] The compounds of the invention as described above can be
synthesized using methods, techniques, and materials known to those
of skill in the art, such as described, for example, in March,
ADVANCED ORGANIC CHEMISTRY 4.sup.th Ed., (Wiley 1992); Carey and
Sundberg, ADVANCED ORGANIC CHEMISTY 3.sup.rd Ed., Vols. A and B
(Plenum 1992), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC
SYNTHESIS 2.sup.nd Ed. (Wiley 1991). Starting materials useful for
preparing compounds of the invention and intermediates thereof are
commercially available from sources, such as Aldrich Chemical Co.
(Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), Maybridge
(Cornwall, England), Asinex (Winston-Salem, N.C.), ChemBridge (San
Diego, Calif.), ChemDiv (San Diego, Calif.), SPECS (Delft, The
Netherlands), Timtec (Newark, Del.), or alternatively can be
prepared by well-known synthetic methods (see, e.g., Harrison et
al., "Compendium of Synthetic Organic Methods", Vols. 1-8 (John
Wiley and Sons, 1971-1996); "Beilstein Handbook of Organic
Chemistry," Beilstein Institute of Organic Chemistry, Frankfurt,
Germany; Feiser et al., "Reagents for Organic Synthesis," Volumes
1-21, Wiley Interscience; Trost et al., "Comprehensive Organic
Synthesis," Pergamon Press, 1991; "Theilheimer's Synthetic Methods
of Organic Chemistry," Volumes 1-45, Karger, 1991; March, "Advanced
Organic Chemistry," Wiley Interscience, 1991; Larock "Comprehensive
Organic Transformations," VCH Publishers, 1989; Paquette,
"Encyclopedia of Reagents for Organic Synthesis," 3d Edition, John
Wiley & Sons, 1995). Other methods for synthesis of the present
compounds and/or starting materials thereof are either described in
the art or will be readily apparent to the skilled artisan.
Alternatives to the reagents and/or protecting groups may be found
in the references provided above and in other compendiums well
known to the skilled artisan. In particular, preparation of the
present compounds may include one or more steps of protection and
de-protection (e.g., the formation and removal of acetal groups).
Guidance for selecting suitable protecting groups can be found, for
example, in Greene & Wuts, "Protective Groups in Organic
Synthesis," Wiley Interscience, 1999. In addition, the preparation
may include various purifications, such as column chromatography,
flash chromatography, thin-layer chromatography (TLC),
recrystallization, distillation, high-pressure liquid
chromatography (HPLC) and the like. Also, various techniques well
known in the chemical arts for the identification and
quantification of chemical reaction products, such as proton and
carbon-13 nuclear magnetic resonance (H and 13C NMR), infrared and
ultraviolet spectroscopy (IR and UV), X-ray crystallography,
elemental analysis (EA), HPLC and mass spectroscopy (MS) can be
used as well. The preparation may also involve any other methods of
protection and de-protection, purification and identification and
quantification that are well known in the chemical arts.
[0048] In one embodiment, the antagonist of the NRP-1/OBR signaling
pathway is a small organic molecule, which impends the binding of
CK2, in particular CK2 alpha to NRP-1 and/or OBR.
[0049] In a particular embodiment, the antagonist of the NRP-1/OBR
signaling pathway is an inhibitor of CK2 gene expression. An
"inhibitor of gene expression" refers to a natural or synthetic
compound that has a biological effect to inhibit the expression of
a gene. In a preferred embodiment of the invention, said inhibitor
of gene expression is a siRNA, an antisense oligonucleotide or a
ribozyme. For example, anti-sense oligonucleotides, including
anti-sense RNA molecules and anti-sense DNA molecules, would act to
directly block the translation of mineralocorticoid receptor mRNA
by binding there to and thus preventing protein translation or
increasing mRNA degradation, thus decreasing the level of
mineralocorticoid receptor, and thus activity, in a cell. For
example, antisense oligonucleotides of at least about 15 bases and
complementary to unique regions of the mRNA transcript sequence
encoding mineralocorticoid receptor can be synthesized, e.g., by
conventional phosphodiester techniques. Methods for using antisense
techniques for specifically inhibiting gene expression of genes
whose sequence is known are well known in the art (e.g. see U.S.
Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091;
6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also
function as inhibitors of expression for use in the present
invention. Mineralocorticoid receptor gene expression can be
reduced by contacting a subject or cell with a small double
stranded RNA (dsRNA), or a vector or construct causing the
production of a small double stranded RNA, such that
mineralocorticoid receptor gene expression is specifically
inhibited (i.e. RNA interference or RNAi). Antisense
oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may
be delivered in vivo alone or in association with a vector. In its
broadest sense, a "vector" is any vehicle capable of facilitating
the transfer of the antisense oligonucleotide, siRNA, shRNA or
ribozyme nucleic acid to the cells and typically cells expressing
mineralocorticoid receptor. Typically, the vector transports the
nucleic acid to cells with reduced degradation relative to the
extent of degradation that would result in the absence of the
vector. In general, the vectors useful in the invention include,
but are not limited to, plasmids, phagemids, viruses, other
vehicles derived from viral or bacterial sources that have been
manipulated by the insertion or incorporation of the antisense
oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
Viral vectors are a preferred type of vector and include, but are
not limited to nucleic acid sequences from the following viruses:
retrovirus, such as moloney murine leukemia virus, harvey murine
sarcoma virus, murine mammary tumor virus, and rous sarcoma virus;
adenovirus, adeno-associated virus; SV40-type viruses; polyoma
viruses; Epstein-Barr viruses; papilloma viruses; herpes virus;
vaccinia virus; polio virus; and RNA virus such as a retrovirus.
One can readily employ other vectors not named but known to the
art.
[0050] In one embodiment, the agent is an antibody. In particular,
the invention embraces antibodies or fragments of antibodies having
the ability to block the interaction between NRP-1 and OBR or the
interaction between NRP-1 and leptin. The antibodies may have
specificity to NRP-1 or OBR. In one embodiment, the antibodies or
fragment of antibodies are directed to all or a portion of the
extracellular domain of OBR. In one embodiment, the antibodies or
fragment of antibodies are directed to an extracellular domain of
NRP-1 or OBR. More particularly this invention provides an antibody
or portion thereof capable of inhibiting binding of OBR and/or to
NRP-1, which antibody binds to an epitope located within a region
of OBR or NRP-1, which region of OBR binds to NRP-1. More
particularly this invention provides an antibody or portion thereof
capable of inhibiting binding of NRP-1 to OBR and/or leptin, which
antibody binds to an epitope located within a region of NRP-1,
which region of NRP-1 binds to OBR and/or leptin.
[0051] In one embodiment of the antibodies or portions thereof
described herein, the antibody is a monoclonal antibody. In one
embodiment of the antibodies or portions thereof described herein,
the antibody is a polyclonal antibody. In one embodiment of the
antibodies or portions thereof described herein, the antibody is a
humanized antibody. In one embodiment of the antibodies or portions
thereof described herein, the antibody is a chimeric antibody. In
one embodiment of the antibodies or portions thereof described
herein, the portion of the antibody comprises a light chain of the
antibody. In one embodiment of the antibodies or portions thereof
described herein, the portion of the antibody comprises a heavy
chain of the antibody. In one embodiment of the antibodies or
portions thereof described herein, the portion of the antibody
comprises a Fab portion of the antibody. In one embodiment of the
antibodies or portions thereof described herein, the portion of the
antibody comprises a F(ab')2 portion of the antibody. In one
embodiment of the antibodies or portions thereof described herein,
the portion of the antibody comprises a Fc portion of the antibody.
In one embodiment of the antibodies or portions thereof described
herein, the portion of the antibody comprises a Fv portion of the
antibody. In one embodiment of the antibodies or portions thereof
described herein, the portion of the antibody comprises a variable
domain of the antibody. In one embodiment of the antibodies or
portions thereof described herein, the portion of the antibody
comprises one or more CDR domains of the antibody.
[0052] As used herein, "antibody" includes both naturally occurring
and non-naturally occurring antibodies. Specifically, "antibody"
includes polyclonal and monoclonal antibodies, and monovalent and
divalent fragments thereof. Furthermore, "antibody" includes
chimeric antibodies, wholly synthetic antibodies, single chain
antibodies, and fragments thereof. The antibody may be a human or
non human antibody. A non human antibody may be humanized by
recombinant methods to reduce its immunogenicity in man.
[0053] Typically, antibodies are prepared according to conventional
methodology. Monoclonal antibodies may be generated using the
method of Kohler and Milstein (Nature, 256:495, 1975). To prepare
monoclonal antibodies useful in the invention, a mouse or other
appropriate host animal is immunized at suitable intervals (e.g.,
twice-weekly, weekly, twice-monthly or monthly) with antigenic
forms of NRP-1, or OBR. The animal may be administered a final
"boost" of antigen within one week of sacrifice. It is often
desirable to use an immunologic adjuvant during immunization.
Suitable immunologic adjuvants include Freund's complete adjuvant,
Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's
Titermax, saponin adjuvants such as QS21 or Quil A, or
CpG-containing immunostimulatory oligonucleotides. Other suitable
adjuvants are well-known in the field. The animals may be immunized
by subcutaneous, intraperitoneal, intramuscular, intravenous,
intranasal or other routes. A given animal may be immunized with
multiple forms of the antigen by multiple routes.
[0054] Briefly, the recombinant NRP-1 or OBR may be provided by
expression with recombinant cell lines, in particular in the form
of human cells expressing NRP-1 or OBR at their surface.
Recombinant forms of OBR or NRP-1 may be provided using any
previously described method. Following the immunization regimen,
lymphocytes are isolated from the spleen, lymph node or other organ
of the animal and fused with a suitable myeloma cell line using an
agent such as polyethylene glycol to form a hydridoma. Following
fusion, cells are placed in media permissive for growth of
hybridomas but not the fusion partners using standard methods, as
described (Coding, Monoclonal Antibodies: Principles and Practice:
Production and Application of Monoclonal Antibodies in Cell
Biology, Biochemistry and Immunology, 3rd edition, Academic Press,
New York, 1996). Following culture of the hybridomas, cell
supernatants are analyzed for the presence of antibodies of the
desired specificity, i.e., that selectively bind the antigen.
Suitable analytical techniques include ELISA, flow cytometry,
immunoprecipitation, and western blotting. Other screening
techniques are well-known in the field. Preferred techniques are
those that confirm binding of antibodies to conformationally
intact, natively folded antigen, such as non-denaturing ELISA, flow
cytometry, and immunoprecipitation.
[0055] Significantly, as is well-known in the art, only a small
portion of an antibody molecule, the paratope, is involved in the
binding of the antibody to its epitope (see, in general, Clark, W.
R. (1986) The Experimental Foundations of Modern Immunology Wiley
& Sons, Inc., New York; Roitt, I. (1991) Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc
regions, for example, are effectors of the complement cascade but
are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab')2 fragment,
retains both of the antigen binding sites of an intact antibody.
Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0056] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, 1986; Roitt,
1991). In both the heavy chain Fd fragment and the light chain of
IgG immunoglobulins, there are four framework regions (FR1 through
FR4) separated respectively by three complementarity determining
regions (CDR1 through CDRS). The CDRs, and in particular the CDRS
regions, and more particularly the heavy chain CDRS, are largely
responsible for antibody specificity.
