Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
BLOOD PLASMA BIOMARKERS FOR BEVACIZUMAB COMBINATION
THERAPIES
FOR TREATMENT OF BREAST CANCER
Field of the Invention
The present invention is directed to methods for identifying which patients
diagnosed with breast
cancer will most benefit from treatment with an anti-cancer therapy comprising
an anti-VEGF
antibody.
Background of the Invention
Angiogenesis contributes to benign and malignant diseases such as cancer
development and,
especially in cancer, is necessary for primary tumor growth, invasiveness and
metastasis. In
order to grow, a tumor must undergo an angiogenic switch. Vascular endothelial
growth factor
(VEGF) is required to induce this angiogenic switch. VEGF and the genes in the
VEGF pathway
are considered important mediators of cancer progression. The VEGF gene family
includes the
VEGF gene, also referred to as VEGFA, homologues to VEGF including, placenta
growth factor
(P1GF), VEGFB, VEGFC, VEGFD, the VEGF receptors, including VEGFR-1 and VEGFR-2
(also referred to as FLT1 and FLK1/KDR, respectively), the VEGF inducers,
including hypoxia-
inducible factors HIF1a, HIF2 a, and the oxygen sensors PHD1, PHD2 and PHD3.
The importance of this pathway in cancer cell growth and metastasis has led to
the development
of anti-angiogenesis agents for use in cancer therapy. These therapies
include, among others,
bevacizumab, pegaptanib, sunitinib, sorafenib and vatalanib. Despite
significantly prolonged
survival obtained with angiogenesis inhibitors, such as bevacizumab, patients
still succumb to
cancer. Further, not all patients respond to angiogenesis inhibitor therapy.
The mechanism
underlying the non-responsiveness remains unknown. Moreover, angiogenesis
inhibitor therapy
is associated with side effects, such as gastrointestinal perforation,
thrombosis, bleeding,
hypertension and proteinuria.
Accordingly, there is a need for methods of determining which patients respond
particular well
to angiogenesis inhibitor therapy.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 2 -
Summary of the Invention
An investigation of the status of biomarkers related to angiogenesis and
tumorigenesis revealed
that the expression levels of E-selectin, ICAM-1 and VEGFR-3 relative to
reference levels
determined in the entire biomarker patient population correlated with an
improved treatment
outcome in breast cancer patients. In particular, patients exhibiting an
increased expression level
of E-selectin, ICAM-1 and VEGFR-3 relative to reference levels determined in
the entire
biomarker patient population, demonstrated a prolonged progression-free
survival in response to
the addition of bevacizumab to the combination chemotherapy of trastuzumab and
docetaxel.
The present invention therefore relates to a method of determining whether a
patient diagnosed
with breast cancer is sensitive to an anti-cancer therapy comprising the
addition of an anti-VEGF
antibody to a chemotherapy regimen, by determing an expression level of at
least one biomarker
which is selected from the group consisting of E-selectin, ICAM-1 and VEGFR-3
in a patient
sample and comparing it with reference levels. The present invention also
relates to a
pharmaceutical composition comprising an anti-VEGF antibody, such as
bevacizumab, for the
treatment of a patient diagnosed with breast cancer and having an increased
expression level of
at least one biomarker which is selected from the group consisting of E-
selectin, ICAM-1 and
VEGFR-3 relative to reference levels. The present invention further relates to
a method for
improving the treatment effect of an anti-cancer therapy comprising a
chemotherapy of a patient
diagnosed with breast cancer by adding an anti-VEGF antibody, such as
bevacizumab, based on
an expression level of at least one biomarker which is selected from the group
consisting of E-
selectin, ICAM-1 and VEGFR-3 in a patient sample.
One embodiment of the invention provides in vitro methods of determining
whether a patient
diagnosed with breast cancer is more or less suitably treated by an anti-
cancer therapy
comprising an anti-VEGF antibody. The methods comprise (a) determining an
expression level
of at least one biomarker which is selected from the group consisting of E-
selectin, ICAM-1 and
VEGFR-3 in a sample derived from a patient diagnosed with breast cancer, and
(b) identifying
the patient as more or less suitably treated by an anti-cancer therapy
comprising an anti-VEGF
antibody based on the expression level in accordance with (a), wherein an
expression level of
said biomarker at or above a reference level indicates that the patient is
more suitably treated
with the anti-cancer therapy, or an expression level of said biomarker below a
reference level
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 3 -
indicates that the patient is less suitably treated with the anti-cancer
therapy. In some
embodiments, whether a patient is suitably treated by an anti-cancer therapy
is determined in
terms of progression-free survival. In some embodiments, the methods further
comprise treating
the patient with the anticancer therapy. In some embodiments, the anti-cancer
therapy comprises
an anti-VEGF antibody, an anti-HER2 antibody and a taxane. In some embodments,
the anti-
VEGF antibody is bevacizumab. In some embodiments, the anti-HER2 antibody is
trastuzumab.
In some embodiments, the taxane is docetaxel or paclitaxel. In some
embodiments, the
expression level of said at least one biomarker is a protein expression level.
In some
embodiments, the sample derived from the patient is a blood plasma sample. In
some
embodiments, the at least one biomarker is E-selectin. In some embodiments,
the at least one
biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-
3.
Another embodiment of the invention provides in vitro methods of selecting a
treatment for a
patient diagnosed with breast cancer. The methods comprise (a) assaying a
biological sample
from the patient, thereby determining that the patient has an expression level
of at least one
biomarker which is selected from the group consisting of E-selectin, ICAM-1
and VEGFR-3 at
or above a reference level, and (b) based on that determination, selecting the
treatment
comprising an anti-cancer therapy comprising an anti-VEGF antibody. In some
embodiments,
whether a patient is suitably treated by an anti-cancer therapy is determined
in terms of
progression-free survival. In some embodiments, the methods further comprise
treating the
patient with the anticancer therapy. In some embodiments, the patient received
no previous
chemotherapeutic or radiation treatment. In some embodiments, the anti-cancer
therapy
comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane. In some
embodments,
the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2
antibody is
trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In
some
embodiments, the expression level of said at least one biomarker is a protein
expression level. In
some embodiments, the sample derived from the patient is a blood plasma
sample. In some
embodiments, the at least one biomarker is E-selectin. In some embodiments,
the at least one
biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-
3.
A further embodiment of the invention provides in vitro methods of determining
whether a
patient diagnosed with breast cancer is sensitive to an anti-cancer therapy
comprising the
addition of an anti-VEGF antibody to a chemotherapy regimen. The methods
comprise (a)
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 4 -
determining an expression level of at least one biomarker which is selected
from the group
consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient diagnosed
with breast cancer, and (b) identifying the patient as sensitive to an anti-
cancer therapy
comprising the addition of an anti-VEGF antibody to a chemotherapy regimen
based on the
expression level in accordance with (a), wherein an expression level of said
biomarker at or
above a reference level indicates that the patient is sensitive to an anti-
cancer therapy comprising
the addition of an anti-VEGF antibody to a chemotherapy regimen. In some
embodiments,
whether a patient is suitably treated by an anti-cancer therapy is determined
in terms of
progression-free survival. In some embodiments, the methods further comprise
treating the
patient with the anticancer therapy. In some embodiments, the patient received
no previous
chemotherapeutic or radiation treatment. In some embodiments, the anti-cancer
therapy
comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane. In some
embodments,
the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2
antibody is
trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In
some
embodiments, the expression level of said at least one biomarker is a protein
expression level. In
some embodiments, the sample derived from the patient is a blood plasma
sample. In some
embodiments, the at least one biomarker is E-selectin. In some embodiments,
the at least one
biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-
3.
Even another embodiment of the invention provides in vitro methods of
selecting an anti-cancer
therapy for a patient diagnosed with breast cancer. The methods comprise (a)
assaying a
biological sample from the patient, thereby determining that the patient has
an expression level
of at least one biomarker which is selected from the group consisting of E-
selectin, ICAM-1 and
VEGFR-3 at or above a reference level, and (b) based on that determination,
selecting the anti-
cancer therapy comprising the addition of an anti-VEGF antibody to a
chemotherapy regimen.
In some embodiments, whether a patient is suitably treated by an anti-cancer
therapy is
determined in terms of progression-free survival. In some embodiments, the
methods further
comprise treating the patient with the anticancer therapy. In some
embodiments, the patient
received no previous chemotherapeutic or radiation treatment. In some
embodiments, the anti-
cancer therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a
taxane. In some
embodments, the anti-VEGF antibody is bevacizumab. In some embodiments, the
anti-HER2
antibody is trastuzumab. In some embodiments, the taxane is docetaxel or
paclitaxel. In some
embodiments, the expression level of said at least one biomarker is a protein
expression level. In
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 5 -
some embodiments, the sample derived from the patient is a blood plasma
sample. In some
embodiments, the at least one biomarker is E-selectin. In some embodiments,
the at least one
biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-
3.
Yet another embodiment of the invention provides pharmaceutical compositions
comprising an
anti-VEGF antibody for the treatment of a patient diagnosed with breast
cancer, wherein the
patient has been identified as more suitably treated by an anti-cancer therapy
comprising an anti-
VEGF antibody in accordance with any of the methods described herein.
Yet another embodiment of the invention provides pharmaceutical compositions
comprising an
anti-VEGF antibody for the treatment of a patient diagnosed with breast
cancer, wherein the
patient is identified as more suitably treated by an anti-cancer therapy
comprising an anti-VEGF
antibody in accordance with any of the methods described herein.
A further embodiment of the invention provides pharmaceutical compositions
comprising an
anti-VEGF antibody for the treatment of a patient diagnosed with breast
cancer, wherein the
patient has been identified as sensitive to an anti-cancer therapy comprising
the addition of an
anti-VEGF antibody to a chemotherapy regimen in accordance with any of the
methods
described herein.
A further embodiment of the invention provides pharmaceutical compositions
comprising an
anti-VEGF antibody for the treatment of a patient diagnosed with breast
cancer, wherein the
patient is identified as sensitive to an anti-cancer therapy comprising the
addition of an anti-
VEGF antibody to a chemotherapy regimen in accordance with any of the methods
described
herein.
Yet another embodiment of the invention provides kits for carrying out any of
the method
described herein. The kits comprise a set of compounds for detecting an
expression level of at
least one biomarker which is selected from the group consisting of E-selectin,
ICAM-1 and
VEGFR-3, the set comprising antibodies capable of specifically binding to said
biomarker.
Even another embodiment of the invention provides methods for improving the
treatment effect
of an anti-cancer therapy comprising a chemotherapy regimen in a patient
diagnosed with breast
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 6 -
cancer by adding an anti-VEGF antibody to the chemotherapy regimen. The
methods comprise
(a) determining an expression level of at least one biomarker which is
selected from the group
consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient diagnosed
with positive breast cancer; (b) identifying the patient as sensitive to an
anti-cancer therapy
comprising the addition of an anti-VEGF antibody to a chemotherapy regimen
based on the
expression level in accordance with (a), wherein an expression level of said
biomarker at or
above a reference level indicates that the patient is sensitive to an anti-
cancer therapy comprising
the addition of an anti-VEGF antibody to a chemotherapy regimen; and (c)
administering an
effective amount of an anti-VEGF antibody in combination with an effective
amount of a
chemotherapy regimen to the patient identified as sensitive to an anti-cancer
therapy comprising
the addition of an anti-VEGF antibody to a chemotherapy in accordance with
(b). In some
embodiments, whether a patient is suitably treated by an anti-cancer therapy
is determined in
terms of progression-free survival. In some embodiments, the methods further
comprise treating
the patient with the anticancer therapy. In some embodiments, the patient
received no previous
chemotherapeutic or radiation treatment. In some embodiments, the anti-cancer
therapy
comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane. In some
embodments,
the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-HER2
antibody is
trastuzumab. In some embodiments, the taxane is docetaxel or paclitaxel. In
some
embodiments, the expression level of said at least one biomarker is a protein
expression level. In
some embodiments, the sample derived from the patient is a blood plasma
sample. In some
embodiments, the at least one biomarker is E-selectin. In some embodiments,
the at least one
biomarker is ICAM-1. In some embodiments, the at least one biomarker is VEGFR-
3.
Detailed Description of the Embodiments
1. Definitions
The terms "administration" or "administering" as used herein mean the
administration of a
pharmaceutical composition, such as an angiogenesis inhibitor, to a patient in
need of such
treatment or medical intervention by any suitable means known in the art.
Nonlimiting routes of
administration include by oral, intravenous, intraperitoneal, subcutaneous,
intramuscular, topical,
intradermal, intranasal or intrabronchial administration (for example as
effected by inhalation).
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 7 -
Particularly preferred in context of this invention is parenteral
administration, e.g., intravenous
administration.
