U.S. patent application number 12/516682 was filed with the patent office on 2010-02-25 for activated her3 as a marker for predicting therapeutic efficacy.
This patent application is currently assigned to U3 PHARMA GMBH. Invention is credited to Mike Rothe, Martin Treder.
Application Number | 20100047829 12/516682 |
Document ID | / |
Family ID | 38961070 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100047829 |
Kind Code |
A1 |
Rothe; Mike ; et
al. |
February 25, 2010 |
ACTIVATED HER3 AS A MARKER FOR PREDICTING THERAPEUTIC EFFICACY
Abstract
The present invention provides methods for the determination of
the activation level of Receptor Tyrosine kinases, e.g.
phosporylated HER3, for the selection of patients for disease
treatment. Methods are also provided for the evaluation of the
biological and pharmacodynamic effects of an active substance
and/or its efficacy in disease treatment, utilizing a tissue sample
from a test subject, for example tumor material or normal tissue
such as skin or hair follicle. Further, methods for the treatment
of HER receptor-associated diseases are disclosed.
Inventors: |
Rothe; Mike; (Krailling,
DE) ; Treder; Martin; (Martinsried, DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
U3 PHARMA GMBH
Martinsried
DE
|
Family ID: |
38961070 |
Appl. No.: |
12/516682 |
Filed: |
November 28, 2007 |
PCT Filed: |
November 28, 2007 |
PCT NO: |
PCT/EP2007/010335 |
371 Date: |
May 28, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60861243 |
Nov 28, 2006 |
|
|
|
Current U.S.
Class: |
435/7.92 ;
435/7.1; 530/387.1 |
Current CPC
Class: |
A61K 39/395 20130101;
C07K 16/32 20130101; C07K 2317/76 20130101; G01N 33/574 20130101;
A61K 2039/505 20130101; G01N 2440/14 20130101; G01N 2333/71
20130101; A61P 17/06 20180101; G01N 2800/205 20130101; G01N 33/5748
20130101; C07K 16/2863 20130101; G01N 2333/91205 20130101; G01N
33/57492 20130101; G01N 2800/52 20130101; G01N 33/57407 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
435/7.92 ;
435/7.1; 530/387.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 28, 2006 |
EP |
06024658.4 |
Claims
1-32. (canceled)
33. A method of identifying the responsiveness of a disease to
treatment with a HER3 modulator comprising (a) obtaining at least
one sample from a subject at risk of or having said disease, (b)
examining the expression and/or activity of at least one HER3
receptor in said sample, and (c) identifying a disease as
responsive if expression and/or activity of the at least one HER3
receptor is detected.
34. The method of claim 33 wherein the disease is a
hyperproliferative disease.
35. The method of claim 34 wherein the hyperproliferative disease
is a tumor disease or psoriasis.
36. The method of claim 35 wherein the tumor disease is selected
from the group consisting of NSCLC, breast, colon, gastric,
melanoma, pancreas or prostate cancer.
37. The method of claim 33 wherein the detection of HER3 receptor
activity comprises a determination of the HER3 receptor
phosphorylation level.
38. The method of claim 37 wherein a phospho-specific antibody is
employed.
39. The method of claim 38 wherein the phospho-specific antibody is
an antibody that recognizes a phosphorylated tyrosine residue in a
HER3 receptor.
40. The method of claim 39 wherein the phospho-specific antibody is
directed against at least one of the tyrosine residues Y1289 or
Y1222 in the HER3 protein.
41. The method of claim 40 wherein the phospho-specific antibody is
the phospho-specific HER3 antibody 21D3 or 50C2.
42. The method of claim 33 wherein step (b) comprises an
immunohistochemical assay, flow cytometry, ELISA or a Western
Blot.
43. The method of claim 33 wherein the sample is a tissue
sample.
44. The method of claim 42 wherein the tissue sample is selected
from the group of solid tissues as from a fresh, frozen and/or
preserved organ or tissue sample or biopsy or aspirate.
45. The method of claim 33 wherein the subject is a mammal.
46. The method of claim 45 wherein the mammal is a human.
47. A method of determining the therapeutic efficacy of a HER3
modulator comprising (a) exposing a subject to at least one HER3
modulator, (b) obtaining at least one sample from the subject, (c)
detecting the activation level of at least one HER3 receptor in
said sample wherein a difference in the activation level of the at
least one HER3receptor is observed as a result of the exposure of
the pharmaceutical composition as compared to the absence of the
exposure to the at least one HER3 modulator.
48. The method of claim 47 wherein the HER3 modulator is an
inhibitor directed against at least one HER3 receptor.
49. The method of claim 47 wherein the detection of activity of a
HER3 receptor is achieved by assessing the phosphorylation
level.
50. The method of claim 47 wherein a phospho-specific antibody is
employed.
51. The method of claim 47 wherein the phospho-specific antibody is
an antibody that recognizes a phosphorylated tyrosine residue in a
HER3 receptor.
52. The method of claim 50 wherein the phospho-specific antibody is
directed against at least on one of the tyrosine residues Y1289 or
Y1222 in the HER3 protein.
53. The method of claim 50 wherein the phospho-specific antibody is
at least one of the phospho-specific HER3 antibodies 21D3 or
50C2.
54. The method of claim 47 wherein the detection step (c) comprises
an immunohistochemical assay, flow cytometry, ELISA or a Western
Blot.
55. The method of claim 47 wherein the sample is a tissue
sample.
56. The method of claim 47 wherein the sample is a normal tissue
sample, e.g. hair follicle sample.
57. The method of claim 47 wherein the subject is a mammal,
particularly a human.
58. An antibody that detects an activated HER3 receptor.
59. The antibody of claim 58 wherein the antibody binds to an
epitope that comprises at least one of the tyrosine residues Y1054,
Y1197, Y1199, Y1222, Y1224, Y1260, Y1262, Y1276, Y1289 and Y1328 of
HER3.
60. Use of an antibody of claim 58 for identifying the
responsiveness of a disease to treatment with a HER3 modulator.
61. A kit for evaluating the activation level of an activated HER3
family member comprising (a) an antibody that detects expression of
a HER3 receptor protein, (b) an antibody that detects activation of
a HER3 receptor member.
62. Use of a HER3 modulator for the manufacture of a medicament for
the treatment of a disease which is associated with HER3 receptor
expression, overexpression and/or activity.
63. The use of claim 62 wherein the expression, overexpression
and/or activity has been determined before administration of the
HER3 modulator.
64. The use of claim 62 wherein the responsiveness of the disease
is tested during the course of the treatment
Description
[0001] The present invention provides methods for the determination
of the activation level of Receptor Tyrosine kinases, e.g.
phosporylated HER3, for the selection of patients for disease
treatment. Methods are also provided for the evaluation of the
biological and pharmacodynamic effects of an active substance
and/or its efficacy in disease treatment, utilizing a tissue sample
from a test subject, for example tumor material or normal tissue
such as skin or hair follicle. Further, methods for the treatment
of HER receptor-associated diseases are disclosed.
[0002] The human epidermal growth factor receptor 3 (HER3, also
known as ErbB3) is a receptor protein tyrosine kinase and belongs
to the epidermal growth factor receptor (EGFR) subfamily of
receptor protein tyrosine kinases, which also includes HER1 (also
known as EGFR), HER2, and HER4 (Plowman et al., Proc. Natl. Acad.
Sci. U.S.A. 87 (1990), 4905-4909; Kraus et al., Proc. Natl. Acad.
Sci. U.S.A, 86 (1989), 9193-9197; and Kraus et al., Proc. Natl.
Acad. Sci. U.S.A. 90 (1993), 2900-2904).
[0003] HER3 has been found to be overexpressed in several types of
cancer such as breast, gastrointestinal and pancreatic cancers.
Interestingly a correlation between the expression of HER2/HER3 and
the progression from a non-invasive to an invasive stage has been
shown (Alimandi et al., Oncogene 10,1813-1821; deFazio et al.,
Cancer 87, 487-498; Naidu et al., Br. J. Cancer 78, 1385-1390).
[0004] These data point out the role of HER3 in the development of
cancer and demonstrate the great potential of HER3 specific target
therapies for the therapy of cancer and other malignancies
characterized by hypersignaling through HER3 and/or its
heterodimerization partners induced signaling pathways (Reviewed in
Citri and Yarden, Nat Reviews Mol Cell Biol, 2006 (7), 505-516;
Shawver et al, Cancer Cell, 2002 (1), 117-123; Yarden and
Sliwkowski, Nat Reviews Mol Cell Biol, 2001 (2),127-137).
