U.S. patent application number 17/036763 was filed with the patent office on 2021-02-11 for methods for enhancing the potency of the immune checkpoint inhibitors.
The applicant listed for this patent is CENTRE HOSPITALIER UNIVERSITAIRE DE TOULOUSE, INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE), Universite Toulouse III - Paul Sabatier. Invention is credited to Nathalie ANDRIEU-ABADIE, Celine COLACIOS VIATGE, Caroline IMBERT, Thierry LEVADE, Nicolas MEYER, Bruno SEGUI.
Application Number | 20210040215 17/036763 |
Document ID | / |
Family ID | 1000005168570 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040215 |
Kind Code |
A1 |
LEVADE; Thierry ; et
al. |
February 11, 2021 |
METHODS FOR ENHANCING THE POTENCY OF THE IMMUNE CHECKPOINT
INHIBITORS
Abstract
The present invention relates to methods for enhancing the
potency of the immune checkpoint inhibitors. In particular, the
present invention relates to a method for enhancing the potency of
an immune checkpoint inhibitor administered to a subject as part of
a treatment regimen, the method comprising administering a
pharmaceutically effective amount of a SK1 inhibitor to a subject
in combination with the immune checkpoint inhibitor.
Inventors: |
LEVADE; Thierry; (TOULOUSE,
FR) ; MEYER; Nicolas; (Toulouse, FR) ;
COLACIOS VIATGE; Celine; (Toulouse, FR) ; IMBERT;
Caroline; (Toulouse, FR) ; ANDRIEU-ABADIE;
Nathalie; (Toulouse, FR) ; SEGUI; Bruno;
(Toulouse, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE)
Universite Toulouse III - Paul Sabatier
CENTRE HOSPITALIER UNIVERSITAIRE DE TOULOUSE |
Paris
Toulouse
Toulouse |
|
FR
FR
FR |
|
|
Family ID: |
1000005168570 |
Appl. No.: |
17/036763 |
Filed: |
September 29, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16073080 |
Jul 26, 2018 |
10822415 |
|
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PCT/EP2017/051812 |
Jan 27, 2017 |
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17036763 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2818 20130101;
A61K 31/426 20130101; A61K 39/3955 20130101; A61K 31/415 20130101;
A61K 31/4245 20130101; A61K 31/137 20130101; G01N 33/56972
20130101; G01N 2800/52 20130101; A61K 31/133 20130101; A61K
2039/505 20130101; A61K 31/40 20130101; G01N 33/574 20130101; A61K
45/06 20130101; A61P 35/00 20180101; A61K 2039/507 20130101; A61K
31/4535 20130101; G01N 2333/70517 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 31/133 20060101 A61K031/133; G01N 33/574 20060101
G01N033/574; G01N 33/569 20060101 G01N033/569; A61P 35/00 20060101
A61P035/00; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395; A61K 31/4535 20060101 A61K031/4535; A61K 31/4245
20060101 A61K031/4245; A61K 31/426 20060101 A61K031/426; A61K
31/415 20060101 A61K031/415; A61K 31/40 20060101 A61K031/40; A61K
31/137 20060101 A61K031/137 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 2016 |
EP |
16305084.2 |
Claims
1. A method for enhancing the potency of an immune checkpoint
inhibitor administered to a subject as part of a treatment regimen,
the method comprising administering to the subject a
pharmaceutically effective amount of a SK1 inhibitor in combination
with the immune checkpoint inhibitor.
2. A method of treating cancer in a subject in need thereof
comprising administering to the subject a therapeutically effective
combination of an immune checkpoint inhibitor with a SK1 inhibitor,
wherein administration of the combination results in enhanced
therapeutic efficacy relative to the administration of the immune
checkpoint inhibitor alone.
3. The method of claim 1 wherein the subject suffers from a cancer
selected from the group consisting of 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; malignant 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; hemangiosarcoma;
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.
4. The method of claim 1 wherein the subject suffers from a
melanoma.
5. The method of claim 1 wherein the subject suffers from a
melanoma resistant to melanoma resistant to BRAF inhibitors.
6. The method of claim 1 wherein the subject suffers from a
melanoma with elevated plasma dehydrogenase (LDH).
7. The method of claim 1 wherein the cancer is characterized by a
low tumor infiltration of CD8+ T cells.
8. The method of claim 1 wherein the SK1 inhibitor is selected from
the group consisting of: ##STR00004##
9. The method of claim 1 wherein the SK1 inhibitor is
N'-[(2-hydroxynaphthalen-1-yl)methylidene]-3-(naphthalen-2-yl)-1H-pyrazol-
e-5-carbohydrazide.
10. The method of claim 1 wherein the SK1 inhibitor is an inhibitor
of SK1 expression.
11. The method of claim 1 wherein the immune checkpoint inhibitor
is an antibody selected from the group consisting of anti-CTLA4
antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2
antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3
antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and
anti-B7H6 antibodies.
12. A method of treating cancer in a subject in need thereof
comprising i) quantifying the density of CD8+ T cells in a tumor
tissue sample obtained from the subject ii) comparing the density
quantified at step i) with a predetermined reference value and iii)
administering to the subject a therapeutically effective
combination of an immune checkpoint inhibitor with a SK1 inhibitor
when the density quantified at step i) is lower than the
predetermined reference value.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for enhancing the
potency of the immune checkpoint inhibitors.
BACKGROUND OF THE INVENTION
[0002] The ability of the immune system to detect and eliminate
cancer was first proposed over 100 years ago. Since then, T cells
reactive against tumor-associated antigens have been detected in
the blood of patients with many different types of cancers,
suggesting a role for the immune system in fighting cancer.
However, tumors can escape host immunity by manipulating the tumor
microenvironment and driving immunosuppression, meaning that
patients cannot mount a potent enough immune response to fully
eliminate cancer cells. The goal of immunotherapy is to restore or
augment antitumor immune responses. An increased understanding of
tumor immunology has led to the identification of novel targets for
new immune-based approaches, including a group of cell-surface
molecules known as immune checkpoint proteins. In particular,
monoclonal antibodies inhibiting CTLA-4 (ipilimumab) or PD-1
(nivolumab, pembrolizumab) have demonstrated significant efficacy
in the treatment of metastatic melanoma, promoting high response
rate and long-lasting tumor control. Despite promising results,
about 40% of patients do not display therapeutic response and a
significant proportion of responders experience tumor relapse in
the 2 years following treatment induction. Moreover, recent
clinical trials combining BRAF and checkpoint inhibitors have shown
high liver toxicity for patients with BRAF-mutated melanoma.
Accordingly, development of novel therapeutic strategies is thus
urgently needed in order to enhance the potency of the immune
checkpoint inhibitor.
[0003] Sphingolipid metabolites, including ceramide, ceramide
1-phosphate, sphingosine, and sphingosine 1-phosphate (S 1P), have
emerged as bioactive signalling molecules that regulate cell
motility, differentiation, proliferation and survival as well as
angiogenesis, inflammation and immunity. It was recently
demonstrated an increased production of S1P in melanoma cells (2,
3). This bioactive sphingolipid metabolite is produced mainly by
sphingosine kinase 1 (SK1), which is overexpressed in human
melanoma tumors compared to nevi (2). In many tumors, S 1P conveys
oncogenic signals as an intracellular second messenger and/or
through the stimulation of a family of G-protein coupled receptors
(S1PR1-5) expressed both on cancer cells and their surrounding
microenvironment (4, 5). In melanoma tumors, dysregulation of S1P
production in cancer cells elicits a fibrotic response in the tumor
microenvironment, which in turn stimulates melanoma cell migration
(2). Additionally, treatment of mice with the S1P receptor
modulator FTY720, which renders cells unresponsive to S1P
activation by sequestering S1PR1 internally, reduced melanoma
progression by inhibiting tumor vascularization (6). These findings
illustrate the paracrine action of melanoma cell-exported S1P
through S1PRs on tumor-stroma interactions. However, recent studies
demonstrate that the SK1/S1P/S1PR axis plays an essential role in
inflammation-associated cancer development (7). Indeed, shRNA-based
downregulation of SK1 or S1PR1 has been shown to block the
persistent activation of the transcription factor STAT3 and the
level of proinflammatory cytokines and reduce cancer progression in
mouse models of inflammation (8, 9). In addition, S1P contributes
to trafficking and effector functions of lymphocytes and other
hematopoietic cells (10). However, the prior art does not suggest
SK1 inhibition could enhance the potency of the immune checkpoint
inhibitors.
SUMMARY OF THE INVENTION
[0004] The present invention relates to methods for enhancing the
potency of the immune checkpoint inhibitors. In particular, the
present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION
[0005] The inventors demonstrated that interfering with
sphingolipid metabolism efficiently impairs tumor progression in
pre-clinical melanoma model and enhances anti-tumor immune response
obtained with the immune checkpoint inhibitor. In particular, the
inventors observed that SK1 downregulation enhances proliferation
and activation of CD8+ T cells within the tumors. Of great interest
is the finding that SK1 knockdown in melanoma enhances the
expression of CTLA-4 and PD-1 on CD8+ TIL, which are both
up-regulated upon T cell activation and exert potent negative
feedback loop on T cell activation. The latter observation
highlights for the first time that melanoma SK1 impairs CD8+ T
cell-dependent immune response. However, the upregulation of both
PD-1 and CTLA-4 on CD8+ T cells is likely involved in the melanoma
immune escape following SK1 knockdown observed at latter time
points. Thus, targeting melanoma SK1 is unlikely sufficient to
trigger total tumor regression. Collectively, the data prompted the
inventors to investigate the combination of SK1 inhibition with the
inhibition of immune checkpoints and demonstrate that said
combination provides synergistic anti-cancer immune responses.
[0006] Accordingly the first object of the present invention
relates to a method of enhancing the proliferation and activation
of tumor infiltrating CD8+ T cells in a patient suffering from
cancer comprising administering to the patient a therapeutically
effective amount of a SK1 inhibitor.
[0007] As used herein, the term "CD8+ T cell" has its general
meaning in the art and refers to a subset of T cells which express
CD8 on their surface. They are MHC class I-restricted, and function
as cytotoxic T cells. "CD8+ T cells" are also called cytotoxic T
lymphocytes (CTL), T-killer cells, cytolytic T cells, or killer T
cells. CD8 antigens are members of the immunoglobulin supergene
family and are associative recognition elements in major
histocompatibility complex class I-restricted interactions. As used
herein, the term "tumor infiltrating CD8+ T cell" refers to the
pool of CD8+ T cells of the patient that have left the blood stream
and have migrated into a tumor.