The term "anti-angiogenesis agent" or "angiogenesis inhibitor" refers to a
small molecular
weight substance, a polynucleotide, a polypeptide, an isolated protein, a
recombinant protein, an
antibody, or conjugates or fusion proteins thereof, that inhibits
angiogenesis, vasculogenesis, or
undesirable vascular permeability, either directly or indirectly. It should be
understood that the
anti-angiogenesis agent includes those agents that bind and block the
angiogenic activity of the
angiogenic factor or its receptor. For example, an anti-angiogenesis agent is
an antibody or other
antagonist to an angiogenic agent as defined throughout the specification or
known in the art,
e.g., but are not limited to, antibodies to VEGF-A or to the VEGF-A receptor
(e.g., KDR
receptor or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such as
GleevecTM (Imatinib
Mesylate). Anti-angiogensis agents also include native angiogenesis
inhibitors, e.g., angiostatin,
endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-
39 (1991); Streit
and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-
angiogenic therapy in
malignant melanoma); Ferrara & Alitalo, Nature Medicine 5:1359-1364 (1999);
Tonini et al.,
Oncogene, 22:6549-6556 (2003) (e.g., Table 2 listing known antiangiogenic
factors); and Sato.
Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 lists anti-angiogenic
agents used in clinical
trials).
The term "antibody" is herein used in the broadest sense and includes, but is
not limited to,
monoclonal and polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies),
chimeric antibodies, CDR grafted antibodies, humanized antibodies, camelized
antibodies, single
chain antibodies and antibody fragments and fragment constructs, e.g., F(ab')2
fragments, Fab-
fragments, Fv-fragments, single chain Fv-fragments (scFvs), bispecific scFvs,
diabodies, single
domain antibodies (dAbs) and minibodies.
The term "VEGF" or "VEGF-A" as used herein refers to, e.g. the 165-amino acid
human
vascular endothelial cell growth factor and related 121-, 189-, and 206- amino
acid human
vascular endothelial cell growth factors, as described by Leung et al. (1989)
Science 246:1306,
and Houck et al. (1991) Mol. Endocrin, 5:1806, together with the naturally
occurring allelic and
processed forms thereof, including the 110- amino acid human vascular
endothelial cell growth
factor generated by plasmin cleavage of VEGF165 as described in Ferrara MoL
Biol. Cell 21:687
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 8 -
(2010). The term "VEGF" also refers to VEGFs from non-human species such as
mouse, rat or
primate. Sometimes the VEGF from a specific species are indicated by terms
such as hVEGF
for human VEGF, mVEGF for murine VEGF, and etc. The term "VEGF" is also used
to refer to
truncated forms of the polypeptide comprising amino acids 8 to 109 or 1 to 109
of the 165-amino
acid human vascular endothelial cell growth factor. Reference to any such
forms of VEGF may
be identified in the present application, e.g., by "VEGF (8-109)," "VEGF (1-
109)" or
"VEGF165." The amino acid positions for a "truncated" native VEGF are numbered
as indicated
in the native VEGF sequence. For example, amino acid position 17 (methionine)
in truncated
native VEGF is also position 17 (methionine) in native VEGF. The truncated
native VEGF has
binding affinity for the KDR and Flt-1 receptors comparable to native VEGF.
"VEGF biological activity" includes binding to any VEGF receptor or any VEGF
signaling
activity such as regulation of both normal and abnormal angiogenesis and
vasculogenesis
(Ferrara and Davis-Smyth (1997) Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol.
Med. 77:527-
543); promoting embryonic vasculogenesis and angiogenesis (Carmeliet et al.
(1996) Nature
380:435-439; Ferrara et al. (1996) Nature 380:439-442); and modulating the
cyclical blood
vessel proliferation in the female reproductive tract and for bone growth and
cartilage
formation (Ferrara et al. (1998) Nature Med. 4:336-340; Gerber et al. (1999)
Nature Med.
5:623-628). In addition to being an angiogenic factor in angiogenesis and
vasculogenesis,
VEGF, as a pleiotropic growth factor, exhibits multiple biological effects in
other
physiological processes, such as endothelial cell survival, vessel
permeability and vasodilation,
monocyte chemotaxis and calcium influx (Ferrara and Davis-Smyth (1997), supra
and Cebe-
Suarez et al. Cell. Mol. Life Sci. 63:601-615 (2006)). Moreover, recent
studies have reported
mitogenic effects of VEGF on a few non-endothelial cell types, such as retinal
pigment
epithelial cells, pancreatic duct cells, and Schwann cells. Guerrin et al.
(1995) J. Cell Physiol.
164:385-394; Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol. 126:125-132;
Sondell et al.
(1999) J. Neurosci. 19:5731-5740.
A "VEGF antagonist" or "VEGF-specific antagonist" refers to a molecule capable
of binding to
VEGF, reducing VEGF expression levels, or neutralizing, blocking, inhibiting,
abrogating,
reducing, or interfering with VEGF biological activities, including, but not
limited to, VEGF
binding to one or more VEGF receptors and VEGF mediated angiogenesis and
endothelial cell
survival or proliferation. Included as VEGF-specific antagonists useful in the
methods of the
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 9 -
invention are polypeptides that specifically bind to VEGF, anti-VEGF
antibodies and antigen-
binding fragments thereof, receptor molecules and derivatives which bind
specifically to VEGF
thereby sequestering its binding to one or more receptors, fusions proteins
(e.g., VEGF-Trap
(Regeneron)), and VEGF121-gelonin (Peregrine). VEGF-specific antagonists also
include
antagonist variants of VEGF polypeptides, antisense nucleobase oligomers
directed to VEGF,
small RNA molecules directed to VEGF, RNA aptamers, peptibodies, and ribozymes
against
VEGF. VEGF-specific antagonists also include nonpeptide small molecules that
bind to VEGF
and are capable of blocking, inhibiting, abrogating, reducing, or interfering
with VEGF
biological activities. Thus, the term "VEGF activities" specifically includes
VEGF mediated
biological activities of VEGF. In certain embodiments, the VEGF antagonist
reduces or inhibits,
by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, the
expression level or
biological activity of VEGF.
An "anti-VEGF antibody" is an antibody that binds to VEGF with sufficient
affinity and
specificity. In certain embodiments, the antibody selected will normally have
a sufficiently
binding affinity for VEGF, for example, the antibody may bind hVEGF with a Kd
value of
between 100 nM-1 pM. Antibody affinities may be determined by a surface
plasmon resonance
based assay (such as the BIAcore assay as described in PCT Application
Publication No.
W02005/012359); enzyme-linked immunoabsorbent assay (ELISA); and competition
assays
(e.g. RIA's), for example. In certain embodiment, the anti-VEGF antibody can
be used as a
therapeutic agent in targeting and interfering with diseases or conditions
wherein the VEGF
activity is involved. Also, the antibody may be subjected to other biological
activity assays, e.g.,
in order to evaluate its effectiveness as a therapeutic. Such assays are known
in the art and
depend on the target antigen and intended use for the antibody. Examples
include the HUVEC
inhibition assay; tumor cell growth inhibition assays (as described in WO
89/06692, for
example); antibody-dependent cellular cytotoxicity (ADCC) and complement-
mediated
cytotoxicity (CDC) assays (US Patent 5,500,362); and agonistic activity or
hematopoiesis assays
(see WO 95/27062). An anti-VEGF antibody will usually not bind to other VEGF
homologues
such as VEGF-B or VEGF-C, nor other growth factors such as P1GF, PDGF or bFGF.
In one
embodiment, anti-VEGF antibody is a monoclonal antibody that binds to the same
epitope as the
monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709. In
another
embodiment, the anti-VEGF antibody is a recombinant humanized anti-VEGF
monoclonal
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 10 -
antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599,
including but
not limited to the antibody known as bevacizumab (BV; AVASTIN ).
The anti-VEGF antibody "Bevacizumab (BV)," also known as "rhuMAb VEGF" or
AVASTIN , is a recombinant humanized anti-VEGF monoclonal antibody generated
according to Presta et al. (1997) Cancer Res. 57:4593-4599. It comprises
mutated human IgG1
framework regions and antigen-binding complementarity-determining regions from
the murine
anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to
its receptors.
Approximately 93% of the amino acid sequence of Bevacizumab, including most of
the
framework regions, is derived from human IgGl, and about 7% of the sequence is
derived
from the murine antibody A4.6.1. Bevacizumab has a molecular mass of about
149,000
daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF
antibodies are
further described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005, the entire
disclosure of which
is expressly incorporated herein by reference.
The term "cancer" refers to the physiological condition in mammals that is
typically
characterized by unregulated cell proliferation. Examples of cancer include
but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma and leukemia. More particular examples
of such
cancers include squamous cell cancer, lung cancer (including small-cell lung
cancer, non-small
cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the
lung), cancer of
the peritoneum, hepatocellular cancer, gastric or stomach cancer (including
gastrointestinal
cancer), pancreatic cancer (including metastic pancreatic cancer),
glioblastoma, cervical cancer,
ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer
(including locally
advanced, recurrent or metastatic HER-2 negative breast cancer and locally
recurrent or
metastatic HER2 positive breast cancer), colon cancer, colorectal cancer,
endometrial or uterine
carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer,
prostate cancer, vulval
cancer, thyroid cancer, hepatic carcinoma and various types of head and neck
cancer, as well as
B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);
small
lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL;
high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small
non-cleaved
cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and
Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic
leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-
transplant
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 11 -
lymphoproliferative disorder (PTLD), as well as abnormal vascular
proliferation associated with
phakomatoses, edema (such as that associated with brain tumors), and Meigs'
syndrome.
Examples of "physiological or patholigical angiogenic abnormalities" include,
but are not
limited to, high grade glioma, glioblastoma, M. Rendu-Osler, von-Hippel-Lindau
diseases,
hemangiomas, psoriasis, Kaposi's sarcoma, ocular neovascularisation,
rheumatoid arthritis,
endometriosis, atherosclerosis, myochardial ischemia, peripheral ischemia,
cerebral ischemia and
wound healing.
The term "chemotherapeutic agent" or "chemotherapy regimen" includes any
active agent that
can provide an anticancer therapeutic effect and may be a chemical agent or a
biological agent,
in particular, that are capable of interfering with cancer or tumor cells.
Particular active agents
are those that act as anti-neoplastic (chemotoxic or chemostatic) agents which
inhibit or prevent
the development, maturation or proliferation of malignant cells. Examples of
chemotherapeutic
agents include alkylating agents such as nitrogen mustards (e.g.,
mechlorethamine,
cyclophosphamide, ifosfamide, melphalan and chlorambucil), nitrosoureas (e.g.,
carmustine
(BCNU), lomustine (CCNU), and semustine (methyl-CCNU)), ethylenimines/
methylmelamines
(e.g., thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),
hexamethylmelamine (HMM, altretamine)), alkyl sulfonates (e.g., busulfan), and
triazines (e.g.,
dacarbazine (DTIC)); antimetabolites such as folic acid analogs (e.g.,
methotrexate,
trimetrexate), pyrimidine analogs (e.g., 5-fluorouracil, capecitabine,
fluorodeoxyuridine,
gemcitabine, cytosine arabino side (AraC, cytarabine), 5-azacytidine, 2,2'-
difluorodeoxycytidine),
and purine analogs (e.g., 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-
deoxycoformycin
(pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-
chlorodeoxyadenosine (cladribine, 2-CdA)); antimitotic drugs developed from
natural products
(e.g., paclitaxel, vinca alkaloids (e.g., vinblastine (VLB), vincristine, and
vinorelbine), docetaxel,
estramustine, and estramustine phosphate), epipodophylotoxins (.e.g.,
etoposide, teniposide),
antibiotics (.e.g, actimomycin D, daunomycin (rubidomycin), daunorubicon,
doxorubicin,
epirubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin),
mitomycinC,
actinomycin), enzymes (e.g., L-asparaginase), and biological response
modifiers (e.g.,
interferon-alpha, IL-2, G-CSF, GM-CSF); miscellaneous agents including
platinum coordination
complexes (e.g., cisplatin, carboplatin, oxaliplatin), anthracenediones (e.g.,
mitoxantrone),
substituted urea (i.e., hydroxyurea), methylhydrazine derivatives (e.g., N-
methylhydrazine
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 12 -
(MIH), procarbazine), adrenocortical suppressants (e.g., mitotane (o,p'-DDD),
aminoglutethimide); hormones and antagonists including adrenocorticosteroid
antagonists (.e.g,
prednisone and equivalents, dexamethasone, aminoglutethimide), progestins
(e.g.,
hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate),
estrogens (e.g.,
diethylstilbestrol, ethinyl estradiol and equivalents thereof); antiestrogens
(e.g., tamoxifen),
androgens (e.g., testosterone propionate, fluoxymesterone and equivalents
thereof),
antiandrogens (e.g., flutamide, gonadotropin-releasing hormone analogs,
leuprolide), non-
steroidal antiandrogens (e.g., flutamide), epidermal growth factor inhibitors
(e.g., erlotinib,
lapatinib, gefitinib) antibodies (e.g., trastuzumab), irinotecan and other
agents such as
leucovorin. For the treatment of locally recurrent or metastatic HER2 positive
breast cancer,
chemotherapeutic agents or chemotherapeutic regimens for administration with
bevacizumab
include capecitabine, paclitaxel, docetaxel and trastuzumab and combinations
thereof (see also
the examples herein provided).
