U.S. patent application number 14/181307 was filed with the patent office on 2014-06-19 for monoclonal and oligoclonal anti-egfr antibodies for use in the treatment of tumors expressing predominantly high affinity egfr ligands or tumors expressing predominantly low affinity egfr ligands.
This patent application is currently assigned to Merrimack Pharmaceuticals, Inc.. The applicant listed for this patent is Merrimack Pharmaceuticals, Inc.. Invention is credited to Raghida BUKHALID, Jeffrey David KEARNS, Ulrik NIELSEN, Shannon WERNER.
Application Number | 20140170668 14/181307 |
Document ID | / |
Family ID | 46682898 |
Filed Date | 2014-06-19 |
United States Patent
Application |
20140170668 |
Kind Code |
A1 |
BUKHALID; Raghida ; et
al. |
June 19, 2014 |
MONOCLONAL AND OLIGOCLONAL ANTI-EGFR ANTIBODIES FOR USE IN THE
TREATMENT OF TUMORS EXPRESSING PREDOMINANTLY HIGH AFFINITY EGFR
LIGANDS OR TUMORS EXPRESSING PREDOMINANTLY LOW AFFINITY EGFR
LIGANDS
Abstract
Disclosed are pharmaceutical preparations for, and methods for
determining, appropriate and effective treatment with therapeutic
agents comprising a single species of anti-EGFR monoclonal antibody
or therapeutic agents comprising a plurality of species of such
antibodies, as well as kits useful for making such
determinations.
Inventors: |
BUKHALID; Raghida; (Melrose,
MA) ; NIELSEN; Ulrik; (Quincy, MA) ; WERNER;
Shannon; (Belmont, MA) ; KEARNS; Jeffrey David;
(Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merrimack Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Merrimack Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
46682898 |
Appl. No.: |
14/181307 |
Filed: |
February 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13488270 |
Jun 4, 2012 |
8691231 |
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14181307 |
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61558945 |
Nov 11, 2011 |
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61504633 |
Jul 5, 2011 |
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61493252 |
Jun 3, 2011 |
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Current U.S.
Class: |
435/6.12 ;
530/387.7 |
Current CPC
Class: |
C07K 16/2863 20130101;
A61P 13/08 20180101; C07K 2317/24 20130101; G01N 33/74 20130101;
A61K 2039/507 20130101; G01N 33/57484 20130101; A61K 2039/505
20130101; C07K 2317/21 20130101; C07K 2317/34 20130101; C07K
2317/56 20130101; A61P 43/00 20180101; C07K 2317/565 20130101; A61P
35/00 20180101; C07K 16/30 20130101; C07K 2317/92 20130101; C07K
14/71 20130101; A61K 9/0019 20130101; C12Q 1/686 20130101; C12Q
2600/106 20130101; A61P 11/00 20180101; C07K 16/32 20130101; C12Q
1/6886 20130101; C07K 2317/55 20130101; G01N 33/57496 20130101;
G01N 2800/52 20130101; A61K 39/39558 20130101; A61K 39/3955
20130101; C07K 2317/76 20130101; G01N 2333/485 20130101; G01N
2333/495 20130101 |
Class at
Publication: |
435/6.12 ;
530/387.7 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 16/32 20060101 C07K016/32 |
Claims
1. A kit for testing a tumor sample to determine levels of both
high and low affinity EGFR ligands in the sample, said kit
comprising a container comprising; a) at least two pairs of high
affinity EGFR ligand-specific polymerase chain reaction (PCR)
primers, b) at least two pairs of low affinity EGFR ligand-specific
PCR primers, and c) at least one reverse transcription PCR (RT-PCR)
reagent.
2. The kit of claim 1, wherein the at least two pairs of high
affinity EGFR ligand-specific polymerase chain reaction primers are
specific to at least two of betacellulin, EGF, HB-EGF or TGF alpha
and each of the at least two pairs of low affinity EGFR
ligand-specific polymerase chain reaction primers are specific to
at least two of amphiregulin, epigen, or epiregulin.
3. The kit of claim 2, wherein the at least two pairs of high
affinity EGFR ligand-specific primers comprise specific primer
pairs for betacellulin, EGF, HB-EGF and TGF alpha and the at least
two pairs of high affinity EGFR ligand-specific primers comprise
specific primer pairs for all of amphiregulin, epigen, and
epiregulin.
4. The kit of claim 2, further comprising at least one fluorescent
reporter molecule.
5. The kit of claim 1, wherein the at least one RT-PCR reagent is
one or more of an RNA-dependent DNA polymerase, a DNA-dependent DNA
polymerase, or a solution comprising at least a micromolar
concentration of each of ATP, GTP, CTP and TTP.
6. The kit of claim 2, wherein the at least one RT-PCR reagent is
one or more of an RNA-dependent DNA polymerase, a DNA-dependent DNA
polymerase, or a solution comprising at least a micromolar
concentration of each of ATP, GTP, CTP and TTP.
7. The kit of claim 5 wherein the primers, the reagents, or both,
have been prepared under current Good Manufacturing Practice (cGMP)
conditions.
8. The kit of claim 5, wherein container further comprises total
RNA isolated from a tumor sample.
9. The kit of claim 8, wherein the tumor sample from which the RNA
is isolated is a fresh, fixed, frozen, or formalin fixed paraffin
embedded (FFPE) sample.
10. The kit of claim 9, wherein the tumor sample is a circulating
tumor cell sample.
11. The kit of claim 9, wherein the tumor sample is a biopsy
sample.
12. The kit of claim 9, wherein the tumor sample is from a tumor of
the skin, central nervous system, head, neck, esophagus, stomach,
colon, rectum, anus, liver, pancreas, bile duct, gallbladder, lung,
breast, ovary, uterus, cervix, vagina, testis, germ cells,
prostate, kidney, ureter, urinary bladder, adrenal, pituitary,
thyroid, bone, muscle or connective tissue.
13. A kit for testing a tumor sample to determine levels of both
high and low affinity EGFR ligands in the sample, said kit
comprising one or more containers, one of said containers
comprising; a) at least two pairs of high affinity EGFR
ligand-specific polymerase chain reaction (PCR) primers, b) at
least two pairs of low affinity EGFR ligand-specific PCR primers,
c) at least one reverse transcription PCR (RT-PCR) reagent, and d)
RNA isolated from a tumor sample.
14. The kit of claim 13, wherein the container comprising a), b),
c), and d) is a 96 well plate.
15. The kit of claim 13, wherein the tumor sample from which the
RNA is isolated is a fresh, fixed, frozen, or formalin fixed
paraffin embedded (FFPE) sample.
16. The kit of claim 15, wherein the tumor sample is a circulating
tumor cell sample.
17. The kit of claim 16, wherein the tumor sample is a biopsy
sample.
18. The kit of claim 15, wherein the tumor sample was from a tumor
of the skin, central nervous system, head, neck, esophagus,
stomach, colon, rectum, anus, liver, pancreas, bile duct,
gallbladder, lung, breast, ovary, uterus, cervix, vagina, testis,
germ cells, prostate, kidney, ureter, urinary bladder, adrenal,
pituitary, thyroid, bone, muscle or connective tissue.
19. The kit of claim 15, wherein the container comprising a), b),
c), and d) is a 96 well plate
20. The kit of claim 18, wherein the container comprising a), b),
c), and d) is a 96 well plate.