[0008] A further object of the present invention relates to a
method for enhancing the potency of an immune checkpoint inhibitor
administered to a subject as part of a treatment regimen, the
method comprising administering to the subject a pharmaceutically
effective amount of a SK1 inhibitor in combination with the immune
checkpoint inhibitor.
[0009] As used herein the term "immune checkpoint protein" has its
general meaning in the art and refers to a molecule that is
expressed by T cells in that either turn up a signal (stimulatory
checkpoint molecules) or turn down a signal (inhibitory checkpoint
molecules) Immune checkpoint molecules are recognized in the art to
constitute immune checkpoint pathways similar to the CTLA-4 and
PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer
12:252-264; Mellman et al., 2011. Nature 480:480-489). Examples of
inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA,
CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The
Adenosine A2A receptor (A2AR) is regarded as an important
checkpoint in cancer therapy because the tumor microenvironment has
relatively high levels of adenosine, which lead to a negative
immune feedback loop through the activation of A2AR. B7-H3, also
called CD276, was originally understood to be a co-stimulatory
molecule but is now regarded as co-inhibitory. B7-H4, also called
VTCN1, is expressed by tumor cells and tumor-associated macrophages
and plays a role in tumor escape. B and T Lymphocyte Attenuator
(BTLA), also called CD272, is a ligand of HVEM (Herpesvirus Entry
Mediator). Cell surface expression of BTLA is gradually
downregulated during differentiation of human CD8+ T cells from the
naive to effector cell phenotype, however tumor-specific human CD8+
T cells express high levels of BTLA. CTLA-4, Cytotoxic
T-Lymphocyte-Associated protein 4 and also called CD152 is
overexpressed on Treg cells serves to control T cell proliferation.
IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme,
a related immune-inhibitory enzymes. Another important molecule is
TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK
cells, generate and activate Tregs and myeloid-derived suppressor
cells, and promote tumor angiogenesis. KIR, Killer-cell
Immunoglobulin-like Receptor, is a receptor for MHC Class I
molecules on Natural Killer cells. LAG3, Lymphocyte Activation
Gene-3, works to suppress an immune response by action to Tregs as
well as direct effects on CD8+ T cells. PD-1, Programmed Death 1
(PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint
is the target of Merck & Co.'s melanoma drug Keytruda, which
gained FDA approval in September 2014. An advantage of targeting
PD-1 is that it can restore immune function in the tumor
microenvironment. TIM-3, short for T-cell Immunoglobulin domain and
Mucin domain 3, expresses on activated human CD4+ T cells and
regulates Th1 and Th17 cytokines. TIM-3 acts as a negative
regulator of Th1/Tc1 function by triggering cell death upon
interaction with its ligand, galectin-9. VISTA. Short for V-domain
Ig suppressor of T cell activation, VISTA is primarily expressed on
hematopoietic cells so that consistent expression of VISTA on
leukocytes within tumors may allow VISTA blockade to be effective
across a broad range of solid tumors.
[0010] As used herein, the term "immune checkpoint inhibitor" has
its general meaning in the art and refers to any compound
inhibiting the function of an immune inhibitory checkpoint protein.
Inhibition includes reduction of function and full blockade.
Preferred immune checkpoint inhibitors are antibodies that
specifically recognize immune checkpoint proteins. A number of
immune checkpoint inhibitors are known and in analogy of these
known immune checkpoint protein inhibitors, alternative immune
checkpoint inhibitors may be developed in the (near) future. The
immune checkpoint inhibitors include peptides, antibodies, nucleic
acid molecules and small molecules. In particular, the immune
checkpoint inhibitor of the present invention is administered for
enhancing the proliferation, migration, persistence and/or cytoxic
activity of CD8+ T cells in the subject and in particular the
tumor-infiltrating of CD8+ T cells of the subject.
[0011] Thus the expression "enhancing the potency of an immune
checkpoint" refers to the ability of the SK1 inhibitor to increase
the ability of the immune checkpoint inhibitor to enhance the
proliferation, migration, persistence and/or cytoxic activity of
CD8+ T cells.
[0012] As used herein, the term "treatment" or "treat" refer to
both prophylactic or preventive treatment as well as curative or
disease modifying treatment, including treatment of patient at risk
of contracting the disease or suspected to have contracted the
disease as well as patients who are ill or have been diagnosed as
suffering from a disease or medical condition, and includes
suppression of clinical relapse. The treatment may be administered
to a subject having a medical disorder or who ultimately may
acquire the disorder, in order to prevent, cure, delay the onset
of, reduce the severity of, or ameliorate one or more symptoms of a
disorder or recurring disorder, or in order to prolong the survival
of a subject beyond that expected in the absence of such treatment.
By "therapeutic regimen" is meant the pattern of treatment of an
illness, e.g., the pattern of dosing used during therapy. A
therapeutic regimen may include an induction regimen and a
maintenance regimen. The phrase "induction regimen" or "induction
period" refers to a therapeutic regimen (or the portion of a
therapeutic regimen) that is used for the initial treatment of a
disease. The general goal of an induction regimen is to provide a
high level of drug to a patient during the initial period of a
treatment regimen. An induction regimen may employ (in part or in
whole) a "loading regimen", which may include administering a
greater dose of the drug than a physician would employ during a
maintenance regimen, administering a drug more frequently than a
physician would administer the drug during a maintenance regimen,
or both. The phrase "maintenance regimen" or "maintenance period"
refers to a therapeutic regimen (or the portion of a therapeutic
regimen) that is used for the maintenance of a patient during
treatment of an illness, e.g., to keep the patient in remission for
long periods of time (months or years). A maintenance regimen may
employ continuous therapy (e.g., administering a drug at a regular
intervals, e.g., weekly, monthly, yearly, etc.) or intermittent
therapy (e.g., interrupted treatment, intermittent treatment,
treatment at relapse, or treatment upon achievement of a particular
predetermined criteria [e.g., pain, disease manifestation,
etc.]).
[0013] In some embodiments, the subject suffers from a cancer. 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 be 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 tract, 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; malignant 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; hemangiosarcoma;
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.
[0014] In some embodiments, the subject suffers from melanoma. As
used herein, "melanoma" refers to a condition characterized by the
growth of a tumor arising from the melanocytic system of the skin
and other organs. Most melanocytes occur in the skin, but are also
found in the meninges, digestive tract, lymph nodes and eyes. When
melanoma occurs in the skin, it is referred to as cutaneous
melanoma. Melanoma can also occur in the eyes and is called ocular
or intraocular melanoma. Melanoma occurs rarely in the meninges,
the digestive tract, lymph nodes or other areas where melanocytes
are found. 40-60% of melanomas carry an activating mutation in the
gene encoding the serine-threonine protein kinase B-RAF (BRAF).
Among the BRAF mutations observed in melanoma, over 90% are at
codon 600, and among these, over 90% are a single nucleotide
mutation resulting in substitution of glutamic acid for valine
(BRAFV600E).
[0015] In some embodiments, the subject suffers from a melanoma
resistant to BRAF inhibitors. As used herein, the term "resistant"
refers to the repeated outbreak of melanoma, or a progression of
the melanoma independently of whether the disease was cured before
said outbreak or progression. As used herein, the term "BRAF
inhibitor" refers to an agent that is capable of inhibiting BRAF
kinase or mutated BRAF kinase activity (one or more mutated forms
of serine-threonine protein kinase B-RAF (BRAF)) (e.g. BRAFV600E).
Accordingly, the term "BRAF inhibitors" encompasses within its
scope a compound that is capable of inhibiting BRAF or its mutated
form; or a compound that is capable of inhibiting V600 mutated form
of BRAF. Examples of BRAF inhibitors include but are not limited to
BAY43-9006 (sorafenib, Bayer), vemurafenib (PLX4032, Plexxikon;
RG7204, RO5185426, Hofmann-LaRoche), GDC-0879 (GlaxoSmithKline),
dabrafenib (GSK21 18436, GlaxoSmithKline), PLX4720
(Hofmann-LaRoche), BMS-908662 (XL281, Bristol-Myers Squibb), LGX818
(Novartis), PLX3603 (RO5212054, Hofmann-LaRoche), ARQ-736 (ArQule),
DP-4978 (Deciphera) or RAF265 (Novartis).
[0016] In some embodiments, the subject suffers from a melanoma
with elevated plasma lactate dehydrogenase (LDH). Plasma LDH can be
considered "elevated" according to the method of the present
invention if it exceeds plasma LDH levels typically found in a
negative control, i.e., a healthy mammal of the same species.
Typically, plasma LDH can be considered "elevated" if it exceeds
about 212 IU/mL. Preferably, plasma LDH is considered "elevated" if
it exceeds about 250 IU/mL. More preferably, plasma LDH is
considered "elevated" if it exceeds about 287 IU/mL.
[0017] Accordingly a further object of the present invention
relates to a method of treating cancer in a subject in need thereof
comprising administering to the subject a therapeutically effective
combination of an immune checkpoint inhibitor with a SK1 inhibitor,
wherein administration of the combination results in enhanced
therapeutic efficacy relative to the administration of the immune
checkpoint inhibitor alone.
[0018] As used herein, the expression "enhanced therapeutic
efficacy," relative to cancer refers to a slowing or diminution of
the growth of cancer cells or a solid tumor, or a reduction in the
total number of cancer cells or total tumor burden. An "improved
therapeutic outcome" or "enhanced therapeutic efficacy" therefore
means there is an improvement in the condition of the patient
according to any clinically acceptable criteria, including, for
example, decreased tumor size, an increase in time to tumor
progression, increased progression-free survival, increased overall
survival time, an increase in life expectancy, or an improvement in
quality of life. In particular, "improved" or "enhanced" refers to
an improvement or enhancement of 1%, 5%, 10%, 25% 50%, 75%, 100%,
or greater than 100% of any clinically acceptable indicator of
therapeutic outcome or efficacy. As used herein, the expression
"relative to" when used in the context of comparing the activity
and/or efficacy of a combination composition comprising the immune
checkpoint inhibitor with the SK1 inhibitor to the activity and/or
efficacy of the immune checkpoint alone, refers to a comparison
using amounts known to be comparable according to one of skill in
the art.