The term "docetaxel" is an anti-neoplastic agent that binds to free tubulin
and promotes the
assembly of tubulin into stable microtubules while simultaneously inhibiting
their assembly.
This leads to the production of microtubule bundles without normal function
and to the
stabilization of microtubules, blocking cells in the M-phase of the cell cycle
and leading to cell
death.
The term "effective amount" refers to an amount of a drug alone or in
combination with other
drug or treatment regimen effective to treat a disease or disorder in a
mammal. In the case of
cancer, the therapeutically effective amount of the drug may reduce the number
of cancer cells;
reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell
infiltration into peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve to some
extent one or more of
the symptoms associated with the disorder. To the extent the drug may prevent
growth and/or
kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer
therapy, efficacy in
vivo can, for example, be measured by assessing the duration of survival,
duration of progression
free survival (PFS), the response rates (RR), duration of response, and/or
quality of life.
The term "expression level" as used herein refers may also refer to the
concentration or amount
of marker/indicator proteins of the present invention in a sample.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 13 -
The term "epitope A4.6.1" refers to the epitope recognized by the anti-VEGF
antibody
bevacizumab (AVASTINO) (see Muller Y et al., Structure 15 September 1998,
6:1153-1167).
In certain embodiments of the invention, the anti-VEGF antibodies include, but
are not limited
to, a monoclonal antibody that binds to the same epitope as the monoclonal
anti-VEGF antibody
A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized anti-VEGF
monoclonal antibody generated according to Presta et al. (1997) Cancer Res.
57:4593-4599.
The "epitope 4D5" is the region in the extracellular domain of HER2 to which
the antibody 4D5
(ATCC CRL 10463) and trastuzumab bind, as described in W02009/154651. This
epitope is
close to the transmembrane domain of HER2, and within Domain IV of HER2, that
being amino
acid residues from about 489-630 - residue numbering without signal peptide.
See Garrett et al
Mol Cell. 1 1 : 495-505 (2003), Cho et al Nature 421 : 756-760 (2003),
Franklin et al Cancer
Cell 5:317-328 (2004), and Plowman et al. Proc. Natl. Acad. Sci 90: 1746- 1750
(1993). To
screen for antibodies which bind essentially to the 4D5 epitope, a routine
cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, Ed
Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping
can be
performed to assess whether the antibody binds essentially to the 4D5 epitope
of HER2 (e.g. any
one or more residues in the region from about residue 529 to, about residue
625, inclusive of the
HER2 extracellular domain, residue numbering including signal peptide).
A "HER receptor" is a receptor protein tyrosine kinase which belongs to the
HER receptor
family and includes EGFR, HER2, HER3 and HER4 receptors. The HER receptor will
generally
comprise an extracellular domain, which may bind an HER ligand and/or dimerize
with another
HER receptor molecule; a lipophilic transmembrane domain; a conserved
intracellular tyrosine
kinase domain; and a carboxyl-terminal signaling domain harboring several
tyrosine residues
which can be phosphorylated. The HER receptor may be a "native sequence" HER
receptor or an
"amino acid sequence variant" thereof. In one embodiment, the HER receptor is
native sequence
human HER receptor. The terms "ErbB1," "HER1 ", "epidermal growth factor
receptor" and
"EGFR" are used interchangeably herein and refer to EGFR as disclosed, for
example, in
Carpenter et al, Ann. Rev. Biochem. 56:881-914 (1987), including naturally
occurring mutant
forms thereof (e.g. a deletion mutant EGFR as in Humphrey et al. PNAS (USA)
87:4207-421 1
(1990)). erbB refers to the gene encoding the EGFR protein product.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 14 -
The expressions "ErbB2" and "HER2" are used interchangeably herein and refer
to the human
antigen. HER2 protein described, for example, in Semba et al., PNAS (USA)
82:6497-6501
(1985) and Yamamoto et al. Nature 319:230-234 (1986) (Genebank accession
number X03363).
The term "erbB2" refers to the gene encoding human ErbB2 and "neu" refers to
the gene
encoding rat pi 85.
An "anti-Her2 antibody" is an antibody that binds to a HER2 receptor.
Optionally, the HER
antibody further interferes with HER2 activation or function. In one
embodiment, an anti-HER2
antibody of the present invention is an anti-HER2 antibody that binds the 4D5
epitope on HER2
polypeptide, or in another embodiment, trastuzumab.
Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-
4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 or trastuzumab (i.e.,
HERCEPTINO) as described in Table 3 of U.S. Patent 5,821,337.
A cancer or cancer cell or tumor that is "HER2 positive" is one which has
significantly higher
levels of a HER receptor protein or gene compared to a noncancerous cell of
the same tissue
type. Such overexpression may be caused by gene amplification or by increased
transcription or
translation. HER receptor overexpression or amplification may be determined in
a diagnostic or
prognostic assay by evaluating increased levels of the HER protein present on
the surface of a
cell (e.g. via an immunohistochemistry assay; IHC). Alternatively, or
additionally, one may
measure levels of HER-encoding nucleic acid in the cell, e.g. via fluorescent
in situ hybridization
(FISH; see W098/45479 published October, 1998), southern blotting, or
polymerase chain
reaction (PCR) techniques, such as quantitative real time PCR (qRT-PCR). One
may also study
HER receptor overexpression or amplification by measuring shed antigen (e.g.,
HER
extracellular domain) in a biological fluid such as serum (see, e.g., U.S.
Patent No. 4,933,294
issued June 12, 1990; W091/05264 published April 18, 1991 ; U.S. Patent
5,401,638 issued
March 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)). Aside
from the above
assays, various in vivo assays are available to the skilled practitioner. For
example, one may
expose cells within the body of the patient to an antibody which is optionally
labeled with a
detectable label, e.g. a radioactive isotope, and binding of the antibody to
cells in the patient can
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 15 -
be evaluated, e.g. by external scanning for radioactivity or by analyzing a
biopsy taken from a
patient previously exposed to the antibody.
The term "metastasis" or "metastatic" refers to the spread of cancer from its
primary site to other
places in the body. Cancer cells can break away from a primary tumor,
penetrate into lymphatic
and blood vessels, circulate through the bloodstream, and grow in a distant
focus (metastasize) in
normal tissues elsewhere in the body. Metastasis can be local or distant.
Metastasis is a
sequential process, contingent on tumor cells breaking off from the primary
tumor, traveling
through the bloodstream, and stopping at a distant site. At the new site, the
cells establish a blood
supply and can grow to form a life-threatening mass. Both stimulatory and
inhibitory molecular
pathways within the tumor cell regulate this behavior, and interactions
between the tumor cell
and host cells in the distant site are also significant.
The terms "oligonucleotide" and "polynucleotide" are used interchangeably and
refer to a
molecule comprised of two or more deoxyribonucleotides or ribonucleotides,
preferably more
than three. Its exact size will depend on many factors, which in turn depend
on the ultimate
function or use of the oligonucleotide. An oligonucleotide can be derived
synthetically or by
cloning. Chimeras of deoxyribonucleotides and ribonucleotides may also be in
the scope of the
present invention.
The term "overall survival (OS)" refers to the length of time during and after
treatment the
patient survives. As the skilled person will appreciate, a patient's overall
survival is improved or
enhanced, if the patient belongs to a subgroup of patients that has a
statistically significant longer
mean survival time as compared to another subgroup of patients.
The term "patient" refers to any single animal, more specifically a mammal
(including such non-
human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows,
pigs, sheep, and
non-human primates) for which treatment is desired. Even more specifically,
the patient herein is
a human.
The term "a patient suffering from" refers to a patient showing clinical signs
in respect to a
disease involving physiological and pathological angiogenesis and/or tumorous
disease, such as
breast cancer, in particular locally recurrent or metastatic HER2 positive
breast cancer.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 16 -
The term "pharmaceutical composition" refers to a sterile preparation that is
in such form as to
permit the biological activity of the medicament to be effective, and which
contains no additional
components that are unacceptably toxic to a subject to which the formulation
would be
administered.
The term "progression-free survival (PFS)" refers to the length of time during
and after treatment
during which, according to the assessment of the treating physician or
investigator, the patient's
disease does not become worse, i.e., does not progress. As the skilled person
will appreciate, a
patient's progression-free survival is improved or enhanced if the patient
belongs to a subgroup
of patients that has a longer length of time during which the disease does not
progress as
compared to the average or mean progression free survival time of a control
group of similarly
situated patients.
The term "polypeptide" relates to a peptide, a protein, an oligopeptide or a
polypeptide which
encompasses amino acid chains of a given length, wherein the amino acid
residues are linked by
covalent peptide bonds. However, peptidomimetics of such proteins/polypeptides
are also
encompassed by the invention wherein amino acid(s) and/or peptide bond(s) have
been replaced
by functional analogs, e.g., an amino acid residue other than one of the 20
gene-encoded amino
acids, e.g., selenocysteine. Peptides, oligopeptides and proteins may be
termed polypeptides.
The terms polypeptide and protein are used interchangeably herein. The term
polypeptide also
refers to, and does not exclude, modifications of the polypeptide, e.g.,
glycosylation, acetylation,
phosphorylation and the like. Such modifications are well described in basic
texts and in more
detailed monographs, as well as in a voluminous research literature. The term
polypeptide also
refers to and encompasses the term "antibody" as used herein.
The term "responsive to" in the context of the present invention indicates
that a subject/patient
suffering, suspected to suffer or prone to suffer breast cancer, in particular
locally recurrent or
metastatic HER2 positive breast cancer, shows a response to a chemotherapy
regimen
comprising bevacizumab. A skilled person will readily be in a position to
determine whether a
person treated with bevacizumab according to the methods of the invention
shows a response.
For example, a response may be reflected by decreased suffering from the
breast cancer, in
particular locally recurrent or metastatic HER2 positive breast cancer, such
as a diminished
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 17 -
and/or halted tumor growth, reduction of the size of a tumor, and/or
amelioration of one or more
symptoms of the cancer. Preferably, the response may be reflected by decreased
or diminished
indices of the metastatic conversion of the breast cancer such as the
prevention of the formation
of metastases or a reduction of number or size of metastases (see, e.g.,
Eisenhauser et al., New
response evaluation criteria in solid tumours: Revised RECIST guideline
(version 1.1) Eur. J.
Cancer 2009 45: 228-247).
The term "reference sample," as used herein, refers to any sample, standard,
or level that is used
for comparison purposes. In one embodiment, a reference sample is obtained
from a healthy
and/or non-diseased part of the body (e.g., tissue or cells) of the same
subject or patient. In
another embodiment, a reference sample is obtained from an untreated tissue
and/or cell of the
body of the same subject or patient. In yet another embodiment, a reference
sample is obtained
from a healthy and/or non-diseased part of the body (e.g., tissues or cells)
of an individual who is
not the subject or patient. In even another embodiment, a reference sample is
obtained from an
untreated tissue and/or cell part of the body of an individual who is not the
subject or patient.
In certain embodiments, a reference sample is a single sample or combined
multiple samples
from the same subject or patient that are obtained at one or more different
time points than when
the test sample is obtained. For example, a reference sample is obtained at an
earlier time point
from the same subject or patient than when the test sample is obtained. Such
reference sample
may be useful if the reference sample is obtained during initial diagnosis of
cancer and the test
sample is later obtained when the cancer becomes metastatic. In certain
embodiments, a
reference sample includes all types of biological samples as defined above
under the term
"sample" that is obtained from one or more individuals who is not the subject
or patient. In
certain embodiments, a reference sample is obtained from one or more
individuals with an
angiogenic disorder (e.g., cancer) who is not the subject or patient. In
certain embodiments, a
reference sample is a combined multiple samples from one or more healthy
individuals who are
not the subject or patient. In certain embodiments, a reference sample is a
combined multiple
samples from one or more individuals with a disease or disorder (e.g., an
angiogenic disorder
such as, for example, cancer) who are not the subject or patient. In certain
embodiments, a
reference sample is pooled RNA samples from normal tissues or pooled plasma or
serum
samples from one or more individuals who are not the subject or patient. In
certain
embodiments, a reference sample is pooled RNA samples from tumor tissues or
pooled plasma
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 18 -
or serum samples from one or more individuals with a disease or disorder
(e.g., an angiogenic
disorder such as, for example, cancer) who are not the subject or patient.