21. A monoclonal anti-EGFR antibody preparation comprising a single
species of monoclonal antibody for treatment of a patient predicted
by a method to have a favorable outcome from treatment with the
monoclonal anti-EGFR antibody preparation by determining whether a
patient having a tumor is predicted to have a favorable outcome,
comprising reduction of growth of the tumor, as a result of
treatment #1 with a monoclonal anti-EGFR antibody preparation
comprising a single species of monoclonal antibody, and as a result
of treatment #2 with an oligoclonal anti-EGFR antibody preparation
comprising a plurality of species of monoclonal anti-EGFR
antibodies, one against each of at least two extracellular epitopes
of EGFR, or, whether the patient is predicted to have the favorable
outcome as a result of treatment #2 but not as a result of
treatment #1; the method comprising: obtaining a biopsy sample of
the tumor and: a) measuring levels of protein or mRNAs coding for
at least two low affinity EGFR ligands selected from amphiregulin,
epigen, or epiregulin in the biopsy sample, b) measuring levels of
protein or mRNAs coding for at least two high affinity EGFR ligands
selected from betacellulin, EGF, HB-EGF or TGF.alpha. in the biopsy
sample, and c) comparing the average level of protein or mRNAs
coding for each of the high affinity EGFR ligands measured in a) to
the average level of protein or mRNAs coding for each of the low
affinity EGFR ligands measured in b); wherein, if the average level
of protein or mRNAs coding for low affinity EGFR ligands measured
in a) is greater than the average level of protein or mRNAs coding
for high affinity EGFR ligands measured in b), the patient is
predicted to have the favorable outcome as a result of treatment #1
and the patient is also predicted to have the favorable outcome as
a result of treatment #2, and if the average level of protein or
mRNAs coding for low affinity EGFR ligands measured in a) is less
than or equal to the average level of protein or mRNAs coding for
high affinity EGFR ligands measured in b), the patient is predicted
to have an unfavorable outcome from treatment #1 and is predicted
to have a favorable outcome from treatment #2.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/488,270, filed on Jun. 4, 2012, which claims the
benefit of U.S. Provisional Application No. 61/493,252, filed on
Jun. 3, 2011, U.S. Provisional Application No. 61/504,633, filed on
Jul. 5, 2011, and U.S. Provisional Application No. 61/558,945,
filed on Nov. 11, 2011, the entire contents of which are
incorporated by reference herein.
BACKGROUND
[0002] The epidermal growth factor receptor (EGFR) is a cell
surface transmembrane receptor of the HER/ErbB receptor family that
transmits signals (including mitogenic signals that drive cell
proliferation) to the interior of a cell when activated, typically
by the binding of any of a number of extracellular ligands such as
epidermal growth factor (EGF). EGFR ligands vary in their affinity
for EGFR and are categorized as either high- or low-affinity
ligands. It is thought that the high- and low-affinity interactions
between EGFR and its ligands activate different signaling pathways.
This signal transmission occurs through a cascade of intracellular
events beginning with protein phosphorylation mediated by receptor
tyrosine kinase activity. EGFR has proven a responsive target for
anti-proliferative (e.g., anti-cancer) drugs, including "small
molecule" tyrosine kinase inhibitor drugs (typically no larger than
700-900 AMU) that may be orally administered as well as monoclonal
antibody based drugs that specifically bind to the extracellular
domain of EGFR. EGFR-targeted monoclonal antibodies are not always
effective against EGFR-expressing tumors. One approach taken with
the aim of improving anti-EGFR antibody efficacy has been to
develop mixtures of anti-EGFR monoclonal antibodies (i.e.,
oligoclonal antibodies) targeted to different sites (epitopes) on
to the extracellular domain of EGFR. See, e.g., PCT Int. Pub. No.
WO/2011/140254 and U.S. Pat. No. 7,887,805. These developments have
created a need to enable the identification of cancer patients
whose tumors have characteristics rendering them unresponsive to
monoclonal anti-EGFR antibodies so that such patients may receive
effective treatment via administration of oligoclonal anti-EGFR
antibodies. The present disclosure answers this need and provides
other benefits.
SUMMARY
[0003] Provided herein are theranostic methods for predicting
responsiveness of tumor cells that express EGFR to therapeutic
agents comprising anti-EGFR antibodies, and methods based on such
predictions for treating patients having such tumors with such
therapeutic agents. Anti-EGFR antibodies such as those monoclonal
and oligoclonal antibodies described in PCT Int. Pub. No.
WO/2011/140254 and corresponding pending U.S. patent application
Ser. No. 13/100,920, in pending U.S. provisional patent
applications Nos. 61/504,633 and 61/558,945, and in U.S. Pat. No.
7,887,805 (the "Oligoclonal Applications"), as well as oligoclonal
mixtures of such antibodies in combination with other anti-EGFR
antibodies, are useful for treatment of cancers, e.g., malignant
(neoplastic) tumors. Examples of cancers include but are not
limited to, carcinoma, adenoma, blastoma, sarcoma, and lymphoma.
More particular examples of such cancers include squamous cell
cancer, small-cell lung cancer, non-small cell lung cancer, gastric
cancer, pancreatic cancer, glial cell tumors such as glioblastoma
and neurofibromatosis, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
melanoma, colorectal cancer, endometrial carcinoma, salivary gland
carcinoma, kidney cancer, renal cancer, prostate cancer, vulvar
cancer, thyroid cancer, hepatic carcinoma and various types of head
and neck cancer. In a particular embodiment, a cancer treated or
diagnosed using the methods disclosed herein is selected from
melanoma, breast cancer, ovarian cancer, renal carcinoma,
gastrointestinal/colon cancer, lung cancer (e.g., NSCLC), and
prostate cancer.
[0004] Accordingly, theranostic methods predicting which tumors
(e.g., malignant tumors) will be responsive to treatment with
oligoclonal anti-EGFR antibodies, but not responsive to treatment
with single monoclonal anti-EGFR antibodies, are provided. Also
provided are kits useful for predicting responsiveness of tumors to
anti-EGFR monoclonal and oligoclonal antibodies and methods of use
thereof.
[0005] In one aspect, a preparation comprising only a single
species of anti-EGFR monoclonal antibody (as opposed to
preparations comprising mixtures of monoclonal antibodies) is
provided for use in treating a patient having a tumor (e.g., a
malignant tumor) that is predicted to have a favorable outcome (as
described below) as a result of treatment #1, i.e., treatment with
the preparation comprising only a single species of monoclonal
antibody, and as a result of treatment #2, i.e., treatment with an
oligoclonal anti-EGFR antibody preparation comprising a plurality
of species of monoclonal anti-EGFR antibodies, one against each of
at least two extracellular epitopes of EGFR, one of which antibody
against at least two extracellular epitopes of EGFR being an
antibody that inhibits ligand binding to EGFR. In another aspect, a
preparation comprising a plurality of species of monoclonal
anti-EGFR antibodies is provided for use in treating a patient
having a tumor that is predicted to have a favorable outcome as a
result of treatment #2, but not as a result of treatment #1.
[0006] Identification of tumors predicted to respond to treatment
#1 and treatment #2, or to treatment #2, but not to treatment #1,
comprises obtaining a biopsy sample of the tumor and: [0007] a)
measuring levels in the sample of at least two (e.g., at least
three or at least four) low affinity EGFR ligands, e.g., selected
from amphiregulin, epigen, or epiregulin, which may, for each
ligand independently, be measured as levels of ligand protein or as
levels of the corresponding RNA species coding for the ligand
protein, in the biopsy sample, [0008] b) measuring levels in the
sample of at least two (e.g., at least three) high affinity EGFR
ligands, e.g., selected from betacellulin, EGF, HB-EGF or
TGF.alpha., which may, for each ligand independently, be measured
as protein levels or as levels of the corresponding RNA species
coding for them, in the biopsy sample, [0009] in one embodiment,
all of the levels measured in a) are protein levels and all of the
levels measured in b) are protein levels; in another embodiment all
of the levels measured in a) are mRNA levels and all of the levels
measured in b) are mRNA levels. [0010] and, [0011] c) comparing the
average level of each of the high affinity EGFR ligands, or of each
corresponding RNA species, measured in a) to the average level of
each of the low affinity EGFR ligands, or of each corresponding RNA
species measured in b) where if the average level of low affinity
EGFR ligands, or RNAs coding for them, measured in a) is greater
than the average level of high affinity EGFR ligands, or RNAs
coding for them, measured in b), the patient is predicted to have
the favorable outcome as a result of treatment #1, as well as as a
result of treatment #2, and if the average level of low affinity
EGFR ligands, or RNAs coding for them, measured in a) is less than
or equal to the average level of high affinity EGFR ligands, or
RNAs coding for them, measured in b), the patient is predicted to
have an unfavorable outcome from treatment #1, but is predicted to
have a favorable outcome from treatment #2.