[0019] In particular, the method of the present invention is
particularly suitable for the treatment of cancer characterized by
a low tumor infiltration of CD8+ T cells. Typically said
tumor-infiltration of CD8+ T cells is determined by any convention
method in the art. For example, said determination comprises
quantifying the density of CD8+ T cells in a tumor sample obtained
from the subject.
[0020] As used herein, the term "tumor tissue sample" means any
tissue tumor sample derived from the patient. Said tissue sample is
obtained for the purpose of the in vitro evaluation. In some
embodiments, the tumor sample may result from the tumor resected
from the patient. In some embodiments, the tumor sample may result
from a biopsy performed in the primary tumor of the patient or
performed in metastatic sample distant from the primary tumor of
the patient. For example an endoscopical biopsy performed in the
bowel of the patient affected by a colorectal cancer. In some
embodiments, the tumor tissue sample encompasses (i) a global
primary tumor (as a whole), (ii) a tissue sample from the center of
the tumor, (iii) a tissue sample from the tissue directly
surrounding the tumor which tissue may be more specifically named
the "invasive margin" of the tumor, (iv) lymphoid islets in close
proximity with the tumor, (v) the lymph nodes located at the
closest proximity of the tumor, (vi) a tumor tissue sample
collected prior surgery (for follow-up of patients after treatment
for example), and (vii) a distant metastasis. As used herein the
"invasive margin" has its general meaning in the art and refers to
the cellular environment surrounding the tumor. In some
embodiments, the tumor tissue sample, irrespective of whether it is
derived from the center of the tumor, from the invasive margin of
the tumor, or from the closest lymph nodes, encompasses pieces or
slices of tissue that have been removed from the tumor center of
from the invasive margin surrounding the tumor, including following
a surgical tumor resection or following the collection of a tissue
sample for biopsy, for further quantification of one or several
biological markers, notably through histology or
immunohistochemistry methods, through flow cytometry methods and
through methods of gene or protein expression analysis, including
genomic and proteomic analysis. The tumor tissue sample can, of
course, be subjected to a variety of well-known post-collection
preparative and storage techniques (e.g., fixation, storage,
freezing, etc.). The sample can be fresh, frozen, fixed (e.g.,
formalin fixed), or embedded (e.g., paraffin embedded).
[0021] In some embodiments, the quantification of density of CD8+ T
cells is determined by immunohistochemistry (IHC). For example, the
quantification of the density of CD8+ T cells is performed by
contacting the tissue tumor tissue sample with a binding partner
(e.g. an antibody) specific for a cell surface marker of said
cells. Typically, the quantification of density of CD8+ T cells is
performed by contacting the tissue tumor tissue sample with a
binding partner (e.g. an antibody) specific for CD8. Typically, the
density of CD8+ T cells is expressed as the number of these cells
that are counted per one unit of surface area of tissue sample,
e.g. as the number of cells that are counted per cm.sup.2 or
mm.sup.2 of surface area of tumor tissue sample. In some
embodiments, the density of cells may also be expressed as the
number of cells per one volume unit of sample, e.g. as the number
of cells per cm3 of tumor tissue sample. In some embodiments, the
density of cells may also consist of the percentage of the specific
cells per total cells (set at 100%) Immunohistochemistry typically
includes the following steps i) fixing the tumor tissue sample with
formalin, ii) embedding said tumor tissue sample in paraffin, iii)
cutting said tumor tissue sample into sections for staining, iv)
incubating said sections with the binding partner specific for the
marker, v) rinsing said sections, vi) incubating said section with
a secondary antibody typically biotinylated and vii) revealing the
antigen-antibody complex typically with avidin-biotin-peroxidase
complex. Accordingly, the tumor tissue sample is firstly incubated
the binding partners. After washing, the labeled antibodies that
are bound to marker of interest are revealed by the appropriate
technique, depending of the kind of label is borne by the labeled
antibody, e.g. radioactive, fluorescent or enzyme label. Multiple
labelling can be performed simultaneously. Alternatively, the
method of the present invention may use a secondary antibody
coupled to an amplification system (to intensify staining signal)
and enzymatic molecules. Such coupled secondary antibodies are
commercially available, e.g. from Dako, EnVision system.
Counterstaining may be used, e.g. H&E, DAPI, Hoechst. Other
staining methods may be accomplished using any suitable method or
system as would be apparent to one of skill in the art, including
automated, semi-automated or manual systems. For example, one or
more labels can be attached to the antibody, thereby permitting
detection of the target protein (i.e the marker). Exemplary labels
include radioactive isotopes, fluorophores, ligands,
chemiluminescent agents, enzymes, and combinations thereof. In some
embodiments, the label is a quantum dot. Non-limiting examples of
labels that can be conjugated to primary and/or secondary affinity
ligands include fluorescent dyes or metals (e.g. fluorescein,
rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g.
rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole)
and bioluminescent proteins (e.g. luciferin, luciferase), haptens
(e.g. biotin). A variety of other useful fluorescers and
chromophores are described in Stryer L (1968) Science 162:526-533
and Brand L and Gohlke J R (1972) Annu. Rev. Biochem. 41:843-868.
Affinity ligands can also be labeled with enzymes (e.g. horseradish
peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes
(e.g. .sup.3H, .sup.14C, .sup.32P, .sup.35S or .sup.125I) and
particles (e.g. gold). The different types of labels can be
conjugated to an affinity ligand using various chemistries, e.g.
the amine reaction or the thiol reaction. However, other reactive
groups than amines and thiols can be used, e.g. aldehydes,
carboxylic acids and glutamine Various enzymatic staining methods
are known in the art for detecting a protein of interest. For
example, enzymatic interactions can be visualized using different
enzymes such as peroxidase, alkaline phosphatase, or different
chromogens such as DAB, AEC or Fast Red. In other examples, the
antibody can be conjugated to peptides or proteins that can be
detected via a labeled binding partner or antibody. In an indirect
IHC assay, a secondary antibody or second binding partner is
necessary to detect the binding of the first binding partner, as it
is not labeled. The resulting stained specimens are each imaged
using a system for viewing the detectable signal and acquiring an
image, such as a digital image of the staining. Methods for image
acquisition are well known to one of skill in the art. For example,
once the sample has been stained, any optical or non-optical
imaging device can be used to detect the stain or biomarker label,
such as, for example, upright or inverted optical microscopes,
scanning confocal microscopes, cameras, scanning or tunneling
electron microscopes, canning probe microscopes and imaging
infrared detectors. In some examples, the image can be captured
digitally. The obtained images can then be used for quantitatively
or semi-quantitatively determining the amount of the marker in the
sample. Various automated sample processing, scanning and analysis
systems suitable for use with immunohistochemistry are available in
the art. Such systems can include automated staining and
microscopic scanning, computerized image analysis, serial section
comparison (to control for variation in the orientation and size of
a sample), digital report generation, and archiving and tracking of
samples (such as slides on which tissue sections are placed).
Cellular imaging systems are commercially available that combine
conventional light microscopes with digital image processing
systems to perform quantitative analysis on cells and tissues,
including immunostained samples. See, e.g., the CAS-200 system
(Becton, Dickinson & Co.). In particular, detection can be made
manually or by image processing techniques involving computer
processors and software. Using such software, for example, the
images can be configured, calibrated, standardized and/or validated
based on factors including, for example, stain quality or stain
intensity, using procedures known to one of skill in the art (see
e.g., published U.S. Patent Publication No. US20100136549). The
image can be quantitatively or semi-quantitatively analyzed and
scored based on staining intensity of the sample. Quantitative or
semi-quantitative histochemistry refers to method of scanning and
scoring samples that have undergone histochemistry, to identify and
quantitate the presence of the specified biomarker (i.e. the
marker). Quantitative or semi-quantitative methods can employ
imaging software to detect staining densities or amount of staining
or methods of detecting staining by the human eye, where a trained
operator ranks results numerically. For example, images can be
quantitatively analyzed using a pixel count algorithms (e.g.,
Aperio Spectrum Software, Automated QUantitatative Analysis
platform (AQUA.RTM. platform), and other standard methods that
measure or quantitate or semi-quantitate the degree of staining;
see e.g., U.S. Pat. Nos. 8,023,714; 7,257,268; 7,219,016;
7,646,905; published U.S. Patent Publication No. US20100136549 and
20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacus
et al. (1997) Analyt Quant Cytol Histol, 19:316-328). A ratio of
strong positive stain (such as brown stain) to the sum of total
stained area can be calculated and scored. The amount of the
detected biomarker (i.e. the marker) is quantified and given as a
percentage of positive pixels and/or a score. For example, the
amount can be quantified as a percentage of positive pixels. In
some examples, the amount is quantified as the percentage of area
stained, e.g., the percentage of positive pixels. For example, a
sample can have at least or about at least or about 0, 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,
31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or more positive pixels as compared to the total
staining area. In some embodiments, a score is given to the sample
that is a numerical representation of the intensity or amount of
the histochemical staining of the sample, and represents the amount
of target biomarker (e.g., the marker) present in the sample.
Optical density or percentage area values can be given a scaled
score, for example on an integer scale. Thus, in some embodiments,
the method of the present invention comprises the steps consisting
in i) providing one or more immunostained slices of tissue section
obtained by an automated slide-staining system by using a binding
partner capable of selectively interacting with the marker (e.g. an
antibody as above described), ii) proceeding to digitalisation of
the slides of step a. by high resolution scan capture, iii)
detecting the slice of tissue section on the digital picture iv)
providing a size reference grid with uniformly distributed units
having a same surface, said grid being adapted to the size of the
tissue section to be analyzed, and v) detecting, quantifying and
measuring intensity of stained cells in each unit whereby the
number or the density of cells stained of each unit is
assessed.
[0022] In some embodiments, the cell density of CD8+ T cells is
determined in the whole tumor tissue sample, is determined in the
invasive margin or centre of the tumor tissue sample or is
determined both in the centre and the invasive margin of the tumor
tissue sample.
[0023] Accordingly a further object of the present invention
relates to a method of treating cancer in a subject in need thereof
comprising i) quantifying the density of CD8+ T cells in a tumor
tissue sample obtained from the subject ii) comparing the density
quantified at step i) with a predetermined reference value and iii)
administering to the subject a therapeutically effective
combination of an immune checkpoint inhibitor with a SK1 inhibitor
when the density quantified at step i) is lower than the
predetermined reference value.