The term "reference level" herein refers to a predetermined value. As the
skilled artisan will
appreciate the reference level is predetermined and set to meet the
requirements in terms of e.g.
specificity and/or sensitivity. These requirements can vary, e.g. from
regulatory body to
regulatory body. It may for example be that assay sensitivity or specificity,
respectively, has to
be set to certain limits, e.g. 80%, 90% or 95%. These requirements may also be
defined in terms
of positive or negative predictive values. Nonetheless, based on the teaching
given in the present
invention it will always be possible to arrive at the reference level meeting
those requirements. In
one embodiment the reference level is determined in healthy individuals. The
reference value in
one embodiment has been predetermined in the disease entity to which the
patient belongs. In
certain embodiments the reference level can e.g. be set to any percentage
between 25% and 75%
of the overall distribution of the values in a disease entity investigated. In
other embodiments the
reference level can e.g. be set to the median, tertiles or quartiles as
determined from the overall
distribution of the values in a disease entity investigated. In one embodiment
the reference level
is set to the median value as determined from the overall distribution of the
values in a disease
entity investigated.
In certain embodiments, the term "increase" or "above" refers to a level above
the reference level
or to an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,
85%, 90%,
95%, 100% or greater, in E-selectin, ICAM-1 or VEGFR-3 level detected by the
methods
described herein, as compared to the E-selectin, ICAM-1 and VEGFR-3 level from
a reference
sample. In certain embodiments, the term increase refers to the increase in E-
selectin, ICAM-1
and VEGFR-3 wherein, the increase is at least about 1.5-, 1.75-, 2-, 3-, 4-, 5-
, 6-, 7-, 8-, 9-, 10-,
15-, 20-, 25-, 30-, 40-, 50-, 60-, 70-, 75-, 80-, 90-, or 100- fold higher as
compared to the E-
selectin, ICAM-1 and VEGFR-3 level e.g. predetermined from a reference sample.
In one
preferred embodiment the term increased level relates to a value at or above a
reference level.
In certain embodiments, the term "decrease" or "below" herein refers to a
level below the
reference level or to an overall reduction of 5%, 10%, 20%, 25%, 30%, 40%,
50%, 60%, 70%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in E-selectin, ICAM-1 and
VEGFR-3
level detected by the methods described herein, as compared to the E-selectin,
ICAM-1 and
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 19 -
VEGFR-3 level from a reference sample. In certain embodiments, the term
decrease refers to the
decrease in E-selectin, ICAM-1 and VEGFR-3 level, wherein the decreased level
is at most
about 0.9-, 0.8-, 0.7-, 0.6-, 0.5-, 0.4-, 0.3-, 0.2-, 0.1-, 0.05-, or 0.01-
fold the E-selectin, ICAM-1
and VEGFR-3 level from the reference sample or lower.
In certain embodiments, the term "at a reference level" refers to a level that
is the same as E-
selectin, ICAM-1 and VEGFR-3 level detected by the methods described herein,
from a
reference sample.
A "recurrent" cancer is one which has regrown, either at the initial site or
at a distant site, after a
response to initial therapy.
The term "sensitive to" in the context of the present invention indicates that
a subject/patient
suffering, suspected to suffer or prone to suffer from breast cancer, in
particular locally recurrent
or metastatic HER2 positive breast cancer, shows in some way a positive
reaction to treatment
with bevacizumab in combination with a chemotherapy regimen. The reaction of
the patient
may be less pronounced when compared to a patient "responsive to" as described
hereinabove.
For example, the patient may experience less suffering associated with the
disease, though no
reduction in tumor growth or metastatic indicator may be measured, and/or the
reaction of the
patient to the bevacizumab in combination with the chemotherapy regimen may be
only of a
transient nature, i.e., the growth of (a) tumor and/or (a) metastasis(es) may
only be temporarily
reduced or halted.
The term "survival" refers to the subject remaining alive, and includes
progression free survival
(PFS) and overall survival (OS). Survival can be estimated by the Kaplan-Meier
method, and
any differences in survival are computed using the stratified log-rank test.
By "extending survival" or "increasing the likelihood of survival" is meant
increasing PFS and/or
OS in a treated subject relative to an untreated subject (i.e. relative to a
subject not treated with a
VEGF antibody), or relative to a control treatment protocol, such as treatment
only with the
chemotherapeutic agent, such as those use in the standard of care for locally
recurrent or
metastatic breast cancer, e.g., capecitabine, taxane, anthracycline,
paclitaxel, docetaxel,
paclitaxel protein-bound particles (e.g., Abraxane0), doxorubicin, epirubicin,
5-fluorouracil,
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 20 -
cyclophosphamide, or trastuzumab (e.g., Herceptin0), or combinations thereof.
In one
embodiment, such standard of care for treating locally recurrent or metastatic
breast cancer is a
treatment combination comprising trastuzumab and docetaxel. Survival is
monitored for at least
about one month, about two months, about four months, about six months, about
nine months, or
at least about 1 year, or at least about 2 years, or at least about 3 years,
or at least about 4 years,
or at least about 5 years, or at least about 10 years, etc., following the
initiation of treatment or
following the initial diagnosis.
The term "hazard ratio (HR)" is a statistical definition for rates of events.
For the purpose of the
invention, hazard ratio is defined as representing the probability of an event
in the experimental
arm divided by the probability of an event in the control arm at any specific
point in time.
"Hazard ratio" in progression free survival analysis is a summary of the
difference between two
progression free survival curves, representing the reduction in the risk of
death on treatment
compared to control, over a period of follow-up.
As used herein, "therapy" or "treatment" refers to clinical intervention in an
attempt to alter the
natural course of the individual or cell being treated, and can be performed
either for prophylaxis
or during the course of clinical pathology. Desirable effects of treatment
include preventing
occurrence or recurrence of disease, alleviation of symptoms, diminishment of
any direct or
indirect pathological consequences of the disease, preventing metastasis,
decreasing the rate of
disease progression, amelioration or palliation of the disease state, and
remission or improved
prognosis.
The term "treatment effect" encompasses the terms "overall survival" and
"progression-free
survival".
The term "E-selectin" refers to an endothelial adhesion molecule that is
induced by various
inflammatory stimuli (Bevilacqua, P. P. et al., Proc. Natl. Acad. Sci. USA 84,
9238-9242 (1987)
; Luscinskas, F.W. et al., J. Immunol. 142 2257- 2263 (1989) ; Kuijpers, T.W.
et al., J. Immunol.
147 1369-1376 (1991)). A cloned gene encoding E-Selectin (ELAM-1) is disclosed
in U. S.
Patent No. 5,081,034. E-selectin is exemplified by UniProtKB Accession Number
P16581 and
Gene ID (NCBI): 6401. The term "E-selectin" encompasses its homologues and
isoforms. In the
context of the invention, the term "E-selectin" also encompasses variants
thereof, as well as
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 21 -
fragments of the sequences, provided that the variant proteins (including
isoforms), homologous
proteins and/or fragments are recognized by one or more E-selectin specific
antibodies, such as
antibody clone BBIG-E5 or 5D11, which are available from R&D Systems.
The term "ICAM-1" refers to intercellular adhesion molecule 1 exemplified by
UniProtKB
Accession Number P05362 and Gene ID (NCBI): 3383. The term "ICAM-1"
encompasses its
homologues and isoforms. In the context of the invention, the term "ICAM-1"
also encompasses
variants thereof, as well as fragments of the sequences, provided that the
variant proteins
(including isoforms), homologous proteins and/or fragments are recognized by
one or more
ICAM-1 specific antibodies, such as antibody clone 11C81 or 14C11, which are
available from
R&D Systems.
The term "VEGFR-3" refers to vascular endothelial growth factor receptor 3
exemplified by
UniProtKB Accession Number P35916 and Gene ID (NCBI): 2324. The term "VEGFR-3"
encompasses its homologues and isoforms. In the context of the invention, the
term "VEGFR-3"
also encompasses variants thereof, as well as fragments of the sequences,
provided that the
variant proteins (including isoforms), homologous proteins and/or fragments
are recognized by
one or more VEGFR-3 specific antibodies, such as antibody clone 54716 or 5B6,
which are
available from R&D Systems and Abnova, respectively.
2. Detailed Embodiments
In the present invention, E-selectin, ICAM-1 and VEGFR-3 were identified as
markers or
predictive biomarkers for survival with an anti-angiogenesis therapy. The
terms "marker",
"biomarker" and "predictive biomarker" can be used interchangeably and refer
to expression
levels of E-selectin, ICAM-1 and VEGFR-3. In one embodiment, E-selectin, ICAM-
1 and
VEGFR-3 can also be used as prognostic biomarkers for breast cancer, more
specifically HER2
positive breast cancer.
Accordingly, the present invention provides an in vitro method of determining
whether a patient
diagnosed with breast cancer is more or less suitably treated by an anti-
cancer therapy
comprising an anti-VEGF antibody, the method comprising:
(a) determining an expression level of at least one biomarker which is
selected from the
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 22 -
group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient
diagnosed with breast cancer, and
(b) identifying the patient as more or less suitably treated by an anti-cancer
therapy
comprising an anti-VEGF antibody based on the expression level in accordance
with (a),
wherein an expression level of said biomarker at or above a reference level
indicates that the
patient is more suitably treated with the anti-cancer therapy, or an
expression level of said
biomarker below a reference level indicates that the patient is less suitably
treated with the anti-
cancer therapy. In one embodiment, whether a patient is suitably treated by an
anti-cancer
therapy is determined in terms of progression-free survival. In one
embodiment, said anti-cancer
therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
The present invention further provides a pharmaceutical composition comprising
an anti-VEGF
antibody for the treatment of a patient diagnosed with breast cancer, wherein
the patient has been
identified as more suitably treated by an anti-cancer therapy comprising an
anti-VEGF antibody
by an in vitro method comprising:
(a) determining an expression level of at least one biomarker which is
selected from the
group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient
diagnosed with breast cancer, and
(b) identifying the patient as more or less suitably treated by an anti-cancer
therapy
comprising an anti-VEGF antibody based on the expression level in accordance
with (a),
wherein an expression level of said biomarker at or above a reference level
indicates that the
patient is more suitably treated with the anti-cancer therapy, or an
expression level of said
biomarker below a reference level indicates that the patient is less suitably
treated with the anti-
cancer therapy. In one embodiment, whether a patient is suitably treated by an
anti-cancer
therapy is determined in terms of progression-free survival. In one
embodiment, said anti-cancer
therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
The present invention further provides a pharmaceutical composition comprising
an anti-VEGF
antibody for the treatment of a patient diagnosed with breast cancer, wherein
the patient is
identified as more suitably treated by an anti-cancer therapy comprising an
anti-VEGF antibody
by an in vitro method comprising:
(a) determining an expression level of at least one biomarker which is
selected from the
group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 23 -
diagnosed with breast cancer, and
(b) identifying the patient as more or less suitably treated by an anti-cancer
therapy
comprising an anti-VEGF antibody based on the expression level in accordance
with (a),
wherein an expression level of said biomarker at or above a reference level
indicates that the
patient is more suitably treated with the anti-cancer therapy, or an
expression level of said
biomarker below a reference level indicates that the patient is less suitably
treated with the anti-
cancer therapy. In one embodiment, whether a patient is suitably treated by an
anti-cancer
therapy is determined in terms of progression-free survival. In one
embodiment, said anti-cancer
therapy comprises an anti-VEGF antibody, an anti-HER2 antibody and a taxane.
The present invention also provides an in vitro method of determining whether
a patient
diagnosed with breast cancer is sensitive to an anti-cancer therapy comprising
the addition of an
anti-VEGF antibody to a chemotherapy regimen, said method comprising:
(a) determining an expression level of at least one biomarker which is
selected from the
group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient
diagnosed with breast cancer, and
(b) identifying the patient as sensitive to an anti-cancer therapy comprising
the addition
of an anti-VEGF antibody to a chemotherapy regimen based on the expression
level in
accordance with (a), wherein an expression level of said biomarker at or above
a reference level
indicates that the patient is sensitive to an anti-cancer therapy comprising
the addition of an anti-
VEGF antibody to a chemotherapy regimen. In one embodiment, whether a patient
is sensitive to
an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a
chemotherapy
regimen is determined in terms of progression-free survival. In one
embodiment, said
chemotherapy regimen comprises an anti-HER2 antibody and a taxane.
The present invention further provides a pharmaceutical composition comprising
an anti-VEGF
antibody for the treatment of a patient diagnosed with breast cancer, wherein
the patient has been
identified as sensitive to an anti-cancer therapy comprising the addition of
an anti-VEGF
antibody to a chemotherapy regimen by an in vitro method comprising:
(a) determining an expression level of at least one biomarker which is
selected from the
group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient
diagnosed with breast cancer, and
(b) identifying the patient as sensitive to an anti-cancer therapy comprising
the addition
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 24 -
of an anti-VEGF antibody to a chemotherapy regimen based on the expression
level in
accordance with (a), wherein an expression level of said biomarker at or above
a reference level
indicates that the patient is sensitive to an anti-cancer therapy comprising
the addition of an anti-
VEGF antibody to a chemotherapy regimen. In one embodiment, whether a patient
is sensitive to
an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a
chemotherapy
regimen is determined in terms of progression-free survival. In one
embodiment, said
chemotherapy regiment comprises an anti-HER2 antibody and a taxane.