[0012] In one embodiment, the at least two low affinity EGFR
ligands is at least three low affinity ligands and the at least two
high affinity EGFR ligands is three high affinity ligands. In
another embodiment, the at least two low affinity EGFR ligands is
four low affinity ligands and the at least two high affinity EGFR
ligands is three high affinity ligands. In another embodiment, the
at least two high affinity EGFR ligands is three high affinity
ligands. In another embodiment, the at least two low affinity EGFR
ligands is four low affinity ligands and the at least two high
affinity EGFR ligands is at least two high affinity ligands.
[0013] In one embodiment, a monoclonal antibody preparation
comprising only a single species of monoclonal antibody is provided
for use in the treatment of a patient predicted to have a favorable
outcome from treatment with either of the monoclonal anti-EGFR
antibody preparation or the oligoclonal anti-EGFR antibody
preparation and the patient is subsequently treated with the
monoclonal preparation. In another embodiment, this patient is
subsequently treated with the oligoclonal anti-EGFR antibody
preparation.
[0014] In yet another embodiment, if the patient is predicted to
have an unfavorable outcome from treatment with the monoclonal
anti-EGFR antibody preparation, the patient is subsequently treated
with the oligoclonal anti-EGFR antibody preparation.
[0015] The monoclonal anti-EGFR antibody preparation comprises a
single monoclonal antibody that may be selected from, e.g.,
cetuximab, zalutumumab, nimotuzumab, matuzumab and panitumumab, all
of which block ligand binding to EGFR, and therefore bind to the
same or overlapping EGFRepitopes (Bin 1) and are not suitable for
use together in oligoclonal antibody preparations. Members of the
plurality of anti-EGFR antibody species in an oligoclonal
preparation separately and uniquely bind to two different
extracellular epitopes of EGFR, may separately and uniquely bind to
at least three extracellular epitopes of EGFR (in some cases no
more than three). Such a plurality may comprise two or three
different species of monoclonal anti-EGFR antibodies, and in some
embodiments no more than three different species. Mixtures
comprising more than one antibody against any one epitope of EGFR
are less preferred. In certain embodiments the oligoclonal
preparations are duos, trios, or fourfold combinations of
antibodies as disclosed in the Oligoclonal Applications. In other
embodiments, the oligoclonal preparations provided herein comprise
one or more of the anti-EGFR antibodies (e.g., of the above duos or
trios or other oligoclonal combinations) that are not Bin 1
antibodies in combination with one of cetuximab, zalutumumab,
nimotuzumab, matuzumab and panitumumab (which are all Bin 1
antibodies).
[0016] In another embodiment, if the patient is predicted to have
an unfavorable outcome from treatment with the monoclonal anti-EGFR
antibody preparation, the patient is subsequently treated with
combination therapy comprising separate administration of at least
two different monoclonal anti-EGFR antibodies. In certain aspects,
the at least two different monoclonal anti-EGFR antibodies are
selected from any of the anti-EGFR antibodies disclosed in the
Oligoclonal Applications, as well as from cetuximab, zalutumumab,
nimotuzumab, matuzumab and panitumumab, provided that one of the
anti-EGFR antibodies is an antibody that inhibits ligand binding to
EGFR.
[0017] In another embodiment the tumor is a tumor of the skin,
central nervous system, head, neck, esophagus, stomach, colon,
rectum, anus, liver, pancreas, bile duct, gallbladder, lung,
breast, ovary, uterus, cervix, vagina, testis, germ cells,
prostate, kidney, ureter, urinary bladder, adrenal, pituitary,
thyroid, bone, muscle or connective tissue.
[0018] In another aspect, a method of treating a tumor in a patient
by administration of a monoclonal anti-EGFR antibody preparation
comprising a single species of monoclonal antibody is provided, the
method comprising, prior to the administration, determining that
the tumor does not have a level of high affinity ligands that is
equal to or greater than the level of low affinity ligands in the
tumor and not administering the monoclonal preparation if the tumor
does have a level of high affinity ligands that is equal to or
greater than the level of low affinity ligands in the tumor.
[0019] In another aspect, a method of treating a tumor in a patient
by administration of an oligoclonal anti-EGFR antibody preparation
comprising a plurality of species of monoclonal anti-EGFR
antibodies, one against each of at least two extracellular epitopes
of EGFR, one of the species of monoclonal anti-EGFR antibody
inhibiting the binding of ligand to EGFR, is provided, the method
comprising, prior to the administration, determining that the tumor
has a level of high affinity ligands that is equal to or greater
than the level of low affinity ligands in the tumor. In one
embodiment, the oligoclonal anti-EGFR antibody preparation is
MM-151.
[0020] Also provided are kits for testing a tumor biopsy sample to
determine levels of both high and low affinity EGFR ligands in the
sample, said kits being comprised by one or more containers
comprising; [0021] a) at least two pairs of high affinity EGFR
ligand-specific polymerase chain reaction (PCR) primers, [0022] b)
at least two pairs of low affinity EGFR ligand-specific PCR
primers, and [0023] c) at least one reverse transcription PCR
(RT-PCR) reagent.
[0024] In one embodiment the at least two pairs of high affinity
EGFR ligand-specific polymerase chain reaction primers are specific
to at least two of betacellulin, EGF, HB-EGF or TGF.alpha. and each
of the at least two pairs of low affinity EGFR ligand-specific
polymerase chain reaction primers are specific to at least two of
amphiregulin, epigen, or epiregulin. In another embodiment the at
least two pairs of high affinity EGFR ligand-specific primers
consist of all of betacellulin, EGF, HB-EGF and TGF.alpha. and the
at least two pairs of high affinity EGFR ligand-specific primers
consist of all of amphiregulin, epigen, and epiregulin. In another
embodiment the kit comprises at least one fluorescent reporter
molecule suitable for use in a real-time RT-PCR assay. In yet
another embodiment the at least one RT-PCR reagent is one or more
of an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase,
a buffer, or a solution comprising at least micromolar
concentrations of each of adenosine triphosphate (ATP), guanosine
triphosphate (GTP), cytidine triphosphate (CTP), and thymidine
triphosphate (TTP). In a further embodiment the one or more
containers comprises either or both of at least one container that
has an internal temperature of below 20.degree. C. and above
0.degree. C., and at least one container that has an internal
temperature of below 0.degree. C. Preferably the contents of all,
or at least one, of the at least one container has been prepared
under cGMP conditions.
[0025] In another aspect, a method is provided for treating a
patient having a cancerous tumor, the method comprising
determining, according to any of the methods described above, if
the patient is predicted to have the favorable outcome as a result
of treatment #1 and as a result of treatment #2 or if the patient
is predicted to have the favorable outcome as a result of treatment
#2 but not as a result of treatment #1, wherein if the patient is
predicted to have a favorable outcome as a result of treatment #1
and as a result of treatment #2, the patient is treated with
treatment #1 or treatment #2; and if the patient is predicted to
have a favorable outcome as a result of treatment #2 but not as a
result of treatment #1, the patient is treated with treatment 2 and
not with treatment #1. Determining whether the patient will have a
favorable outcome may be accomplished by use of any of the
theranostic methods described above, which may be accomplished by
use of a kit described above.