[0024] Typically, the predetermined reference value correlates with
the survival time of the subject. Those of skill in the art will
recognize that OS survival time is generally based on and expressed
as the percentage of people who survive a certain type of cancer
for a specific amount of time. Cancer statistics often use an
overall five-year survival rate. In general, OS rates do not
specify whether cancer survivors are still undergoing treatment at
five years or if they've become cancer-free (achieved remission).
DSF gives more specific information and is the number of people
with a particular cancer who achieve remission. Also,
progression-free survival (PFS) rates (the number of people who
still have cancer, but their disease does not progress) includes
people who may have had some success with treatment, but the cancer
has not disappeared completely. As used herein, the expression
"short survival time" indicates that the patient will have a
survival time that will be lower than the median (or mean) observed
in the general population of patients suffering from said cancer.
When the patient will have a short survival time, it is meant that
the patient will have a "poor prognosis". Inversely, the expression
"long survival time" indicates that the patient will have a
survival time that will be higher than the median (or mean)
observed in the general population of patients suffering from said
cancer. When the patient will have a long survival time, it is
meant that the patient will have a "good prognosis".
[0025] In some embodiments, the predetermined value is a threshold
value or a cut-off value. Typically, a "threshold value" or
"cut-off value" can be determined experimentally, empirically, or
theoretically. A threshold value can also be arbitrarily selected
based upon the existing experimental and/or clinical conditions, as
would be recognized by a person of ordinary skilled in the art. For
example, retrospective measurement of cell densities in properly
banked historical patient samples may be used in establishing the
predetermined reference value. The threshold value has to be
determined in order to obtain the optimal sensitivity and
specificity according to the function of the test and the
benefit/risk balance (clinical consequences of false positive and
false negative). Typically, the optimal sensitivity and specificity
(and so the threshold value) can be determined using a Receiver
Operating Characteristic (ROC) curve based on experimental data.
For example, after quantifying the density of CD8+ T cells in a
group of reference, one can use algorithmic analysis for the
statistic treatment of the measured densities in samples to be
tested, and thus obtain a classification standard having
significance for sample classification. The full name of ROC curve
is receiver operator characteristic curve, which is also known as
receiver operation characteristic curve. It is mainly used for
clinical biochemical diagnostic tests. ROC curve is a comprehensive
indicator that reflects the continuous variables of true positive
rate (sensitivity) and false positive rate (1-specificity). It
reveals the relationship between sensitivity and specificity with
the image composition method. A series of different cut-off values
(thresholds or critical values, boundary values between normal and
abnormal results of diagnostic test) are set as continuous
variables to calculate a series of sensitivity and specificity
values. Then sensitivity is used as the vertical coordinate and
specificity is used as the horizontal coordinate to draw a curve.
The higher the area under the curve (AUC), the higher the accuracy
of diagnosis. On the ROC curve, the point closest to the far upper
left of the coordinate diagram is a critical point having both high
sensitivity and high specificity values. The AUC value of the ROC
curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic
result gets better and better as AUC approaches 1. When AUC is
between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7
and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the
accuracy is quite high. This algorithmic method is preferably done
with a computer. Existing software or systems in the art may be
used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1
medical statistical software, SPSS 9.0, ROCPOWER.SAS,
DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0
(Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
[0026] In some embodiments, the predetermined reference value is
determined by carrying out a method comprising the steps of a)
providing a collection of tumor tissue samples from subject
suffering from the cancer of interest; b) providing, for each tumor
tissue sample provided at step a), information relating to the
actual clinical outcome for the corresponding patient (i.e. the
duration of the disease-free survival (DFS) and/or the overall
survival (OS)); c) providing a serial of arbitrary quantification
values; d) quantifying the density of CD8+ T cells for each tumor
tissue sample contained in the collection provided at step a); e)
classifying said tumor tissue samples in two groups for one
specific arbitrary quantification value provided at step c),
respectively: (i) a first group comprising tumor tissue samples
that exhibit a quantification value for level that is lower than
the said arbitrary quantification value contained in the said
serial of quantification values; (ii) a second group comprising
tumor tissue samples that exhibit a quantification value for said
level that is higher than the said arbitrary quantification value
contained in the said serial of quantification values; whereby two
groups of tumor tissue samples are obtained for the said specific
quantification value, wherein the tumor tissue samples of each
group are separately enumerated; f) calculating the statistical
significance between (i) the quantification value obtained at step
e) and (ii) the actual clinical outcome of the patients from which
tumor tissue samples contained in the first and second groups
defined at step f) derive; g) reiterating steps f) and g) until
every arbitrary quantification value provided at step d) is tested;
h) setting the said predetermined reference value as consisting of
the arbitrary quantification value for which the highest
statistical significance (most significant) has been calculated at
step g). For example the density of CD8+ T cells has been assessed
for 100 tumor tissue samples of 100 patients. The 100 samples are
ranked according to the density of CD8+ T cells. Sample 1 has the
highest density and sample 100 has the lowest density. A first
grouping provides two subsets: on one side sample Nr 1 and on the
other side the 99 other samples. The next grouping provides on one
side samples 1 and 2 and on the other side the 98 remaining samples
etc., until the last grouping: on one side samples 1 to 99 and on
the other side sample Nr 100. According to the information relating
to the actual clinical outcome for the corresponding cancer
patient, Kaplan Meier curves are prepared for each of the 99 groups
of two subsets. Also for each of the 99 groups, the p value between
both subsets was calculated. The predetermined reference value is
then selected such as the discrimination based on the criterion of
the minimum p value is the strongest. In other terms, the density
of CD8+ T cells corresponding to the boundary between both subsets
for which the p value is minimum is considered as the predetermined
reference value. It should be noted that the predetermined
reference value is not necessarily the median value of cell
densities. Thus in some embodiments, the predetermined reference
value thus allows discrimination between a poor and a good
prognosis with respect to DFS and OS for a patient. Practically,
high statistical significance values (e.g. low P values) are
generally obtained for a range of successive arbitrary
quantification values, and not only for a single arbitrary
quantification value. Thus, in one alternative embodiment of the
invention, instead of using a definite predetermined reference
value, a range of values is provided. Therefore, a minimal
statistical significance value (minimal threshold of significance,
e.g. maximal threshold P value) is arbitrarily set and a range of a
plurality of arbitrary quantification values for which the
statistical significance value calculated at step g) is higher
(more significant, e.g. lower P value) are retained, so that a
range of quantification values is provided. This range of
quantification values includes a "cut-off" value as described
above. For example, according to this specific embodiment of a
"cut-off" value, the outcome can be determined by comparing the
density of CD8+ T cells with the range of values which are
identified. In some embodiments, a cut-off value thus consists of a
range of quantification values, e.g. centered on the quantification
value for which the highest statistical significance value is found
(e.g. generally the minimum p value which is found).
[0027] A further object of the present invention relates to a
method of treating cancer in a patient in need thereof comprising
administering to the patient a therapeutically effective amount of
a SK-1 inhibitor in combination with a cancer vaccine.
[0028] As used herein, the term "cancer vaccine" has its general
meaning in the art and refers to a composition capable of inducing
active immunity against at least one cancer antigen. The cancer
vaccine can result in a production of antibodies or simply in the
activation of certain cells, in particular antigen-presenting
cells, T lymphocytes (in particular T-CD8+ cells) and B
lymphocytes. The cancer vaccine can be a composition for
prophylactic purposes or for therapeutic purposes or both. As used
herein the term "antigen" refers to a molecule capable of being
specifically bound by an antibody or by a T cell receptor (TCR) if
processed and presented by MHC molecules. The term "antigen", as
used herein, also encompasses T-cell epitopes. An antigen is
additionally capable of being recognized by the immune system
and/or being capable of inducing a humoral immune response and/or
cellular immune response leading to the activation of B- and/or
T-lymphocytes. An antigen can have one or more epitopes or
antigenic sites (B- and T-epitopes). As used herein, the term
"cancer antigen" refers to an antigen that is characteristic of a
tumor tissue. There are multiple types of cancer vaccines.
Non-limiting examples of cancer vaccines include tumor cell
vaccines, antigen vaccines, dendritic cell vaccines, DNA vaccines,
and vector based vaccines.
[0029] Typically, the cancer vaccine of the present invention
comprises a tumor-associated antigen ("TAA") or nucleic acid
sequence (e.g. DNA) that encodes for a tumor-associated antigen.
Numerous tumor-associated antigens are known in the art. Exemplary
tumor-associated antigens include, but are not limited to, 5 alpha
reductase, alpha-fetoprotein, AM-1, APC, April, BAGE, beta-catenin,
Bell 2, bcr-abl, CA-125, CASP-8/FLICE, Cathepsins, CD 19, CD20,
CD21, CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55,
CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, CGFR, EMBP,
Dna78, farnesyl transferase, FGF8b, FGF8a, FLK-1/KDR, folic acid
receptor, G250, GAGE-family, gastrin 17, gastrin-releasing hormone,
GD2/GD3/GM2, GnRH, GnTV, GP1, gp100/Pmel17, gp-100-in4, gp15,
gp75/TRP-1, hCG, heparanse, Her2/neu, HMTV, Hsp70, hTERT, IGFR1,
IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT,
MAGE-family, mammaglobin, MAP 17, melan-A/MART-1, mesothelin, MIC A
B, MT-MMPs, mucin, NY-ESO-1, osteonectin, p15, P170/MDR1, p53,
p97/melanotransferrin, PAI-1, PDGF, uPA, PRAME, probasin,
progenipoientin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX-family,
STAT3, STn, TAG-72, TGF-alpha, TGF-beta, Thymosin-beta-15,
TNF-alpha, TYRP-, TYRP-2, tyrosinase, VEGF, ZAG, pl6INK4, and
glutathione-S-transferase.
[0030] In some embodiments, the vaccine is a DNA vaccine. Vectors
can be engineered to contain specific DNAs that can be injected
into a subject which leads to the DNA being taken up by cells. Once
the cells take up the DNA, the DNA will program the cells to make
specific antigens, which can then provoke the desired immune
response.