The present invention further provides a pharmaceutical composition comprising
an anti-VEGF
antibody for the treatment of a patient diagnosed with breast cancer, wherein
the patient is
identified as sensitive to an anti-cancer therapy comprising the addition of
an anti-VEGF
antibody to a chemotherapy regimen by an in vitro method comprising:
(a) determining an expression level of at least one biomarker which is
selected from the
group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient
diagnosed with breast cancer, and
(b) identifying the patient as sensitive to an anti-cancer therapy comprising
the addition
of an anti-VEGF antibody to a chemotherapy regimen based on the expression
level in
accordance with (a), wherein an expression level of said biomarker at or above
a reference level
indicates that the patient is sensitive to an anti-cancer therapy comprising
the addition of an anti-
VEGF antibody to a chemotherapy regimen. In one embodiment, whether a patient
is sensitive to
an anti-cancer therapy comprising the addition of an anti-VEGF antibody to a
chemotherapy
regimen is determined in terms of progression-free survival. In one
embodiment, said
chemotherapy regiment comprises an anti-HER2 antibody and a taxane.
The present invention also provides a method for improving the treatment
effect of an anti-
cancer therapy comprising a chemotherapy regimen in a patient diagnosed with
breast cancer by
adding an anti-VEGF antibody to the chemotherapy regimen, the method
comprising:
(a) determining an expression level of at least one biomarker which is
selected from the
group consisting of E-selectin, ICAM-1 and VEGFR-3 in a sample derived from a
patient
diagnosed with positive breast cancer;
(b) identifying the patient as sensitive to an anti-cancer therapy comprising
the addition
of an anti-VEGF antibody to a chemotherapy regimen based on the expression
level in
accordance with (a), wherein an expression level of said biomarker at or above
a reference level
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 25 -
indicates that the patient is sensitive to an anti-cancer therapy comprising
the addition of an anti-
VEGF antibody to a chemotherapy regimen; and
(c) administering an effective amount of an anti-VEGF antibody in combination
with an
effective amount of a chemotherapy regimen to the patient identified as
sensitive to an anti-
cancer therapy comprising the addition of an anti-VEGF antibody to a
chemotherapy in
accordance with (b). In one embodiment, whether a patient is sensitive to an
anti-cancer therapy
comprising the addition of an anti-VEGF antibody to a chemotherapy regimen is
determined in
terms of progression-free survival. In one embodiment, said chemotherapy
regimen comprises an
anti-HER2 antibody and a taxane.
In one embodiment, said determination of the biomarker is carried out either
a) by contacting the
sample with an agent that specifically binds to the biomarker, thereby forming
a complex
between the agent and the biomarker, detecting the amount of complex formed,
thereby
measuring the level of the biomarker; or b) by amplifying the biomarker
present in the sample
and detecting the amplified biomarker with an agent that specifically binds to
the amplified
biomarker, thereby measuring the level of the biomarker.
In one embodiment, said patient is diagnosed with HER2 positive breast cancer,
more
specifically, locally recurrent or metastatic HER2 positive breast cancer. In
one embodiment,
said patient received no previous chemotherapeutic or radiation treatment.
In one embodiment, said anti-VEGF antibody binds the A4.6.1 epitope. More
specifically, said
anti-VEGF antibody is bevacizumab.
In one embodiment, said taxane is docetaxel or paclitaxel, more specifically,
docetaxel.
In one embodiment, said anti-HER2 antibody binds the 4D5 epitope. More
specifically, said
anti-HER2 antibody is trastuzumab.
In one embodiment, said expression level is a protein expression level.
In one embodiment, said sample is a blood plasma sample.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 26 -
In one embodiment, said expression level is an expression level of E-selectin.
In one
embodiment, said expression level is an expression level of ICAM-1. In one
embodiment, said
expression level is an expression level of VEGFR-3.
In the context of the herein described invention, the expression levels, in
particular protein
expression levels, of E-selectin, ICAM-1 and VEGFR-3, may be considered
separately, as
individual markers, or in groups of two or more, as an expression profile or
marker panel. In the
context of the herein described invention an expression profile or marker
panel wherein the
expression profiles of two or more markers may be considered together may also
be referred to
as a combined expression level. For example, the expression levels of two or
more markers may
be added together and compared to a similarly determined control combined
expression level.
Therefore, the methods of the invention encompass determination of an
expression profile,
including a combined expression level, based on the expression level of one or
more of the
markers.
In the context of the herein described invention, and in accordance with the
appended illustrative
example, for consideration of E-selectin, ICAM-1 and VEGFR-3 separately, the
following
values were used as the corresponding high or low expression value of the
marker: High E-
selectin (> 36.9 ng/mL), Low E-selectin (< 36.9 ng/mL), High ICAM-1 (?210
ng/mL), Low
ICAM-1 (< 210 ng/mL), High VEGFR-3 (> 10.6 ng/mL), Low VEGFR-3 (< 10.6 ng/mL).
These
levels were determined as the sample median, as per a prospective analysis
plan. Additionally,
optimized levels constituting the cut-off value between high and low
expression of a particular
marker may be determined by varying the cut-off until the subset of patients
above and below
the cut-off satisfy a relevant statistical optimality criterion. For example,
an optimal cut-point
may be chosen to maximize the differences in treatment Hazard Ratio between
the subset above
and below, or to maximize treatment effect in one sub-group, or any other
relevant statistical
criterion. The skilled person will, however, understand that the expression
level of the particular
marker and, therefore, what constitutes a high or low expression level may
vary by patient and
by patient population. Accordingly, the skilled person will understand that
when using detection
methods other than those described in the appended illustrative example and
studying patients
and patient populations other than those described in the appended
illustrative example, what the
skilled person considers a high and/or low expression level for a particular
biomarker may vary
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 27 -
from the values herein described. Given the methods herein described, the
skilled person can
determine what constitutes a high and/or low level of expression of a
particular biomarker.
In one embodiment, said reference level of E-selectin, ICAM-1 and VEGFR-3 is
set to the
median value of concentrations in samples derived from a patient group. In one
example, said
reference level of E-selectin is about 36.9 ng/mL in plasma. In one example,
said reference level
of ICAM-1 is about 210 ng/mL in plasma. In one example, said reference level
of VEGFR-3 is
about 10.6 ng/mL in plasma. In one embodiment, said reference level of E-
selectin, ICAM-1 and
VEGFR-3 is determined by a quartile analysis of samples derived from a patient
group. In one
embodiment, said reference level of E-selectin, ICAM-1 and VEGFR-3 is set to
the 1st quartile
value in order of increasing concentrations in samples derived from a patient
group. In one
example, said reference level of E-selectin is about 26.7 ng/mL in plasma. In
one example, said
reference level of ICAM-1 is about 170.4 ng/mL in plasma. In one example, said
reference level
of VEGFR-3 is about 7.8 ng/mL in plasma. In one embodiment, said reference
level of E-
selectin, ICAM-1 and VEGFR-3 is set to the 3rd quartile value in order of
increasing
concentrations in samples derived from a patient group. In one example, said
reference level of
E-selectin is about 49.1 ng/mL in plasma. In one example, said reference level
of ICAM-1 is
about 272.2 ng/mL in plasma. In one example, said reference level of VEGFR-3
is about 13.0
ng/mL in plasma.
As the skilled artisan will appreciate there are many ways to use the
measurements of two or
more markers in order to improve the diagnostic question under investigation.
In a quite simple,
but nonetheless often effective approach, a positive result is assumed if a
sample is positive for at
least one of the markers investigated.
However, a combination of markers may also be evaluated. The values measured
for markers of
a marker panel (or a combined expression level), e.g. for E-selectin, ICAM-1
and VEGFR-3,
may be mathematically combined and the combined value may be correlated to the
underlying
diagnostic question. Marker values may be combined by any appropriate state of
the art
mathematical method. Well-known mathematical methods for correlating a marker
combination
to a disease or to a treatment effect employ methods like, discriminant
analysis (DA) (i.e. linear-,
quadratic-, regularized-DA), Kernel Methods (i.e. SVM), Nonparametric Methods
(i.e. k-
Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based Methods
(i.e. Logic
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 28 -
Regression, CART, Random Forest Methods, Boosting/Bagging Methods),
Generalized Linear
Models (i.e. Logistic Regression), Principal Components based Methods (i.e.
SIMCA),
Generalized Additive Models, Fuzzy Logic based Methods, Neural Networks and
Genetic
Algorithms based Methods. The skilled artisan will have no problem selecting
an appropriate
method to evaluate a marker combination of the present invention. The method
used in
correlating marker combinations in accordance with the invention herein
disclosed with, for
example improved overall survival, progression free survival, responsiveness
or sensitivity to
addition of bevacizumab to chemotherapeutic agents/chemotherapy regimen and/or
the
prediction of a response to or sensitivity to bevacizumab (in addition to one
or more
chemotherapeutic agents/chemotherapy regimen) is selected from DA (i.e. Linear-
, Quadratic-,
Regularized Discriminant Analysis), Kernel Methods (i.e. SVM), Nonparametric
Methods (i.e.
k-Nearest-Neighbor Classifiers), PLS (Partial Least Squares), Tree-Based
Methods (i.e. Logic
Regression, CART, Random Forest Methods, Boosting Methods), or Generalized
Linear Models
(i.e. Logistic Regression). Details relating to these statistical methods are
found in the following
references: Ruczinski, I., et al, J. of Computational and Graphical
Statistics, 12 (2003) 475-511;
Friedman, J. H., J. of the American Statistical Association 84 (1989) 165-175;
Hastie, Trevor,
Tibshirani, Robert, Friedman, Jerome, The Elements of Statistical Learning,
Springer Series in
Statistics, 2001; Breiman, L., Friedman, J. H., Olshen, R. A., Stone, C. J.
(1984) Classification
and regression trees, California: Wadsworth; Breiman, L., Random Forests,
Machine Learning,
45 (2001) 5-32; Pepe, M. S., The Statistical Evaluation of Medical Tests for
Classification and
Prediction, Oxford Statistical Science Series, 28 (2003); and Duda, R. O.,
Hart, P. E., Stork, D.
G., Pattern Classification, Wiley Interscience, 2nd Edition (2001).
Accordingly, the invention herein disclosed relates to the use of an optimized
multivariate cut-
off for the underlying combination of biological markers and to discriminate
state A from state
B, e.g. patients responsive to or sensitive to an anti-cancer therapy
comprising the addition of
bevacizumab to a chemotherapy regimen from patients that are poor responders
to the addition of
bevacizumab therapy to a chemotherapy regimen. In this type of analysis the
markers are no
longer independent but form a marker panel or a combined expression level.
3. Detection of Expression Levels of E-selectin, ICAM-1 or VEGFR-3
The expression level of one or more of the markers E-selectin, ICAM-1 and
VEGFR-3 may be
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 29 -
assessed by any method known in the art suitable for determination of specific
protein levels in a
patient sample and is preferably determined by an immunoassay method, such as
ELISA,
employing antibodies specific for one or more of E-selectin, ICAM-1 and VEGFR-
3. Such
methods are well known and routinely implemented in the art and corresponding
commercial
antibodies and/or kits are readily available. For example, commercially
available antibodies/test
kits for E-selectin, ICAM-1 and VEGFR-3 can be obtained from R&D Systems as
clone BBIG-
E5 and 5D11, from R&D systems as clone 11C81 and 14C11 and from R&D Systems
and
Abnova as clone 54716 and 5B6, respectively. Preferably, the expression levels
of the
marker/indicator proteins of the invention are assessed using the reagents
and/or protocol
recommendations of the antibody or kit manufacturer. The skilled person will
also be aware of
further means for determining the expression level of one or more of E-
selectin, ICAM-1 and
VEGFR-3 by immunoassay methods. Therefore, the expression level of one or more
of the
markers/indicators of the invention can be routinely and reproducibly
determined by a person
skilled in the art without undue burden. However, to ensure accurate and
reproducible results, the
invention also encompasses the testing of patient samples in a specialized
laboratory that can
ensure the validation of testing procedures.
E-selectin, ICAM-1 and VEGFR-3 protein or nucleic acids can be detected using
any method
known in the art. For example, tissue or cell samples from mammals can be
conveniently
assayed for, e.g., proteins using Westerns and ELISAs, mRNAs or DNAs from a
genetic
biomarker of interest using Northern, dot-blot, or polymerase chain reaction
(PCR) analysis,
array hybridization, RNase protection assay, or using DNA SNP chip
microarrays, which are
commercially available, including DNA microarray snapshots. For example, real-
time PCR (RT-
PCR) assays such as quantitative PCR assays are well known in the art. In an
illustrative
embodiment of the invention, a method for detecting mRNA from a genetic
biomarker of interest
in a biological sample comprises producing cDNA from the sample by reverse
transcription
using at least one primer; amplifying the cDNA so produced; and detecting the
presence of the
amplified cDNA. In addition, such methods can include one or more steps that
allow one to
determine the levels of mRNA in a biological sample (e.g., by simultaneously
examining the
levels a comparative control mRNA sequence of a "housekeeping" gene such as an
actin family
member). Optionally, the sequence of the amplified cDNA can be determined.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 30 -
Many references are available to provide guidance in applying the above
techniques (Kohler et
al., Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980);
Tijssen, Practice
and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); Campbell,
Monoclonal
Antibody Technology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal Hybridoma
Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982); and
Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-1 58 (CRC Press, Inc.,
1987).