[0057] It is now well-established in the art that the non CDR
regions of a mammalian antibody may be replaced with similar
regions of conspecific or heterospecific antibodies while retaining
the epitopic specificity of the original antibody. This is most
clearly manifested in the development and use of "humanized"
antibodies in which non-human CDRs are covalently joined to human
FR and/or Fc/pFc' regions to produce a functional antibody.
[0058] This invention provides in certain embodiments compositions
and methods that include humanized forms of antibodies. As used
herein, "humanized" describes antibodies wherein some, most or all
of the amino acids outside the CDR regions are replaced with
corresponding amino acids derived from human immunoglobulin
molecules. Methods of humanization include, but are not limited to,
those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089,
5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated
by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and
WO 90/07861 also propose four possible criteria which may used in
designing the humanized antibodies. The first proposal was that for
an acceptor, use a framework from a particular human immunoglobulin
that is unusually homologous to the donor immunoglobulin to be
humanized, or use a consensus framework from many human antibodies.
The second proposal was that if an amino acid in the framework of
the human immunoglobulin is unusual and the donor amino acid at
that position is typical for human sequences, then the donor amino
acid rather than the acceptor may be selected. The third proposal
was that in the positions immediately adjacent to the 3 CDRs in the
humanized immunoglobulin chain, the donor amino acid rather than
the acceptor amino acid may be selected. The fourth proposal was to
use the donor amino acid reside at the framework positions at which
the amino acid is predicted to have a side chain atom within 3 A of
the CDRs in a three dimensional model of the antibody and is
predicted to be capable of interacting with the CDRs. The above
methods are merely illustrative of some of the methods that one
skilled in the art could employ to make humanized antibodies. One
of ordinary skill in the art will be familiar with other methods
for antibody humanization.
[0059] In one embodiment of the humanized forms of the antibodies,
some, most or all of the amino acids outside the CDR regions have
been replaced with amino acids from human immunoglobulin molecules
but where some, most or all amino acids within one or more CDR
regions are unchanged. Small additions, deletions, insertions,
substitutions or modifications of amino acids are permissible as
long as they would not abrogate the ability of the antibody to bind
a given antigen. Suitable human immunoglobulin molecules would
include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A
"humanized" antibody retains a similar antigenic specificity as the
original antibody. However, using certain methods of humanization,
the affinity and/or specificity of binding of the antibody may be
increased using methods of "directed evolution", as described by Wu
et al., J. Mol. Biol. 294:151, 1999, the contents of which are
incorporated herein by reference.
[0060] Fully human monoclonal antibodies also can be prepared by
immunizing mice transgenic for large portions of human
immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat.
Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and
references cited therein, the contents of which are incorporated
herein by reference. These animals have been genetically modified
such that there is a functional deletion in the production of
endogenous (e.g., murine) antibodies. The animals are further
modified to contain all or a portion of the human germ-line
immunoglobulin gene locus such that immunization of these animals
will result in the production of fully human antibodies to the
antigen of interest. Following immunization of these mice (e.g.,
XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal
antibodies can be prepared according to standard hybridoma
technology. These monoclonal antibodies will have human
immunoglobulin amino acid sequences and therefore will not provoke
human anti-mouse antibody (KAMA) responses when administered to
humans.
[0061] In vitro methods also exist for producing human antibodies.
These include phage display technology (U.S. Pat. Nos. 5,565,332
and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat.
Nos. 5,229,275 and 5,567,610). The contents of these patents are
incorporated herein by reference.
[0062] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab') 2 Fab, Fv and
Fd fragments; chimeric antibodies in which the Fc and/or FR and/or
CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced
by homologous human or non-human sequences; chimeric F(ab')2
fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or
light chain CDR3 regions have been replaced by homologous human or
non-human sequences; chimeric Fab fragment antibodies in which the
FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have
been replaced by homologous human or non-human sequences; and
chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or
CDR2 regions have been replaced by homologous human or non-human
sequences. The present invention also includes so-called single
chain antibodies.
[0063] The various antibody molecules and fragments may derive from
any of the commonly known immunoglobulin classes, including but not
limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are
also well known to those in the art and include but are not limited
to human IgGl, IgG2, IgG3 and IgG4.
[0064] In another embodiment, the antibody according to the
invention is a single domain antibody. The term "single domain
antibody" (sdAb) or "VHH" refers to the single heavy chain variable
domain of antibodies of the type that can be found in Camelid
mammals which are naturally devoid of light chains. Such VHH are
also called "Nanobody.RTM.". According to the invention, sdAb can
particularly be llama sdAb.
[0065] In some embodiments, the antibody is a bispecific antibody.
The term "bispecific antibody" has its general meaning in the art
and refers to any antibody consisting of one binding site for a
first target antigen and a second binding site for a second target
antigen. In particular, a bispecific antibody according the
invention antibody consists of one binding site for NRP-1 and a
second binding site for OBR. Methods for making bispecific
antibodies are known in the art. Traditional production of
full-length bispecific antibodies is based on the coexpression of
two immunoglobulin heavy chain-light chain pairs, where the two
chains have different specificities (see, e.g., Milstein et al.,
1983, Nature 305:537-39). Because of the random assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules, of
which only one has the correct bispecific structure. Similar
procedures are disclosed in International Publication No. WO
93/08829, and in Traunecker et al., 1991, EMBO J. 10:3655-59. Other
examples of bispecific antibodies include Bi-specific T-cell
engagers (BiTEs) that are a class of artificial bispecific
monoclonal antibodies. BiTEs are fusion proteins consisting of two
single-chain variable fragments (scFvs) of different antibodies, or
amino acid sequences from four different genes, on a single peptide
chain of about 55 kilodaltons. Other bispecific antibodies those
described in WO2006064136. In particular the bispecific antibody is
a Fab format described in WO2006064136 comprising one VH or VHH
specific for NRP-1 and one VH or VHH specific for OBR.
[0066] In some embodiments, the antagonist is a polypeptide. In a
particular embodiment the polypeptide is a functional equivalent of
NRP-1 or OBR. As used herein, a "functional equivalent of NRP-1 or
OBR is a compound which is capable of binding to OBR or NRP-1
respectively, thereby preventing its interaction with OBR or NRP-1
or leptin, respectively. The term "functional equivalent" includes
fragments, mutants, and muteins of NRP-1 or OBR. The term
"functionally equivalent" thus includes any equivalent of NRP-1 or
OBR obtained by altering the amino acid sequence, for example by
one or more amino acid deletions, substitutions or additions such
that the protein analogue retains the ability to bind to OBR or
NRP-1 respectively. Amino acid substitutions may be made, for
example, by point mutation of the DNA encoding the amino acid
sequence. Typically, functional equivalents include molecules that
bind OBR or NRP-1 and comprise all or a portion of the
extracellular domains of NRP-1 or OBR respectively.
[0067] The functional equivalents include soluble forms of NRP-1 or
OBR. A suitable soluble form of these proteins, or functional
equivalents thereof, might comprise, for example, a truncated form
of the protein from which the transmembrane domain has been removed
by chemical, proteolytic or recombinant methods.
[0068] Typically, the functional equivalent is at least 80%
homologous to the corresponding protein. In a preferred embodiment,
the functional equivalent is at least 90% homologous as assessed by
any conventional analysis algorithm such as for example, the Pileup
sequence analysis software (Program Manual for the Wisconsin
Package, 1996).
[0069] The term "a functionally equivalent fragment" as used herein
also may mean any fragment or assembly of fragments of NRP-1 or OBR
that binds to OBR or NRP-1 respectively. Accordingly the present
invention provides a polypeptide capable of inhibiting binding of
OBR to NRP-1, and Leptin to NRP-1, which polypeptide comprises
consecutive amino acids having a sequence which corresponds to the
sequence of at least a portion of an extracellular domain of OBR,
and/or Leptin which portion binds to NRP-1. In one embodiment, the
polypeptide corresponds to an extracellular domain of OBR. The
present invention also provides a polypeptide capable of inhibiting
binding of NRP-1 to OBR an/or Leptin, which polypeptide comprises
consecutive amino acids having a sequence which corresponds to the
sequence of at least a portion of an extracellular domain of NRP-1,
which portion binds to OBR or Leptin. In one embodiment, the
polypeptide corresponds to an extracellular domain of NRP-1 or OBR
or Leptin.
[0070] Functionally equivalent fragments may belong to the same
protein family as the human NRP-1 or OBR identified herein. By
"protein family" is meant a group of proteins that share a common
function and exhibit common sequence homology. Homologous proteins
may be derived from non-human species. Preferably, the homology
between functionally equivalent protein sequences is at least 25%
across the whole of amino acid sequence of the complete protein.
More preferably, the homology is at least 50%, even more preferably
75% across the whole of amino acid sequence of the protein or
protein fragment. More preferably, homology is greater than 80%
across the whole of the sequence. More preferably, homology is
greater than 90% across the whole of the sequence. More preferably,
homology is greater than 95% across the whole of the sequence.
[0071] The polypeptides of the invention may be produced by any
suitable means, as will be apparent to those of skill in the art.
In order to produce sufficient amounts of the polypeptides for use
in accordance with the present invention, expression may
conveniently be achieved by culturing under appropriate conditions
recombinant host cells containing the polypeptide of the invention.
Preferably, the polypeptide is produced by recombinant means, by
expression from an encoding nucleic acid molecule. Systems for
cloning and expression of a polypeptide in a variety of different
host cells are well known. When expressed in recombinant form, the
polypeptide is typically generated by expression from an encoding
nucleic acid in a host cell. Any host cell may be used, depending
upon the individual requirements of a particular system. Suitable
host cells include bacteria mammalian cells, plant cells, yeast and
baculovirus systems. Mammalian cell lines available in the art for
expression of a heterologous polypeptide include Chinese hamster
ovary cells. HeLa cells, baby hamster kidney cells and many others.
Bacteria are also preferred hosts for the production of recombinant
protein, due to the ease with which bacteria may be manipulated and
grown. A common, preferred bacterial host is E coli.
[0072] In specific embodiments, it is contemplated that
polypeptides used in the therapeutic methods of the present
invention may be modified in order to improve their therapeutic
efficacy. Such modification of therapeutic compounds may be used to
decrease toxicity, increase circulatory time, or modify
biodistribution. For example, the toxicity of potentially important
therapeutic compounds can be decreased significantly by combination
with a variety of drug carrier vehicles that modify
biodistribution. In example adding dipeptides can improve the
penetration of a circulating agent in the eye through the blood
retinal barrier by using endogenous transporters.
[0073] A strategy for improving drug viability is the utilization
of water-soluble polymers. Various water-soluble polymers have been
shown to modify biodistribution, improve the mode of cellular
uptake, change the permeability through physiological barriers; and
modify the rate of clearance from the body. To achieve either a
targeting or sustained-release effect, water-soluble polymers have
been synthesized that contain drug moieties as terminal groups, as
part of the backbone, or as pendent groups on the polymer
chain.