[0005] Agents and methods capable of treating HER3 associated
diseases have been described before. For example anti-HER3
antibodies described in WO 03/013602 are reported to induce
accelerated receptor internalization and to reduce tumor cell
proliferation and migration. In U.S. Pat. No. 5,968,511
(corresponding to WO 97/135885) HER3 antibodies were found to
reduce ligand-induced formation of HER2/HER3 heterodimers. WO
00/078347 discloses methods for arresting or inhibiting cell
growth, comprising preventing or reducing HER2/HER3 heterodimer
formation, for example, by administering a combination of an
anti-HER2 antibody, e.g. Herceptin, and an anti-HER3 antibody, e.g,
antibody 105.5 purchased from Neomarkers.
[0006] Based on the increasing implication of uncontrolled signal
transduction in many pathological conditions including cancer, a
principle aim of medical/pharmaceutical drug development is the
development of individual or targeted therapies for the treatment
of diseases. Such specific therapies may e.g. comprise therapeutic
antibodies, small molecule inhibitors, nucleic acid interference,
and the administration of an individually selected or dosed
pharmaceutical composition.
[0007] Most of these so called target specific therapies
predominantly affect a single target. Thus it is critical in modern
drug development to identify those patients responsive to the
target specific therapy.
[0008] A very prominent example is the therapeutic antibody
Herceptin that is directed against the receptor tyrosine kinase
HER2. This particular antibody has been approved for the treatment
of breast cancer, a tumor indication which is associated with an
amplification of the HER2 gene in about 20% of cases causing
overexpression of the corresponding protein. In order to
differentiate those 20% of patients which would benefit from the
antibody therapy from the 80% that would not, a diagnostic assay,
HercepTest, has been developed.
[0009] However, such assays including HercepTest only detect the
amount of the targeted protein, whereas often it is the activity of
the protein that is actually causing the cellular signal
deregulation and subsequent malignancy. For example, the HercepTest
only predicts a successful patient response in approximately 30% of
the cases when Herceptin is used as a single agent (Leyland-Jones,
Lancet Oncol (2002) March;3(3):137-44). This low predictive rate is
observed even though all of the patients treated are judged to be
overexpressing HER2, demonstrating the significant limitations of
this type of diagnostic assay and the need for identifying better
biomarkers of responsiveness to therapy.
[0010] Another critical step during drug development is the
selection of the dose for therapeutic agents. Usually, in case of
non-targeted conventional drugs the assumption of the maximally
tolerated dose is used. This same principle, however, does not
apply for targeted therapies, where an optimal biologic dose would
be preferred instead. In fact the definition of the optimal dose to
be administered may be defined by pharmacodynamic or -kinetic
parameters and the determination of the efficacy on the target
molecule (Albanell et al., 2002, J. Clin. Oncol. 20, 110-124).
Therefore, it is desirable to have a robust test system to
determine pharmacodynamic parameters, such as for example
sufficient solubility and stability of a compound that allows
delivery to the site of action in sufficient concentration,
metabolic stability so that the compound is not cleared from the
body so rapidly that it does not have a chance to be an effective
pharmacological agent, or pharmacokinetics that allow the compound
to reach a desired plasma/serum concentration.
[0011] Successful development, approval and use of targeted drugs
will often depend in large part upon the ability of the developer
or clinician to determine before and during treatment the
activation status of the specific protein which the drug is
targeted against. Another aspect of pharmacodynamic correlation is
the dose response for a given therapeutic and the desirable
(treatment) and undesirable (adverse events) effects. Careful
assessment of the risk-benefit-ratio of a given new drug
(-combination) will lead to a tolerable administration and a
successful completion of the therapeutic intervention. The
assessment of potential resistance markers after completion of
treatment is the final aspect of pharmacodynamic effects that would
influence the decision on further treatment.
[0012] However, in clinical routine it is difficult to assess the
biological and pharmacodynamic effects of therapeutic agents. In
general, pharmacodynamic effects can be measured through extensive
imaging and radioactive labeling of substance or substrate (e.g.
PET, CT) and the read-out is to be compared with the observed side
effects and clinical efficacy. For many therapeutics (including
targeted therapeutics) so-called surrogate markers for biological
efficacy (PD markers) have been defined and are followed during the
course of therapy. However, these markers don't indicate the direct
biological effect of the therapeutic on normal and/or cancerous
cells and therefore may be subject to off-target effects and
activation/deactivation through external (not target specific)
pathways. Examples for these markers are CA-125, KI67, PTEN, and
.beta.HCG. A desirable marker would be specific to the pathway and
the therapeutic (targeted) intervention, easily accessible and
analyzable without intra- and/or intersubject variability.
[0013] In a specific case, the functional role and expression of
epidermal growth factor, EGF, and its cognate receptor, EGFR, in
the skin were correlated with the pharmacological side effects of
anti-EGFR therapy such as skin rash and hair loss (Lacouture et al.
(2006), Nat. Rev. Cancer 6, 803-812). In particular, by using a
sample derived from adult skin keratinocytes as surrogate marker
tissue, treatment of tumor patients with for example the EGFR
inhibitor, ZD1839, can be monitored by analyzing the inhibition of
EGFR tyrosine phosphorylation through immunohistochemical methods
(Albanell et al., 2002), supra).
[0014] Nevertheless, presently applied methods for determination of
pharmacodynamic and-kinetic parameters are of limited use. Whereas
traditional methods are often too broad for individual therapies,
other methods such as the detection of EGFR are target
restricted.
[0015] Thus, the technical problem underlying the present invention
was to provide a rapid, quantitative, reproducible, and inexpensive
assay that is compatible with current clinical laboratory
instrumentation and which is suitable for determination of the
activation and/or expression level of HER receptors.
[0016] The solution of the above problems is achieved by providing
the embodiments characterized in the claims.
[0017] According to the present invention, a method for the
determination of the sensitivity or responsiveness of a disease to
a HER modulator or to a combination of at least one HER modulator
with a further agent is provided. For example, based on the
surprising finding that the sensitivity of tumor cell growth to
inhibition by a HER3 modulator correlates with HER3 receptor
activation, e.g. phosphorylation, methods and procedures have been
devised for predicting the responsiveness of a subject to treatment
with a HER modulator.
[0018] The results presented in the examples herein demonstrate
that tumor cells, such as BxPC3 (pancreas cancer), A431 (epithelial
carcinoma) or A549 (lung carcinoma) grown in vitro express HER3 and
show basal HER3 phosphorylation. Further experiments validated
these initial findings in a majority of the examined tumor cell
lines. Interestingly, examination of tumor xenograft models treated
with HER3 inhibitors showed that those tumors arising from tumor
cell lines with HER3 expression and elevated basal HER3
phosphorylation, e.g. T47D (breast cancer), BxPC3 (pancreas
cancer), HT-29 (colon cancer) and CaLu-3 (NSCLC) are particularly
responsive to treatment protocols targeting a HER3 receptor. The
data indicate that HER receptor activation, e.g. phosphorylation,
may be a general biological switch that predefines the level of
responsiveness of a disease to HER modulators. Thus, activation of
a HER receptor such as HER3 is indicative of a disorder that is
particularly sensitive to treatment with a HER modulator.
[0019] Accordingly, a first aspect of the invention relates to a
method for determining whether a disease is responsive to treatment
with a HER modulator, by obtaining at least one sample from a
subject at risk of or having said disease, examining the expression
and/or activity of at least one HER receptor in a cellular assay,
and identifying a disease as responsive if expression and/or
activity of at least one HER receptor is detected.
[0020] The term "HER receptor" is intended to mean a HER1 protein,
e.g. human HER1/EGFR (Acc-Nr. Swiss Prot P00533), a HER2 protein,
e.g. human HER2 (Acc-Nr. Swiss Prot P04626), a HER3 protein, e.g.
human HER3 (Acc-Nr. Swiss Prot P21860) or a HER4 protein (Acc-Nr.
Swiss Prot Q155503). Preferably, the HER receptor is a HER3
protein, more preferably the human HER3 protein.
[0021] In another preferred aspect the present invention relates to
the use of a modulator that affects a HER receptor selected from
the group of HER1, HER2, HER3 or HER4. In particular, a modulator
that affects the activity of HER3, e.g. human HER3, is
preferred.
[0022] The term "HER modulator" is intended to mean a compound or
drug that acts either on the nucleic acid level or on the protein
level to directly or indirectly modulate HER receptor activity.