[0026] In certain aspects of the above methods, the oligoclonal
anti-EGFR antibody preparation is a composition comprising a trio
of anti-EGFR antibodies comprising a first antibody, a second
antibody and a third antibody, wherein (i) the first antibody is,
or competes for binding to EGFR with, or binds to the same epitope
as, an antibody selected from the group consisting of ca, cb and
cc; (ii) the second antibody is, or competes for binding to EGFR
with, or binds to the same epitope as, an antibody selected from
the group consisting of cd, ce and cf; and (iii) the third antibody
is, or competes for binding to EGFR with, or binds to the same
epitope as, an antibody selected from the group consisting of cg,
ch, ci, cj and ck, wherein ca, cb, cd, ce, cf, cg, ch, ci, cj, and
ck are each disclosed in PCT Int. Pub. No. WO/2011/140254 and
corresponding pending U.S. patent application Ser. No.
13/100,920.
[0027] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIGS. 1A-1C: Phospho-EGF receptor and phospho-ERK signaling
inhibition by single and pairwise combinations of Bin 1+Bin 2 or
Bin 1+Bin 3 antibodies and comparisons with other known anti-EGFR
antibodies such as cetuximab, nimotuzumab, and zalutumumab. FIG. 1A
shows inhibition of ERK activation by the Bin1/2 antibodies cb and
cd. FIG. 1B shows the inhibition of EGFR activation by the Bin1/2
antibodies and cd. FIG. 1C shows the inhibition of ERK activation
by the Bin1/3 antibodies cb and ch. Lines depict a five parameter
logistic fit to the data from each combination.
[0029] FIGS. 2A-2G: Inhibition of ligand-mediated tumor cell
signaling in A431 cells preincubated with varying concentrations of
anti-EGFR monoclonal antibodies cb (Bin1), cd (Bin2), cetuximab,
zalutumumab, or nimotuzumab; as well as the oligoclonal combination
of cb+cd; for 2 hrs. After incubation cells were stimulated with an
EGFR ligand (8 nanomolar final concentration) for about 10 minutes.
Figures show ELISA analysis of phospho-ERK (pERK) production
(y-axis) as a function of antibody concentration (x-axis, in Log
Molar concentration) after stimulation with the ligands
amphiregulin (FIG. 2A), epigen (FIG. 2B), epiregulin (FIG. 2C),
betacellulin (FIG. 2D), epidermal growth factor (EGF, FIG. 2E),
heparin-binding EGF-like growth factor (HB-EGF, FIG. 2F), or
transforming growth factor .alpha. (TGF-.alpha., FIG. 2G). A431
cells incubated in the absence of anti-EGFR antibodies but with the
ligand indicated in each graph (+Lig) or without ligand stimulation
(-Lig) were used as positive and negative controls,
respectively.
[0030] FIGS. 3A-3G: Inhibition of ligand-mediated tumor cell
signaling in A431 cells preincubated with varying concentrations of
anti-EGFR monoclonal antibodies cb (Bin1), ch (Bin3), cetuximab,
zalutumumab, or nimotuzumab; as well as the oligoclonal combination
of cb+ch; for 2 hrs. After pre-incubation with antibodies, cells
were stimulated with an EGFR ligand (8 nanomolar final
concentration) for 10 minutes. Figures show ELISA analysis of
phospho-ERK (pERK) production (y-axis) as a function of antibody
concentration (x-axis, in Log Molar concentration) after
stimulation with the ligands amphiregulin (FIG. 3A), epigen (FIG.
3B), epiregulin (FIG. 3C), betacellulin (FIG. 3D), epidermal growth
factor (EGF, FIG. 3E), heparin-binding EGF-like growth factor
(HB-EGF, FIG. 3F), or transforming growth factor .alpha.
(TGF-.alpha., FIG. 3G). A431 cells incubated in the absence of
anti-EGFR antibodies but with the ligand indicated in each graph
(+Lig) or without ligand stimulation (-Lig) were used as positive
and negative controls, respectively.
[0031] FIGS. 4A-4L: Inhibition of high affinity EGFR
ligand-mediated tumor cell proliferation. H322M cells (FIGS.
4A-4D), H1975 cells (FIGS. 4E-4H), and LIM1215 cells (FIGS. 4I-4L)
were treated with varying concentrations of anti-EGFR monoclonal
and oligoclonal antibodies in the presence of EGFR ligands. Cells
were treated with 200 ng/ml amphiregulin (AREG) (FIGS. 4A, 4E, and
4I), 50 ng/ml EGF (FIGS. 4B, 4F, and 4J), 50 ng/ml TGF.alpha.
(FIGS. 4C, 4G, and 4K) or 90 ng/ml HB-EGF (FIGS. 4D, 4H, and 4L) in
the presence of varying concentrations of MM-151 (open circles or
cetuximab (CTX, solid squares; Bristol-Myers Squibb). Cells treated
with ligand (+Lig, upward arrow) or without ligand (-Lig, downward
arrow) in the absence of antibody treatment served as controls. The
y-axes represent cell viability as the fraction of the viability of
the amphiregulin-treated control cells and the x-axes represent
antibody concentration in Log (Molar).
[0032] FIGS. 5A-5L: Effect of EGFR high affinity ligand titration
on cell responsiveness to anti-EGFR inhibitors in vitro. The
non-small cell lung cancer (NSCLC) lines H322M (FIGS. 5A-5D),
HCC827 (FIGS. 5E-5H), and H1975 (FIGS. 5I-5L) were tested. Controls
were growth in media with amphiregulin alone (+AREG, 200 ng/ml) or
EGF alone as a control (+EGF, 20 ng/ml) or no added ligand (-Lig).
Treatments were with varying concentrations (0.1-1 .mu.M final
concentration) of MM-151 or cetuximab (CTX) in the following
conditions: amphiregulin alone (200 ng/ml, FIGS. 5A, 5E, and 5I); a
1000:1 amphiregulin:EGF ratio (0.2 ng/ml EGF, FIGS. 5B, 5F, and
5J); a 100:1 amphiregulin:EGF ratio (2 ng/ml EGF, FIGS. 5C, 5G, and
5K); and a 10:1 amphiregulin:EGF ratio (20 ng/ml EGF, FIGS. 5D, 5H,
and 5L). The y-axes represent cell viability as a fraction of the
viability of the AREG-treated control cells, whereas and the x-axes
represent antibody concentration in Log (Molar).
[0033] FIG. 6: Effect of EGFR ligand concentration on phopho-ERK
cell signaling. The epidermoid cancer cell line A431 was treated
with media alone ("No Inhibitor"), MM-151 (100 nM) or cetuximab
(100 nM) for 2 hrs, followed by the addition of various
concentrations of EGF (0.16 ng/ml, 0.8 ng/ml, 4.0 ng/ml, 20 ng/ml,
100 ng/ml) or AREG (0.48 ng/ml, 2.4 ng/ml, 12 ng/ml, 60 ng/ml, 300
ng/ml), alone or in combination. Cells were incubated with the
various EGF and AREG ligand combinations for 10 minutes, lysed, and
levels of ERK phosphorylation measured by phospho-ERK ELISA.
DETAILED DESCRIPTION
I. Definitions
[0034] The terms "EGFR," and "EGF receptor" are used
interchangeably herein to refer to human EGFR protein (also
referred to as ErbB1 or HER1); see UniProtKB/Swiss-Prot entry
P00533.
[0035] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Such antibodies may
be obtained, e.g. from hybridomas or by recombinant expression.
Antigen binding fragments (including scFvs) of such immunoglobulins
are also encompassed by the term "monoclonal antibody" as used
herein. Monoclonal antibodies are highly specific, generally being
directed against a single epitope on a single antigen site, e.g.,
on the extracellular domain of EGFR. Monoclonal antibodies include
chimeric antibodies - whose variable regions derive from a first
animal species (e.g., mouse) and whose constant regions derive from
a second animal species (e.g., human), human antibodies and
humanized antibodies.
[0036] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration to a
subject, an antibody or antibody pair or trio disclosed herein, for
example, a subject having a disorder associated with EGFR dependent
signaling or predisposed to having such a disease or disorder, in
order to prevent, cure, delay, reduce the severity of, or
ameliorate one or more symptoms of the disease or disorder or
recurring disease or disorder, or in order to prolong the survival
of a subject beyond that expected in the absence of such
treatment.