[0074] Polyethylene glycol (PEG) has been widely used as a drug
carrier, given its high degree of biocompatibility and ease of
modification. Attachment to various drugs, proteins, and liposomes
has been shown to improve residence time and decrease toxicity. PEG
can be coupled to active agents through the hydroxyl groups at the
ends of the chain and via other chemical methods; however, PEG
itself is limited to at most two active agents per molecule. In a
different approach, copolymers of PEG and amino acids were explored
as novel biomaterials which would retain the biocompatibility
properties of PEG, but which would have the added advantage of
numerous attachment points per molecule (providing greater drug
loading), and which could be synthetically designed to suit a
variety of applications. Those of skill in the art are aware of
PEGylation techniques for the effective modification of drugs. For
example, drug delivery polymers that consist of alternating
polymers of PEG and tri-functional monomers such as lysine have
been used by VectraMed (Plainsboro, N.J.). The PEG chains
(typically 2000 daltons or less) are linked to the a- and e-amino
groups of lysine through stable urethane linkages. Such copolymers
retain the desirable properties of PEG, while providing reactive
pendent groups (the carboxylic acid groups of lysine) at strictly
controlled and predetermined intervals along the polymer chain. The
reactive pendent groups can be used for derivatization,
cross-linking, or conjugation with other molecules. These polymers
are useful in producing stable, long-circulating pro-drugs by
varying the molecular weight of the polymer, the molecular weight
of the PEG segments, and the cleavable linkage between the drug and
the polymer. The molecular weight of the PEG segments affects the
spacing of the drug/linking group complex and the amount of drug
per molecular weight of conjugate (smaller PEG segments provides
greater drug loading). In general, increasing the overall molecular
weight of the block co-polymer conjugate will increase the
circulatory half-life of the conjugate. Nevertheless, the conjugate
must either be readily degradable or have a molecular weight below
the threshold-limiting glomular filtration (e.g., less than 60
kDa).
[0075] In addition, to the polymer backbone being important in
maintaining circulatory half-life, and biodistribution, linkers may
be used to maintain the therapeutic agent in a pro-drug form until
released from the backbone polymer by a specific trigger, typically
enzyme activity in the targeted tissue. For example, this type of
tissue activated drug delivery is particularly useful where
delivery to a specific site of biodistribution is required and the
therapeutic agent is released at or near the site of pathology.
Linking group libraries for use in activated drug delivery are
known to those of skill in the art and may be based on enzyme
kinetics, prevalence of active enzyme, and cleavage specificity of
the selected disease-specific enzymes. Such linkers may be used in
modifying the protein or fragment of the protein described herein
for therapeutic delivery.
[0076] In one embodiment, the agent is an aptamer specific for
NRP-1 or OBR and thus impends the formation of the NRP-1/OBR
complex. Aptamers are a class of molecule that represents an
alternative to antibodies in term of molecular recognition.
Aptamers are oligonucleotide or oligopeptide sequences with the
capacity to recognize virtually any class of target molecules with
high affinity and specificity. Such ligands may be isolated through
Systematic Evolution of Ligands by EXponential enrichment (SELEX)
of a random sequence library. The random sequence library is
obtainable by combinatorial chemical synthesis of DNA. In this
library, each member is a linear oligomer, eventually chemically
modified, of a unique sequence.
[0077] In some embodiments, the antagonists of the invention are
used in combination with a chemotherapeutic agent for the treatment
of cancer. Chemotherapeutic agents include, but are not limited to
alkylating agents such as thiotepa and cyclosphosphamide; alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone); a camptothecin
(including the synthetic analogue topotecan); bryostatin;
callystatin; CC-1065 (including its adozelesin, carzelesin and
bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammall and calicheamicin omegall; dynemicin, including dynemicin
A; bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antiobiotic chromophores, aclacinomysins, actinomycin,
authrarnycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; amino levulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and
doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum coordination complexes such
as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;
vinorelbine; novantrone; teniposide; edatrexate; daunomycin;
aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1);
topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0078] By a "therapeutically effective amount" is meant a
sufficient amount of the antagonist of the invention to treat the
diseases at a reasonable benefit/risk ratio applicable to any
medical treatment.
[0079] It will be understood that the total daily usage of the
compounds of the present invention will be decided by the attending
physician within the scope of sound medical judgment. The specific
therapeutically effective dose level for any particular subject
will depend upon a variety of factors including the disease being
treated and the severity of the disorder; activity of the specific
compound employed; the specific composition employed, the age, body
weight, general health, sex and diet of the subject; the time of
administration, route of administration, and rate of excretion of
the specific compound employed; the duration of the treatment;
drugs used in combination or coincidential with the specific
compound employed; and like factors well known in the medical arts.
For example, it is well within the skill of the art to start doses
of the compound at levels lower than those required to achieve the
desired therapeutic effect and to gradually increase the dosage
until the desired effect is achieved. However, the daily dosage of
the products may be varied over a wide range from 0.01 to 1,000 mg
per adult per day. Typically, the compositions contain 0.01, 0.05,
0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500
mg of the active ingredient for the symptomatic adjustment of the
dosage to the subject to be treated. A medicament typically
contains from about 0.01 mg to about 500 mg of the active
ingredient, preferably from 1 mg to about 100 mg of the active
ingredient. An effective amount of the drug is ordinarily supplied
at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body
weight per day, especially from about 0.001 mg/kg to 7 mg/kg of
body weight per day.
[0080] The antagonists of the invention are administered as a
formulation in association with one or more pharmaceutically
acceptable excipients to form pharmaceutical composition.
[0081] As used herein, the term "Pharmaceutically" or
"pharmaceutically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to a mammal, especially a
human, as appropriate. A pharmaceutically acceptable carrier or
excipient refers to a non-toxic solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any
type.
[0082] Pharmaceutical compositions suitable for the delivery of
compounds of the present invention and methods for their
preparation will be readily apparent to those skilled in the
art.
[0083] In the pharmaceutical compositions of the present invention
for oral, sublingual, subcutaneous, intramuscular, intravenous,
transdermal, local or rectal administration, the active principle
(i.e. a antagonist of the invention), alone or in combination with
another active principle, can be administered in a unit
administration form, as a mixture with conventional pharmaceutical
supports, to animals and human beings. Suitable unit administration
forms comprise oral-route forms such as tablets, gel capsules,
powders, granules and oral suspensions or solutions, sublingual and
buccal administration forms, aerosols, implants, subcutaneous,
transdermal, topical, intraperitoneal, intramuscular, intravenous,
subdermal, transdermal, intrathecal and intranasal administration
forms and rectal administration forms.
[0084] In particular, the pharmaceutical compositions contain
vehicles which are pharmaceutically acceptable for a formulation
capable of being injected. These may be in particular isotonic,
sterile, saline solutions (monosodium or disodium phosphate,
sodium, potassium, calcium or magnesium chloride and the like or
mixtures of such salts), or dry, especially freeze-dried
compositions which upon addition, depending on the case, of
sterilized water or physiological saline, permit the constitution
of injectable solutions.
[0085] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and storage and
must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0086] Solutions comprising compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0087] Antagonists of the invention can be formulated into a
composition in a neutral or salt form as above described.
[0088] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetables oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin.
[0089] Sterile injectable solutions are prepared by incorporating
the antagonists of the invention in the required amount in the
appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various
sterilized active ingredients into a sterile vehicle which contains
the basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0090] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically 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.
[0091] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual
subject.
[0092] Antagonists of the invention may be formulated within a
therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or
about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10
milligrams per dose or so. Multiple doses can also be
administered.
[0093] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g. tablets or
other solids for oral administration; liposomal formulations; time
release capsules; and any other form currently used.
[0094] In some embodiments, the antagonist of the NRP-1/OBR
signaling pathway is combined with an anti-VEGF agent of the
treatment of cancer.
[0095] As used herein an "anti-VEGF agent" refers to a molecule
that inhibits VEGF-mediated angiogenesis, vasculogenesis, or
undesirable vascular permeability. For example, an anti-VEGF
therapeutic may be an antibody to or other antagonist of VEGF. An
"anti-VEGF antibody" is an antibody that binds to VEGF with
sufficient affinity and specificity to be useful in a method of the
invention. An anti-VEGF antibody will usually not bind to other
VEGF homologues such as VEGF-B or VEGF-C, or other growth factors
such as P1GF, PDGF or bFGF. A preferred anti-VEGF antibody is a
monoclonal antibody that binds to the same epitope as the
monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma
ATCC.RTM. HB 10709 and is a high-affinity anti-VEGF antibody. A
"high-affinity anti-VEGF antibody" has at least 10-fold better
affinity for VEGF than the monoclonal anti-VEGF antibody A4.6.1.
Preferably the anti-VEGF antibody is a recombinant humanized
anti-VEGF monoclonal antibody fragment generated according to WO
98/45331, including an antibody comprising the CDRs or the variable
regions of Y0317. More preferably, anti-VEGF antibody is the
antibody fragment known as ranibizumab (LUCENTIS.RTM.). The
anti-VEGF antibody ranibizumab is a humanized, affinity-matured
anti-human VEGF Fab fragment. Ranibizumab is produced by standard
recombinant technology methods in E. coli expression vector and
bacterial fermentation. Ranibizumab is not glycosylated and has a
molecular mass of -48,000 daltons. See WO98/45331 and U.S.
2003/0190317. Anti-VEGF agents include but are not limited to
bevacizumab (rhuMab VEGF, Avastin.RTM., Genentech, South San
Francisco Calif.), ranibizumab (rhuFAb V2, Lucentis.RTM.,
Genentech), pegaptanib (Macugen.RTM., Eyetech Pharmaceuticals, New
York N.Y.), sunitinib maleate (Sutent.RTM., Pfizer, Groton
Conn.)
[0096] A further object of the present invention relates to a
method for the treatment of cancer in a subject in need thereof
comprising administering the subject with a therapeutically
effective amount of an anti-VEGF agent and with an therapeutically
effective amount of a CK2 inhibitor as above described.
[0097] A further object of the present invention relates to a
method for preventing or reducing the adaptive-evasive response
(e.g. metastases, in particular micrometastases) induced by a
prolonged exposure to anti-VEGF treatment in a subject suffering
from a cancer comprising administering the subject with a
therapeutically effective amount of an antagonist of the NRP-1/OBR
signaling pathway. In particular, the NRP-1/OBR/Leptin signaling
pathway is a CK2 inhibitor. In particular, the subject suffers from
breast cancer.
[0098] The present invention also relates to a method for screening
a plurality of test substances useful for the prevention or
treatment of a disease selected from the group consisting of
cancers, obesity and obesity related diseases, cachexia, anorexia,
ureteral obstructive kidney disease, autoimmune diseases and
infectious diseases comprising the steps consisting of (a) testing
each of the test substances for its ability to inhibit the
NRP-1/OBR/Leptin signaling pathway and (b) and positively selecting
the test substances capable of inhibiting said pathway.
[0099] In some embodiments, step (a) of the screening method may
consist in determining whether the test substances i) inhibits the
formation of the complex between NRP-1 with OBR and/or Leptin, in
particular inhibits the interaction between the extracellular
domains of NRP1 and OBR ii) inhibits the phosphorylation of OBR and
NRP1 induced by CK2, iii) inhibits the nuclear translocation of the
NRP-1/OBR complex and iv) inhibits the inhibits the expression of
the genes which are expressed under control of the NRP-1/OBR/Leptin
signaling pathway.
[0100] Any method suitable for the screening of protein-protein
interactions is suitable. Whatever the embodiment of the screening
method, the complete NRP-1 protein, the complete OBR protein or the
complete CK2.alpha. protein may be used as the binding partners.