[0031] In some embodiments, the vaccine consists of a recombinant
virus that encodes or express a cancer antigen. In some
embodiments, the recombinant virus is a poxvirus expressing a tumor
antigen and more particularly an orthopoxvirus such as, but not
limited to, a vaccinia virus, a Modified Vaccinia Ankara (MVA)
virus, or MVA-BN. Examples of vaccinia virus strains are the
strains Temple of Heaven, Copenhagen, Paris, Budapest, Dairen, Gam,
MRIVP, Per, Tashkent, TBK, Tom, Bern, Patwadangar, BIEM, B-15,
Lister, EM-63, New York City Board of Health, Elstree, Ikeda and
WR. A preferred vaccinia virus (W) strain is the Wyeth (DRYVAX)
strain (U.S. Pat. No. 7,410,644). Another preferred W strain is a
modified vaccinia virus Ankara (MVA) (Sutter, G. et al. [1994],
Vaccine 12: 1032-40). Another preferred W strain is MVA-BN.
Examples of MVA virus strains that are useful in the practice of
the present invention and that have been deposited in compliance
with the requirements of the Budapest Treaty are strains MVA 572,
deposited at the European Collection of Animal Cell Cultures
(ECACC), Vaccine Research and Production Laboratory, Public Health
Laboratory Service, Centre for Applied Microbiology and Research,
Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom, with the
deposition number ECACC 94012707 on Jan. 27, 1994, and MVA 575,
deposited under ECACC 00120707 on Dec. 7, 2000. MVA-BN, deposited
on Aug. 30, 2000 at the European Collection of Cell Cultures
(ECACC) under number V00083008, and its derivatives, are additional
exemplary strains. In some embodiments, the invention encompasses
the use of recombinant orthopoxviruses, preferably a vaccinia virus
(W), a Wyeth strain, ACAM 1000, AC AM 2000, MVA, or MVA-BN for
cancer therapy. Recombinant orthopoxviruses are generated by
insertion of heterologous sequences into an orthopoxvirus. In some
embodiments, the recombinant poxvirus expressing a tumor antigen is
an avipoxvirus, such as but not limited to a fowlpox virus. The
term "avipoxvirus" refers to any avipoxvirus, such as Fowlpoxvirus,
Canarypoxvirus, Uncopoxvirus, Mynahpoxvirus, Pigeonpoxvirus,
Psittacinepoxvirus, Quailpoxvirus, Peacockpoxvirus,
Penguinpoxvirus, Sparrowpoxviras, Starlingpoxviras and
Turkeypoxviras. Preferred avipoxviruses are Canarypoxvirus and
Fowlpoxvirus.
[0032] In some embodiments, the vaccine composition comprises at
least one population of antigen presenting cells that present the
selected antigen. The antigen-presenting cell (or stimulator cell)
typically has an MHC class I or II molecule on its surface, and in
one embodiment is substantially incapable of itself loading the MHC
class I or II molecule with the selected antigen. Preferably, the
antigen presenting cells are dendritic cells. Suitably, the
dendritic cells are autologous dendritic cells that are pulsed with
the antigen of interest (e.g. a peptide). T-cell therapy using
autologous dendritic cells pulsed with peptides from a tumor
associated antigen is disclosed in Murphy et al. (1996) The
Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32,
272-278. Thus, in some embodiments, the vaccine composition
containing at least one antigen presenting cell is pulsed or loaded
with one or more antigenic peptides. As an alternative the antigen
presenting cell comprises an expression construct encoding an
antigenic peptide. The polynucleotide may be any suitable
polynucleotide and it is preferred that it is capable of
transducing the dendritic cell, thus resulting in the presentation
of a peptide and induction of an immune response.
[0033] In some embodiments, the vaccine composition include one or
more adjuvants. Adjuvants are substances that non-specifically
enhance or potentiate the immune response (e.g., immune responses
mediated by CD8-positive T cells and helper-T (TH) cells to an
antigen, and would thus be considered useful in the medicament of
the present invention. Suitable adjuvants include, but are not
limited to, 1018 ISS, aluminum salts, AMPLIVAX.RTM., AS15, BCG,
CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived
from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod
(ALDARA.RTM.), resiquimod, ImuFact IMP321, Interleukins as IL-2,
IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivatives
thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune.RTM.,
LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312,
Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,
water-in-oil and oil-in-water emulsions, OK-432, OM-174,
OM-197-MP-EC, ONTAK, OspA, PepTel.RTM. vector system, poly(lactid
co-glycolid) [PLG]-based and dextran microparticles, talactoferrin
SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF
trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which is
derived from saponin, mycobacterial extracts and synthetic
bacterial cell wall mimics, and other proprietary adjuvants such as
Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or
GM-CSF are preferred. Several immunological adjuvants (e.g., MF59)
specific for dendritic cells and their preparation have been
described previously (Allison and Krummel, 1995). Also cytokines
may be used. Several cytokines have been directly linked to
influencing dendritic cell migration to lymphoid tissues (e.g.,
TNF-), accelerating the maturation of dendritic cells into
efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,
IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated
herein by reference in its entirety) and acting as immunoadjuvants
(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich
et al., 1996).
[0034] A further object of the present invention relates to a
cancer vaccine comprising an immunoadjuvant together with one or
more cancer antigens, for inducing an immune response against said
one or more cancer antigens wherein the immunoadjuvant is a SK-1
inhibitor.
[0035] The term "sphingosine kinase-1" or "SK1" refers to an enzyme
that catalyzes the transformation of sphingosine to
sphingosine-1-phosphate (S1P), i.e., phosphorylates sphingosine
into S1P. Properties and activities of SK1, e.g., protein sequence
of SK1, structural properties of SK1, biochemical properties of
SK1, and regulation of SK1, are described in Taha et al. (2006,
Journal of Biochemistry and Molecular Biology, 39(2): 113-131).
Thus, as used herein the term "SK1 inhibitor" refers to any
compound that is capable to inhibit SK1 expression or activity. As
used herein the term `SK1 activity" refers to the production,
release, expression, function, action, interaction or regulation of
SK1, including, e.g., temporal, site or distribution aspects. The
activity of SK1 includes modifications, e.g., covalent or
non-covalent modifications of SK1 polypeptide, covalent or
non-covalent modifications that SK1 induces on other substances,
changes in the distribution of SK1 polypeptide, and changes that
SK1 induces on the distribution of other substances. Any aspect of
SK1 activity can be evaluated. Methods and techniques known to
those skilled in the art can be found in various references, e.g.,
Ausubel et al., ed., Current Protocols in Mol. Biology, New York:
John Wiley & Sons, 1990; Sambrook et al., Mol. Cloning, Cold
Spring Harbor Laboratory Press, New York, N.Y. (1989). Examples of
SK1 activity that can be evaluated include binding activity of SK1
polypeptide to a binding molecule; the effect of SK1 polypeptide on
the posttranslational modification or stability of a target gene;
the level of SK1 protein; the level of SK1 mRNA; or the level of
SK1 modification, e.g., phosphorylation, acetylation, methylation,
carboxylation or glycosylation. By binding molecule is meant any
molecule to which SK1 can bind, e.g., a nucleic acid, e.g., a DNA
regulatory region, a protein, a metabolite, a peptide mimetic, a
non-peptide mimetic, an antibody, or any other type of ligand.
Binding can be shown, e.g., by electrophoretic mobility shift
analysis (EMSA), by the yeast or mammalian two-hybrid or
three-hybrid assays, by competition with dimethylspingosine
photoaffinity label or biotin-SK1 binding. Transactivation of a
target gene by SK1 can be determined, e.g., in a transient
transfection assay in which the promoter of the target gene is
linked to a reporter gene, e.g., .beta.-galactosidase or
luciferase, and co-transfected with a SK1 expression vector. Levels
of SK1 protein, mRNA or modification, can, e.g., be measured in a
sample, e.g., a tissue sample, e.g., endothelial cells in blood
vessels, T and B lymphocytes from blood or lymph organs, heart,
muscle or bone joints. In some embodiments, the evaluations are
done in vitro; in other embodiments the evaluations are done in
vivo.
[0036] SK1 inhibitors are well known to the skilled person. For
example the skilled person may easily identify such inhibitors from
the following patent publications: WO2003105840, WO2006138660,
WO2010033701, WO2010078247, WO2010127093, WO2011020116,
WO2011072791, WO2012069852, WO2013119946, WO2014118556 and
WO2014157382.