The expression level of one or more of E-selectin, ICAM-1 and VEGFR-3 may be
assessed in a
patient sample that is a biological sample. The patient sample may be a blood
sample, blood
serum sample or a blood plasma sample. Methods of obtaining blood samples,
blood serum
samples and blood plasma samples are well known in the art. The patient sample
may be
obtained from the patient prior to or after neoadjuvant therapy or prior to or
after adjuvant
therapy.
4. Methods of Treatment
In the context of the present invention, bevacizumab is to be administered in
addition to or as a
co-therapy or co-treatment with one or more chemotherapeutic agents
administered as part of
standard chemotherapy regimen as known in the art. Examples of agents included
in such
standard chemotherapy regimens include 5-fluorouracil, leucovorin, irinotecan,
gemcitabine,
erlotinib, capecitabine, taxanes, such as docetaxel and paclitaxel, interferon
alpha, vinorelbine,
and platinum-based chemotherapeutic agents, such as, carboplatin, cisplatin
and oxaliplatin. As
demonstrated in the appended illustrative example, the addition of bevacizumab
effected an
increase in the progression free survival in the patients and/or patient
population defined and
selected according to the expression level of one or more of E-selectin, ICAM-
1 and VEGFR-3.
Thus, bevacizumab may be combined with a chemotherapy regimen, such as
docetaxel therapy
as demonstrated in the appended illustrative example.
Common modes of administration include parenteral administration as a bolus
dose or as an
infusion over a set period of time, e.g., administration of the total daily
dose over 10 min., 20
min., 30 min., 40 min., 50 min., 60 min., 75 min., 90 min., 105 min., 120
min., 3 hr., 4 hr., 5 hr.
or 6 hr. For example, 2.5 mg/kg of body weight to 15 mg/kg of body weight
bevacizumab
(Avastin ) can be administered every week, every 2 weeks or every 3 weeks,
depending on the
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 31 -
type of cancer being treated. Examples of dosages include 2.5 mg/kg of body
weight, 5 mg/kg
of body weight, 7.5 mg/kg of body weight, 10 mg/kg of body weight and 15 mg/kg
of body
weight given every week, every 2 weeks or every 3 weeks. Further examples of
dosages are 5
mg/kg of body weight every 2 weeks, 10 mg/kg every 2 weeks, 7.5 mg/kg of body
weight every
3 weeks and 15 mg/kg of body weight every 3 weeks. In the context of the
herein described
invention, low dose bevacizumab includes, for example, dosages of 2.5 mg/kg of
body weight
every week, 5 mg/kg of body weight every 2 weeks and 7.5 mg/kg of body weight
every 3
weeks. In the context of the herein described invention, high dose bevacizumab
includes, for
example, dosages of 5 mg/kg of body weight every week, 10 mg/kg of body weight
every 2
weeks and 15 mg/kg of body weight every 3 weeks.
The skilled person will recognize that further modes of administration of
bevacizumab are
encompassed by the invention as determined by the specific patient and
chemotherapy regimen,
and that the specific mode of administration and therapeutic dosage are best
determined by the
treating physician according to methods known in the art.
The patients selected according to the methods of the present invention are
treated with
bevacizumab in combination with a chemotherapy regimen, and may be further
treated with one
or more additional anti-cancer therapies. In certain aspects, the one or more
additional anti-
cancer therapy is radiation.
5. Kit
The present invention also relates to a diagnostic composition or kit
comprising oligonucleotides
or polypeptides suitable for the determination of expression levels of one or
more of E-selectin,
ICAM-1 and VEGFR-3. As detailed herein, oligonucleotides such as DNA, RNA or
mixtures of
DNA and RNA probes may be of use in detecting mRNA levels of the
marker/indicator proteins,
while polypeptides may be of use in directly detecting protein levels of the
marker/indicator
proteins via specific protein-protein interaction. In preferred aspects of the
invention, the
polypeptides encompassed as probes for the expression levels of one or more of
E-selectin,
ICAM-1 and VEGFR-3, and included in the kits or diagnostic compositions
described herein, are
antibodies specific for these proteins, or specific for homologues and/or
truncations thereof.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 32 -
Accordingly, in a further embodiment of the present invention provides a kit
useful for carrying
out the methods herein described, comprising oligonucleotides or polypeptides
capable of
determining the expression level of one or more of E-selectin, ICAM-1 and
VEGFR-3. The
oligonucleotides may comprise primers and/or probes specific for the mRNA
encoding one or
more of the markers/indicators described herein, and the polypeptides comprise
proteins capable
of specific interaction with the marker/indicator proteins, e.g.,
marker/indicator specific
antibodies or antibody fragments.
In addition to the methods described above, the invention also encompasses
further
immunoassay methods for assessing or determining the expression level of one
or more of E-
selectin, ICAM-1 and VEGFR-3, such as by Western blotting and ELISA-based
detection. As is
understood in the art, the expression level of the marker/indicator proteins
of the invention may
also be assessed at the mRNA level by any suitable method known in the art,
such as Northern
blotting, real time PCR, and RT PCR. Immunoassay- and mRNA-based detection
methods and
systems are well known in the art and can be deduced from standard textbooks,
such as
Lottspeich (Bioanalytik, Spektrum Akademisher Verlag, 1998) or Sambrook and
Russell
(Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY,
U.S.A., 2001).
The described methods are of particular use for determining the expression
levels of E-selectin,
ICAM-1 and VEGFR-3 in a patient or group of patients relative to control
levels established in a
population diagnosed with breast cancer, in particular locally recurrent or
metastatic HER2
positive breast cancer.
The expression level of one or more of E-selectin, ICAM-1 and VEGFR-3, can
also be
determined on the protein level by taking advantage of immunoagglutination,
immunoprecipitation (e.g., immunodiffusion, immunelectrophoresis, immune
fixation), western
blotting techniques (e.g., (in situ) immuno cytochemistry,
affinitychromatography, enzyme
immunoassays), and the like. Amounts of purified polypeptide in solution may
also be
determined by physical methods, e.g. photometry. Methods of quantifying a
particular
polypeptide in a mixture usually rely on specific binding, e.g., of
antibodies. Specific detection
and quantitation methods exploiting the specificity of antibodies comprise for
example
immunoassay methods. For example, concentration/amount of marker/indicator
proteins of the
present invention in a patient sample may be determined by enzyme linked-
immunosorbent
assay (ELISA). Alternatively, Western Blot analysis or immunostaining can be
performed.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 33 -
Western blotting combines separation of a mixture of proteins by
electrophoresis and specific
detection with antibodies. Electrophoresis may be multi-dimensional such as 2D
electrophoresis.
Usually, polypeptides are separated in 2D electrophoresis by their apparent
molecular weight
along one dimension and by their isoelectric point along the other direction.
As mentioned above, the expression level of the marker/indicator proteins
according to the
present invention may also be reflected in an increased expression of the
corresponding gene(s)
encoding E-selectin, ICAM-1 and VEGFR-3. Therefore, a quantitative assessment
of the gene
product prior to translation (e.g. spliced, unspliced or partially spliced
mRNA) can be performed
in order to evaluate the expression of the corresponding gene(s). The person
skilled in the art is
aware of standard methods to be used in this context or may deduce these
methods from standard
textbooks (e.g. Sambrook, 2001, loc. cit.). For example, quantitative data on
the respective
concentration/amounts of mRNA encoding one or more of E-selectin, ICAM-1 and
VEGFR-3
can be obtained by Northern Blot, Real Time PCR and the like.
In a further aspect of the invention, the kit of the invention may
advantageously be used for
carrying out a method of the invention and could be, inter alia, employed in a
variety of
applications, e.g., in the diagnostic field or as a research tool. The parts
of the kit of the
invention can be packaged individually in vials or in combination in
containers or multicontainer
units. Manufacture of the kit follows preferably standard procedures which are
known to the
person skilled in the art. The kit or diagnostic compositions may be used for
detection of the
expression level of one or more of E-selectin, ICAM-1 and VEGFR-3 in
accordance with the
herein-described methods of the invention, employing, for example,
immunohistochemical
techniques described herein.
For use in the detection methods described herein, the skilled person has the
ability to label the
polypeptides, for example antibodies, or oligonucleotides encompassed by the
present invention.
As routinely practiced in the art, hybridization probes for use in detecting
mRNA levels and/or
antibodies or antibody fragments for use in immunoassay methods can be
labelled and visualized
according to standard methods known in the art, nonlimiting examples of
commonly used
systems include the use of radiolabels, enzyme labels, fluorescent tags,
biotin-avidin complexes,
chemiluminescence, and the like.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 34 -
The present invention is further illustrated by the following non-limiting
illustrative example.
Example 1 - Bevacizumab in combination with trastuzumab/docetaxel compared
with
trastuzumab/docetaxel alone as first line treatment for patients with HER2
positive locally
recurrent or metastatic breast cancer ¨ AVEREL study
The primary objective of the clinical trial disclosed herein was to compare
Progression Free
Survival (PFS) in patients randomized to bevacizumab in combination with
trastuzumab /
docetaxel versus patients randomized to trastuzumab / docetaxel alone. The
secondary
objectives were to evaluate Overall Survival (OS); Best Overall Response (OR);
Duration of
Response (DR); Time to Treatment Failure (TTF); Safety and tolerability of
combining
bevacizumab with trastuzumab and docetaxel; and finally Quality of Life.
Specifically, the study described herein were to determine (1) that
bevacizumab at 15 mg/kg
every 3 weeks + trastuzumab at 8 mg/kg loading dose followed by 6 mg/kg every
3 weeks until
disease progression + docetaxel 100 mg/m2 every 3 weeks for a minimum of 6
Cycles confers a
positive treatment effect on the primary variable of PFS when compared to
trastuzumab 8 mg/kg
loading dose followed by 6 mg/kg every 3 weeks until disease progression +
docetaxel 100
mg/m2 every 3 weeks for a minimum of 6 Cycles; and (2) that bevacizumab at 15
mg/kg every 3
weeks + trastuzumab 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks
until disease
progression + docetaxel 100 mg/m2 every 3 weeks for a minimum of 6 Cycles has
an acceptable
safety profile.
Study Design
The trial was a randomized, open label, 2-arm, multicentre, phase III study.
Patients were
randomly assigned to treatment groups on a 1:1 basis through a central
randomization process.
A block design randomization procedure was used. In order to avoid an
imbalance of important
prognostic factors in the patient population between the two treatment arms,
patients were
stratified, according to the following criteria:
= Prior adjuvant/neo-adjuvant taxane/ time to relapse since last dose of
adjuvant/neoadjuvant chemotherapy. Patients were initially stratified for
prior treatment with
taxanes (Yes versus No). If 'no prior taxanes', a second stratification was
performed i.e. never
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 35 -
received adjuvant/neo-adjuvant chemotherapy or relapse? 12 months since last
dose of
chemotherapy versus < 12 months since last dose of chemotherapy.
= Trastuzumab as part of adjuvant treatment versus no trastuzumab;
= Hormone receptor (ER/PgR) status (positive versus negative); and
= Measurable disease (Yes versus No)
Patients were randomized to one of the following two arms:
= Arm A: Trastuzumab 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks
until disease
progression + Docetaxel 100 mg/m2 every 3 weeks for a minimum of 6 Cycles (or
up to disease
progression or unacceptable toxicity, whichever occurs first). After 6 Cycles
with no progression
or toxicity docetaxel may be continued for additional Cycles at the discretion
of the investigator.
= Arm B: Trastuzumab 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks
until disease
progression + Docetaxel 100 mg/m2 every 3 weeks for a minimum of 6 Cycles (or
up to disease
progression or unacceptable toxicity, whichever occurs first). After 6 Cycles
with no progression
or toxicity docetaxel may be continued for additional Cycles at the discretion
of the investigator
+ Bevacizumab 15 mg/kg every 3 weeks until disease progression.
CA 02871385 2014-10-23
WO 2014/001232
PCT/EP2013/063094
- 36 -
Table 1: Study Overview and Dosing Regimen
Screening/Baseline Drug Treatment Period Follow-up
Screening Day Drug Treatment Period Day Follow-up Day
-28 to -1 Each treatment Cycle is 21 Clinical assessment
after last
days in length dose of study treatment
Trastuzumab loading dose of At:
8 mg/kg was administered on
-1 month [(Day 28) for safety]
Day 1 of Cycle 1, 24 hours
after last dose of study
prior to the first dose of
treatment
bevacizumab and/or
docetaxel. Then a dose of 6
-3 months after last dose of
mg/kg was administered on
study treatment
Day 1 of each 3-weekly Cycle
until disease progression,
Thereafter every 3 months
unacceptable toxicity
(requiring discontinuation of
study treatment) or
withdrawal of patient's
consent.