Alternatively, fragments of NRP-1 protein, OBR protein and
CK2.alpha. that include the site of interaction may be used as the
binding partners.
[0101] In some embodiments step a) of the screening method of the
invention consists of the following steps: [0102] a1) bringing into
contact the test substance to be tested with a mixture of a first
NRP-1 polypeptide or a substantially homologous or substantially
similar amino acid sequence thereof and (2) a second OBR
polypeptide or a substantially homologous or substantially similar
amino acid sequence thereof and/or (3) a Leptin polypeptide or
substantially homologous or substantially similar amino acid
sequence thereof [0103] a2) determining the ability of said test
substance to modulate the binding between said NRP-1 polypeptide
and said second OBR polypeptide and/or determining the ability of
said test substance to modulate the binding between said NRP-1
polypeptide and said second Leptin polypeptide.
[0104] In some embodiments step a) of the screening method of the
invention consists of the following steps: [0105] a1) bringing into
contact the test substance to be tested with a mixture of a first
NRP-1 or OBR polypeptide or a substantially homologous or
substantially similar amino acid sequence thereof and (2) a second
CK2.alpha. polypeptide or a substantially homologous or
substantially similar amino acid sequence thereof [0106] a2)
determining the ability of said test substance to modulate the
binding between said NRP-1 or OBR polypeptide and said second
CK2.alpha. polypeptide.
[0107] In some embodiments step a) of the screening method of the
invention consists of the following steps: [0108] a1) bringing into
contact the test substance to be tested with a mixture of a first
NRP-1 or Leptin polypeptide or a substantially homologous or
substantially similar amino acid sequence thereof and (2) a second
CK2 alpha polypeptide or a substantially homologous or
substantially similar amino acid sequence thereof [0109] a2)
determining the ability of said test substance to modulate the
binding between said NRP-1 or Leptin polypeptide and said second
CK2 alpha polypeptide.
[0110] In one embodiment the step a2) consists in generating
physical values which illustrate or not the ability of said test
substance to modulate the interaction between said first
polypeptide and said second polypeptide and comparing said values
with standard physical values obtained in the same assay performed
in the absence of the said test substance. The "physical values"
that are referred to above may be of various kinds depending of the
binding assay that is performed, but notably encompass light
absorbance values, radioactive signals and intensity value of
fluorescence signal. If after the comparison of the physical values
with the standard physical values, it is determined that the said
test substance modulates the binding between said first polypeptide
and said second polypeptide, then the candidate is positively
selected at step b).
[0111] In some embodiments, the compounds that inhibit the
interaction between (i) the NRP-1 polypeptide and (ii) the OBR
polypeptide or (iii) the Leptin polypeptide encompass those
compounds that bind either to the NRP-1 polypeptide or to OBR
polypeptide or to Leptin polypeptide and (ii) the CK2.alpha.
polypeptide encompass those compounds that bind either to the NRP-1
or OBR polypeptide or to CK2.alpha. polypeptide, provided that the
binding of the said compounds of interest then inhibits the
interaction between NRP-1 and OBR or Leptin.
[0112] In some embodiments, the compounds that inhibit the
interaction between (i) the NRP-1 or OBR or Leptin polypeptide and
(ii) the CK2 alpha polypeptide encompass those compounds that bind
either to the NRP-1 or OBR polypeptide or to CK2alpha polypeptide,
provided that the binding of the said compounds of interest then
inhibits the interaction between NRP-1 and OBR and/or functionality
of this complex.
[0113] In some embodiments, any polypeptide of the invention
suitable for the screening method (i.e. NRP-1, OBR or CK2.alpha.
polypeptides) is labelled with a detectable molecule for the
screening purposes.
[0114] According to the invention, said detectable molecule may
consist of any compound or substance that is detectable by
spectroscopic, photochemical, biochemical, immunochemical or
chemical means. For example, useful detectable molecules include
radioactive substance (including those comprising .sup.32P,
.sup.35S, .sup.3H, or .sup.125I), fluorescent dyes (including
5-bromodesosyrudin, fluorescein, acetylaminofluorene or
digoxigenin), fluorescent proteins (including GFPs and YFPs), or
detectable proteins or peptides (including biotin, polyhistidine
tails or other antigen tags like the HA antigen, the FLAG antigen,
the c-myc antigen and the DNP antigen).
[0115] According to the invention, the detectable molecule is
located at, or bound to, an amino acid residue located outside the
said amino acid sequence of interest, in order to minimise or
prevent any artefact for the binding between said polypeptides or
between the test substance and or any of said polypeptides.
[0116] In another particular embodiment, the polypeptides of the
invention are fused with a GST tag (Glutathione S-transferase). In
this embodiment, the GST moiety of the said fusion protein may be
used as detectable molecule. In the said fusion protein, the GST
may be located either at the N-terminal end or at the C-terminal
end. The GST detectable molecule may be detected when it is
subsequently brought into contact with an anti-GST antibody,
including with a labelled anti-GST antibody. Anti-GST antibodies
labelled with various detectable molecules are easily commercially
available.
[0117] In another particular embodiment, the polypeptides of the
invention are fused with a poly-histidine tag. Said poly-histidine
tag usually comprises at least four consecutive hisitidine residues
and generally at least six consecutive histidine residues. Such a
polypeptide tag may also comprise up to 20 consecutive histidine
residues. Said poly-histidine tag may be located either at the
N-terminal end or at the C-terminal end. In this embodiment, the
poly-histidine tag may be detected when it is subsequently brought
into contact with an anti-poly-histidine antibody, including with a
labelled anti-poly-histidine antibody. Anti-poly-histidine
antibodies labelled with various detectable molecules are easily
commercially available.
[0118] In a further embodiment, the polypeptides of the invention
are fused with a protein moiety consisting of either the DNA
binding domain or the activator domain of a transcription factor.
Said protein moiety domain of transcription may be located either
at the N-terminal end or at the C-terminal end. Such a DNA binding
domain may consist of the well-known DNA binding domain of LexA
protein originating form E. Coli. Moreover said activator domain of
a transcription factor may consist of the activator domain of the
well-known Gal4 protein originating from yeast.
[0119] In one embodiment of the screening method according to the
invention, the first polypeptide and the second polypeptide as
described above, comprise a portion of a transcription factor. In
said assay, the binding together of the first and second portions
generates a functional transcription factor that binds to a
specific regulatory DNA sequence, which in turn induces expression
of a reporter DNA sequence, said expression being further detected
and/or measured. A positive detection of the expression of said
reporter DNA sequence means that an active transcription factor is
formed, due to the binding together of said first polypeptide and
second polypeptide.
[0120] Usually, in a two-hybrid assay, the first and second portion
of a transcription factor consist respectively of (i) the DNA
binding domain of a transcription factor and (ii) the activator
domain of a transcription factor. In some embodiments, the DNA
binding domain and the activator domain both originate from the
same naturally occurring transcription factor. In some embodiments,
the DNA binding domain and the activator domain originate from
distinct naturally occurring factors, while, when bound together,
these two portions form an active transcription factor. The term
"portion" when used herein for transcription factor, encompass
complete proteins involved in multi protein transcription factors,
as well as specific functional protein domains of a complete
transcription factor protein.
[0121] Therefore in one embodiment of the invention, step a) of the
screening method of the invention comprises the following
steps:
[0122] (1) providing a host cell expressing: [0123] a first fusion
polypeptide between (i) the first polypeptide as define above and
(ii) a first protein portion of transcription factor [0124] a
second fusion polypeptide between (i) the second polypeptide as
defined above and (ii) a second portion of a transcription
factor
[0125] said transcription factor being active on DNA target
regulatory sequence when the first and second protein portion are
bound together and
[0126] said host cell also containing a nucleic acid comprising (i)
a regulatory DNA sequence that may be activated by said active
transcription factor and (ii) a DNA report sequence that is
operatively linked to said regulatory sequence
[0127] (2) bringing said host cell provided at step 1) into contact
with a test substance to be tested
[0128] (3) determining the expression level of said DNA reporter
sequence.
[0129] The expression level of said DNA reporter sequence that is
determined at step (3) above is compared with the expression of
said DNA reporter sequence when step (2) is omitted. A different
expression level of said DNA reporter sequence in the presence of
the test substance means that the said test substance effectively
modulates the binding between the NRP-1 polypeptide and the OBR
polypeptide and that said test substance may be positively selected
a step b) of the screening method.
[0130] Suitable host cells include, without limitation, prokaryotic
cells (such as bacteria) and eukaryotic cells (such as yeast cells,
mammalian cells, insect cells, plant cells, etc.). However
preferred host cell are yeast cells and more preferably a
Saccharomyces cerevisiae cell or a Schizosaccharomyces pombe
cell.
[0131] Similar systems of two-hybrid assays are well known in the
art and therefore can be used to perform the screening method
according to the invention (see. Fields et al. 1989; Vasavada et
al. 1991; Fearon et al. 1992; Dang et al., 1991, Chien et al. 1991,
U.S. Pat. No. 5,283,173, U.S. Pat. No. 5,667,973, U.S. Pat. No.
5,468,614, U.S. Pat. No. 5,525,490 and U.S. Pat. No. 5,637,463).
For instance, as described in these documents, the Gal4 activator
domain can be used for performing the screening method according to
the invention. Gal4 consists of two physically discrete modular
domains, one acting as the DNA binding domain, the other one
functioning as the transcription-activation domain. The yeast
expression system described in the foregoing documents takes
advantage of this property. The expression of a Gal1-LacZ reporter
gene under the control of a Gal4-activated promoter depends on the
reconstitution of Gal4 activity via protein-protein interaction.
Colonies containing interacting polypeptides are detected with a
chromogenic substrate for .beta.-galactosidase. A compete kit
(MATCHMAKER.TM.) for identifying protein-protein interactions is
commercially available from Clontech.
[0132] So in one embodiment, the first polypeptide as above defined
is fused to the DNA binding domain of Gal4 and the second
polypeptide as above defined is fused to the activation domain of
Gal4.
[0133] The expression of said detectable marker gene may be
assessed by quantifying the amount of the corresponding specific
mRNA produced. However, usually the detectable marker gene sequence
encodes for detectable protein, so that the expression level of the
said detectable marker gene is assessed by quantifying the amount
of the corresponding protein produced. Techniques for quantifying
the amount of mRNA or protein are well known in the art. For
example, the detectable marker gene placed under the control of
regulatory sequence may consist of the .beta.-galactosidase as
above described.
[0134] In another one embodiment, step a) comprises a step of
subjecting to a gel migration assay the mixture of the first
polypeptide and the second polypeptide as above defined, with or
without the test substance to be tested and then measuring the
binding of the said polypeptides altogether by performing a
detection of the complexes formed between said polypeptides. The
gel migration assay can be carried out as known by the one skilled
in the art.
[0135] Therefore in one embodiment of the invention, step a) of the
screening method of the invention comprises the following
steps:
[0136] (1) providing the first polypeptide and the second
polypeptide as defined above
[0137] (2) bringing into contact the test substance to be tested
with said polypeptides
[0138] (3) performing a gel migration assay a suitable migration
substrate with said polypeptides and said test substance as
obtained at step (2)
[0139] (4) detecting and quantifying the complexes formed between
said polypeptides on the migration assay as performed at step
(3).