Direct or indirect modulation includes activation or inhibition of
HER receptor activity or HER receptor signal transduction pathway.
Preferably, the modulation includes an inhibition.
[0023] The modulator of HER receptor activity may act on the
nucleic acid level, either on the transcription or on the gene
itself. On the gene level said modulator may cause a partial or
complete gene inactivation, for example by gene disruption.
Reducing or inhibiting transcription may comprise application of
effector nucleic acids, such as antisense molecules, for example
DNA or RNA molecules or RNA analogues, ribozymes, small
double-stranded RNA molecules capable of RNA interference (siRNA)
or microRNAs. Further, precursor RNA molecules of siRNA or DNA
molecules encoding the latter may be suitable.
[0024] Effector molecules may be directly introduced into a cell or
generated within a cell by transcription from suitable nucleic acid
templates. Production and uses of effector nucleic acids are
extensively discussed in the literature and are widely known and
available to one skilled in the art.
[0025] In another embodiment, the HER modulator may act on the
protein level by at least partially inhibiting HER receptor
mediated signal transduction. For example the modulator may block
the ligand induced activation of a HER receptor. By a ligand is
meant a polypeptide that binds to and/or activates a HER receptor.
Preferred examples of ligands are selected from the group of:
TABLE-US-00001 AMPR (amphiregulin) NM 001657 BTC (betacellulin) NM
001729 DTR (diphtheria toxin receptor (heparin-binding NM 001945
epidermal growth factor-like growth factor)) EGF (epidermal GF,
beta-urogastrone) NM 001963 EREG (epiregulin) ##STR00001## NRG1
(neuregulin 1) NM 013957 NRG2 (neuregulin 2) NM 013982 NRG3
(neuregulin 3) AL 096706 NRG4 (neuregulin 4) ##STR00002## TGFA
(transforming growth factor, alpha) NM 003236
[0026] Particularly preferred are neuregulin 1 isoforms encoded by
the neuregulin 1 gene.
[0027] Accordingly, such a modulator may act by occupying the
ligand binding site or a portion thereof of the HER receptor,
thereby making the receptor inaccessible to its natural ligand so
that its normal biological activity is prevented or reduced. In
this embodiment, ligand muteins capable of binding to the receptor,
but unable to induce signal transduction, or antibodies directed
against ligands are examples of HER modulators. Suitable types of
antibodies are discussed in detail below.
[0028] In another aspect the modulator interferes with ligand
dependent or independent formation of HER receptor oligomers, e.g.
hetero-oligomers or homo-oligomers. An HER receptor hetero-oligomer
herein is a non-covalently associated oligomer comprising at least
two different HER receptors. A HER receptor homo-oligomer is a
non-covalently associated oligomer that comprises at least two HER
receptors of the same. Examples of such HER oligomers include, but
are not limited to HER1/HER1, HER1/HER2, HER1/HER3, HER1/HER4,
HER2/HER2, HER2/HER3, HER2/HER4, HER3/HER4, HER4/HER4. Moreover,
preferred hetero-oligomers may comprise one, two or more HER2
receptors combined with a different HER receptor, such as HER1,
HER3, or HER4. Other proteins, such as a cytokine receptor subunit
(e.g., gp130) or other receptor tyrosine kinases such as the IGF-1R
may also be included in the hetero-oligomer.
[0029] A reduction of HER receptor mediated signal transduction may
be further caused by a downregulation from the membrane and/or
degradation of HER receptor resulting in an at least partial
disappearance of HER molecules from the cell surface or by a
stabilization of HER molecules on the cell surface in a
substantially inactive form, i.e, a form which exhibits a lower
signal transduction compared to the non-stabilized form.
[0030] Alternatively, a reduction of HER mediated signal
transduction may also be caused by influencing, e.g. decreasing or
inhibiting, the binding of a signal transduction molecule, e.g
PI3K, Shc or Grb7 to HER-3, of GRB2 to HER-2, of GRB2 to SHC, or by
inhibiting AKT phosphorylation, PYK2 tyrosine phosphorylation or
ERK2 phosphorylation. Negative regulators, such as PTPs or
proteases, could also be influenced.
[0031] In another aspect the HER modulator may be an antibody or a
fragment thereof, directed against a HER receptor. The antibody may
be a monoclonal or polyclonal antibody, as well as a recombinant
antibody, e.g. single chain antibody or a fragment thereof, which
contains at least one antigen-binding site, an antibody fragment
such as a Fab, Fab' or F(ab').sub.2 fragment or a recombinant
fragment such as a scFv fragment and a humanized antibody or a
human antibody. For therapeutic purposes, particularly for the
treatment of a candidate in need thereof, the application of
chimeric antibodies, humanized antibodies or human antibodies is
especially preferred.
[0032] In a preferred embodiment of the present invention an
anti-HER3 antibody is selected from the group consisting of
antibody 105.5 (Chen et al, JBC 1996, 271 (3) 7620-9), SGP-1
(Rajkumar et al, The Breast 1995, 4 84-91), H3 90.6 (Chen et al,
JBC 1996, 271 (3) 7620-9), 1B4C3 and 2D1D12 (PCT/EP02/08938) or one
of the human anti-HER3 antibodies disclosed in U.S. 60/755,022. An
anti-HER2 antibody is selected from the group consisting of
Trastuzumab, Pertuzumab, Herceptin-geldanamycin,
213-bi-Herceptin-alpha conjugate, Herceptin-DM1 and an anti-HER1
antibody is selected from the group consisting of Panitumumab,
Cetuximab, Matuzumab, Erbitux-paclitaxel conjugate, Erbitux-MMC
(mitomycinc) and LA22-MMC.
[0033] Another example of a modulator in terms of the methods of
the present invention is a scaffold protein, having an antibody
like binding activity that binds to a HER family member. Within the
context of the present invention, the term "scaffold protein", as
used herein, means a polypeptide or protein with exposed surface
areas in which amino acid insertions, substitutions or deletions
are highly tolerable. Examples of scaffold proteins that can be
used in accordance with the present invention are protein A from
Staphylococcus aureus, the bilin binding protein from Pieris
brassicae or other lipocalins, ankyrin repeat proteins, and human
fibronectin (reviewed in Binz and Pluckthun, (2005) Curr Opin
Biotechnol, 16, 459-69). Engineering of a scaffold protein can be
regarded as grafting or integrating an affinity function onto or
into the structural framework of a stably folded protein. Affinity
function means a protein binding affinity according to the present
invention. A scaffold can be structurally separable from the amino
acid sequences conferring binding specificity. In general, proteins
appearing suitable for the development of such artificial affinity
reagents may be obtained by rational, or most commonly,
combinatorial protein engineering techniques such as panning
against a HER family member, either purified protein or protein
displayed on the cell surface, for binding agents in an artificial
scaffold library displayed in vitro, skills which are known in the
art (Binz and Pluckthun, 2005, supra). In addition, a scaffold
protein having an antibody like binding activity can be derived
from an acceptor polypeptide containing the scaffold domain, which
can be grafted with binding domains of a donor polypeptide to
confer the binding specificity of the donor polypeptide onto the
scaffold domain containing the acceptor polypeptide. Insertion can
be accomplished by various methods known to those skilled in the
art including, for example, polypeptide synthesis, nucleic acid
synthesis of an encoding amino acid as well by various forms of
recombinant methods well known to those skilled in the art.
[0034] Reducing or inhibiting of HER activity on the protein level
may be also achieved by application of low molecular weight
inhibitors. Examples of low molecular weight inhibitors may include
organic compounds, organometallic compounds, salts of organic and
organometallic compounds, saccharides, amino acids, and
nucleotides. Low molecular weight inhibitors further include
molecules that would otherwise be considered biological molecules,
except their molecular weight is preferably not greater than 600,
more preferably not greater than 450. Thus, low molecular weight
inhibitors may also be lipids, oligosaccharides, oligopeptides, and
oligonucleotides and their derivatives. These molecules are merely
called low molecular weight inhibitors because they typically have
molecular weights not greater than 600 and the term shall not be
construed as restricted to a specific molecular weight. Low
molecular weight inhibitors include compounds that are found in
nature as well as synthetic compounds.