[0037] Commercially available pharmaceutical anti-EGFR antibodies
include cetuximab, panitumumab and nimotuzumab (which is not yet
available in the US market). Other pharmaceutical anti-EGFR
antibodies include zalutumumab, and matuzumab, which are in
development. Still other anti-EGFR antibodies include those
disclosed in the Oligoclonal Applications, e.g., the antibodies
disclosed below.
[0038] P1X is a human IgG1 having a heavy chain variable region
comprising SEQ ID NO: 1 and a light chain variable region
comprising SEQ ID NO: 2;
[0039] P2X is a human IgG1 having a heavy chain variable region
comprising SEQ ID NO: 3 and a light chain variable region
comprising SEQ ID NO: 4; and
[0040] P3X is a human IgG1 having a heavy chain variable region
comprising SEQ ID NO: 5 and a light chain variable region
comprising SEQ ID NO: 6.
[0041] "MM-151" indicates a triple combination of P1X+P2X+P3X at a
P1X:P2X:P3X molar ratio of 2:2:1.
TABLE-US-00001 TABLE 1 Exemplary Antibodies P1X V.sub.H
MGFGLSWLFLVAILKGVQC SEQ ID NO: 1 QVQLVQSGAEVKKPGSSVKV
SCKASGGTFSSYAISWVRQA PGQGLEWMGSIIPIFGTVNY AQKFQGRVTITADESTSTAY
MELSSLRSEDTAVYYCARDP SVNLYWYFDLWGRGTLVTVSS P1X V.sub.L
MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 2 DIQMTQSPSTLSASVGDRVT
ITCRASQSISSWWAWYQQKP GKAPKLLIYDASSLESGVPS RFSGSGSGTEFTLTISSLQP
DDFATYYCQQYHAHPTTFGG GTKVEIK P2X V.sub.H MGFGLSWLFLVAILKGVQC SEQ ID
NO: 3 QVQLVQSGAEVKKPGSSVKV SCKASGGTFGSYAISWVRQA
PGQGLEWMGSIIPIFGAANP AQKSQGRVTITADESTSTAY MELSSLRSEDTAVYYCAKMG
RGKVAFDIWGQGTMVTVSS P2X V.sub.L MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 4
DIVMTQSPDSLAVSLGERAT INCKSSQSVLYSPNNKNYLA WYQQKPGQPPKLLIYWASTR
ESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYGS PITFGGGTKVEIK P3X V.sub.H
MGFGLSWLFLVAILKGVQC SEQ ID NO: 5 QVQLVQSGAEVKKPGASVKV
SCKASGYAPTSYGINWVRQA PGQGLEWMGWISAYNGNTYY AQKLRGRVTMTTDTSTSTAY
MELRSLRSDDTAVYYCARDL GGYGSGSVPFDPWGQGTLVTVSS P3X V.sub.L
MGTPAQLLFLLLLWLPDTTG SEQ ID NO: 6 EIVMTQSPATLSVSPGERAT
LSCRASQSVSSNLAWYQQKP GQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQS
EDFAVYYCQDYRTWPRRVFG GGTKVEIK zalutumumab QVQLVESGGGVVQPGRSLRLS SEQ
ID NO: 7 V.sub.H CAASGFTFSTYGMHWVRQAPG KGLEWVAVIWDDGSYKYYGDS
VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCARDGITMVR GVMKDYFDYWGQGTLVTVSS
zalutumumab AIQLTQSPSSLSASVGDRVTI SEQ ID NO: 8 V.sub.L
TCRASQDISSALVWYQQKPGK APKLLIYDASSLESGVPSRFS GSESGTDFTLTISSLQPEDFA
TYYCQQFNSYPLTFGGGTKVE IK Nimotuzumab QVQLQQPGAELVKPGASVKLS SEQ ID
NO: 9 V.sub.H CKASGYTFTNYYIYWVKQRPG QGLEWIGAGINPTSGGSNFNE
KFKTKATLTVDESSTTAYMQL SSLTSEDSAVYYCTRQGLWFD SDGRGFDFWGQGTTLTVSS
Nimotuzumab DVLMTQIPLSLPVSLGDQASISC SEQ ID NO: 10 V.sub.L
RSSQNIVHSNGNTYLDWYLQKP GQSPNLLIYKVSNRESGVPDRF
RGSGSGTDFTLKISRVEAEDLGV YYCFQYSHVPWTFGGGTKLEIK
[0042] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0043] The term "sample" refers to tissue, body fluid, or a cell
(or a fraction of any of the foregoing) taken from a patient.
Normally, the tissue or cell will be removed from the patient, but
in vivo diagnosis is also contemplated. In the case of a solid
tumor, a tissue sample can be taken from a surgically removed tumor
and prepared for testing by conventional techniques. In the case of
lymphomas and leukemias, lymphocytes, leukemic cells, or lymph
tissues can be obtained (e.g., leukemic cells from blood) and
appropriately prepared. Other samples, including urine, tears,
serum, plasma, cerebrospinal fluid, feces, sputum, cell extracts
etc. can also be useful for particular cancers.
[0044] Various aspects of the disclosure are described in further
detail in the following subsections.
II. Outcomes
[0045] A patient having a tumor predicted by the methods disclosed
herein to have a favorable outcome following treatment with a
monoclonal or oligoclonal anti-EGFR antibody, and who is then
treated accordingly, may exhibit one of the following responses to
therapy: [0046] Pathologic complete response (pCR): absence of
invasive cancer following primary systemic treatment. [0047]
Complete Response (CR): Disappearance of all target lesions. [0048]
Partial Response (PR): At least a 30% decrease in the sum of
dimensions of target lesions, taking as reference the baseline sum
diameters; or [0049] Stable Disease (SD): Neither sufficient
shrinkage to qualify for partial response, nor sufficient increase
to qualify for progressive disease, taking as reference the
smallest sum diameters while on study.
[0050] In exemplary outcomes, patients treated as disclosed herein
may experience improvement in at least one sign of cancer.
[0051] In one embodiment the patient so treated exhibits pCR, CR,
PR, or SD.
[0052] In another embodiment, the patient so treated experiences
tumor shrinkage and/or decrease in growth rate, i.e., suppression
of tumor growth. In another embodiment, unwanted cell proliferation
is reduced or inhibited. In yet another embodiment, one or more of
the following can occur: the number of cancer cells can be reduced;
tumor size can be reduced; cancer cell infiltration into peripheral
organs can be inhibited, retarded, slowed, or stopped; tumor
metastasis can be slowed or inhibited; tumor growth can be
inhibited; recurrence of tumor can be prevented or delayed; one or
more of the symptoms associated with cancer can be relieved to some
extent.
[0053] In other embodiments, such improvement is measured by a
reduction in the quantity and/or size of measurable tumor lesions.
Measurable lesions are defined as those that can be accurately
measured in at least one dimension (longest diameter is to be
recorded) as .gtoreq.10 mm by CT or MRI scan (e.g., CT scan slice
thickness no greater than 5 mm), 10 mm caliper measurement by
clinical exam or >20 mm by chest X-ray. The size of non-target
lesions can also be measured for improvement. In one embodiment,
lesions can be measured on x-rays or CT or MRI images.
[0054] In other embodiments, cytology or histology can be used to
evaluate responsiveness to a therapy. The cytological confirmation
of the neoplastic origin of any effusion that appears or worsens
during treatment when the measurable tumor has met criteria for
response or stable disease can be considered to differentiate
between response or stable disease (an effusion may be a side
effect of the treatment) and progressive disease.
[0055] In some embodiments, a beneficial response to therapy is
indicated by at least one therapeutic effect selected from the
group consisting of reduction in size of a tumor, reduction in
number of metastatic lesions appearing over time, complete
remission, partial remission, stable disease, increase in overall
response rate, or a pathologic complete response.