[0140] The presence or the amount of the complexes formed between
the polypeptides are then compared with the results obtained when
the assay is performed in the absence of the test substance to be
tested.
[0141] The detection of the complexes formed between the said two
polypeptides may be easily performed by staining the migration gel
with a suitable dye and then determining the protein bands
corresponding to the protein analysed since the complexes formed
between the first and the second polypeptides possess a specific
apparent molecular weight. Staining of proteins in gels may be done
using any well known methods in the art. Suitable gels are well
known in the art but it is preferred to use non denaturant gels. In
a general manner, western blotting assays are well known in the art
and have been widely described.
[0142] In a particular embodiment, the protein bands corresponding
to the polypeptides submitted to the gel migration assay can be
detected by specific antibodies. It may used both antibodies
directed against the first polypeptides (e.g. NRP-1 polypeptides)
and antibodies specifically directed against the second
polypeptides (e.g. OBR polypeptides).
[0143] In another embodiment, the said two polypeptides are
labelled with a detectable antigen as above described. Therefore,
the proteins bands can be detected by specific antibodies directed
against said detectable antigen. Preferably, the detectable antigen
conjugates to the first polypeptide is different from the antigen
conjugated to the second polypeptide. For instance, the first
polypeptide can be fused to a GST detectable antigen and the second
polypeptide can be fused with the HA antigen. Then the protein
complexes formed between the two polypeptides may be quantified and
determined with antibodies directed against the GST and HA antigens
respectively.
[0144] In another embodiment, step a) included the use of an
optical biosensor such as described by Edwards et al. (1997) or
also by Szabo et al. (1995). This technique allows the detection of
interactions between molecules in real time, without the need of
labelled molecules. This technique is indeed based on the surface
plasmon resonance (SPR) phenomenon. Briefly, a first protein
partner is attached to a surface (such as a carboxymethyl dextran
matrix). Then the second protein partner is incubated with the
previously immobilised first partner, in the presence or absence of
the test substance to be tested. Then the binding including the
binding level, or the absence of binding between said protein
partner is detected. For this purpose, a light beam is directed
towards the side of the surface area of the substrate that does not
contain the sample to be tested and is reflected by said surface.
The SPR phenomenon causes a decrease in the intensity of the
reflected light with a combination of angle and wavelength. The
binding of the first and second protein partner causes a change in
the refraction index on the substrate surface, which change is
detected as a change in the SPR signal.
[0145] In another one embodiment of the invention, the screening
method includes the use of affinity chromatography. Test substances
for use in the screening method above can also be selected by any
immunoaffinity chromatography technique using any chromatographic
substrate onto which (i) the first polypeptide or (ii) the second
polypeptide as above defined, has previously been immobilised,
according to techniques well known from the one skilled in the art.
Briefly, the NRP-1 polypeptide or the OBR polypeptide as above
defined may be attached to a column using conventional techniques
including chemical coupling to a suitable column matrix such as
agarose, Affi Gel.RTM., or other matrices familiar to those of
skill in the art. In some embodiment of this method, the affinity
column contains chimeric proteins in which the NRP-1 polypeptide or
OBR polypeptide as above defined, is fused to
glutathion-s-transferase (GST). Then a test substance is brought
into contact with the chromatographic substrate of the affinity
column previously, simultaneously or subsequently to the other
polypeptide among the said first and second polypeptide. The after
washing, the chromatography substrate is eluted and the collected
elution liquid is analysed by detection and/or quantification of
the said later applied first or second polypeptide, so as to
determine if, and/or to which extent, the test substance has
modulated the binding between (i) first polypeptide and (ii) the
second polypeptide.
[0146] In another one embodiment of the screening method according
to the invention, the first polypeptide and the second polypeptide
as above defined are labelled with a fluorescent molecule or
substrate. Therefore, the potential alteration effect of the test
substance to be tested on the binding between the first polypeptide
and the second polypeptide as above defined is determined by
fluorescence quantification.
[0147] For example, the first polypeptide and the second
polypeptide as above defined may be fused with auto-fluorescent
polypeptides, as GFP or YFPs as above described. The first
polypeptide and the second polypeptide as above defined may also be
labelled with fluorescent molecules that are suitable for
performing fluorescence detection and/or quantification for the
binding between said polypeptides using fluorescence energy
transfer (FRET) assay. The first polypeptide and the second
polypeptide as above defined may be directly labelled with
fluorescent molecules, by covalent chemical linkage with the
fluorescent molecule as GFP or YFP. The first polypeptide and the
second polypeptide as above defined may also be indirectly labelled
with fluorescent molecules, for example, by non covalent linkage
between said polypeptides and said fluorescent molecule. Actually,
said first polypeptide and second polypeptide as above defined may
be fused with a receptor or ligand and said fluorescent molecule
may be fused with the corresponding ligand or receptor, so that the
fluorecent molecule can non-covalently bind to said first
polypeptide and second polypeptide. A suitable receptor/ligand
couple may be the biotin/streptavifin paired member or may be
selected among an antigen/antibody paired member. For example, a
polypeptide according to the invention may be fused to a
poly-histidine tail and the fluorescent molecule may be fused with
an antibody directed against the poly-histidine tail.
[0148] As already specified, step a) of the screening method
according to the invention encompasses determination of the ability
of the test substance to modulate the interaction between the first
polypeptide and the second polypeptide as above defined by
fluorescence assays using FRET. Thus, in a particular embodiment,
the first polypeptide as above defined is labelled with a first
fluorophore substance and the second polypeptide is labelled with a
second fluorophore substance. The first fluorophore substance may
have a wavelength value that is substantially equal to the
excitation wavelength value of the second fluorophore, whereby the
bind of said first and second polypeptides is detected by measuring
the fluorescence signal intensity emitted at the emission
wavelength of the second fluorophore substance. Alternatively, the
second fluorophore substance may also have an emission wavelength
value of the first fluorophore, whereby the binding of said and
second polypeptides is detected by measuring the fluorescence
signal intensity emitted at the wavelength of the first fluorophore
substance.
[0149] The fluorophores used may be of various suitable kinds, such
as the well-known lanthanide chelates. These chelates have been
described as having chemical stability, long-lived fluorescence
(greater than 0.1 ms lifetime) after bioconjugation and significant
energy-transfer in specificity bioaffinity assay. Document U.S.
Pat. No. 5,162,508 discloses bipyridine cryptates. Polycarboxylate
chelators with TEKES type photosensitizers (EP0203047A1) and
terpyridine type photosensitizers (EP0649020A1) are known. Document
WO96/00901 discloses diethylenetriaminepentaacetic acid (DPTA)
chelates which used carbostyril as sensitizer. Additional DPT
chelates with other sensitizer and other tracer metal are known for
diagnostic or imaging uses (e.g., EP0450742A1).
[0150] In a preferred embodiment, the fluorescence assay performed
at step a) of the screening method consists of a Homogeneous Time
Resolved Fluorescence (HTRF) assay, such as described in document
WO 00/01663 or U.S. Pat. No. 6,740,756, the entire content of both
documents being herein incorporated by reference. HTRF is a TR-FRET
based technology that uses the principles of both TRF
(time-resolved fluorescence) and FRET. More specifically, the one
skilled in the are may use a HTRF assay based on the time-resolved
amplified cryptate emission (TRACE) technology as described in
Leblanc et al. (2002). The HTRF donor fluorophore is Europium
Cryptate, which has the long-lived emissions of lanthanides coupled
with the stability of cryptate encapsulation. XL665, a modified
allophycocyanin purified from red algae, is the HTRF primary
acceptor fluorophore. When these two fluorophores are brought
together by a biomolecular interaction, a portion of the energy
captured by the Cryptate during excitation is released through
fluorescence emission at 620 nm, while the remaining energy is
transferred to XL665. This energy is then released by XL665 as
specific fluorescence at 665 nm. Light at 665 nm is emitted only
through FRET with Europium. Because Europium Cryptate is always
present in the assay, light at 620 nm is detected even when the
biomolecular interaction does not bring XL665 within close
proximity.
[0151] Therefore in one embodiment, step a) of the screening method
may therefore comprises the steps consisting of:
[0152] (1) bringing into contact a pre-assay sample comprising:
[0153] a first polypeptide fused to a first antigen, [0154] a
second polypeptide fused to a second antigen [0155] a test
substance to be tested
[0156] (2) adding to the said pre assay sample of step (2): [0157]
at least one antibody labelled with a European Cryptate which is
specifically directed against the first said antigen [0158] at
least one antibody labelled with XL665 directed against the second
said antigen
[0159] (3) illuminating the assay sample of step (2) at the
excitation wavelength of the said European Cryptate
[0160] (4) detecting and/or quantifying the fluorescence signal
emitted at the XL665 emission wavelength
[0161] (5) comparing the fluorescence signal obtained at step (4)
to the fluorescence obtained wherein pre assay sample of step (1)
is prepared in the absence of the test substance to be tested.
[0162] If at step (5) as above described, the intensity value of
the fluorescence signal is different (lower or higher) than the
intensity value of the fluorescence signal found when pre assay
sample of step (1) is prepared in the absence of the test substance
to be tested, then the test substance may be positively selected at
step b) of the screening method.
[0163] Antibodies labelled with a European Cryptate or labelled
with XL665 can be directed against different antigens of interest
including GST, poly-histidine tail, DNP, c-myx, HA antigen and FLAG
which include. Such antibodies encompass those which are
commercially available from CisBio (Bedfors, Mass., USA), and
notably those referred to as 61GSTKLA or 61HISKLB respectively.
[0164] The test substances that have been positively selected at
the end of any one of the embodiments of the in vitro screening
which has been described previously in the present specification
may be subjected to further selection steps in view of further
assaying its properties on NRP-1 phosphorylation, OBR
phosphorylation, effects on the gene expression mediated by the
NRP-1/OBR/Leptin signaling pathway or cellular functions mediated
by the NRP-1/OBR/Leptin signaling pathway (e.g. cell migration).
Thus the screening method of the present invention further
comprises the steps of screening the compounds positively selected
at the end of step i) for their abilities to inhibit i) NRP-1
phosphorylation induced by CK2, to inhibit the OBR phosphorylation
induced by CK2, to inhibit the translocation of the nuclear
NRP-1/OBR complex, to inhibit the gene expression mediated by the
NRP-1/OBR/Leptin signaling pathway or to inhibit the cellular
functions mediated by the NRP-1/OBR/Leptin signaling pathway (e.g.
cell migration).
[0165] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0166] FIG. 1: Endogenous NRP-1/OBR detection in breast cancer cell
line. A-NRP-1 and OBR mRNA detection in MDA-MB231 positive cells
compared to T47D negative cells. B. NRP-1/OBR complex detection by
Proximity Ligation Assay technology (PLA: Duolink) In whole cell
(red dot) and in the nucleus (white dot: merge between red dot and
dapi) http://www.olink.com/products/duolink/how-use-duolink.
NRP-1/OBR complex detection after NRP-1 overexpression in T47Dneg
Cells alone or with OBR overexpression: a very low NRP-1/OBR
complex detection in empty vector condition (pcDNA3). An increase
of NRP-1/OBR complex detection when NRP-1 is overexpressed alone
(pcDNA3-NRP-1) or with OBR (pcDNA3NRP-1/OBR). No change in OBR
overexpression alone compared to empty vector indicating the
specificity of the signal of NRP-1/OBR complex. D. NRP-1/OBR
detection by co-immuno-precipitation: in the cytoplasmic fraction
(C) and in nuclear fraction (N) in MDA-MB231 compared toT 470.