[0035] In one embodiment, the HER modulator is a low molecular
weight inhibitor that inhibits cell growth. In another embodiment,
the HER modulator is a low molecular weight inhibitor that inhibits
at least partially HER mediated signal transduction. A variety of
low molecular weight inhibitors directed against HER receptors have
been described. For example in one embodiment of the present
invention the low molecular weight inhibitor is one of the group
comprising Gefitinib, Erlotinib, Lapatinib, BIBW2992, AV412. In
another embodiment the low molecular weight inhibitor belongs to
the group of indirect HER modulators such as kahahalide F (Janmaat
et al, 2005) or estrogen receptor inhibitors such as tamoxifen.
[0036] The invention also encompasses combinations of HER
modulators, e.g. HER modulators directed against the same receptor,
e.g. HER3, or HER modulators directed against different HER
receptors, e.g. HER3 and HER1, HER3 and HER2, and HER3 and HER4.
For example, combinations of antibodies may be used.
[0037] The present invention further relates to a method for
determining responsiveness of disorder to the administration of at
least one modulator of a HER receptor and/or a further agent as
described in detail below.
[0038] The active ingredient, e.g. the HER modulator is usually
administered as a pharmaceutical composition. The composition may
be in solid, liquid or gaseous form and may be, inter alia, in a
form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an)
aerosol(s). Said composition may comprise at least one, e.g. two,
three, four, or five active compounds.
[0039] The pharmaceutical composition is useful for the treatment
of a disease as referred to below. In a preferred embodiment, said
disease is a hyperproliferative disease, an inflammatory disease or
a neurodegenerative disease. The hyperproliferative disease may
comprise, but is not limited to psoriasis or breast, lung, colon,
kidney, lymphoma, skin, ovary, prostate, pancreas, esophagus,
barret, stomach, bladder, cervix, liver, thyroid cancer, soft
tissue sarcoma, melanoma or other hyperplastic or neoplastic
diseases associated with HER receptor expression, overexpression
and/or activation.
[0040] As indicated above, the pharmaceutical composition may
comprise at least one further active agent. Examples for additional
active agents, which may be used in accordance with the present
invention, are antibodies or low molecular weight inhibitors of
other receptor protein kinases, such as IGF-1R, or c-met, receptor
ligands such as vascular endothelial factor (VEGF), cytotoxic or
anti-neoplastic agents, such as doxorubicin, platinum compounds
such as cis-platin or carboplatin, cytokines, antisense molecules,
aptamers, or siRNA molecules. Many antineoplastic agents are
presently known in the art. The cytotoxic or antineoplastic agent
may be selected from the group of therapeutic proteins including,
but not limited to, antibodies or immunomodulatory proteins, or
from the group of small molecule inhibitors or chemotherapeutic
agents consisting of mitotic inhibitors, kinase inhibitors,
alkylating agents, anti-metabolites, intercalating antibiotics,
growth factor inhibitors, cell cycle inhibitors, enzymes,
topoisomerase inhibitors, histone deacetylase inhibitors,
anti-survival agents, biological response modifiers, anti-hormones,
e.g. anti-androgens, and anti-angiogenesis agents. When the
anti-neoplastic agent is radiation, treatment can be achieved
either with an internal (brachytherapy BT) or external (external
beam radiation therapy: EBRT) source.
[0041] The term "disease" when used in the present invention shall
mean any condition that would benefit from a medical treatment or
that is associated with an abnormal HER receptor expression,
activation and/or signal transduction. This includes chronic and
acute diseases or diseases including those pathological conditions
which predispose the candidate to the disease in question. A
preferred disease to be treated in accordance with the present
invention is a hyperproliferative disease. A hyperproliferative
disease as mentioned above includes any neoplasia, i.e. any
abnormal and/or uncontrolled new growth of tissue. The term
"uncontrolled new growth of tissue" as used herein may depend upon
a dysfunction and/or loss of growth regulation. A
hyperproliferative disease further includes tumor diseases and/or
cancer, such as metastatic or invasive cancers. In a particular
preferred embodiment of the method of the present invention, said
hyperproliferative disease is in brain, central nervous system,
soft-tissue sarcoma, hematological malignancies, oral cavity, head
and neck, breast, lung, colon, gastric, kidney, lymphoma, skin,
ovary, prostate, pancreas, esophagus, bladder, cervix, liver,
thyroid cancer, melanoma, cancer of unknown origin, or other
hyperplastic or neoplastic diseases associated with HER receptor
expression, overexpression and/or activation, e.g.
hyperphosphorylation.
[0042] A disease which is associated with the expression or
overexpression of a HER receptor, is a disease with cells
comprising on their cell surface a HER receptor protein and/or a
ligand binding to a HER receptor. For example a disease which
"expresses" a HER family member is one which has significantly
higher levels of an HER receptor, such as HER3, at the cell surface
thereof, compared to a healthy cell of the same tissue type. Such
expression may be caused by gene amplification or by increased
transcription or translation. HER receptor expression may be
determined in a diagnostic or prognostic assay by evaluating levels
of the HER protein present on the surface of a cell (e.g., via
immunohistochemistry; 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 WO 98/45479 published
October, 1998), Southern blotting, or polymerase chain reaction
(PCR) techniques, such as real time quantitative PCR (RT-PCR).
Expression of the HER ligand, may be determined diagnostically by
evaluating levels of the ligand (or nucleic acid encoding it) in
the patient by various diagnostic assays such as DNA arrays,
Northern blotting, FISH, Southern blotting, PCR or protein based
assays described above. In addition the presence of various
N-terminal HER3 isoforms or serum concentrations of shed receptor
domains may be evaluated when practicing the present invention.
[0043] Aside from the above assays, various other 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 labelled with a detectable label, e.g., a radioactive
isotope, and binding of the antibody to cells in the patient can be
evaluated, e.g., by external scanning for radioactivity or by
analyzing a biopsy taken from a patient previously exposed to the
antibody.
[0044] In a further aspect of the invention, the disease may be
associated with HER activation. Activation of a HER family member
may generally involve formation of HER oligomers, followed by
activation of the intrinsic receptor kinase activity, the binding
of intracellular second messenger molecules to the receptor and/or
modification, e.g. tyrosine phosphorylation, of the HER receptor
and/or the second messenger molecules, which leads to specific
biologic responses, as for example cell proliferation, cell
migration or anti-apoptosis.
[0045] Another aspect of the present invention is concerned with a
method for determining and/or predicting the sensitivity of a
disease or condition associated with HER receptor mediated signal
transduction to a HER modulator, optionally in combination with a
further agent, comprising analyzing a sample by detecting the
expression and/or activity of a HER receptor in that sample.
Preferably, the method comprises detecting the expression and/or
activity of a HER3 receptor. More preferably, the method comprises
detecting the activity, e.g. the degree of the phosphorylation of a
HER3 receptor.
[0046] For example, according to the present invention, the method
may be used for the detection of a HER receptor in a cell, for the
determination of HER receptor concentration in subjects suffering
from a disease as mentioned above or for the staging of said
disease in a subject. In order to stage the progression of a
disease in a subject under study, or to characterize the response
of the subject to a course of therapy, the amount of the HER
receptor present in the sample and/or its activation level is
determined in a tissue sample, taken from the subject. The amount
so identified correlates with a stage of progression or a stage of
therapy identified in the various populations of diagnosed
subjects, thereby providing a determination of the disease stage in
the subject under study. The amount and/or activity of the HER
receptor present in the disease tissue may be assessed by
immunohistochemistry, ELISA or antibody arrays including
phospho-specific antibodies using HER receptor and/or other signal
transduction antibodies. Other suitable methods may include
bead-based technologies such as Luminex bead assays and proteomics
approaches (2-D gels, MS analysis etc). Cellular preparations with
methodical prerequisites such as phosphatase inhibitors
(ortho-Vanadate, Suramine, H.sub.2O.sub.2 or specific inhibitors)
as would be the case with phosphatase inhibitor tablets, could be
envisioned as part of the quantification of phospho-specific
antigen/epitopes.
[0047] Other parameters of diagnostic interest and which may form
part of the present invention are the oligomerization state as well
as the oligomerization partners of a HER receptor. Protein
analytical methods to determine those parameters are well known in
the art and are among others western blot and immunoprecipitation
techniques, FACS analysis, chemical crosslinking, bioluminescence
resonance energy transfer (BRET), fluorescence resonance energy
transfer (FRET) and the like (e.g. Price et al, Methods in
Molecular Biology, 218: 255-268 (2002) or the eTag technology (WO
05/03707, WO 04/091384, WO 04/011900).