Docetaxel initial dose of 100
mg/m2 was administered on
Day 2 of Cycle 1 and
thereafter the same dose was
administered on Day 1 of each
3-weekly Cycle for a
minimum of 6 Cycles.
Bevacizumab initial dose of
15 mg/kg was administered on
Day 2 of Cycle 1 and
thereafter the same dose was
administered on Day 1 of each
3-weekly Cycle until
progression, unacceptable
toxicity (requiring
discontinuation of study
treatment) or withdrawal of
patient's consent.
Length of Study
424 patients were recruited from 60 centers over approximately 29 months and
followed for
about 26 months for the primary endpoint (PFS).
End of Study
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 37 -
This was an event driven trial. The analysis of the primary endpoint was
performed when 310
events were confirmed in the 424 patients randomized. An additional analysis
of overall survival
took place approximately 36 months after randomization of the last patient and
the trial ended at
this point. End of study occurred at the date of the last visit of the last
patient participating in this
trial which coincided with the final Overall Survival analysis which took
place approximately 36
months after randomization of the last patient.
After this clinical cut-off for the final Overall Survival analysis, patients
who benefited from
study treatment could continue to receive bevacizumab until disease
progression.
Number of Patients/ Assignment to Treatment Groups 424 patients were
randomized 1:1 into
two arms of the trial:
= 205 HER2 positive patients in the trastuzumab / docetaxel arm (Arm A);
and
= 205 HER2 positive patients in the bevacizumab plus trastuzumab /
docetaxel treatment arm
(Arm B).
Patients were randomly assigned to treatment groups. Patients received their
first dose of study
treatment on the day of randomization, but no later than 5 working days after
randomization.
Under no circumstances were patients who enrolled in this study permitted to
be re-randomised
to this study and enrolled for a second course of treatment.
Eligible patient enrollment criteria
Pre- and postmenopausal female and male patients with locally recurrent or
metastatic HER2
positive breast cancer (excluding primary tumor-T4d-Inflammatory carcinoma).
Patients could
have received prior radiotherapy for metastatic breast cancer (MBC) provided
that it was
completed 3 weeks prior to randomization and no more than 30% of marrow-
bearing bone was
irradiated. Prior adjuvant radiotherapy was allowed provided it finished at
least 6 months before
randomization.
Patients who received trastuzumab in the adjuvant setting were allowed to be
enrolled, provided
that? 6 months had elapsed since last adjuvant administration of trastuzumab.
Patients had to
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 38 -
have an adequate Left Ventricular Ejection Function at baseline defined as
LVEF not below 50%
as measured by either echocardiography or MUGA. Patients who were treated with
anthracyclines for adjuvant disease could have been included into the study if
the maximum
cumulative dose was less/equal to 360 mg/m2 of doxorubicin or 720 mg/m2 of
epirubicin.
Patients had to have a histologically or cytologically confirmed HER2
positive, pre- or
postmenopausal adenocarcinoma of the breast with measurable or non measurable
locally
recurrent or metastatic disease. Patients had to have a good performance
status (ECOG 0-1),
normal liver, renal and bone marrow functions, and be free of other serious
diseases, which could
affect compliance with the protocol or the interpretation of results. They
could not be at
increased risk of GI perforation, hypertension, proteinuria and wound healing
complications,
thromboembolism or haemorrhage. Patients with metastatic CNS disease or spinal
cord
compression caused by metastasis were not eligible. Patients could not have
had another primary
tumor within the last 5 years (except for adequately treated CIS of the
cervix, squamous
carcinoma of the skin or basal cell skin cancer). Pregnant or lactating
females were excluded.
Full anticoagulation at study entry was allowed as long as the patient had
been on a stable level
of anticoagulation for at least two weeks at the time of randomization.
Eligibility Critieria:
1. Patients age? 18 years;
2. Able to comply with the protocol;
3. ECOG PS of< 1;
4. Life expectancy of? 12 weeks;
5. Pre- or postmenopausal patients with histologically or cytologically
confirmed breast
cancer (adenocarcinoma) with measurable or non-measurable, locally recurrent
or
metastatic lesions (excluding primary tumor-T4d-Inflammatory carcinoma), who
were
candidates for chemotherapy. Locally recurrent disease could not be amenable
to
resection with curative intent. ER/PgR and HER2 status had to have been
documented;
6. Patients must have had HER2 protein overexpression (3+) as determined by
immunohistochemistry (IHC); or amplification of HER2/c-erbB2 as determined by
fluorescent in situ hybridization (FISH) or chromogenic in situ hybridization
(CISH), of
the primary tumor or a metastasis confirmed by the central laboratory prior to
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 39 -
ramdomization. Confirmation of HER2 positivity of the primary tumor by the
central
laboratory was not required in this trial for the patients who previously
participated in
Roche or Genentech sponsored trials of adjuvant trastuzumab where HER2 status
has
been centrally confirmed. (e.g. the HERA, BCIRG006, NSABP B31, or
Intergroup/NCCTG/H2061s trials);
7. Patients who received trastuzumab in the adjuvant setting were eligible as
long as they
did not relapsed within 6 months after the last dose of trastuzumab;
8. Patients who were treated with anthracyclines in adjuvant or neo-adjuvant
setting are
only eligible if they received their last dose > 6 months prior to
randomization. The
maximum cumulative dose must not have exceeded 360 mg/m2 for doxorubicin and
720
mg/m2 for epirubicin;
9. Patients who were treated with a taxane are only eligible if they
received their last
adjuvant or neo-adjuvant chemotherapy > 12 months prior to randomization;
10. Baseline Left Ventricular Ejection Fraction (LVEF) not below 50% measured
by either
echocardiography or MUGA;
11. The use of full-dose oral or parenteral anticoagulants was permitted as
long as the
patient was on a stable level of anticoagulation for at least two weeks at the
time of
randomization:
= Patients on heparin treatment that had a baseline aPTT between 1.5 - 2.5
times
ULN or patients value before starting heparin treatment
= Patients on low molecular weight heparins (LMWH) that received daily dose
of
1.5
= 2 mg/kg (of enoxaparin) or appropriate doses of the correspondent
anticoagulant,
according to package insert
= Patients on coumarin derivatives that had an INR between 2.0 and 3.0
assessed at
baseline in two consecutive measurements 1-4 days apart
= Patients not receiving anticoagulant medication must have had an INR <
1.5 and
aPTT < 1.5 times ULN within 7 days prior to randomization.
The Exclusion Criteria:
1. Previous chemotherapy for metastatic or locally recurrent breast cancer.
Prior hormonal
therapy was allowed but must have been discontinued at least 2 weeks prior
randomization.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 40 -
2. Previous radiotherapy for treatment of metastatic breast cancer was not
allowed in case:
= More than 30% of marrow-bearing bone had been irradiated
= The last fraction of radiotherapy had been administered within 3 weeks
prior to
randomization
Prior adjuvant radiotherapy for breast cancer was allowed, provided it stopped
at least 6
months prior to randomization.
3. Other primary tumor (including primary brain tumors) within the last 5
years prior to
randomization, except for adequately treated carcinoma in situ of the cervix,
squamous
carcinoma of the skin, or adequately controlled limited basal cell skin
cancer.
4. Evidence of spinal cord compression or current evidence of CNS metastasis.
CT or MRI
scan of the brain was mandatory (within 4 weeks prior to randomization) in
case of
clinical suspicion of brain metastasis.
5. History or evidence upon physical/neurological examination of CNS
disease (unrelated to
cancer) (unless adequately treated with standard medical therapy) e.g.
uncontrolled
seizures.
6. Major surgical procedure, open biopsy or significant traumatic injury
within 28 days
prior to study treatment start, or anticipation of the need for major surgery
during the
course of the study treatment.
7. Existing peripheral neuropathy > CTC Grade 2 at randomization.
8. Inadequate bone marrow function: ANC < 1.5 x 109/L, Platelet count < 100 x
109/L and
Hb < 9 g/dL.
9. Inadequate liver function:
= serum (total) bilirubin > ULN
= AST and ALT > 2.5 x ULN
= AST or ALT >1.5 x ULN concurrent with serum alkaline phosphatase levels >
2.5 x
ULN at baseline
10. Inadequate renal function:
i. Serum Creatinine > 2.0 mg/dL or 177 [tmol/L
ii. Urine dipstick for proteinuria > 2+. Patients with? 2+ proteinuria on
dipstick
urinalysis at baseline should undergo 24 hours urine collection and must
demonstrate
<1 g of protein/24 hr.
11. Chronic daily treatment with corticosteroids (dose of > 10 mg/day
methylpredniso lone
equivalent) (excluding inhaled steroids).
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 41 -
12. Chronic daily treatment with aspirin (> 325 mg / day) or clopidogrel (> 75
mg / day).
13. Uncontrolled hypertension (systolic > 150 mm Hg and/or diastolic > 100 mm
Hg) or
clinically significant (i.e. active) cardiovascular disease: CVA/stroke (< 6
months prior to
randomization), myocardial infarction (< 6 months prior to randomization),
unstable
angina, New York Heart Association (NYHA, Appendix 6) Class 2 or greater
Congestive
Heart Failure, or serious cardiac arrhythmia requiring medication
14. History or evidence of inherited bleeding diathesis or coagulopathy with
the risk of
bleeding.
15. History of abdominal fistula, gastrointestinal perforation, or intra-
abdominal abscess
within 6 months prior to randomization.
16. Active infection requiring i.v. antibiotics at randomization.
17. Serious non-healing wound, peptic ulcer, or bone fracture.
18. Evidence of any other disease, metabolic or psychological dysfunction,
physical
examination finding, or clinical laboratory finding giving reasonable
suspicion of a
disease or condition that contraindicates use of an investigational drug, or
that may affect
patient compliance with study routines, or place the patient at high risk from
treatment
complications.
19. Pregnant or lactating females. Serum pregnancy test to be assessed within
7 days prior to
study treatment start, or within 14 days with a confirmatory urine pregnancy
test within 7
days prior to study treatment start.
20. Patients of childbearing potential (women < 2 years after last
menstruation) not using
effective non-hormonal means of contraception (intrauterine contraceptive
device, barrier
method of contraception in conjunction with spermicidal jelly or surgically
sterile).
21. Current or recent (within 30 days prior to starting study treatment)
treatment with another
investigational drug or participation in another investigational study.
22. Known hypersensitivity to any of the study drugs or excipients.
23. Hypersensitivity to Chinese hamster ovary cell products or other
recombinant human or
humanized antibodies.
Results:
The improvement in investigator-assessed PFS, which was not stratified and not
censored for
non-protocol therapy (NPT), was calculated to be 2.8 months longer than that
of the control arm
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 42 -
of the study, as described above. Specifically, as summarized in Table 2, the
median PFS was
13.7 months for the control group (Arm A) versus 16.5 months for the
"bevacizumab" group
(Arm B), where the hazard ratio was 0.82 at a 95% CI 0.65,1.02), p-values =
0.0775.
Table 2: Investigator-Assessed PFS
Trast+Doc Trast+Doc+Bv
(n=208) (n=216)
No. of subjects with an event 154 (74.0%) 153 (70.8%)
Progression free survival (months)
Median 13.7 16.5
(95% CI) (11.4, 16.3) (14.1, 19.1)
HR (95% CI) 0.82 (0.65, 1.02)
p-value (log-rank) 0.0775
Stratified analysis
HR (95% CI) 0.76 (0.60, 0.96)
p-value (log-rank) 0.0216
Further, the independent review committee (IRC) assessed PFS results, which
were stratified and
censored for NPT, was statistically significant. Specifically, as summarized
on Table 3, the
median PFS was 13.9 months for the control group (Arm A) versus 16.8 months
for the
"bevacizumab" group (Arm B), where the hazard ratio was 0.72 at a 95% CI
(0.54, 0.94), p-
values = 0.0162. Overall, the median "Arm B" or "bevacizumab" group's PFS was
2.9 months
longer than that of the control arm of the study.
Table 3: Independent Review Committee (IRC) PFS
Trast+Doc Trast+Doc+Bv
(n=208) (n=216)
No. of subjects with an event 114 (54.8%) 111 (51.4%)
Progression-free survival (months)
Median 13.9 16.8
(95% CI) (11.2, 16.7) (14.1, 19.5)
Stratified analysis
HR (95% CI) 0.72 (0.54, 0.94)
p-value (log-rank) 0.0162
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 43 -
The objective response rate (ORR) results of the study, both investigator-
assessed results (INV)
and independent review committee (IRC) assessed results are shown on Table 4.
Specifically for
the INV-assessed ORR, the study showed a 69.9% ORR in the control arm versus a
74.3% in the
"bevacizumab" or Arm B. The difference between the two is 4.43%, at a 95% CI (-
5.2%,
14.0%), with a p-value of 0.3492. For the IRC-assessed ORR, the study showed a
65.9% ORR
in the control arm versus a 76.5% in the "bevacizumab" or Arm B. The
difference between the
two is 10.59%, at a 95% CI (1.0%, 20.2%), with a p-value of 0.0265.