[0167] FIG. 2. NRP-1/OBR complex is dependent of the activity of
Protein-kinase CK2: A. NRP-1 and OBR are both a substrate of the
holoenzyme CK2: In vitro phosphorylation of the recombinant full
length NRP-1 and the length OBR indicate that the CK2.beta. subunit
is indispensable for the CK2 activity. B. Detection of the NRP-1
and CK2.beta. subunit interaction: By using the PLA technology, the
in vitro data showing the implication of CK2.beta. subunit in the
phosphorylation of NRP-1 by CK2 are in part confirmed in MDA-MB231
cells by the detection of CK2.beta. interaction with NRP-1 as
indicated by the duolink (white dot). C. inhibition of the
NRP-1/OBR by CX4945, the ATP competitive inhibitor of the CK2
(Phase II Clinical Trial): MDA-MB231 treatment with the CX4945 (5
.mu.M) during 4 h induces a decrease in NRP-1/OBR complex detection
by PLA (red dot) and its nuclear localization (white dot) under the
leptin stimulation (10 nM). D. inhibition of the NRP-1/OBR by the
simultaneous CK2.alpha. and CK2.alpha.' expression silencing using
siRNA that confirms the CK2 implication In the NRP-1/OBR complex
formation and nuclear localization.
[0168] FIG. 3. NRP1/OBR complex formation inhibited by two
alosteric inhibitors of Protein-kinase CK2 in skin fibroblast of
healthy donor and anorexic patient. The NRP-1/OBR complex (Red dot)
is detected by de Proximity Ligation assay technology (PLA or
Ouolink). Cells were grown in DMEM plus 20% of fetal bovine serum+
Sodium pyruvate+ LGiutamate and penicillin and streptomycin. Cells
were treated or not with D3.1 or M4 CK2 inhibitor during 4 hours.
Cells were washed with PBS1X and fixed with iced acetone and
rehydrated with PBS before blockage and staining with a Goat
ant-Human leptin and Rabbit anti-Neuropilin-1 and Mouse anti-Leptin
receptor OBR. Leptin was detected using a donkey and Goat Alexa 488
(Green) and NRP-1 and OBR with anti-Rabbit-PLA minus and
antimouse-PLA plus as described in the link indicated above. The
NRP-1/OBR complex is inhibited by D3.1 and M4 treatment. The D3.1
seems more efficient. Interestingly, Leptin is also inhibited by
CK2 inhibitors.
[0169] FIG. 4. Leptin and NRP-1/OBR complex detection in the
Peripheral Blood Mononuclear Cells (PBMC) of the anorexic patient:
Leptin and NRP-1/OBR complex are detected in PBMC freshly isolated
from heparinized whole blood by use of Ficoll density gradient
centrifugation. As it was observed in MDA-MB231 cells breast cancer
cell line and in fibroblasts from healthy donor and anorexic
patient, NRP-1/OBR complex is detected in the nucleus. Cells.
NRP1/OBR complex is also detected in macrophages of Healthy donor
(data not shown). As is proposed in the patent, NRP-1/OBR could be
a target in immune system failure implicating exacerbated Leptin
and NRP-1/OBR signaling pathway.
[0170] FIG. 5. NRP-1, OBR and leptin interactions confirmation
using a Bio-Layer Interferometry (BLI) technology
(http://www.fortebio.com/bli-technology.html): A-Leptin (OB)
interaction to OBR is used as a positive control. Human
neuropilin-1 (hNRP-1) interact with human OBR with a high affinity
and specifically with human leptin (hOB) but not with murine leptin
(mOB). VEGF165 binding to hNRP-1 is used as a positive control.
B-Leptin and Avastin association increases MDA-MB231 cells
transmigration through 80 .mu.m pore Insert. Avastin treatment may
prevent a negative feedback control by VEGF of the pro-angiogenic
signaling pathway of Leptin. VEGF binds to OBR which confirms our
hypothesis that should be more investigated.
EXAMPLE 1
Neuropilin-1 (NRP-1) Induces Breast Cancer Metastasis Through its
New Partners Leptin/OBR Complex: Identification of Nuclear NRP-1
Associated to Genes Sequences with RNA Polymerase II and
Transcription Factor Binding Site
[0171] In Vitro and In Vivo Implication of NRP-1 and Leptin in
Breast Cancer Cell Line Migration
[0172] First MBA-MB-231 and T47D breast cancer cells line were
selected for their high and undetectable NRP-1 expression
respectively and the expression of Leptin receptor (OBR). In order
to investigate NRP-1 and Leptin association in breast cancer cell
line migration, we proceeded to NRP-1 expression silencing in
MDA-MB-231 using either siRNA or shRNA approaches or NRP-1
overexpression in T47D. In contrast to several published data, we
were unable to observe MDA-MB-231 and T47D-NRP-1 proliferation
under Leptin stimulation. Surprisingly, NRP-1 repression by RNA
silencing induces MDA-MD-231 proliferation. In contrast Leptin
induced MDA-MB-231 migration that was decreased in siNRP-1
conditions. The in vitro data were confirmed in vivo by MDA-MB-231
and T47D xenografts in Nude mice. Modulation of NRP-1 expression
either by shRNA silencing in MDA-MB-231 or by overexpression in
T47D induced an increase of proliferation and a decrease of lymph
node infiltration by MDA-MB-231-shNRP-1 and a decrease of
proliferation and an increase of lymph node infiltration by
T47D-NRP-1-RFP in mice treated with Leptin.
[0173] Leptin Induces NRP-1/OBR Complex Formation Resulting in OBR
Oligomerization (Duolink, HTRF and BRET) and Nuclear Translocation
of the Complex.
[0174] To confirm a direct association of NRP-1 and Leptin in
breast cancer cell migration, we investigated the NRP-1 and OBR
interaction by using proximity ligation assay technology (Duolink)
in MDA-MB-231 and in T47D-NRP-1. Since OBR is a member of the class
I cytokine receptor family, we asked the question if the formation
of the complex NRP-1/OBR can or not be modulated by Leptin. This
question was investigated endogenously in MDA-MB-231 or by
transient transfection of T47D either by NRP-1 or by NRP-1/OBR or
OBR alone. After 24 h of transfection, cells were serum starved for
16 h then stimulated with Leptin (10 nM). After 3 h, the
stimulation was stopped by removing the medium, cells was first
washed then fixed and NRP-1/OBR complex formation was analyzed by
Duolink in situ Proximity Ligation Assay (PLA) and by confocal
microscopy detection. Co-localization was analyzed using JACoP
(ImageJ; National Institutes of Health, Bethesda, Md.) software.
Co-localization between molecules was indicated by a positive
Pearson coefficient (r). First, the expression of NRP-1 in
transfected T47D was confirmed by RT-PCR. NRP-1 forms a complex
with endogenous OBR as it is shown by the Duolink detection as red
dot in cells transfected with pcDNA3-NRP-1. The Duolink signal
increased in the T47D co-transfected with NRP-1 and OBR. The
Duolink signals in T47D transfected with either the empty vector
pcDNA3 or with the OBR expressing vector were similar. The same
results are observed in MDA-MB-231. Surprisingly, the Duolink
signal was detected in the nucleus as shown in white dot. By using
subcellular protein fractionation kit, NRP-1 and OBR localization
in cytoplasmic (C), membrane (M), in the total nuclear extract (N)
or in soluble nuclear (SN) and chromatin bound fractions (CB) was
analyzed by western blotting either in total lysate or after
coimmunoprecipitation. The purity of fraction was assessed by the
detection of specific proteins of each fraction such as Hsp90 for
the C fraction, SP1 and HDAC2 for the SN and CB fractions and
specifically the Calreticulin for endoplasmic reticulum (ER) in
order to detect any contamination by the ER membrane fraction in
the nuclear fractions. As suspected, OBR was coimmunoprecipitated
with NRP-1 in the cytoplasmic (C) and in the nuclear fraction (N)
of MDA-MB-231 but not in T47D. As well as Phosphorylated OBR
(P-OBR), NRP-1 was Detected in Cytoplasmic Fraction and in Nuclear
Fractions SN and CB of 16 h Serum starved MDA-MB-231 and stimulated
with 10 nM of Leptin during 3 h at 37.degree. C. The P-OBR and
NRP-1 coimmunoprecipitated in the nuclear fractions, increasing
with Leptin stimulation as observed by confocal microscopy.
[0175] NRP-1/OBR Complex Formation and its Nuclear Translocation
Implicate NRP-1 Phosphorylation by Protein-Kinase CK2.
[0176] Beside the modulation of the NRP-1/OBR complex formation by
Leptin and OBR phosphorylation, we postulated a possible
phosphorylation of NRP-1 that can regulate the translocation of the
complex to the nucleus. Two putative phosphorylation sites of NRP-1
have been reported with no more investigation. The first one at the
extracellular B domain as reported by Shintani et al in 2009 with
no antibody available and the second one at the Threonine 916
located in the cytoplasmic domain as it was reported by Kyle et al
in 2011. Since a specific antibody against P-NRP-1 at the T916 was
available, we investigated by coimmunoprecipitation the detection
of P-NRP-1 in MDA-MB-231 after serum starvation and stimulation by
Leptin (10 nM). Surprisingly, NRP-1 is phosphorylated upon Leptin
stimulation and is located in the nucleus either in the soluble
fraction or in chromatin bound fraction with P-OBR. To confirm this
phosphorylation state, we investigated the possible implication of
a Serine/threonine Kinase CK2 using one of its first chemical
inhibitor (DRB). In serum starved condition, P-NRP-1 was detected
as polarized spot in the cytoplasm and the nucleus of MDA-MB-231.
Interestingly, in the MDA-MB-231 stimulated with Leptin (10 nM),
P-NRP-1 was detected as diffuse staining in the nucleus. DRB
treatment (50 .mu.M) of Leptin-stimulated cells led not only to the
inhibition of the diffuse nuclear P-NRP-1 staining but also to the
decrease of P-NRP-1 polarized spot observed in unstimulated cells.
The inhibition was specific since, in stimulated condition combined
with DMSO at the same dilution of DRB, we were able to observe a
diffuse nuclear P-NRP-1 staining.
[0177] NRP-1 phosphorylation by CK2 was confirmed in vitro using
recombinant protein and .sup.32P-ATP. In contrast to soluble form
of NRP-1 (data not shown), only full length of NRP-1 was
phosphorylated in a CK2.alpha. and CK2.beta. dependent manner.
Since NRP-1 phosphorylation seems to be induced not only with CK2
but also in response to Leptin stimulation we asked the question if
NRP-1/OBR complex stability can be also affected by CK2
inhibition.
[0178] The investigation was assessed on MDA-MB-231 stably
transfected with lentiviral expressing shNRP-1 plasmid and on T47D
stably transduced with retroviral expressing vector for NRP-1-RFP
stimulated or not with Leptin combined or not with CK2 inhibitors
(DRB or TBB).
[0179] As attempted, NRP-1/OBR complex was detected by confocal
microscopy after by Duolink in situ Proximity Ligation Assay (PLA)
in MDA-MB-231 stimulated with Leptin (10 nM). This detection was
also observed in MDA-MB-231-shGFP but very less in
MDA-MB-231-shNRP-1 confirming NRP-1 association with OBR.