[0048] The kinase activity can be measured by capturing the kinase
in the cell lysate by an antibody with immunoprecipitated and is
then subjected to kinase activity reactions in the presence of
.sup.32P-.gamma.-TP. The activity of the kinase in the reaction is
analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) and autoradiography. Alternatively, in vitro
kinase assays can be performed with non-radioactive detection
methods (e.g. CST kinase assays) or synthetic peptides that can
serve as substrates for a HER receptor, such as HER3, can be
spotted on arrays for estimating HER kinase activity.
[0049] In another aspect of the present invention the activation
level of a HER receptor correlates with the activation status of a
second messenger molecule involved in HER receptor mediated signal
transduction. Thus one embodiment of the present invention refers
to a method for identifying the responsiveness of a disease to
treatment with a HER modulator, by obtaining at least one sample
from a subject at risk of or having said disease, examining the
expression and/or activity of at least one molecule involved in HER
receptor mediated signal transduction in a cellular assay, and
identifying a disease as responsive if expression and/or activity
of at least one a molecule involved in HER receptor mediated signal
transduction is detected. Preferably, the expression and/or
activity of HER3, optionally in combination with other HER
receptors, is examined.
[0050] "Signaling pathway" or "signal transduction" refers to a
series of molecular events usually beginning with the interaction
of a cell surface receptor with an extracellular ligand or with the
binding of an intracellular molecule to a phosphorylated site of a
cell surface receptor, e.g. a HER receptor, that triggers a series
of molecular interactions, wherein the series of molecular
interactions results in a regulation of gene expression in the
nucleus of a cell. The terms "intracellular molecule", "second
messenger molecule", "molecule involved in HER receptor mediated
signaling" or "substrate of HER receptor" are used interchangeably
herein and refer to molecules involved in HER-mediated signaling
pathways as for example reviewed in Citri and Yarden, Nat Reviews
Mol Cell Biol, 2006 (7), 505-516; Shawver et al, Cancer Cell, 2002
(1), 117-123; Yarden and Sliwkowski, Nat Reviews Mol Cell Biol,
2001 (2), 127-137. Exemplary molecules that may be part of a HER
receptor mediated signaling pathway include, but are not limited
to, PI3K proteins, AKT proteins, Grb2 proteins, Grb7 proteins, Shc
proteins, Gab-1 proteins, Sos proteins, Src proteins, Cbl proteins,
PLCy proteins, Shp2 proteins, GAP proteins, Vav proteins, Nck
proteins and Crk proteins.
[0051] In a preferred embodiment of the present invention the
phosphorylation state of one of the HER receptors or their
substrates can be assessed as a measure of expression and
activation of the receptor. As is well known in the art,
phosphorylation of a HER receptor indicates that the receptor has
been activated and is the mechanism for transducing the downstream
signal.
[0052] Phosphorylation of one or multiple tyrosine residues in a
HER receptor or in one or more of its substrates can be analysed
using various tyrosine phosphorylation assays. For example HER
receptors or their substrates may be immunoprecipitated with
specific antibodies from lysates of cells expressing HER receptors
and their substrates and then assayed for tyrosine phosphorylation
activity using a phosphotyrosine monoclonal antibody (which is
optionally conjugated with a detectable label). In a preferred
embodiment tyrosine phosphorylation of HER receptors and their
substrates is detected by using phospho-specific antibodies. In a
particular embodiment said phospho-specific antibody is selected
from the group comprising phospho-specific HER3 antibodies 21D3
(Y1289, Cell Signalling Technology, USA) and 50C2 (Y1222, Cell
Signalling Technology, USA), as well as pEGFR, pHER2, pHER4,
pIGF-1R, pAkt, pErk, pBad, pp70-S6K, pGSK, p-src, pPyk2, with all
relevant phosphotyrosines in a given protein being covered
here.
[0053] In general, the term "phospho-specific antibody" is meant to
represent either a polyclonal or a monoclonal antibody that binds
to a phosphorylated epitope in a HER receptor and/or a second
messenger molecule associated with HER mediated signal
transduction. For example the phosphorylated epitope may include at
least one phosphorylated serin-residue. In a preferred aspect of
the present invention the phosphorylated epitope may include at
least one phosphorylated tyrosine residue. In a particular
preferred embodiment of the present invention the phospho-tyrosine
residue is selected from the group consisting of Y1054, Y1197,
Y1199, Y1222, Y1224, Y1260, Y1262, Y1276, Y1289 and Y1328 in the
HER3 protein (numbering according to Kraus et al, PNAS 1989 (86)
9193-9197). The term also encompasses a phospho-specific
recombinant antibody, e.g. single chain antibody or a fragment
thereof, which contains at least one antigen-binding site, an
antibody fragment such as a Fab, Fab' or F(ab').sub.2 fragment or a
recombinant fragment such as a scFv fragment and a humanized
antibody or a human antibody directed against a phosphorylated
epitope in a HER receptor and/or a molecule associated with HER
mediated signal transduction.
[0054] Phospho-specific polyclonal antibodies can be obtained by
methods well known in the art. For example any animal, which is
known to produce antibodies can be immunized with a phospho-HER
receptor polypeptide. Antibody containing sera is isolated from the
immunized animal and is screened for the presence of antibodies
with the desired specificity using methods as for example, ELISA or
FACS.
[0055] Methods for the production of monoclonal antibodies produced
by the hybridoma method are first described by Kohler et al.,
Nature, 256:495 (1975). Monoclonal antibodies can also be produced
by recombinant DNA methods (see, for example, U.S. Pat. No.
4,816,567) or may be isolated from phage antibody libraries using
the techniques described in Clackson et al., Nature, 352:624-628
(1991) and Marks et al., J. Mol. Biol., 222:581-597 (1% I), for
example.
[0056] Humanized forms of the antibodies may be generated according
to the methods known in the art such as chimerization or CDR
grafting. The present invention also relates to a hybridoma or
recombinant cell line, which produces the above described
monoclonal antibodies or binding fragments thereof.
[0057] A disease which is responsive to treatment shows
statistically significant improvement in response to a HER
modulator treatment when compared to no treatment or treatment with
placebo in a recognized animal model or a human clinical trial. The
terms "treat" or treatment" refer to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) an undesired physiological change or
disorder, such as the development of a hyperproliferative disease,
e.g. cancer. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or s slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0058] The present invention provides a method of treating a
subject in need thereof, comprising determining expression and/or
activation of a HER receptor in said subject, and administering to
a subject in which HER receptor expression and/or activation has
been determined, a therapeutically effective amount of a HER
modulator and optionally at least one further agent. Preferably,
activation of the HER receptor is determined. More preferably, the
HER receptor is HER3.
[0059] Depending on the type of the HER modulator, type and
severity of the condition to be treated, about 0.01-10000 mg of the
HER modulator may be administered to a patient in need thereof,
e.g. by one or more separate administrations or by continuous
infusion. A typical daily dosage might range from about 0.001 mg/kg
to about 1000 mg/kg or more, depending on the factors mentioned
above. For repeated administrations over several days or longer,
depending on the condition to be treated, the treatment is
sustained until a desired suppression of disease symptoms
occurs.
[0060] The dose of the at least one antineoplastic agent
administered depends on a variety of factors. These are, for
example, the nature of the agent, the tumor type or the route of
administration. It should be emphasized that the present invention
is not limited to any dose.
[0061] Furthermore the present invention provides additional
methods and procedures to evaluate the therapeutic efficacy of a
HER modulator or a pharmaceutical composition comprising a HER
modulator and/or at least one further agent.
[0062] Determination of the pharmacodynamics of a modulator
targeting a HER receptor and/or a HER receptor mediated signaling
pathway may involve immunohistochemical staining with
phospho-specific antibodies of samples of diseased tissue, e.g.
tumor tissue, in order to quantitate the activation level of HER
receptors and/or related second messenger molecules.
[0063] Surprisingly, it was found that relevant pharmacodynamic
parameters, e.g. the activation level of a HER3 receptor, may also
be determined in primary, i.e. non-diseased normal tissue samples.
This allows to establish a rapid, quantitative, reproducible, and
inexpensive assay that is compatible with current clinical
laboratory instrumentation, wherein the presence of HER3
particularly in its activated form in primary human tissues may be
determined, e.g. by immunohistochemistry.
[0064] The results presented in the examples herein below
demonstrate that human tumor cells express HER3. Surprisingly, very
strong HER3 expression and/or activity was also detected in hair
follicles. Whereas the expression of total HER3 was located
predominantly in the cytoplasm, phosphorylated, i. e. activated
HER3 was almost exclusively associated with cell surface
membranes.