[0037] In some embodiments, the SK1 inhibitor is selected from the
group consisting of 3-(4-Chloro-phenyl)-adamantane-1-carboxylic
acid isopropylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylic
acid cyclopropylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylic
acid (2-ethylsulfanyl-ethyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid phenylamide;
Adamantane-1-carboxylic acid (4-hydroxy-phenyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(4-hydroxy-phenyl)-amide; Acetic acid
4-{[3-(4-chloro-phenyl)-adamantane-1-carbonyl]-amino}-phenyl ester;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(2,4-dihydroxy-phenyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(3-hydroxymethyl-phenyl)-amide; Adamantane-1-carboxylic acid
(4-cyanomethyl-phenyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(4-cyanomethyl-phenyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-tert-butyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-methylsulfanyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3-trifluoromethyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-trifluoromethyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3,5-bis-trifluoromethyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3-fluoro-5-trifluoromethyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
2-fluoro-4-trifluoromethyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3,5-difluoro-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3,4-difluoro-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3,4,5-trifluoro-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3-chloro-4-fluoro-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-fluoro-3-trifluoromethyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
2-chloro-4-fluoro-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-chloro-3-trifluoromethyl-ben .SIGMA. ylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3-aminomethyl-2,4,5,6-tetrachloro-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[1-(4-chloro-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[1-(4-bromo-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-methanesulfonyl-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-dimethylamino-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-trifluoromethoxy-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3-trifluoromethoxy-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-phenoxy-benzylamide; Adamantane-1-carboxylic acid
3,4-dihydroxy-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
3,4-dihydroxy-benzylamide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid phenethyl-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[2-(4-fluoro-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[2-(4-bromo-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[2-(4-hydroxy-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
4-phenoxy-benzylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylic
acid [2-(3-bromo-4-methoxy-phenyl)-ethyl]-amide;
Adamantane-1-carboxylic acid
[2-(3,4-dihydroxy-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[2-(3,4-dihydroxy-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(2-benzo[1,3]dioxol-5-yl-ethyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[2-(3-phenoxy-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[2-(4-phenoxy-phenyl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(3-phenyl-propyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(biphenyl-4-ylmethyl)-amide; Adamantane-1-carboxylic acid
(1-methyl-piperidin-4-yl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(1-methyl-piperidin-4-yl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(4-methyl-piperazin-1-yl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(3-tert-butylamino-propyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(3-pyrrolidin-1-yl-propyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[3-(2-oxo-pyrrolidin-1-yl)-propyl]-amide; Adamantane-1-carboxylic
acid [2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(2-morpholin-4-yl-ethyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(2-piperazin-1-yl-ethyl)-amide; Adamantane-1-carboxylic acid
(pyridin-4-ylmethyl)-amide;
3-(4-Fluoro-phenyl)-adamantane-1-carboxylic acid
(pyridin-4-ylmethyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(pyridin-4-ylmethyl)-amide; Adamantane-1-carboxylic acid
(pyridin-4-ylmethyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(2-pyridin-4-yl-ethyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(3-imidazol-1-yl-propyl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(2-methyl-1H-indol-5-yl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(1H-tetrazol-5-yl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(9-ethyl-9H-carbazol-3-yl)-amide; Adamantane-1-carboxylic acid
[4-(4-chloro-phenyl)-thiazol-2-yl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
[4-(4-chloro-phenyl)-thiazol-2-yl]-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
benzothiazol-2-ylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylic
acid (5-chloro-benzooxazol-2-yl)-amide;
3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid
(9H-purin-6-yl)-amide;
[3-(4-Chloro-phenyl)-adamantane-1-ylmethyl]-isopropyl-amine
4-{[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-amino}-phenol;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-trifluoromethyl-benzyl)-ami-
ne;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(2-fluoro-4-trifluoromethyl-
-benzyl)-amine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-fluoro-3-trifluoromethyl-be-
nzyl)-amine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-trifluoromethoxy-benzyl)-am-
ine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-[2-(3-phenoxy-phenyl)-ethy-
l]-amine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(1-methyl-piperidin-4-
-yl)-amine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-methyl-piperazin-1-yl)-amin-
e;
N-tert-Butyl-N'-[3-(4-chloro-phenyl)-adamantan-1-ylmethyl]-propane-1,3--
diamine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(3-pyrrolidin-1-yl-pro-
pyl)-amine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-[2-(1-methyl-pyrrolidin-2-yl)--
ethyl]-amine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(2-morpholin-4-yl-ethyl)-amine-
;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-pyridin-4-ylmethyl-amine;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(9-ethyl-9H-carbazol-3-yl)-ami-
ne;
[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-[5-(4-chloro-phenyl)-thiazo-
l-2-yl]-amine; 1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethylamine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-isopropyl-amine;
Phenyl-[1-(3-phenyl-adamantan-1-yl)-ethyl]-amine;
{1-[3-(4-Fluoro-phenyl)-adamantan-1-yl]-ethyl}-.rho.henyl-amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-phenyl-amine;
(1-Adamantan-1-yl-ethyl)-benzyl-amine;
Benzyl-[1-(3-phenyl-adamantan-1-yl)-ethyl]-amine;
Benzyl-{1-[3-(4-fluoro-phenyl)-adamantan-1-yl]-ethyl}-amine;
Benzyl-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amine;
(4-tert-Butyl-benzyl)-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amin-
e;
[1-(4-Bromo-phenyl)-ethyl]-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethy-
l}-amine;
(1-Adamantan-1-yl-ethyl)-[2-(4-bromo-phenyl)-ethyl]-amine;
[2-(4-Bromo-phenyl)-ethyl]-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-
-amine; (1-Adamantan-1-yl-ethyl)-(1-methyl-piperidin-4-yl)-amine;
(1-Methyl-piperidin-4-yl)-[1-(3-phenyl-adamantan-1-yl)-ethyl]-amine;
{1-[3-(4-Fluoro-phenyl)-adamantan-1-yl]-ethyl}-(1-methyl-piperidin-4-yl)--
amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(1-methyl-piperidin--
4-yl)-amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(4-methyl-piperazin-1-yl)--
amine;
{1-[3-(Phenyl)-adamantan-1-yl]-ethyl}-pyridin-4-ylmethyl-amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(6-chloro-pyridin-3-ylmeth-
yl)-amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(2-pyridin-4-yl--
ethyl)-amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(3H-imidazol-4-ylmethyl)-a-
mine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(2-methyl-1H-indol-5--
yl)-amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(9-ethyl-9H-carb-
azol-3-yl)-amine;
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(9-ethyl-9H-carbazol-3-ylm-
ethyl)-amine; 9-Ethyl-9H-carbazole-3-carboxylic acid
{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amide;
1-{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-3-(4-chloro-3-trifluorom-
ethyl-phenyl)-urea;
1-{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-3-(4-chloro-3-trifluorom-
ethyl-phenyl)-urea;
(4-Bromo-thiophen-2-ylmethyl)-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-eth-
yl}-amine; and
{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(4-phenyl-thiophen-2-ylmet-
hyl)-amine
[0038] In some embodiments, the SK1 inhibitor of the present
invention is selected from the group consisting of:
##STR00001## ##STR00002##
[0039] In some embodiments, the SK1 inhibitor is
N'-[(2-hydroxynaphthalen-1-yl)methylidene]-3-(naphthalen-2-yl)-1H-pyrazol-
e-5-carbohydrazide having the formula of:
##STR00003##
[0040] In some embodiments, the SK1 inhibitor is an inhibitor of
SK1 expression. An "inhibitor of 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 SK1
mRNA by binding thereto and thus preventing protein translation or
increasing mRNA degradation, thus decreasing the level of SK1, 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 SK1 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. SK1 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 SK1 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
SK1. 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.
[0041] In some embodiments, the immune checkpoint inhibitor is an
antibody selected from the group consisting of anti-CTLA4
antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2
antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3
antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and
anti-B7H6 antibodies.
[0042] As used herein, the term "antibody" is thus used to refer to
any antibody-like molecule that has an antigen binding region, and
this term includes antibody fragments that comprise an antigen
binding domain such as Fab', Fab, F(ab')2, single domain antibodies
(DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv,
Fd, linear antibodies, minibodies, diabodies, bispecific antibody
fragments, bibody, tribody (scFv-Fab fusions, bispecific or
trispecific, respectively); sc-diabody; kappa(lamda) bodies
(scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv
tandems to attract T cells); DVD-Ig (dual variable domain antibody,
bispecific format); SIP (small immunoprotein, a kind of minibody);
SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART
(ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody
mimetics comprising one or more CDRs and the like. The techniques
for preparing and using various antibody-based constructs and
fragments are well known in the art (see Kabat et al., 1991,
specifically incorporated herein by reference). Diabodies, in
particular, are further described in EP 404,097 and WO 93/11161;
whereas linear antibodies are further described in Zapata et al.
(1995). Antibodies can be fragmented using conventional techniques.
For example, F(ab')2 fragments can be generated by treating the
antibody with pepsin. The resulting F(ab')2 fragment can be treated
to reduce disulfide bridges to produce Fab' fragments. Papain
digestion can lead to the formation of Fab fragments. Fab, Fab' and
F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers,
minibodies, diabodies, bispecific antibody fragments and other
fragments can also be synthesized by recombinant techniques or can
be chemically synthesized. Techniques for producing antibody
fragments are well known and described in the art. For example,
each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall
et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and
Young et al., 1995 further describe and enable the production of
effective antibody fragments. In some embodiments, the antibody of
the present invention is a single chain antibody. As used herein
the term "single domain antibody" has its general meaning in the
art and 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 single domain antibody
are also "nanobody.RTM.". For a general description of (single)
domain antibodies, reference is also made to the prior art cited
above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct.
12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003,
21(11):484-490; and WO 06/030220, WO 06/003388.
[0043] In some embodiments, the antibody is a humanized antibody.
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.
[0044] In some embodiments, the antibody is a fully human antibody.
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. 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.
[0045] In some embodiments, the antibody comprises human heavy
chain constant regions sequences but will not deplete CD8+ T cells
to which they are bound and preferably do not comprise an Fc
portion that induces antibody dependent cellular cytotoxicity
(ADCC). As used herein, the term "depleting", with respect to CD8+
T cells means a process, method, or compound that can kill,
eliminate, lyse or induce such killing, elimination or lysis, so as
to negatively affect the number of CD8+ T cells present in a sample
or in a subject. The terms "Fc domain," "Fc portion," and "Fc
region" refer to a C-terminal fragment of an antibody heavy chain,
e.g., from about amino acid (aa) 230 to about aa 450 of human gamma
heavy chain or its counterpart sequence in other types of antibody
heavy chains (e.g., .alpha., .delta., .epsilon. and .mu. for human
antibodies), or a naturally occurring allotype thereof. Unless
otherwise specified, the commonly accepted Kabat amino acid
numbering for immunoglobulins is used throughout this disclosure
(see Kabat et al. (1991) Sequences of Protein of Immunological
Interest, 5th ed., United States Public Health Service, National
Institute of Health, Bethesda, Md.). In some embodiments the
antibody of the present invention does not lead, directly or
indirectly, to the depletion of CD8+ T cells (e.g. do not lead to a
10%, 20%, 50%, 60% or greater elimination or decrease in number
CD8+ T cells). In some embodiments, the antibody of the present
invention does not comprise an Fc domain capable of substantially
binding to a FcgRIIIA (CD16) polypeptide. In some embodiments, the
antibody of the present invention lacks an Fc domain (e.g. lacks a
CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4
isotype. In some embodiments, the antibody of the present invention
consists of or comprises a Fab, Fab', Fab'-SH, F (ab') 2, Fv, a
diabody, single-chain antibody fragment, or a multispecific
antibody comprising multiple different antibody fragments. In some
embodiments, the antibody of the present invention is not linked to
a toxic moiety. In some embodiments, one or more amino acids
selected from amino acid residues can be replaced with a different
amino acid residue such that the antibody has altered C2q binding
and/or reduced or abolished complement dependent cytotoxicity
(CDC). This approach is described in further detail in U.S. Pat.
No. 6,194,551 by Idusogie et al.
[0046] Examples of anti-CTLA-4 antibodies are described in U.S.
Pat. Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157;
6,682,736; 6,984,720; and 7,605,238. One anti-CTLA-4 antibody is
tremelimumab, (ticilimumab, CP-675,206). In some embodiments, the
anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010) a
fully human monoclonal IgG antibody that binds to CTLA-4.
[0047] Examples of PD-1 and PD-L1 antibodies are described in U.S.