Interestingly the NRP-1/OBR complex formation was inhibited by CK2
inhibitors but not by DMSO. The same effect on NRP-1/OBR complex
formation was observed in T47D stably transduced with
NRP-1-RFP.
[0180] The results were confirmed by silencing CK2 subunits by
siRNA in MDA-MB-231. As observed in TBB and DBB treatment, a
decrease of CK2 expression induced a decrease of NRP-1/OBR complex
formation in MDA-MB-231-siCK2.alpha. and MDA-MB-231-siCK2.alpha.'
but not in MDA-MB-231-wt and MDA-MB-231-siGFP.
[0181] Since both NRP-1/OBR complex formation and NRP-1
phosphorylation were abolished by CK2 inhibition, we supposed that
OBR signaling pathway could be also affected by CK2 inhibition. As
suspected, Leptin induced OBR and STAT3 phosphorylation increase in
the cytoplasmic and nuclear fractions that was inhibited by TBB
treatment in a dose dependent manner.
[0182] NRP-1 Chip-Seq Analysis of Sample Generated from MDA-MB-231
and Transcriptome Analysis of RNA Extracts from T47D-NRP-1
Xenograft LED to Identify Genes Implicated in Breast Cancer
Metastasis with Sequences Containing Binding Site of RNA Polymerase
II and Transcription Factors and Increased Expression
Respectively.
[0183] On the basis of the NRP-1 detection in the nucleus and
especially in the chromatin bound fraction, we performed a Chip-Seq
in 16 h serum starved MDA-MB-231 and treated with increasing doses
of Leptin (0, 2 and 10 nM) for 3 hours. Chromatin
immunoprecipitation (ChIP) assay was conducted with EZ-Magna
ChIP.TM. A using a purified polyclonal rabbit anti-NRP-1 (a
generous gift of Alex Kolodkin team). The samples are designed as
follow: input mix comprises input of non stimulated cells (NS),
cells stimulated with 2 nM of Leptin and cells stimulated with 10
nM of Leptin. For the IgG control, we analyzed a mixt of chip with
IgG control of NS, cells stimulated with 2 nM of leptin and cells
stimulated with 10 nM of Leptin. The Chip-Seq analysis can be
summarized in 1) mapping of reads on complete genome of Homo
Sapiens (GRCh37), 2) peak detection and coverage by detecting peaks
on the reference genome and computing their coverage in mapped
reads consisted in finding region enriched in one of the IP sample
(NS, 2 nM and 10 nM of leptin) but absent in both control samples
(Input mix and IgG mixt) and 3) normalization and comparison of
libraries. The number and percentage of mapped reads for each
library were 23'226'278 (79.66%) for NS Chip sample, 25'247'185
(76.13%) for 2 nM Chip sample and 29'694'919 (75.27%) for 10 nM
Chip sample. The peak detection was performed with the software
SEQMONK. A total of 20'495 peaks were detected and annotated with
the closest gene located within 10 kb of each peak. We detected
3,844 peaks at a distance >10 kb from a gene, 13,893 peaks
overlapping a gene, 1,336 peaks downstream from a gene <10 kb
and 1,422 peaks upstream from a gene >10 kb. Interestingly the
number of detected peaks increased with the concentration of
Leptin. We consider 3181 genes found in the annotations of the
20'495 peaks from all samples. We then map this set of genes to
Gene Ontology terms (GO) database and Pathways from Reactome
database, and extract the 50 most represented terms and pathways
from this databases. More interestingly, identified pathways are
globally known to implicate Leptin and NRP-1 such as signal
transduction, metabolism, immune system, axon guidance and
metabolism of lipids.
[0184] By using ENCODE data at UCSC website, we compared the NRP-1
Chip-Seq to reported transcription factors Chip-Seq. The matched
sequences led to identify a binding sequence of 140 transcription
factors beside RNA polymerase II (Pol2). The main enriched
sequences that overlap the starting site of the genes are those
containing binding site of transcription factors known to form a
complex with Pol2 such CTCF or those known to be a partner of each
other such as c-Myc and Max. Interestingly, the count of the
sequence increased with Leptin stimulation but with the maximum
reached at 2 nM of Leptin but no significant increase between 2 nM
and 10 nM of Leptin. The detection of the Pol2 binding sequence
raised the question if NRP-1 forms a complex with Pol2. By using an
antibody against a carboxy-terminal domain (CTD) of Pol2, we were
able to confirm an interaction between NRP-1 and RNA Pol2 as it is
shown by Duolink in situ Proximity Ligation Assay with the
increasing signal in MDA-MB-231 stimulated with 10 nM of Leptin
compared to unstimulated cells. The Duolink results were confirmed
by the co-immunoprecipitation of Pol2 with NRP-1 using a Rabbit
polyclonal anti-NRP-1.
[0185] In order to give a sense to these results in terms of
possible implication of NRP-1/OBR/Leptin signaling pathways in the
induction of the gene expressions implicated in cell migration, we
analyzed a transcriptome of xenografts. Although the Chip-Seq was
realized on MDA-MB-231, we opted to realize a transcriptome of T47D
overexpressing NRP-1-RFP xenografts compared to T47D-wt and
T47D-RFP treated or not with Leptin regarding to the lymph node
infiltration induced by the NRP-1 overexpression and its increase
by Leptin treatment. To determine any direct association of NRP-1
in gene expression, we realize first a global analysis of
transcriptome of T47D-NRP-1 xenografts treated and not treated with
Leptin (n=8) compared to T47D-wt and T47D-RFP xenografts treated
and not treated with Leptin (n=8). By using Ingenuity Pathways
Analysis (IPA), 32 genes implicated in cell movement, invasion and
migration of breast cancer cell lines were increased by NRP-1
overexpression and 5 genes were decreased. The maximum increase was
observed for lysyl oxidase gene (LOX, fold increase 2.46, p=0.008)
and interestingly, the majority of these genes were enriched in the
Chip-Seq of MDA-MB-231 as and some of them contained peaks
corresponding to transcription factor binding sequence such as
SERPINE1 (plasminogen activator inhibitor type1 gene, fold increase
1.64; p=0.019) for Pol2 binding sequence and BCAR1 (breast cancer
anti-estrogen resistance 1 gene, fold increase 1.72; p=0.003) for
CTCF binding sequence. The gene expression decrease is observed for
TNFSF10 (tumor necrosis factor (ligand) superfamily, member 10,
fold decrease 3.05, p=0.034) enriched also in the Chip-Seq but with
no transcription factor binding sequence association. The analysis
of gene expression in Leptin treated T47D-NRP-1-RFP compared to no
treated xenografts, highlighted 66 genes with 31 genes enriched in
the Chip-Seq of MDA-MB-231 and that were not detected in T47D-wt
and T47D-RFP treated or not with Leptin. Only 4 genes enriched in
the Chip-Seq contained peaks associated to transcription factor
binding site such as FUS (fused in sarcoma, fold increase 1.79;
p=0.045) for Pol2 and C12orf45 (chromosome 12 open reading frame,
fold increase 1.29; p=0.03) for CTCF. The Ingenuity Pathway
Analysis led to confirm that some genes overexpressed by the
expression of NRP-1 in T47D were related to Leptin and CK2 networks
such as SERPINE1, IGFBP3 and CD44. Since Plasminogen Activator
Inhibitor 1 (PAI.1) related to SERPINE1 gene has been associated to
breast cancer invasion and metastasis, we evaluated PAI.1
expression by immunostaining in T47D-NRP-1-RFP xenografts compared
to T47D-wt and T47D-RFP. Interestingly, we observe an increase
staining of PAI.1 in T47D-NRP-1-RFP xenografts compared to T47D-wt
and T47D-RFP and this increase correlated with Leptin
treatment.
[0186] On the basis of the association of NRP-1 overexpression in
T47D in the infiltration of lymph node by T47D-NRP-1-RFP and in the
expression increase of genes reported to be implicated in lymph
node infiltration in breast cancer such as N-myc downstream
regulated 1 (NDRG1, fold increase 1.81; p=0.006) which was also
enriched in the Chip-Seq with peaks related to the transcription
factor binding sequence of CTCF and p300, we assessed NRP-1/OBR
complex by Duolink in situ Proximity Ligation Assay in infiltrated
lymph node by T47D-NRP-1-RFP. Interestingly, we were able to detect
T47D-NRP-1-RFP cells as shown in red du to the RFP expression
either in treated or not treated xenograft by Leptin but with an
increase of red cells in treated condition. NRP-1/OBR complex
represented by white dot was detected as well as in treated or not
treated cells with nuclear localization indicating NRP-1/OBR
complex signaling in the infiltrated lymph node.
Discussion
[0187] Neuropolin-1 Induced Breast Cancer Migration and Lymph Node
Infiltration Through Leptin/OBR Complex.
[0188] Several studies have associated NRP-1 in tumor progression
and metastasis independently of its known ligand such as VEGFs and
Semaphorins. On the basis of our published data on adipocytes role
in the regulation of granulopoiesis through NRP-1 and on the
growing connection between obesity and cancer progression, we
postulated that the adipokine Leptin and its main receptor OBR may
be a new partner of NRP-1. To confirm this hypothesis, we opted to
investigate NRP-1 and Leptin association in breast cancer cell line
migration. MDA-MB-231 and T47D cell lines were selected for high
and undetectable expression of NRP-1 respectively but expressing
OBR at different level.
[0189] Leptin induced in vitro MDA-MB-231 migration that was
decreased by NRP-1 repression using siRNA. Lel Xu et al have
reported a direct evidence of NRP-1 up-regulation in tumors treated
by anti-VEGF (Bevacizumab). Interestingly, Leptin increased
MDA-MB-231 migration induced by 10 .mu.g/ml of Bevacizumab. This
observation can be a first explanation of breast cancer therapy
failure (increase of Progression survival while decreasing overall
survival) by Bevacizumad and that was revoked by the FDA (Food and
Drugs Administration, US).
[0190] This need to be more investigated and may be a new strategy
for combined anti-Leptin and anti-VEGF therapies in breast cancer.
As demonstrated by lymph node infiltration, NRP-1 implication in
breast cancer cell line migration was confirmed in vivo by its
overexpression in T47D cell lines known for their negative
expression of NRP-1 and by shNRP-1 silencing in MDA-MB-231 known
for their high NRP-1 expression and aggressiveness. Leptin
treatment significantly increased lymph node infiltration by
T47D-NRP-1-RFP stained for the human KL1. However, in contrast to
MDA-MB-231-shGFP, Leptin treatment did not increase lymph node
infiltration by MDA-MB-231-shNRP-1 as it was evaluated by KL1
staining. In vitro and in vivo data are a direct evidence of NRP-1
and Leptin association in breast cancer cell line migration. This
implication of Leptin and NRP-1 are in agreement of published data
but with no association between Leptin and NRP-1. Interestingly
other studies in other area have reported Leptin, OBR and NRP-1 but
with no any evidence of their association as a signaling complex,
for example the expression of Leptin and NRP-1 in a case of
idiopathic choroidal neovascularization and NRP-1 expression
alteration in OBR deficient mice (db/db).