[0065] This finding supported the idea that the presence of
activated, e.g. phosphorylated, HER3 in such tissues could be used
for an easy and rapid determination of the efficacy of a HER
modulator when administered to a subject. For example at least
partially reduction of HER3 receptor activation indicates a
therapeutically effective amount of said modulator. Conversely no
difference in HER3 receptor activity upon treatment with a HER3
modulator correlates with ineffective therapeutic treatment. Thus
these findings can form the basis of a new and efficient method for
monitoring HER3 receptor directed therapy. Furthermore, hair
follicle biopsies could serve as a pharmacodynamic marker for
monitoring HER3 modulator directed treatment.
[0066] Accordingly the present invention provides a method for
determining the therapeutic efficacy of the treatment of a HER
receptor, particularly a HER3 receptor- associated disease with a
HER modulator and/or a further active agent comprising exposing a
subject to the HER modulator and/or the further active agent,
obtaining at least one sample from the subject, detecting the
activation level of the HER receptor in said sample wherein a
difference in the activation level of HER is observed as a result
of the exposure to the HER modulator and/or the further active
agent as compared to the absence of the exposure to the HER
modulator and/or the further active agent.
[0067] The term "sample" as embraced by the present invention
preferably means the use of a tissue sample for the detection of an
activated form of a HER family member or quantification of HER
receptor expression. The HER receptor is preferably HER3. The
activation level is preferably the degree of phosphorylation.
[0068] The term "tissue sample" is meant to include a collection of
cells obtained from a tissue of a subject or patient, preferably
containing nucleated cells with protein material. The four main
human tissues are (1) epithelium; (2) the connective tissues,
including blood vessels, bone and cartilage; (3) muscle tissue; and
(4) nerve tissue. The source of the tissue sample may be selected
from the group comprising of solid tissues as from a fresh, frozen
and/or preserved organ or tissue sample or biopsy or aspirate. The
present invention also includes the use of samples derived from
blood or any blood constituents, bodily fluids such as cerebral
spinal fluid, amniotic fluid, peritoneal fluid, or interstitial
fluid and cells from any time in gestation or development of the
subject. The tissue sample may also be primary or cultured cells or
cell lines. The tissue sample may contain compounds which are not
naturally intermixed with the tissue in nature such as
preservatives, anticoagulants, buffers, fixatives, nutrients,
antibiotics, or the like.
[0069] For use in the present invention the tissue sample is may be
a single part or piece of a tissue sample, e.g., a thin slice of
tissue or cells cut or micro-dissected from a tissue sample.
Generally, tissue arrays can be formalin-fixed tissue samples cut
into thin sections and mounted on silanised glass slides that can
be used for expression analysis and cellular localization on a
protein, RNA or DNA level. In a preferred embodiment at least 10
samples are mounted on one silanised glass slide. In a more
preferred embodiment at least 20 samples are mounted on one
silanised glass slide. In a most preferred embodiment 40 or more
samples are mounted on one silanised glass slide.
[0070] The tissue may be fixed (i.e. preserved) by conventional
methods known to one skilled in the art. In order to preserve
cellular morphology tissue can be fixed in 4% neutral buffered
formalin for 16-20 hours and embedded in paraffin.
[0071] In a preferred embodiment of the present invention the
tissue sample is a hair follicle sample which can be obtained by
using a punch biopsy procedure. Suitable areas to be biopsied are
the forearm, upper extremity and torso. The selected sites should
have visible hair growing.
[0072] The size of the biopsy can vary between 2 and 8 mm, whenever
possible a specimen with at least 3.5 mm diameter should be
harvested. The skin is cleansed and anesthetized. A small needle is
used to administer the anesthetic to limit discomfort. The lines of
least skin tension should be identified for the area to be
biopsied. For example, on the arm, these lines run perpendicular to
the long axis of the extremity. The incision line created by the
suturing after the biopsy is performed will be oriented parallel to
the lines of least skin tension. Physicians who cannot recall the
line orientation for a specific body area should consult the widely
published drawings of these lines. The skin is stretched
perpendicular to the lines of least skin tension. When the skin
relaxes after the biopsy is performed, an elliptical-shaped wound
remains that is oriented in the same direction as the lines of
least skin tension. On the arm, the skin is stretched along the
long axis of the extremity. The punch biopsy instrument is held
vertically over the skin and rotated downward using a twirling
motion. Once the instrument has penetrated the dermis into the
subcutaneous fat, or once the instrument reaches the hub, it is
removed. The cylindrical skin specimen is elevated with the
anesthesia needle. The use of forceps is discouraged because these
instruments may cause crush artifacts. The specimen is then cut
free from the subcutaneous tissues. The cut is made below the level
of the dermis. The wound is closed, if necessary, with one or two
interrupted nylon sutures: 5-0 nylon is used for most non-facial
areas, and 6-0 nylon for most facial areas. The skin specimen is
immediately transferred into buffer medium and processed further
for (protein) analysis.
[0073] In a particular preferred embodiment suitable areas for the
hair collection are the scalp (posterior neck region), the eyebrows
and the eyelashes. The number of individual hairs collected can
vary between 2 and 6, whenever possible at least 4 individual hair
(follicles) should be harvested. Without further anesthesia, the
hairs are pulled from the regions previously described. The hairs
are inspected for intactness of the shaft and follicle and the
suitable specimen will be individually mounted on slides for
further processing and protein analysis.
[0074] In order to preserve phospho-epitopes in fixed and
paraffin-embedded material, tissue samples have to be processed as
quickly as possible; i.e. as soon as the surgeon has removed the
biopsy material, it needs to be fixed/frozen and subsequently
processed. The fixation solutions to be used may depend on the
specific phospho-epitopes that are to be analyzed.
[0075] The term "therapeutic efficacy" refers to the amount of a
HER modulator and/or further agent effective to at least partially
block HER receptor activation. The therapeutically effective amount
shows beneficial or clinical results as mentioned before. In a
preferred embodiment, the therapeutically effective amount may
reduce the number of cancer cells, reduce the tumor size, inhibit
at least partially cancer cell infiltration into peripheral organs
and tumor metastasis, inhibit at least partially tumor growth
and/or relieve at least partially one or more of the symptoms
associated with the cancer.
[0076] Thus the present invention also provides a method for
determining the therapeutic efficacy of a HER modulator and/or a
further agent in a subject by using the HER receptor activation
level as a surrogate marker.
[0077] As used herein the term "subject" is meant to be an
individual or a patient, either treated or untreated with a HER
modulator or pharmaceutical composition comprising a HER modulator
and at least one further agent, for any purpose. The term "subject"
may also include animals, preferably mammals such as mouse, rat,
rabbit, dog, pig and nonhuman primates, e.g. cynomolgous monkey,
chimpanzee that are treated with a HER modulator. The term patient
refers to a human in need of a treatment with a HER modulator
and/or at least one further agent. Preferably the human is in need
of such a treatment to treat a hyperproliferative disease, e.g. any
neoplastic disease or cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0078] FIGS. 1a, b show basal phosphorylation of HER3 in tumor cell
lines;
[0079] FIG. 2a shows basal phosphorylation and expression of HER3
in breast;
[0080] FIG. 2b shows basal phosphorylation and expression of HER3
in lung cancer cell lines;
[0081] FIG. 2c shows basal phosphorylation and expression of HER3
in colon cancer cell lines;
[0082] FIG. 2d shows basal phosphorylation and expression of HER3
in pancreas cancer cell lines;
[0083] FIG. 2e shows basal phosphorylation and expression of HER3
in gastric cancer cell lines;
[0084] FIG. 2f shows basal phosphorylation and expression of HER3
in melanoma cancer cell lines;
[0085] FIG. 2g shows basal phosphorylation and expression of HER3
in prostate cancer cell lines;
[0086] FIG. 3: Correlation between HER3 and pHER3 expression in
vitro in all cell lines analysed;
[0087] FIG. 4a shows basal phosphorylation and expression of HER3
in selected cancer cell lines;
[0088] FIG. 4b: Correlation between pHER3 expression and
sensitivity to anti-HER3 treatment;
[0089] FIG. 5a: HER3 expression in human hair follicles.
Immunostaining and peroxidase detection of human hair follicles
using monoclonal HER3 antibody;
[0090] FIG. 5b: HER3 phosphorylation in human hair follicles.
Immunostaining and peroxidase detection of pHER3 human hair
follicles using monoclonal antibody 21D3 showing high levels of
membranous HER3 phosphorylation.