III. Pharmaceutical Compositions
[0056] Pharmaceutical compositions for use in the methods provided
for herein are commercially available anti-EGFR compositions, e.g.,
of cetuximab, panitumumab and nimotuzumab, as well as the various
pharmaceutical compositions provided in the Oligoclonal
Applications.
IV. Use of Oligoclonal Antibodies
[0057] Provided herein are methods of determining whether or not a
monoclonal anti-EGFR antibody preparation comprising only a single
species of anti-EGFR antibody should be used to treat a tumor. Use
of oligoclonal anti-EGFR antibodies for the treatment of a disease
associated with high-affinity EGFR ligand-driven signaling is also
provided, as are methods of use of oligoclonal anti-EGFR antibodies
for the treatment of tumor comprising protein or mRNA levels of at
least two high-affinity EGFR ligands that are higher than levels in
the tumor of at least two low-affinity EGFR ligands. Cancers
treated in accordance with the methods provided include melanoma,
breast cancer, ovarian cancer, renal carcinoma, gastrointestinal
cancer, gastro-esophageal junction cancer, colon cancer, lung
cancer, pancreatic cancer, skin cancer, head and neck cancer
glioblastoma, prostate cancer and other solid and/or metastatic
tumors.
[0058] The monoclonal or oligoclonal antibody can be administered
alone or with another therapeutic agent that acts in conjunction
with or synergistically with the oligoclonal antibody to treat the
disease associated with EGFR-mediated signaling.
[0059] Also provided are kits for testing a tumor sample, e.g., a
tumor biopsy sample or a circulating tumor cell, to determine
levels of both high and low affinity EGFR ligands in the sample,
said kits being comprised by one or more containers comprising;
[0060] a) at least two pairs of high affinity EGFR ligand-specific
polymerase chain reaction (PCR) primers, [0061] b) at least two
pairs of low affinity EGFR ligand-specific PCR primers, and [0062]
c) at least one reverse transcription PCR (RT-PCR) reagent.
[0063] In another embodiment, the kit may further contain
instructions for use in determining how to treat a tumor in a
patient following determination of levels of high and low affinity
ligands in a sample of the tumor. The kit may include an indication
of the intended use of the contents of the kit (e.g., in the form
of a label or other printed or recorded matter).
[0064] Other embodiments are described in the following
non-limiting Examples.
[0065] The present invention is further illustrated by the
following examples which should not be construed as further
limiting. The contents of Sequence Listing, figures and all
references, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
EXAMPLES
Materials and Methods
[0066] Throughout the examples, the following materials and methods
are used unless otherwise stated.
[0067] In general, unless otherwise indicated, conventional
techniques of chemistry, molecular biology, recombinant DNA
technology, immunology (especially, e.g., antibody technology), and
standard techniques in polypeptide preparation are used. See, e.g.,
Sambrook, Fritsch and Maniatis, Molecular Cloning Cold Spring
Harbor Laboratory Press (1989); Antibody Engineering Protocols
(Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996);
Antibody Engineering: A Practical Approach (Practical Approach
Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A
Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); and
Current Protocols in Molecular Biology, eds. Ausubel et al., John
Wiley & Sons (1992).
[0068] Pulverization of Tumor Cells
[0069] A cryopulverizer (COVARIS Inc.) is used for the
pulverization of tumors. Tumors are stored in special bags
(pre-weighed before the addition of the tumor) and placed in liquid
nitrogen while handling them. For small tumors, 200 .mu.L of Lysis
buffer is first added to the bag containing the tumor, frozen in
liquid nitrogen and then pulverized to improve the recovery of the
tumor from the bag. Pulverized tumors are transferred to 2 mL
EPPENDORF tubes and placed in liquid nitrogen until ready for
further processing.
[0070] Lysis of Tumor Cells
[0071] Tumors are lysed in Lysis buffer supplemented with protease
and phosphatase inhibitors. Lysis Buffer is added to the tumor
aliquots in a final concentration of about 62.5 mg/mL. Tumor
samples are homogenized by vortexing for 30 sec and incubating on
ice for about 30 min. The lysates are spun for about 10 min in
Qiagen QIASHREDDER columns for further homogenization of the
samples. Cleared lysates are aliquoted into fresh tubes for further
processing.
[0072] Measurement of Inhibition of EGFR Ligand-Mediated
Phosphorylation of ERK in Tumor Cells
[0073] Inhibition of ligand-mediated tumor cell signaling is
investigated as follows: A431 (ATCC CRL-1555.TM.) epidermoid
carcinoma cells are seeded at a density of 35,000 cells/well or
17,500 cells per half well in 96 well tissue culture plates and
grown in DMEM medium supplemented with antibiotics, 2 mM
L-glutamine and 10% fetal bovine serum (FBS) for 24 hours at
37.degree. C. and 5% carbon dioxide. Cells are serum starved in 1%
FBS medium with antibiotics and 2 mM L-glutamine for about 20 hours
at 37.degree. C. and 5% carbon dioxide. Cells are then treated as
described below in each Example. Cells are washed with ice-cold PBS
and lysed in 50 .mu.l ice-cold Lysis buffer (Mammalian Protein
Extraction Lysis Reagent (M-PER, Pierce, Thermo Scientific product
#78505) amended with 150 mM NaCl and protease inhibitor cocktail
(Sigma, P714)) by incubating on ice for 30 minutes. Lysates are
either analyzed immediately by ELISA for phospho-ERK (a downstream
effector of EGFR) or frozen at '180.degree. C. until use.
[0074] ELISA Assays
[0075] For the phospho-EGFR sandwich ELISA, 96-half well GREINER
high binding plates (Cat. #675077; GREINER BIO-ONE, Monroe, N.C.)
are coated with 50 .mu.L of an EGFR antibody (4 .mu.g/ml final
concentration; EGFR Ab-11, Clone: 199.12, without BSA and azide,
Fisher Scientific, cat #MS396P1ABX), and incubated overnight at
room temperature. Next morning, plates are washed 3 times with 100
.mu.l/well PBST (0.05% Tween-20) on a BIOTEK plate washer. Plates
are subsequently blocked for about 1 hour at room temperature with
2% BSA in PBS. The plates are washed 3 times with 100 .mu.l/well
PBST (0.05%Tween-20) on the BIOTEK plate washer. Cell lysates (50
.mu.l) or standards (pEGFR pY1068 ELISA kit, R&D Systems, cat
#DYC3570) diluted in 50% Lysis buffer and 1%BSA-PBS (per the
manufacturer's recommendations) are added to the plates in
duplicates and incubated for 2 hrs at room temperature or overnight
at 4.degree. C. with shaking. Plates are then washed 3 times with
100 .mu.l/well in the BIOTEK plate washer with PBST (PBS with
0.05%Tween-20). About 50 .mu.l of a detection antibody (pEGFR
pY1068 ELISA kit, R&D Systems, cat #DYC3570) conjugated to
horseradish peroxidase (HRP) diluted (as per manufacturer's
instructions) in 2% BSA, PBS is added and incubated for about 2
hour at room temperature. The plate is washed 3 times with 100
.mu.l/well in the BIOTEK plate washer with PBST (0.05%Tween-20).
About 50 .mu.L of SUPERSIGNAL PICO ELISA substrate is added and the
plate is read using an Envision (Perkin Elmer) plate reader. The
data are analyzed and duplicate samples are averaged and error bars
are used to represent the standard deviation between the two
replicates.
[0076] The phospho-ERK ELISA is performed similarly to the
phospho-EGFR ELISA with the following changes: Human pERK ELISA
DUOSET kit is purchased from R&D Systems (cat #DYC1018-5) and
used as recommended by the manufacturer. The data are analyzed by
subtracting background signal, regressing to a recombinant standard
supplied by the manufacturer, and back-calculating the data (BCD)
to correct for dilution factors. Duplicate samples are averaged and
error bars are used when indicated to represent the standard
deviation between two replicates.