Pat. Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149,
and PCT Published Patent Application Nos: WO03042402, WO2008156712,
WO2010089411, WO2010036959, WO2011066342, WO2011159877,
WO2011082400, and WO2011161699. In some embodiments, the PD-1
blockers include anti-PD-L1 antibodies. In certain other
embodiments the PD-1 blockers include anti-PD-1 antibodies and
similar binding proteins such as nivolumab (MDX 1106, BMS 936558,
ONO 4538), a fully human IgG4 antibody that binds to and blocks the
activation of PD-1 by its ligands PD-L1 and PD-L2; lambrolizumab
(MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody
against PD-1; CT-011 a humanized antibody that binds PD-1; AMP-224
is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559
(MDX-1105-01) for PD-L1 (B7-H1) blockade.
[0048] Other immune-checkpoint inhibitors include lymphocyte
activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig
fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211).
Other immune-checkpoint inhibitors include B7 inhibitors, such as
B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody
MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834).
Also included are TIM3 (T-cell immunoglobulin domain and mucin
domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med.
207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94).
As used herein, the term "TIM-3" has its general meaning in the art
and refers to T cell immunoglobulin and mucin domain-containing
molecule 3. The natural ligand of TIM-3 is galectin 9 (Gal9).
Accordingly, the term "TIM-3 inhibitor" as used herein refers to a
compound, substance or composition that can inhibit the function of
TIM-3. For example, the inhibitor can inhibit the expression or
activity of TIM-3, modulate or block the TIM-3 signaling pathway
and/or block the binding of TIM-3 to galectin-9. Antibodies having
specificity for TIM-3 are well known in the art and typically those
described in WO2011155607, WO2013006490 and WO2010117057.
[0049] In some embodiments, the immune checkpoint inhibitor is an
IDO inhibitor. Examples of IDO inhibitors are described in WO
2014150677. Examples of IDO inhibitors include without limitation
1-methyl-tryptophan (IMT), .beta.-(3-benzofuranyl)-alanine,
.beta.-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan,
6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyl tryptophan,
6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan,
indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate,
5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin,
5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-Amino-naphtoic
acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin
derivative, a thiohydantoin derivative, a .beta.-carboline
derivative or a brassilexin derivative. Preferably the IDO
inhibitor is selected from 1-methyl-tryptophan,
.beta.-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan,
3-Amino-naphtoic acid and .beta.-[3-benzo(b)thienyl]-alanine or a
derivative or prodrug thereof.
[0050] As used herein the term "co-administering" as used herein
means a process whereby the combination of the SK1 inhibitor and
the immune checkpoint inhibitor, is administered to the same
patient. The SK1 inhibitor and the immune checkpoint inhibitor may
be administered simultaneously, at essentially the same time, or
sequentially. If administration takes place sequentially, the SK1
inhibitor is administered before the immune checkpoint inhibitor.
The SK1 inhibitor and the immune checkpoint inhibitor need not be
administered by means of the same vehicle. The SK1 inhibitor and
the immune checkpoint inhibitor may be administered one or more
times and the number of administrations of each component of the
combination may be the same or different. In addition, the SK1
inhibitor and the immune checkpoint inhibitor need not be
administered at the same site.
[0051] As used herein, the term "therapeutically effective
combination" as used herein refers to an amount or dose of a SK1
inhibitor together with the amount or dose of the immune checkpoint
inhibitor that is sufficient to treat the disease (e.g. cancer).The
amount of the SK1 inhibitor in a given therapeutically effective
combination may be different for different individuals and
different tumor types, and will be dependent upon the one or more
additional agents or treatments included in the combination. The
"therapeutically effective amount" is determined using procedures
routinely employed by those of skill in the art such that an
"improved therapeutic outcome" results. It will be understood,
however, that the total daily usage of the compounds and
compositions 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
disorder 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 coincidental with
the specific polypeptide 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.
[0052] According to the invention, the SK1 inhibitor and the immune
checkpoint inhibitor are administered to the subject in the form of
a pharmaceutical composition. Typically, the SK1 inhibitor and the
immune checkpoint inhibitor may be combined with pharmaceutically
acceptable excipients, and optionally sustained-release matrices,
such as biodegradable polymers, to form therapeutic compositions.
"Pharmaceutically" or "pharmaceutically acceptable" refer 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. In the pharmaceutical compositions of the
present invention for oral, sublingual, subcutaneous,
intramuscular, intravenous, transdermal, local or rectal
administration, the active principle, 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. Typically, 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. 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. Solutions comprising
compounds of the invention 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. The SK1 inhibitor and the immune checkpoint
inhibitor can be formulated into a composition in a neutral or salt
form. Pharmaceutically acceptable salts include the acid addition
salts (formed with the free amino groups of the protein) and which
are formed with inorganic acids such as, for example, hydrochloric
or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. 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. Sterile injectable solutions are prepared
by incorporating the active compounds in the required amount in the
appropriate solvent with several 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 typical 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. The
preparation of more, or highly concentrated solutions for direct
injection is also contemplated, where the use of DMSO as solvent is
envisioned to result in extremely rapid penetration, delivering
high concentrations of the active agents to a small tumor area.
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. 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. 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.
[0053] 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
[0054] FIG. 1A-D. Downregulation of SK1 reduces melanoma tumor
development in mice. (A), SK1 enzymatic activity was measured in
Yumm cells stably transfected with a control (shCtrl) or two
SK1-targeted shRNA: shSK1 and shSK1(2). Enzyme activity (calculated
as pmol/min/mg of proteins) in cells transfected with shSK1 is
compared to that of shCtrl cells. Data are means.+-.sem of 3
independent experiments. (B-D), shCtrl or shSK1 Yumm cells (3.105)
were injected in the dermis of wild-type (WT) C57BL/6 mice (B and
C) or CD8-deficient mice (D). Tumor volume was determined at the
indicated days (B and D) or at the end of the experiment (C, day
26). Growth profiles are presented as mean of tumor volume.+-.SEM
and are representative of at least two independent experiments. (B
and C n=12 per group, D n=8 per group). Samples were compared using
Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001.
[0055] FIG. 2A-C. Downregulation of SK1 increases CD8+ T cell
proliferation and activation and inversely, reduces Treg. shCtrl or
shSK1 Yumm murine melanoma cells were injected in C57BL/6 mice and
TILs were analyzed on day 11 by using flow cytometry. (A) CD8+,
Foxp3+ CD4+ (Treg) and Foxp3- CD4+ T cells percentages and
CD8+/Treg ratio in tumors at day 11. (B and C) The proportion of
CD8+ (B) and Treg cells (C) expressing Ki67, PD-1 and CTLA-4 was
evaluated. Each symbol represents an independent tumor (n=9 mice
per group). Results are representative of at least 2 independent
experiments. Samples were compared using Mann-Whitney test.
*p<0.05, **p<0.01, ***p<0.001.
[0056] FIG. 3A-D. Downregulation of SK1 in tumor cells enhances the
efficacy of anti-CTLA-4 or anti-PD-1 therapy. Mice were challenged
with 3.105 shCtrl or shSK1 Yumm cells on day 0, and then treated
with control antibody (upper panels), .alpha.-CTLA-4 (days 7, 10
and 13) or/and .alpha.-PD-1 (days 5, 7 and 10). (A and B)
Individual curves are depicted for each tumor (n=6-11 mice per
group). Inserts: numbers indicate percentage of tumor-free mice at
day 25. (C) Cumulative survival curves (Logrank test: *p<0.05;
**p<0.01; ***p<0.001). (D) CD8/Treg ratio in tumors at day 11
are represented as Tukey box (n=10 per group). Results are
representative of at least 2 independent experiments. Samples were
compared using Mann-Whitney test. *p<0.05, **p<0.01,
***p<0.001.
[0057] FIG. 4A-B. Pharmacological inhibition of SK1 synergizes with
CTLA-4 blockade to eradicate melanoma tumors. Mice were challenged
with 3.105 untransfected Yumm cells on day 0, and then treated or
not with SKI-I on days 5, 7, 10, 13 and 15. Control antibody or
.alpha.-CTLA-4 was injected on days 7, 10 and 13. (A) Tumor volumes
determined at the indicated days for individual tumors are depicted
(n=6 mice per group). Inserts: numbers indicate percentage of
tumor-free mice at day 30. (B) Cumulative survival curves (Logrank
test: *p<0.05).
[0058] FIG. 5A-B. Pharmacological inhibition of SK1 synergizes with
anti-PD-1 blockade to reduce tumor growth in melanoma and colon
carcinoma. (A) Mice were challenged with 3.105 untransfected Yumm
cells on day 0, and then treated or not with SKI-I on days 5, 7,
10, 13 and 15. Control antibody or .alpha.-PD-1 was injected on
days 5, 7 and 10 (n=11-12 mice per group). (B) Mice were inoculated
with 3.105 MC38 cells on day 0, and then treated or not with SKI-I
on days 5, 7, 10, 13 and 15. Control antibody or .alpha.-PD-1 was
injected on days 7 and 10 (n=4-6 mice per group). Tumor volume
(means.+-.sem for each group) was determined at the indicated days.
Samples were compared using Mann-Whitney test. *p<0.05.
EXAMPLE
[0059] Material & Methods
[0060] Cell Culture
[0061] Yumm murine melanoma cells, which harbor BRAFV600E mutation,
Pten and Cdkn2a deletion [1] were kindly provided by Dr. S.
Tartare-Deckert (INSERM U1065 Nice, France). Yumm cells were grown
as monolayers in OptiMEM media supplemented with 3%
heat-inactivated fetal calf serum (FCS) in the presence of 5% CO2
in a humidified atmosphere at 37.degree. C. To guarantee cell line
authenticity, Yumm cell lines were used for a limited number of
passages and routinely tested for the expression of
melanocyte-lineage proteins such as MelanA/MART1. MC38 cells were
kindly provided by Drs T. Chardes et A. Pelegrin (INSERM U1194,
Montpellier, France) and were cultured in DMEM containing 10% FCS,
2 mM glutamine, 0.1 mM non essential amino acids, 1 mM sodium
pyruvate and 10 mM Hepes.