[0091] FIG. 6: HER3 phosphorylation in human normal tissues.
Immunostaining and peroxidase detection of pHER3 human normal
tissue using monoclonal antibody 21D3 showing high levels of
membranous HER3 phosphorylation. Shown is GI tract (left), testis
(middle) and epithelium of the bladder (right).
[0092] FIG. 7: Immunohistochemical staining with rabbit monoclonal
anti-pHer3 antibody (Cell Signalling 21D3, Lot 4 1:650 dilution,
0.074 ug/ml) on FFPE sections of BxPC3 xenografts, 20.times..
[0093] (A) and (B) Tumour after administration of control IgG1 500
.mu.g/mouse
[0094] (C) and (D) Tumour after administration of antibody U3-1287
500 .mu.g/mouse
[0095] Stainings were done in duplicate on three independent
xenografts.
[0096] FIG. 8: Immunohistochemical staining with rabbit monoclonal
anti-pHer3 antibody (Cell Signalling 21D3, Lot 4 1:650 dilution,
0.074 ug/ml) on FFPE sections of BxPC3 xenografts, 20.times..
[0097] (A) Tumour after administration of antibody U3-1287 25
.mu.g/mouse [0098] (B) Tumour after administration of antibody
U3-1287 100 .mu.g/mouse [0099] (C) Tumour after administration of
antibody U3-1287 200 .mu.g/mouse [0100] (D) Tumour after
administration of antibody U3-1287 500 .mu.g/mouse [0101] (E)
Tumour after administration of control IgG1 500 .mu.g/mouse.
[0102] Stainings were done in duplicate on three independent
xenografts.
[0103] FIG. 9: Immunohistochemical staining with mouse monoclonal
anti-Her3 antibody (Dako-H3-IC, 1:250 dilution, 0.52 ug/ml) on FFPE
sections of BxPC3 xenografts, 20.times..
[0104] (A) and (B) Tumour after administration of control IgG1 500
.mu.g/mouse
[0105] (C) and (D) Tumour after administration of antibody U3-1287
500 .mu.g/mouse
[0106] Stainings were done in duplicate on three independent
xenografts.
[0107] FIG. 10: Immunohistochemical staining with mouse monoclonal
anti-Her3 antibody (Dako-H3-IC, 1:250 dilution, 0.52 ug/ml) on FFPE
sections of BxPC3 xenografts, 20.times.. [0108] (A) Tumour after
administration of antibody U3-1287 25 .mu.g/mouse [0109] (B) Tumour
after administration of antibody U3-1287 100 .mu.g/mouse [0110] (C)
Tumour after administration of antibody U3-1287 200 .mu.g/mouse
[0111] (D) Tumour after administration of antibody U3-1287 500
.mu.g/mouse [0112] (E) Tumour after administration of control IgG1
500 .mu.g/mouse
[0113] Stainings were done in duplicate on three independent
xenografts.
[0114] FIG. 11: Immunohistochemical staining with rabbit monoclonal
anti pHer3 antibody (Cell Signalling 21D3, Lot 4 1:8000 dilution,
0.006 ug/ml) on FFPE sections Calu-3 xenografts, 40.times..
[0115] (A) and (B) Tumour after administration of control IgG1 25
mg/kg 72 h
[0116] (C) and (D) Tumour after administration of antibody U3-1287
25 mg/kg 72 h
[0117] Stainings were done in duplicate on five independent
xenografts.
EXAMPLES
[0118] The detection of basal phosphorylation of HER3 was conceived
to underlie autocrine receptor activation and represent a selection
marker for potentially suitable models in the use of HER3-directed
therapeutic intervention. To this end, several cell lines were
chosen and analysed for their phospho-HER3 content in the presence
or absence of serum. An initial experiment showed that the
pancreatic tumor cell line Bx-PC3 contains high levels of basally
phosphorylated, i. e. activated HER3 in serum-starved and unstarved
cells, indicating that Bx-PC3 may be a suitable model for an
anti-HER3 therapeutic approach (FIG. 1a).
[0119] Additional experiments confirmed the finding in Bx-PC3 cells
and extended the observation of basal HER3 phosphorylation to A549
and A431 cells (FIG. 1b).
[0120] Subsequently, based on these findings more cell lines were
analyzed systematically and extended to tumor cell lines of 7
different cancer indications (breast, lung, colon, pancreas,
prostate, gastric, melanoma) (FIG. 2a-g).
[0121] Overall phosphorylated, i. e. activated HER3 was detected in
approx. 2/3 of the examined tumor cell lines. No significant
difference between serum and serum-starved phosphorylation could be
detected (FIG. 3a, b).
[0122] The hypothesis that the presence of phosphorylated HER3 in
tumor cell lines in vitro implies and predicts responsiveness to
HER3-directed intervention was tested in subsequent in vivo studies
using cell lines such as Bx-PC3, HT-144, and T47D among others.
From these studies, in vivo efficacy was correlated with pHER3
expression in vitro, suggesting that activated HER3 would serve as
a surrogate marker for therapy (FIG. 4a, b).
[0123] In order to apply the results obtained from in vitro western
blot analysis and in vivo animal xenograft experiments to a
therapeutically relevant scenario, we investigated the presence of
HER3 and its activated form in primary human tissues by
immunohistochemistry. Expression of HER3 was detected in a variety
of tumor samples, including a prominent presence in melanoma. In
contrast, HER3 expression was not detected in normal skin,
but--surprisingly--was very strong in hair follicles (FIG. 5a,
b).
[0124] Whereas the expression of total HER3 was located
predominantly in the cytoplasm, phosphorylated, i. e. activated
HER3 was almost exclusively associated with cell surface membranes.
This finding supported the idea that the presence of phosphorylated
HER3 in such tissues could be used for selecting tumor patients
responsive to anti-HER3 therapy. Furthermore, as well as monitoring
HER3-directed therapy hair follicle biopsies could serve as a
pharmacodynamic marker for monitoring HER3-directed treatment.
Activated HER3 was also detected in a number of additional normal
human tissues, including the GI tract, testis and bladder (FIG.
6).
[0125] A reduction of membrane staining intensity, a reduction of
tumour cells compared to whole cell number in the tumour and a
reduction of pHer3 positive cells compared to whole cell number in
the tumour was found after administration of anti-HER3 antibody
(FIGS. 7, 8 and 11).
[0126] Reduction of staining intensity and reduction of Her3
positive cells correlates with reduction in tumour volume (FIGS. 9
and 10).
[0127] The role of HER3 in normal skin has not been characterized
previously. RNA expression was previously detected in postnatal
skin (Kraus et al, 1989) Thus, our present analysis represents the
first description in this respect. Surprisingly, we found that HER3
and its activated form are expressed in the hair follicles and in
cells of the eccrine and sebaceous glands. This was not expected
since the preferred partner of HER3, HER2, has not been reported to
be expressed in these tissues This opens up the use of activated
HER3 for patients selection etc. In contrast to activated EGFR,
activated HER3 is not located intracellularly, but predominantly
membranous. Expression of (activated) HER3 was also not observed in
normal keratinocytes, where expression of EGFR is widespread
(Expression of HER3 is rather low in keratinocytes (Laux et al,
2006). Thus, use of HER3 for diagnosis/selection and therapy may
not only provide a regimen with less severe side effects compared
to EGFR therapy which causes prominent skin rash, but may prove to
be very useful for the monitoring of combination therapy.
[0128] HER3 Phosphorylation in Tumor Cell Lines
[0129] Cells were seeded in 6-well dishes overnight, serum-starved
or cultivated with 10% FCS-containing growth medium for 24 hours
and treated with lysis buffer for 20 minutes. Lysate was cleared by
centrifugation for 30 min and HER3 was immunoprecipitated from
crude lysate with a specific anti-HER3 monoclonal antibody (1B4C3).
Immunoprecipitates were incubated for 4 hours at 4.degree. C.,
washed three times with 1.times. HNTG (50 mM Hepes pH 7.5, 150 mM
NaCl, 10% Glycerine, 1 mM EDTA pH 8.0, 0.1% Triton X-100) and
denatured with 3.times. Laemmli buffer containing b-mercaptoethanol
for 5 min at 100.degree. C. The protein samples were separated by
7.5% SDS-PAGE, transferred to nitrocellulose membrane and incubated
with anti-phosphotyrosine (4G10) or anti-pHER3 (21D3).