Example 1
Phospho-EGF Receptor and Phospho-ERK Signaling Inhibition by Single
and Pairwise Combinations of Bin 1+Bin 2 or Bin 1+Bin 3 Antibodies
and Comparisons with Each of Individual Monoclonal Antibodies
Cetuximab, Nimotuzumab, and Zalutumumab
[0077] A431 cells were treated with single antibodies or antibody
pairs and their ability to inhibit EGFR-dependent signaling was
compared to that each of cetuximab, nimotuzumab, and zalutumumab.
Cells were incubated with varying concentrations of anti-EGFR
antibodies for 2 hrs, and then stimulated with an EGFR ligand for
10 minutes at 37.degree. C. and 5% carbon dioxide. The seven
recombinant human EGFR ligands used individually were 100 ng/ml
amphiregulin ("AREG," R&D Systems, cat #262-AR/CF), 100 ng/ml
betacellulin (R&D Systems, cat #261-CE-050/CF), EGF
(PreproTech, cat #AF-100-15), 220 ng/ml epigen (epithelial mitogen
homolog, PreproTech, cat #100-51), 150 ng/ml epiregulin (R&D
Systems, cat #1195-EP/CF), 90 ng/ml HB-EGF (heparin-binding
EGF-like growth factor, PreproTech, cat #100-47), and 50 ng/ml
TGF.alpha. (transforming growth factor alpha, R&D Systems, cat
#239-A). ELISA measurements were performed as described above for
pERK and pEGFR signaling and the results are shown in FIGS. 1A-C.
Only mixtures of Bin1/Bin2 antibodies cb and cd (FIG. 1A) and
Bin1/Bin3 antibodies cb and ch (FIG. 1C) were effective at
completely inhibiting phospho-ERK signaling when compared to
cetuximab, nimotuzumab, and zalutumumab, as well as to individual
components cb, cd, and ch. All antibodies, including the mixtures,
were effective at complete inhibition of Phospho-EGF receptor
signaling, with the exception of nimotuzumab (FIG. 1B).
Example 2
Phospho-ERK Signaling Inhibition by Single and Pairwise
Combinations of Bin 1, Bin 2, and Bin 3 Antibodies and Comparisons
with Cetuximab, Nimotuzumab, and Zalutumumab
[0078] Single antibodies cb, cd, and ch, or pairs of cb and cd or
cb and ch, (as described above in Example 1) were used to treat
A431 cells at indicated total concentrations, and their ability to
inhibit EGFR ligand-dependent signaling was compared to that of
each single anti-EGFR antibodies cetuximab, nimotuzumab, and
zalutumumab at the same concentrations. Cells were incubated with
antibody for 2 hours followed by stimulation with EGFR ligand for
10 minutes. Seven EGFR ligands were used individually: amphiregulin
(100 ng/ml), betacellulin (100 ng/ml), EGF (50 ng/ml), epigen (220
ng/ml), epiregulin (150 ng/ml), HB-EGF (90 ng/ml), and TGF.alpha.
(50 ng/ml). Experiments were performed as described above and the
results are shown in FIGS. 2A-G and 3A-G. Individually, cb and cd,
as well as well cetuximab, nimotuzumab, and zalutumumab, were
effective at inhibiting phospho-ERK signaling (i.e., inhibiting
phosphorylation of ERK1 and ERK2) in response to the three ligands
with low affinity for EGF receptor (amphiregulin, epigen, and
epiregulin), but not in response to the four ligands with high
affinity for EGF receptor (betacellulin, EGF, HB-EGF, and
TGF.alpha.). Only oligoclonal mixtures of Bin1/Bin2 antibodies cb
and cd (FIGS. 2A-G) and Bin1/Bin3 antibodies cb and ch (FIGS. 3A-G)
were effective at essentially completely inhibiting phospho-ERK
signaling in response to all seven (both high- and low-affinity)
EGFR ligands when compared to individual components of the
mixtures, cb, cd, and ch and the other tested individual monoclonal
antibodies, cetuximab, nimotuzumab, and zalutumumab.
Example 3
Effect of EGFR Ligand Concentration on Phospho-ERK Cell
Signaling
[0079] Inhibition of tumor cell signaling in vitro is analyzed by
the methods described above or minor variations thereof. The
epidermoid cancer cell line A431 was treated with media alone ("No
Inhibitor"), MM-151 (100 nM) or cetuximab (100 nM) for 2 hrs,
followed by the addition of various concentrations of EGF (0.16
ng/ml, 0.8 ng/ml, 4.0 ng/ml, 20 ng/ml, 100 ng/ml) or AREG (0.48
ng/ml, 2.4 ng/ml, 12 ng/ml, 60 ng/ml, 300 ng/ml), alone or in
combination, as shown in FIG. 6. Cells were incubated with the
various EGF and AREG ligand combinations for 10 minutes, lysed, and
levels of ERK phosphorylation measured by phospho-ERK ELISA and
analyzed as indicated in the methods. FIG. 6 shows MM-151 and
cetuximab-mediated modulation of ERK signaling represented as
fraction of the highest signal across all treatments. Phospho-ERK
signaling is inhibited by cetuximab in A431 cells under
low-affinity EGFR ligand (AREG) stimulation, but become
increasingly resistant to inhibition upon the addition of
increasing amounts of the high-affinity EGFR ligand, EGF (middle
panel), while inhibition of signaling by MM-151 is largely
maintained under all conditions (lower panel).
Example 4
Inhibition of Tumor Cell Proliferation in the Presence of High or
Low Affinity EGFR Ligands
Inhibition of Tumor Cell Proliferation In Vitro
[0080] Inhibition of cellular proliferation of cells expressing
EGFR is examined in vitro as follows: H322M (NCI, Frederick, Md.
21701), H1975 (ATCC CRL-2868.TM.), and LIM1215 (Cell Bank
Australia, NSW 2145) cancer cells are separately seeded in 96 well
tissue culture plates at 5,000 cells per well and grown in
RPMI-1640 medium supplemented with antibiotics, 2 mM L-glutamine
and 10% fetal calf serum (FCS) (H322M and H1975) or RPMI-1640
medium supplemented with 25 mM HEPES, antibiotics, 2 mM
L-glutamine, 10% FCS, 0.6 .mu.g/ml insulin, 1 .mu.g/ml
hydrocortisone and 10 .mu.M thioglycerol (LIM1215) for 24 hours at
37 degrees Celsius and 5% carbon dioxide. Medium is then switched
to RPMI-1640 with antibiotics, 2 mM L-glutamine, 1% FBS (for H322M
and H1975) or RPMI-1640 with 25 mM HEPES, antibiotics, 2 mM
L-glutamine, 1% FCS, 0.6 .mu.g/ml Insulin, 1 .mu.g/ml
hydrocortisone and 10 .mu.M thioglycerol (for LIM1215) supplemented
with 200 ng/ml AREG, 50 ng/ml EGF, 50 ng/ml TGF.alpha. or 90 ng/ml
HB-EGF in the presence of varying concentrations of MM-151 or
cetuximab (Bristol-Myers Squibb). Cell viability is measured 72
hours post-treatment using the CellTiter-Glo.RTM. (CTG) Luminescent
Viable Cell Number Assay (Promega Corporation) according to
manufacturer's instructions. The CTG assay measures the number of
viable cells in culture based upon quantitation of ATP present,
which is an indicator of metabolically active cells. Control
treatments include cells treated with 1% FCS-containing medium (as
detailed above) in the presence ("+Lig") or absence ("-Lig") of the
respective ligand treatment. Viable cell numbers are plotted in
GraphPad Prism (GraphPad Software, La Jolla, Calif.) as a fraction
of the respective ligand ("+Lig") treatment control.