Table 4: Objective Response Rate (ORR)
Trast+Doc Trast+Doc+Bv
(n=176) (n=183)
INV
No. of patients with overall response 123 (69.9%) 136 (74.3%)
CR 1O(5.7%) 1O(5.5%)
PR 113 (64.2%) 126 (68.9%)
95% CI for overall response (62.5, 76.6) (67.4, 80.5)
Difference in overall response rates 4.43
95% CI for difference (-5.2, 14.0)
P-value 0.3492
IRC
116 (65.9%) 140 (76.5%)
No. of patients with overall response
2(1.1%) 2(1.1%)
CR
114 (64.8%) 138 (75.4%)
PR
(58.4, 72.9) (69.7, 82.4)
95% CI for overall response
Difference in overall response rates 10.59
95% CI for difference (1.0, 20.2)
P-value 0.0265
Interim overall survival (OS) results of the study is summarized in Table 5,
which showed that
the median survival rate was 38.3 months in the control arm versus 38.5 months
in the
"bevacizumab" Arm B, with a hazard ratio (HR) of 1.01, at a 95% CI (0.74,
1.38), and a p-value
of 0.9543.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 44 -
Table 5: Interim Overall Survival (OS)
Trast+Doc Trast+Doc+Bv
(n=208) (n=216)
No. of subjects who died 78 (37.5%) 81 (37.5%)
Duration of overall survival (months)
Median 38.3 38.5
(95% CI) (34.3, NR) (32.1, NR)
HR (95% CI) 1.01 (0.74, 1.38)
p-value (log-rank) 0.9543
Stratified analysis
HR (95% CI) 0.94 (0.68, 1.30)
p-value (log-rank) 0.7078
Further, a preliminary assessment of safety demonstrated that there were no
new safety signals
shown by patients in this study.
EXAMPLE 2 - Exploratory biomarker analysis in AVEREL study
Patients and Immunochemical Methods
Blood plasma baseline samples were available for analysis from 162 patients in
this trial.
Blood Plasma Analysis
Blood samples for biomarker discovery and validation were collected from
consenting patients
in study B020231. Blood samples (approx 20 mL in total) were collected at
baseline (after
randomization but before the first administration of study medication) and at
time of disease
progression.
A total of 4.9 mLs of blood were drawn into a S-monovette (EDTA) tube. They
were mixed
immediately thereafter by gentle invertion of the tube and were centrifuged
within 30 minutes at
approximately 1500g in a centrifuge (room temperature for 10 minutes).
Immediately hereafter,
supernatant plasma was aliquoted in a clear polypropylene 5mL transfer tube.
Thereafter,
plasma was aliquoted into 2 plastic storage tubes (approximately 1.25 ml
each). Samples were
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 45 -
stored in an upright position at -70 C. In some cases, samples were stored at -
20 C for up to one
month and then transferred to -70 C.
Samples were used for measurement of levels of Interleukin-8 (I1-8), Inter-
Cellular Adhesion
Molecule 1 (ICAM-1), VEGFA, VEGF-C, VEGF receptor-1 (VEGFR1), VEGF Receptor 2
(VEGFR2), VEGF receptor-3 (VEGFR3), basic Fibroblast Growth Factor (bFGF),
Platelet
Derived Growth Factor-C (PDGF-C), and E-SELECTIN using an Immunological
MultiParameter Chip Technique (IMPACT) from Roche Diagnostics GmbH.
IMPACT Multiplex Assay Technology
Roche Professional Diagnostics (Roche Diagnostics GmbH) has developed a
multimarker
platform under the working name IMPACT (Immunological MultiParameter Chip
Technique).
This technology was used for the measurement of the protein markers mentioned
above in the
"blood plasma analysis" section. The technology is based on a small
polystyrene chip
manufactured by procedures as disclosed in EP 0939319 and EP 1610129. The chip
surface was
coated with a streptavidin layer, onto which the biotinylated antibodies were
then spotted for
every assay. For each marker, spots of antibodies were loaded in a vertical
line onto the chip.
During the assay, the array was probed with specimen samples containing the
specific analytes.
The plasma volume required per specimen for measuring all markers on one chip
was 20 1 for
chip 1 and 8 iut for chip 2 and chip 3 (see below). The sample volume was
applied together with
incubation buffer (50 mM HEPES pH 7.2, 150 mM NaC1, 0.1% Thesit, 0.5% bovine
serum
albumin and 0.1% Oxypyrion as a preservative agent) to give a total reaction
volume of 40 1 per
chip. After incubation for 12 minutes and washing of the chip using a washing
buffer (5 mM
Tris pH 7.9, 0.01% Thesit and 0.001% Oxypyrion) the digoxigenylated detection
antibody mix
was added (40 iut of incubation buffer including a mix of the analyte-specific
antibodies labeled
with Digoxigenin) and was incubated for an additional 6 minutes to bind onto
the captured
analytes. The second antibody was finally detected after washing with 40 iut
of a reagent buffer
(62.5 mM TAPS pH 8.7, 1.25 M NaC1, 0.5% bovine serum albumin, 0.063% Tween 20
and
0.1% Oxypyrion) including an anti-digoxigenin antibody conjugate coupled to
fluorescent latex.
Using this label, 10 individual binding events in a single spot could be
detected, resulting in very
high sensitivity down to the limn concentration. Chips were transported into
the detection
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 46 -
unit, and a charge coupled device (CCD) camera generated an image that was
transformed into
signal intensities using dedicated software. Individual spots were
automatically located at
predefined positions and quantified by image analysis. For each marker, lines
of 10-12 spots
were loaded on the chips, and the concentration of the markers was calculated
as mean of at least
spots from the respective line on the chip.The advantages of the technology
are the ability of
multiplexing up to 10 parameters in a sandwich or competitive format. The
calibrators and
patient samples were measured in duplicate. One run was designed to contain a
total of 100
determinations, including calibrators and 2 multi-controls as a run control.
Since some of the
selected analytes react with each other (i.e VEGFA with VEGFR1 or VEGRF2 ),
the analytes
were divided on three different chips as follows:
Chip 1: VEGFA, VEGF-C, PDGF-C
Chip 2: VEGFR1, VEGFR2, VEGFR3,I1-8, bFGF,
Chip 3: E-selectin, ICAM-1
Table 6. Antibodies used for the present assays
Capture Detection
Analyte Manufacturer Manufacturer
antibody antibody
<E-
<E-Selectin>M-
E-selectin Selectin>M- R&D Systems R&D Systems
5D11
BBIG-E5
<ICAM-1>M- <ICAM-1>M-
ICAM-1 R&D Systems R&D Systems
11C81 14C11
<VEGF- <VEGF-R3>M-
VEGFR-3 R&D Systems Abnova
R3>M-54716 5B6
Statistical Analysis
Sample median was used to dichotomize biomarker values as low (below median)
or high (at or
above median).
Hazard Ratio of treatment effect in sub-group of patients with high or low
biomarker levels were
estimated with proportional hazard cox regression analysis.
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 47 -
In addition, proportional hazard cox regressions was used to evaluate the
association between
biomarker level and treatment effect. The model included the following
covariates: trial
treatment, biomarker level, binary stratification factors (ER/PgR status,
measurable disease at
baseline, Prior adjuvant/neo-adjuvant taxane/time to relapse since the last
dose of adjuvant/neo-
adjuvant chemotherapy, prior Trastuzumab neoadjuvant therapy), interaction
term of treatment
by biomarker level. Wald test for the interaction term was used to determine
the association
between biomarker level and treatment effect. P-value below 0.05 was
considered significant.
STATISTICAL METHODS
Analysis Populations
A biomarker evaluable population was defined in this study, consisting of all
patients who
received any component of study medication and had marker levels at baseline
for any of the
following biomarkers assessed as described above and with commercially
available antibodies:
VEGF-A, VEGF-C, VEGF-R1, VEGF-R2, E-selectin, VEGFR-3, IL-8, bFGF, PDGF-C,
ICAM-
1.
Adjustment for multiplicity
In order to avoid inflation of the type I error a hierarchy was applied to the
biomarkers as shown
below. Each biomarker was tested at the two-sided 5% a level, and only if
statistical significance
was met, the next biomarker in the hierarchy was tested.
1. VEGF-A
2. VEGF-R2
3. ICAM-1
4. bFGF
5. IL-8
6. VEGF-R1
7. PDGF-C
8. VEGF-C
9. VEGFR-3
10. E-selectin
CA 02871385 2014-10-23
WO 2014/001232 PCT/EP2013/063094
- 48 -
Efficacy Analyses
PFS and OS have been defined as specified in the Data Reporting Analysis
Manual (DRAM) for
Study B020231. The analyses of biomarker data has been based on the PFS
(Investigator
assessed) data at the time of final PFS analysis. The sample median biomarker
concentration was
used as the cut point to group patients (high vs. low levels of
concentration).
Analyses of Progression Free
The following analyses have been performed for PFS on the biomarker evaluable
population:
= Median PFS (95% CI) estimated from Kaplan-Meier curves for patients with
low and
patients with high biomarker levels
= Unstratified (& stratified for PFS) HR (95% CI) from the Cox model for
patients
with low and patients with high biomarker levels
= Unstratified HR (95% CI) from Cox model by quartile of biomarker levels
= An interaction test for biomarker evaluable patients has been performed
to assess
whether the biomarker is predictive of treatment benefit on PFS with
bevacizumab for
patients with HER2 positive locally recurrent or metastatic breast cancer,
using a Cox
model that includes baseline biomarker levels (as a binary variable
dichotomized as high
and low at the sample median), treatment, baseline prognostic factors and the
interaction
term (baseline biomarker level by treatment).
CA 02871385 2014-10-23
WO 2014/001232
PCT/EP2013/063094
- 49 -
Results
Blood Plasma Markers
The baseline descriptive statistics of the biomarkers are presented in Table
7.
Table 7: Descriptive Statistics of Biomarker Values (Baseline)
E-selectin ICAIVI-1 VEGFR-3
(ng/mL) (ng/mL) (ng/mL)
min 9.3 92.2 3.5
qu 25% 26.7 170.4 7.8
median 36.9 210 10.6
qu 75% 49.1 272.2 13.0
max 212.9 749.9 43.5
mean 41.4 239.5 10.8
sd 25.08 106.27 4.30
CA 02871385 2014-10-23
WO 2014/001232
PCT/EP2013/063094
- 50 -
Table 8 presents the results of the analysis of the association of E-selectin,
ICAM-1 and
VEGFR-3 with treatment effect on Investigator assessed progression free
survival.
Table 8
TH BV+TH
Events/ Median, Events/ Median, Interaction
Subgroup pts, n mo pts, n mo HR (95% CI) p
value
All 64/82 11.2 66/80 16.5 0.78
(0.56 1.11)
E-selectin L 31/44 16.4 30/36 16.5 1.01
(0.61 1.68)
0.241
H 32/37 8.2 35/43 16.6 0.57
(0.35 0.92)
ICAM-1 L 30/45 16.4 29/35 16.5 1.13
(0.68 1.89)
0.017
H 33/36 10.3 36/44 16.2 0.49
(0.30 0.80)
VEGFR-3 L 35/48 12.2 25/32 15.3 0.88
(0.52 1.47)
0.051
H 28/33 11.0 40/47 16.6 0.65
(0.40 1.06)
'Model includes prognostic factors
T: docetaxel, H: trastuzumab, BV: bevacizumab, L: low, H: high
CA 02871385 2014-10-23
WO 2014/001232
PCT/EP2013/063094
- 51 -
In this analysis, for E-selectin, High E-selectin (> 36.9 ng/mL), Low E-
selectin (< 36.9 ng/mL),
and for ICAM-1, High ICAM-1 (> 210 ng/mL), Low ICAM-1 (< 210 ng/mL), for VEGFR-
3,
High VEGFR-3 (> 10.6 ng/mL), Low VEGFR-3 (< 10.6 ng/mL) were used.
High baseline ICAM-1 correlated statistically significantly with greater PFS
benefit from
bevacizumab at a=0.05. VEGFR-3 and E-selectin showed potential predictive
value. Therefore,
ICAM-1, E-selectin and VEGFR-3 can be independent predictive biomarkers for
bevacizumab
treatment effect on Progression Free Survival.
The effect of ICAM-1 on PFS was also apparent in the exploratory quartile
analysis of ICAM-1
(Table 9).
Table 9: Quartile analysis of PFS according to baseline ICAM-1 concentration
Median (months)
Quartile Events/N BV+TH TH HR (95%
CI)
1st 28/40 12.7 17.2 1.54 (0.72-
3.31)
2nd 31/40 20.0 14.7 0.84 (0.41-
1.75)
3rd 34/40 16.4 10.5 0.59 (0.30-
1.17)
4th 35/40 16.2 8.6 0.35 (0.17-
0.73)