Phosphoproteins were detected with anti-mouse-POD (for 4G10) or
anti-rabbit-POD (for 21D3) secondary antibodies. The membranes were
stripped and reprobed with anti-HER3 antibody (sc-285).
[0130] HER3 Phosphorylation in Tissue Samples
[0131] Using a microtome, 2-4 .mu.m thin sections were cut, mounted
on silanized glass slides and dried at 60.degree. C. for 30 minutes
and at 38.degree. C. overnight. Deparaffinisation and rehydration
of the specimen was achieved by incubating 2.times.5 minutes in
Xylol, 2.times.2 minutes in 100% ethanol and 2 minutes each in 96%,
80% and 70% ethanol. After rinsing 20 seconds in distilled water,
the slides were incubated for two minutes in PBS. For antigen
retrieval the specimens were incubated in a steamer, containing a
cuvette filled with 1 mM EDTA pH 8.0 at 96-98.degree. C. for 20
minutes. The slides were cooled down for 20 min at RT, then washed
5 minutes in A. dest. Except for incubation with primary antibody
pHer3, the following steps were performed at room temperature:
phosphotyrosine Endogenous peroxidases were blocked for 20 minutes
in RE7101 (3 drops per section, Novocastra). Sections were then
washed 5 minutes in A. dest. and 5 minutes in TBS buffer.
Unspecific background staining was blocked by incubation with 10%
goat serum in PBS for 20 minutes. Solution was tapped off and
sections were incubated with monoclonal antibody rabbit-anti-pHer3
(10 .mu.g/ml (Lot #3), Cell Signaling) overnight at 4.degree. C. in
a humidified chamber (1:40 in Dako dilution buffer). As IgG isotype
control IgG rabbit absorbed (15 g/L, X0936 Dako) was used (1:50.000
in Dako dilution buffer). To remove the antibody, the slides were
washed 2.times.5 minutes with TBS/TWEEN 0.05% and 1.times.5 minutes
with TBS. Post Primary Block (RE7111, Novocastra) was added (3
drops per sections) for 30 minutes, followed by washing as before.
Then NovoLink Polymer RE7112 (3 drops per section, Novocastra) were
added, incubated for 30 minutes and removed in a washing step as
before. Staining was achieved by incubation with 100 .mu.l
DAB-substrate-chromogen-solution for 10 minutes. In a last step,
the slides were rinsed three times in fresh distilled water,
counterstained with Harris' hematoxylin and covered with a glass
slide.
[0132] Xenograft Experiments
[0133] The anti-tumor efficacy of a HER modulator were evaluated in
human xenograft tumor studies. In these studies, human tumors were
grown as xenografts in immunocompromised mice and therapeutic
efficacy was measured by the degree of tumor growth inhibition in
response to administrations of the HER modulator. In order to
determine, whether a HER modulator, as defined in forgoing
paragraphs, at least partially interferes with tumor growth of
human cancer cells in vivo, cells were implanted in nude/nude or
SCID mice, using protocols known to the skilled artisan (Sausville
and Burger, (2006), Cancer Res. 66, 3351-3354). For example tumor
cells were injected under the skin of nude mice, resulting in
subcutaneous tumor growth on the back of the animals. Treatment was
either started at the time of tumor cell implantation or when
tumors had reached a defined size, e.g. a mean volume of 20-50
mm.sup.3. Prior to first treatment, mice were randomized to assure
uniform tumor volumes (mean, median and standard deviation) across
treatment groups. Typical dosing regimen included weekly
administrations of 25 mg/kg of the HER modulator into the
interpeneum. The first treatment included a loading dose of 50
mg/kg. Mice in control arms received agents, e.g. doxorubicin
(pharmaceutical grade) with known cytostatic or cytotoxic activity
against the human tumor cells.
[0134] Detection of HER3 Phosphorylation in Human Patient
Tissues
[0135] For the selection of patients amenable for an anti-HER3 mAb
treatment, the HER3 receptor activation will be measured via IHC in
cellular samples (tumor material at time of diagnosis, fresh tumor
material prior to treatment, normal tissue) derived from a patient
deemed to be a candidate for an anti-HER3 mAb treatment. The
cellular sample will be achieved through various methods of
biopsies (e.g. punch, brush, incisional, core) or other methods
(e.g. plucking of hair and air follicles, buccal swab). The
harvested tissue material will be processed, fixed and analyzed for
presence of pHER3 (qualitative assay) and the relative amount of
pHER3 (quantitative assay) via immunohistochemistry or other
applicable methods (e.g. rtPCR, WB). An activation score for pHER3
will be calculated and the subject will be enrolled in the clinical
study/treatment routine accordingly.
[0136] Assessment of the Efficacy of a HER3 Inhibitor
[0137] The efficacy of an anti HER3 antibody in reducing HER3
receptor activation and/or HER3 mediated signal transduction can be
assessed in cellular samples derived from a subject that has been
treated with said anti HER3 antibody. The cellular samples can be
retrieved in the previously described way, the timing of the
samples is dependent on the treatment duration, schedule and follow
up of therapy, but at least 2 samples will be taken (one at
treatment start and one at maximum response). The quantitative and
qualitative measurements for the 2 time points will be compared and
the pharmacodynamic effect will be calculated from the delta/shift
of values for the HER3 receptor activation. Normal tissue (e.g.
skin, hair follicles) will serve as surrogate tissue for the tumor
tissue, since the normal tissue may be easier accessible for the
clinical routine diagnostic.
[0138] Development of Prognostic Index for Subjects Amenable to
Anti-HER3 mAb Therapy
[0139] For patients that have received an anti-HER3 mAb treatment,
the outcome of the treatment will be correlated with the level of
HER3 phosphorylation and the modulation of the
phosphorylation/activation over time. The resulting prognostic
index will be compared with standard indices (e.g. tumor grade,
stage, patient demographics, treatment) and it will be determined
whether pHER3 can serve as a superior marker for efficacy of the
treatment, prognostic index for outcome, variabilities in response
to the treatment or recurrence of the disease. Ultimately HER3
phosphorylation may become a new surrogate marker for the
assessment of a risk-benefit score or a positive/negative prognosis
with respect to anti-HER3 mAb therapy and other targeted or
classical antineoplastic therapies.
[0140] Clinical Study to Identify Cancer Patients for Treatment
with an Anti HER3 Antibody
[0141] A cellular sample comprising normal and/or cancer cells is
obtained from a subject deemed eligible for the treatment. The
following methods are used in routine clinical practice to retrieve
a tissue sample: swab (buccal, nasal swab), cuts (finger nails, toe
nails), fine needle aspiration, punch biopsy, brush biopsy, scratch
biopsy, biopsy using pincers or other surgical instruments,
aspiration (e.g. blood, bone marrow), puncture (e.g. ascites,
pleural effusion, cerebrospinal fluid), (micro-derm) abrasive
cytology, incision, surgical removal of organ parts or whole
anatomical structures (bloc resection, tumor excision, lumpectomy),
radiation assisted surgical procedure (gamma-knife surgery, laser
assisted surgery), lavage (e.g. broncho-alveolar lavage, abdominal
lavage), external drainage of organs (e.g. hydrocephalus,
nephrostomy, T-drain bile duct). Any other method known in clinical
practice for harvesting of tissue samples can be used as well. The
biological sample is analyzed for HER3 phosphorylation, e.g., by
immunoprecipitation or Western blot analysis, and/or for the
presence of HER2/HER3 and/or HER3/HER4 heterodimers by any of the
techniques described above.
[0142] Clinical Study to Monitor Efficacy of Treatment with a HER3
Modulator
[0143] Patients with solid tumors (e.g. lung, colorectal, breast
cancer) will undergo at least 2 biopsies for the assessment of the
pharmacodynamic effects of an anti-HER3 mAb treatment evaluated
through changes/modulations in the HER3 phosphorylation. At study
entry, patients will be stratified for the pHER3 level and at the
time of maximum clinical response, a second tissue sample will be
taken from the patient. The samples will be analyzed for pHER3
expression (quantitative and qualitative) and the results are
correlated with other parameters and clinical outcome. A rise in
pHER3 activation may be considered as progression or non-response,
whereas a decrease of pHER3 may be considered response to therapy.
Patients with at least a stabilization of pHER3 levels (increase
.ltoreq.25% from baseline) will continue on treatment with
anti-HER3 mAb therapy, patients with an increase of pHER3>25%
from baseline will be considered as progressive and treatment with
anti-HER3 mAb therapy will be discontinued.
* * * * *