[0191] Neuropilin-1 Forms a Complex with OBR and Induces its
Oligomerization Through a Direct Binding of Leptin to OBR andbut
not to NRP-1.
[0192] A direct association of NRP-1 and Leptin to breast cancer
cell lines migrations was confirmed by the detection of NRP-1/OBR
complex in MDA-MB-231 and T47D overexpressing NRP-1.
[0193] By using a Duolink PLA, we showed that a direct interaction,
between NRP-1 and OBR, takes place under Leptin stimulation
compared to serum starved and unstimulated cells. This observation
is in agreement with OBR propriety as a member of the class I
cytokine receptor family which dimerization is dependent on ligand
binding. The NRP-1/OBR complex was confirmed by
coimmunoprecipitation of NRP-1 and OBR endogenously in MDA-MB-231
and in transfected Hela cells with either OBR-GFP and/or NRP-1.
Immunoprecipitation of OBR-GFP using a specific anti-GFP antibody
led to NRP-1 detection in NRP-1 and OBR-GFP co-transfection
condition but not in Hela cells transfected with NRP-1 alone
confirming the specificity of this complex.
[0194] NRP-1 has been reported to bind directly VEGF increasing
thus the binding affinity of VEGF to its receptor VEGFR2 (ref) and
Sema3A enabling thus its binding to Plexin 3A. Our study led us to
highlight a new mechanism of NRP-1 function. In contrast to VEGF
and Sema3A, Leptin is unable to bind directly to NRP-1 as it is
demonstrated by HTRF and confocal microscopy. (show data of direct
binding of leptin to NRP1) However, Leptin binding to OBR induces
NRP-1/OBR complex formation resulting in physical proximity between
NRP-1 and Leptin as it was demonstrated by the energy transfer from
NRP-1 to Leptin in HTRF analysis and OBR oligomerization following
NRP-1 binding as demonstrated by BRET analysis. NRP-1 repression by
siNRP-1 decreased the BRET signal and OBR oligomerization. The
consequence of OBR oligomerization can be linked to the
acceleration and the increase of leptin/OBR signaling by NRP-1
overexpression as it was demonstrated in PAEC-NRP-1 cells compared
to PAEC-wt. The increase of OBR signaling by NRP-1 can explain the
metastatic property acquired by T47D overexpressing NRP-1 as
demonstrated by the lymph node infiltration and its decrease in
MDA-MB-231-shNRP-1.
[0195] NRP-1/OBR Complex Formation and its Nuclear Translocation
Depend on NRP-1 Phosphorylation by Protein-Kinase CK2.
[0196] The Duolink PLA assay revealed by confocal microscopy showed
NRP-1/OBR complex in the nucleus as represented in white dot
resulting from co-localization analysis of the Duolink in red dot
and the DAPI staining of the nucleus using Jacop software. It is
for the first time that membrane complex receptors are reported to
translocate to the nucleus. This surprising result was confirmed by
inhibiting nuclear export of NRP-1/OBR complex using leptomycin B.
MDA-MB-231 co-treated with Leptin and leptomycin B showed increased
NRP-1/OBR complex detection by Duolink in the nucleus than
MDA-MB-231 treated with Leptin alone. The localization of NRP-1 and
OBR was confirmed by the detection of NRP-1 and OBR in the nuclear
fraction as well as in the soluble form than in the bound to the
chromatin. This localization is specific since we do not detect
calreticulin that can be from any contamination by the endoplasmic
reticulum membrane. Even tough at this level, the function of NRP-1
in the nucleus was not identify, we suggested that nuclear
translocation of the NRP-1/OBR complex should be regulated by their
phosphorylation. As reported in the literature, NRP-1 can be a
target of CK2. By using chemical CK2 inhibitors TBB or DRB, we were
able to confirm not only that NRP-1 is a target for CK2-mediated
phosphorylation but that this phosphorylation was indispensable for
the NRP-1/OBR complex stability and its nuclear translocation as it
was demonstrated by using a specific anti-P-NRP-1 and by the
Duolink. The immunostaining of P-NRP-1 clearly show NRP-1
translocation depends on its phosphorylated state since in serum
starved and unstimulated MDA-MB-231, P-NRP-1 is concentrated in the
nuclear periphery. In stimulated condition, we can observe an
accumulation of P-NRP-1 in the nucleus while this localization is
inhibited by CK2 inhibitors.
[0197] To confirm the association of NRP-1 phosphorylation with the
NRP-1/OBR complex formation, we tested a clinically used CK2
inhibitor CX4945 and we repressed CK2.alpha. and CK2.alpha.'
subunit in MDA-MB-231 that have shown the same results obtained
with TBB and DRB inhibitors. It was already demonstrated that CK2
phosphorylation activity is implicated in the nuclear translocation
of phosphorylated proteins with importin (ref). It might be the
case of NRP-1.
[0198] Neuropilin-1 Chip-Seq Revealed its Association with RNA
Polymerase II (Pol2) and Transcription Factors CTCF and p300: Is
NRP-1 a Transcription Factor of a Super Activator?
[0199] The association of NRP-1 in in vitro and in vivo breast
cancer cell migration and its detection in the nucleus of
MDA-MB-231 and of T47D-NRP-1-RFP and principally in the chromatin
bound fraction raised the question whether NRP-1 can be associated
to gene sequences implicated in cell movement, migration and
metastasis. The NRP-1 Chip-Seq analysis has clearly shown a
correlation between Leptin stimulation and peak number increase
which confirmed the association of NRP-1 and OBR signaling. More
interestingly, the enriched peaks corresponded to genes related to
Leptin functions, which can be another argument for NRP-1/OBR
action. Sequence comparison of NRP-1 Chip-Seq and transcription
factor Chip-Seq data from the ENCODE project lead to identify
sequence binding of RNA polymerase II (Pol2) and transcription
factors. The main enriched sequence were for CTCF, TPB (TATA
Binding Protein), TAF1 (Transcription initiation factor TFIID
subunit 1 or TBP-associated factor 250 kDa), and p300.
Interestingly, the number of enriched sequences corresponding to
CTCF binding increased approximately at the same level than Pol2
binding sequences which concords with the already reported studies
that made a link between Pol2 and CTCF (MOLECULAR AND CELLULAR
BIOLOGY, March 2007, p. 1631-1648; Nature, vol 4 7 9|3 Nov. 2011).
The same conclusion can be made for TBP and TAF1 (ref). All these
observation conducted us to conclude on the possible role of NRP-1
as transcription factor or activator or super-activator factor but
more investigations are needed to confirm this.
[0200] Transcriptome Analysis of T47D Stably Overexpressing NRP-1
Revealed Genes Enriched in NRP-1 Chipseq from MDA-MB-231 and
Implicated in Cell Movement and Breast Cancer Metastasis.
[0201] It was clearly admitted that NRP-1 overexpression results in
larger tumors and in Cell motility. However, the molecular
mechanisms associated to cell migration and metastasis are not
fully elucidated and NRP-1 function independently of its known
ligand such as VEGF is also reported. Mikael Klagsbrun team has
already shown that the increase in tumor cell migration after NRP-1
overexpression is independent of VEGF and they postulated that
NRP-1 overexpression induces downstream genes that are responsible
for enhancing cell motility (FASEB. 2000, vol 14).
[0202] In our study, by NRP-1 overexpression in T47D and repression
in MDA-MB-231 we also observe a correlation between NRP-1
expression and Tumor volume increase or decrease, respectively.
Nevertheless, T47D overexpressing NRP-1 xenograft has tumor growth
decrease when treated with Leptin as it was observed for the
MDA-MB231. This tumor growth decrease induced by Leptin treatment
was accompanied by lymph node infiltration that were not observed
in NRP-1 repression condition in MDA-MB-231-shNRP-1.
[0203] This observation can lead to conclude that NRP-1 induces
tumor growth independently of Leptin/OBR pathway and that NRP-1/OBR
complex signaling is in a favor of cell movement and migration
induction. The implication of Leptin in tumor growth and metastasis
has already been studied but the published data are contradictory
depending on cancer cell type and model studies. In human colon
cancer model, Aparicio T et al have shown that leptin stimulates
colon cancer cell proliferation in vitro but does not promote their
growth in vivo in two xenograft model (Gut 2005; 54:1136-1145). In
our case, in vitro leptin implication in cell growth has been
evaluated by different methods. By MTT assay and (H3)-thymidine
incorporation and by BrdU incorporation. In all case we were not
able to observe a significant increase of cell proliferation and
this was confirmed in vivo. In other hand in vitro and in vivo
studies have clearly shown leptin-induced cell migration that was
induced of repressed by NRP-1 overexpression in T47D or repression
in MDA-MB231. On the basis on this observation we focused our
transcriptome analysis on the identification of genes that were
already associated with breast cancer metastasis and lymph nod
infiltration and that are increased by NRP-1 overexpression and by
leptin treatment in T47D and more essentially those identified by
NRP-1 Chipseq analysis of MDA-MB231. Even the maximum of the fold
increase induced by NRP-1 overexpression in T47D was 2,46,
interestingly approximately 72% of genes increased by NRP-1
overexpression were enriched by NRP-1 Chipseq and 17% of these
enriched genes contained sequences corresponding to RNA polymerase
II (Pol2) and transcription factors CTCF, Rad21, SRF, ERalpha and
CEBPB binding sequence. Interestingly, these genes were already
demonstrated to be modulated either by leptin or by NRP-1. For
example SERPINE1 a gene expressing PAI.1 (Plasminogen Activator
Inhibitor-1) has been shown to be up-regulated in endothelial cells
(Prachi Singh, BBRC, 2010) and its overexpression has been found in
many obesity-related types of cancer and is associated with the
progression of breast, endometrial, colorectal, thyroid, renal, and
prostate cancer (Cancer Epidemiol Biomarkers Prev 2009 voir biblio
97-102). It remain to demonstrate clearly the implication of
NRP-1/OBR complex action on SERPINE1 promoter activity by
identifying the essential sequence of NRP-1/OBR complex in
association with Pol2 since the enriched sequence by NRP-1 Chipseq
contained Pol2 binding site. Two genes attracted our attention,
BCAR1 (Breast cancer anti-estrogen resistance protein 1/p130 (Cas)
and PTK2 (Protein tyrosine Kinase 2) that are not only increased by
NRP-1 overexpression in T47D but they were enriched by NRP-1
Chipseq with sequences containing transcription factor binding site
of CTCF, Rad21, SRF and CEBPB. Interestingly, NRP-1 was
demonstrated to regulate endothelial and tumor cell chemotactic
migration through p130Cas) pathway (Evans et Al, MOL. CELL. BIOL.,
2011).
[0204] In conclusion, this study clearly shows NRP-1 as a new co
receptor of Leptin and OBRa discovery of leptin receptor (OBR) as a
new partner of NRP-1 and that this complex is associated with
breast cancer cell growth arrest but increase of lymph node
infiltration. More interestingly, this study highlighted an
eventual role of NRP-1 and/or NRP-1/OBR complex as transcription
factor or activator or super-activator in the regulation of gene
expression by interacting with RNAPol2 as demonstrated here and may
be with other transcription factors such as CTCF, P300 and TAF1
that remain to be demonstrated. More interestingly, this new data
may open a wide field of investigation of NRP-1/OBR complex in
cancer, obesity, immune system and diabetes.
REFERENCES
[0205] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure.
* * * * *
References