[0062] Cell Transfection
[0063] Yumm cells were co-transfected, in a 1:10 ratio, with the
pEGFP-N empty vector and a SK1 shRNA (shSK1 or shSK1(2)) plasmid
(shRNA from Thermoscientific) or a control non-targeting shRNA
(shCtrl) plasmid (pLK01, Addgene). In brief, 500,000 cells were
seeded in T25 cell culture flasks. Plasmids were diluted in OptiMEM
(Thermofisher) medium without serum. Cells were transfected with 10
.mu.g shRNA oligomer using Lipofectamine 2000 reagent (Invitrogen)
according to the manufacturer's instructions. Transfected cells
were selected with 0.4 mg/ml G418 and 1.5 .mu.g/ml puromycin and
GFP-expressing cells were sorted by FACS. Stable transfectants were
maintained in media containing 1 .mu.g/ml puromycin; for the
experiments, cells were cultured in medium without puromycin.
[0064] SK1 Enzymatic Assay
[0065] SK1 activity was determined as described (Lavieu, Scarlatti
et al. 2008) with minor modifications.
[0066] Tumor Cell Injections and Treatments in Mice
[0067] Animal experiments were conducted in accordance with
national and international policies, and our protocol was approved
by the Regional Ethics Committee of Midi-Pyrenees. 3.105 of Yumm
cell lines (Untransfected, shCtrl, shSK1 or shSK1(2)) were
intradermally injected into the flank of 7-week-old C57BL/6 mice
(Charles River, L'Arbresle, France). CD8-deficient C57BL/6 mice
were a gift from Prof. J. van Meerwijk (INSERM U1043, Toulouse,
France). Tumor volume was calculated using a caliper at the
indicated days as described (Albinet, Bats et al. 2014). For
combination experiments involving shCtrl or shSK1 Yumm cells, mice
were challenged intradermally (i.d.) with 3.105 cells on day 0 on
their right flank. Mice were then injected i.p. three times with
anti-CTLA-4 (200 .mu.g per mouse on D7 and 100 .mu.g per mouse on
D10 and D13), and/or with anti-PD-1 or isotype control antibody
(200 .mu.g per mouse on D5, D7 and D10). Tumor volumes were
measured every 2-3 days. Anti-CTLA-4 (9H10), anti-PD-1 (RMP1-14)
and isotype control (2A3) were purchased from BioXcell.
[0068] For SKI-I treatment, 5 days after Yumm or MC38 cell
implantation, mice were treated or not with 50 mg/kg SKI-I
(N'-[(2-hydroxynaphthalen-1-yl)
methylidene]-3-(naphthalen-2-yl)-1H-pyrazole-5-carbohydrazide,
Enamine) in 50 .mu.l of a mixture of DMSO (10%), Cremophor (5%),
Tween-80 (5%) and glucose (80%) (i.p.). Mice received additional
treatments of SKI-I on days 7, 10, 13 and 15. Mice with Yumm tumors
were injected i.p. with anti-CTLA-4 or anti-PD-1 or control
antibody as described above. For MC38 tumors, mice were injected
i.p. two times with anti-PD-1 (100 .mu.g per mouse on D7 and
D10).
[0069] Analysis of Leukocyte Content in Tumors
[0070] Yumm cells (3.105) were intradermally injected into C57BL/6
mice. At day 11, mice were sacrificed and tumors were collected and
digested with Mouse Tumor Dissociation kit and GentleMacs
(Miltenyi). Cells were counted and stained with the indicated
antibodies and LIVE/DEAD reactive dyes (Invitrogen) prior to flow
cytometry analysis (BD LSRFortessa X-20). Analyses were restricted
to viable cells and performed using anti-mouse CD45 (BD
Biosciences), anti-mouse Thy1 (Biolegend), anti-mouse CD8
(Biolegend), anti-mouse CD4 (BD Biosciences), anti-mouse Foxp3
(eBioscience), anti-mouse Ki-67 (BD Bioscience), anti-mouse PD-1
(eBioscience) or anti-mouse CTLA-4 (eBioscience). Isotype controls
were from BD Biosciences, Biolegend or eBioscience.
[0071] Statistical Analyses
[0072] Data were analysed using GraphPad Prism (GraphPad Software,
San Diego, Calif.). Results are expressed as means.+-.sem.
Student's t test was used for statistical comparisons among groups
and differences were considered statistically significant when
p<0.05 (*, p<0.05; **, p<0.01; ***, p<0.001). Tumor
survival data were analyzed with the Kaplan-Meier method. The
log-rank test was used to compare survival curves for different
subgroups on univariate analyses.
[0073] Results
[0074] SK1 Downregulation Reduces Tumor Growth and Enhances
Antitumor Responses to Melanoma
[0075] In order to evaluate the effect of SK1 in a syngeneic
C57BL/6 mouse model of melanoma, we used a transplantable tumor
cell line (Yumm cells) established from a BrafV600E/+; Pten-/-;
CDKN2A-/- mouse (Pencheva, Buss et al. 2014). We generated stable
SK1 knockdown Yumm cells, by using a shRNA-mediated silencing
technology. We obtained two puromycin-resistant cell lines; shSK1
and shSK1(2), exhibiting a markedly reduced enzymatic activity of
SK1 (around 60% inhibition) (FIG. 1A). Then, Yumm cells, SK1
knockdown or not (shCtrl) for SK1, were intradermally injected in
C57BL/6 mice, and tumor growth was monitored. The tumor growth of
shSK1 and shSK1(2) Yumm cells was significantly lower than that of
shCtrl Yumm cells (FIGS. 1B and C). Interestingly, a tumor
regression after day 11 was observed in WT mice injected with ShSK1
cells that could reflect an increased anti-melanoma immune
response. However, this effect was unlikely sufficient to obtain a
long-lasting immune response, presumably due to immune escape
mechanisms. Importantly, SK1 knockdown failed to impair Yumm
melanoma growth in CD8-deficient mice (FIG. 1D).
[0076] To evaluate the impact of SK1 downregulation on the
composition of intratumoral lymphocyte infiltrate, we analyzed
Tumor Infiltrating Lymphocytes (TIL) on day 11. Of interest was the
finding that SK1 downregulation increased the proportion of CD8+ T
cells and decreased the proportion of Foxp3+ CD4+ T cells (Treg)
leading to a 4-fold increase in CD8/Treg ratio (FIG. 2A). Moreover,
the analysis of TIL proliferation (as evaluated by monitoring Ki67
expression) and activation (as evaluated by PD-1 and CTLA-4
expression) showed that SK1 knockdown significantly increased CD8+
T cell proliferation and activation (FIG. 2B) and inversely,
decreased Treg proliferation as well as CTLA-4 expression (FIG.
2C).
[0077] SK1 Downregulation Improves the Response to
Immunotherapy
[0078] Given that SK1 downregulation was associated with an
increase of tumor activated CD8+ T cells, we hypothesized that SK1
inhibition may improve the efficacy of Immune Checkpoint Inhibitors
(ICI, e.g., anti-CTLA-4 and anti-PD-1). As shown in FIG. 3, whereas
anti-CTLA-4 or/and anti-PD-1 treatment alone had limited effects on
established Yumm tumors (FIG. 3A), SK1 silencing dramatically
enhanced the response to anti-CTLA-4 or anti-PD-1 treatment,
leading to tumor rejection in 100% and 67% of the animals,
respectively (FIG. 3B). Moreover, SK1 downregulation significantly
improved overall survival (FIG. 3C). Indeed, this combination
(ICI+SK1 silencing) induced durable cures in 100% and 42% of the
mice treated with anti-CTLA-4 and anti-PD-1, respectively, 2 months
after cessation of therapy, suggesting the establishment of an
effective immunological memory. Interestingly, amongst the
long-term survivors, none of them developed a tumor upon a second
melanoma cell injection, indicating that they were fully vaccinated
against this melanoma cell line (data not shown). This enhanced
response to ICI was associated with an increased CD8/Treg ratio in
tumors (FIG. 3D). Of note, the CD8/Treg ratio is impressively
increased in the tumors of Yumm ShSK1+anti-CTLA-4 group (Fold
Change=16), this could explain the total tumor regression observed
when using this combination.
[0079] Synergistic Anti-Cancer Immune Response of Immune Checkpoint
Blockade and SK1 Pharmacological Inhibition.
[0080] To further confirm the potency of the combined therapy based
on SK1 downregulation and ICI, we used SKI-I, a pharmacological
inhibitor of SK1 (French, Schrecengost et al. 2003). Our data show
that, whereas CTLA-4 blockade alone led to no tumor rejection at
all, the combination of SKI-I+anti-CTLA-4 greatly synergized,
resulting to total rejection in 67% of mice (FIG. 4A) and improved
the overall survival (FIG. 4B). To confirm our observation with
anti-PD-1, mice harboring Yumm tumors were treated with SKI-I
combined or not with anti-PD-1. As shown in FIG. 5A, SKI-I enhanced
the efficacy of anti-PD-1. Importantly, this effect was also
observed in mice inoculated with MC38 colon carcinoma (FIG.
5B).
[0081] Collectively our data indicate that greater therapeutic
success will be achieved by combining immune checkpoint blockade
with agents that modulate the oncogenic SK1/S1P pathway.
Interfering with sphingolipid metabolism may facilitate the
development of novel avenues for therapeutic intervention in
melanoma as well as in other cancer types.
REFERENCES
[0082] 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.
[0083] Albinet, V., M. L. Bats, A. Huwiler, P. Rochaix, C.
Chevreau, B. Segui, T. Levade and N. Andrieu-Abadie (2014). "Dual
role of sphingosine kinase-1 in promoting the differentiation of
dermal fibroblasts and the dissemination of melanoma cells."
Oncogene 33(26): 3364-3373.
[0084] French, K. J., R. S. Schrecengost, B. D. Lee, Y. Zhuang, S.
N. Smith, J. L. Eberly, J. K. Yun and C. D. Smith (2003).
"Discovery and evaluation of inhibitors of human sphingosine
kinase." Cancer Res 63(18): 5962-5969.
[0085] Lavieu, G., F. Scarlatti, G. Sala, S. Carpentier, T. Levade,
R. Ghidoni, J. Botti and P. Codogno (2008). "Sphingolipids in
macroautophagy." Methods Mol Biol 445: 159-173.
[0086] Pencheva, N., C. G. Buss, J. Posada, T. Merghoub and S. F.
Tavazoie (2014). "Broad-spectrum therapeutic suppression of
metastatic melanoma through nuclear hormone receptor activation."
Cell 156(5): 986-1001.
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