Results
[0081] The non-small cell lung cancer (NSCLC) lines H322M and H1975
and colon cancer cell line LIM1215 were treated with varying
concentrations of MM-151or cetuximab (0.1-1 .mu.M final
concentration). Potent inhibition of growth of H322M, H1975 and
LIM1215 cells was obtained over a range of MM-151 concentrations in
the presence of high affinity EGFR ligands (EGF, TGF.alpha.,
HB-EGF), but not in the presence of cetuximab or in assay medium
alone (1% FCS)--FIG. 4 (B-D, F-H, and J-L). Potent inhibition of
growth of H322M, H1975 and LIM1215 cells was obtained over a range
of concentrations for both MM-151 and cetuximab, but not by assay
medium alone (1% FCS) in the presence of the low affinity EGFR
ligand amphiregulin (AREG)--FIG. 4 (A, E and I). These data
demonstrate the ability of the MM-151 oligoclonal mixture to
inhibit tumor cell proliferation in vitro in response to both
high-affinity (EGF, TGF.alpha., HB-EGF) and low-affinity (AREG)
ligands, whereas cetuximab is only potently effective in cells
treated with low-affinity (AREG) ligand.
Example 5
Effects of EGF Ligand Concentration on Cell Proliferation
[0082] Using methods essentially as described in the preceding
Example, non-small cell lung cancer (NSCLC) cell lines H322M,
HCC827 and H1975 were treated with AREG alone (200 ng/ml) or with
AREG plus increasing amounts of EGF (0.2, 2, 20 ng/ml) in the
presence of varying concentrations of MM-151or cetuximab (0.1-1
.mu.M final concentration).
Results
[0083] The NSCLC cell lines respond to cetuximab under low-affinity
EGFR ligand stimulation (AREG), but become increasingly
unresponsive to treatment upon the addition of increasing amounts
of the high-affinity EGFR ligand EGF, while sensitivity to MM-151
is largely maintained (see FIGS. 5A-5L).
Example 6
Assays and Kits
Measurement of EGFR Family Ligand Expression Levels by RT-qPCR
[0084] Measurement of EGFR ligand expression in tumor biopsy
samples by real-time quantitative polymerase chain reaction
(RT-qPCR) of DNAs reverse transcribed from RNAs is carried out as
follows:
[0085] Total RNA is isolated from patient biopsy/tumor samples,
e.g., by commercially available standard methods. The method of
total RNA isolation may be any method (including conventional
methods) suitable for use with the type of patient biopsy sample
being tested, e.g., fresh, fixed, frozen, formalin fixed paraffin
embedded (FFPE), etc. Total RNA is then converted to cDNA using the
gene specific primers described below and Qiagen.RTM. OneStep
RT-PCR reagents and protocol (Cat. #210210, Qiagen, Germantown,
Md.). The cDNA is then used for RT-qPCR using the following gene
specific primers as TaqMan.RTM. probe sets obtained from Applied
Biosystems (Carlsbad, Calif.) along with reagents and equipment
from the same source, all as described below: [0086] 1. TaqMan.RTM.
Gene Expression Assay, Gene Name: betacellulin, Assay ID:
Hs01101201_ml [0087] 2. TaqMan.RTM. Gene Expression Assay, Gene
Name: transforming growth factor, alpha, Assay ID: Hs00608187_ml
[0088] 3. TaqMan.RTM. Gene Expression Assay, Gene Name:
heparin-binding EGF-like growth factor, Assay ID: Hs00181813_ml
[0089] 4. TaqMan.RTM. Gene Expression Assay, Gene Name: epiregulin,
Assay ID: Hs00914313_ml [0090] 5. TaqMan.RTM. Gene Expression
Assay, Gene Name: amphiregulin, Assay ID: Hs00950669_ml [0091] 6.
TaqMan.RTM. Gene Expression Assay, Gene Name: epidermal growth
factor, Assay ID: Hs01099999_ml. [0092] 7. TaqMan.RTM. Gene
Expression Assay, Gene Name: epithelial mitogen homolog (epigen),
Assay ID Hs02385425_ml. 5 .mu.l of diluted cDNA is mixed with 10
.mu.l of TaqMan.RTM. Fast Advanced Master Mix (Cat. #4444556), 2
.mu.l of the above primer probe set and 3 .mu.l of water in a
MicroAmp.RTM. Fast Optical 96-Well Reaction Plate (Cat. #4366932).
The plate is then placed in a Viia.TM. 7 RT-qPCR machine and a
thermal cycling program completed as described in the manufacturers
protocol. Data collection and analysis is carried out using the
Viia.TM. 7-RUO-Software (Applied Biosystems).
[0093] Also see US Patent Publication Nos. 20030165952,
20040009489, 20050095634, 20050266420, 20070141587, 20070141588,
20070141589, 20080182255, 20090125247, 20090280490, 20100221754 and
20110086349, and U.S. Pat. Nos. 6,750,013, 6,808,888, 6,939,670,
6,964,850, 6,692,916, 7,081,340, 7,171,311, 7,526,387, 7,569,345,
7,622,251, 7,871,769, 7,838,224, 7,858,304, 7,930,104, and
8,071,286.
Equivalents
[0094] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments described herein. Such
equivalents are intended to be encompassed by the following claims.
Any combination of the embodiments disclosed in the any plurality
of the dependent claims is contemplated to be within the scope of
the disclosure.
Sequence CWU 1
1
101140PRTHomo sapiens 1Met Gly Phe Gly Leu Ser Trp Leu Phe Leu Val
Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe 35 40 45 Ser Ser Tyr Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met
Gly Ser Ile Ile Pro Ile Phe Gly Thr Val Asn Tyr Ala 65 70 75 80 Gln
Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90
95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110 Tyr Tyr Cys Ala Arg Asp Pro Ser Val Asn Leu Tyr Trp Tyr
Phe Asp 115 120 125 Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
130 135 140 2127PRTHomo sapiens 2Met Gly Thr Pro Ala Gln Leu Leu
Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Asp Ile
Gln Met Thr Gln Ser Pro Ser Thr Leu Ser 20 25 30 Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser 35 40 45 Ile Ser
Ser Trp Trp Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60
Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser 65
70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser 85 90 95 Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr His 100 105 110 Ala His Pro Thr Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 115 120 125 3138PRTHomo sapiens 3Met Gly Phe
Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val
Gln Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25
30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe
35 40 45 Gly Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu 50 55 60 Glu Trp Met Gly Ser Ile Ile Pro Ile Phe Gly Ala
Ala Asn Pro Ala 65 70 75 80 Gln Lys Ser Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Met
Gly Arg Gly Lys Val Ala Phe Asp Ile Trp 115 120 125 Gly Gln Gly Thr
Met Val Thr Val Ser Ser 130 135 4133PRTHomo sapiens 4Met Gly Thr
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp
Thr Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25
30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser
35 40 45 Val Leu Tyr Ser Pro Asn Asn Lys Asn Tyr Leu Ala Trp Tyr
Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr
Gly Ser Pro Ile Thr Phe Gly Gly Gly Thr 115 120 125 Lys Val Glu Ile
Lys 130 5142PRTHomo sapiens 5Met Gly Phe Gly Leu Ser Trp Leu Phe
Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35 40 45 Thr Ser Tyr
Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu
Trp Met Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Tyr Tyr Ala 65 70
75 80 Gln Lys Leu Arg Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr
Ser 85 90 95 Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Leu Gly Gly Tyr Gly
Ser Gly Ser Val Pro 115 120 125 Phe Asp Pro Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 130 135 140 6128PRTHomo sapiens 6Met Gly Thr
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp
Thr Thr Gly Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser 20 25
30 Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
35 40 45 Val Ser Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr
Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Ser Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Asp Tyr Arg 100 105 110 Thr Trp Pro Arg Arg Val
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 7125PRTHomo
sapiens 7Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Asp Asp Gly Ser
Tyr Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe 100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
8107PRTHomo sapiens 8Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Val Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 9123PRTHomo
sapiens 9Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30 Tyr Ile Tyr Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Thr Ser Gly
Gly Ser Asn Phe Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu
Thr Val Asp Glu Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg
Gln Gly Leu Trp Phe Asp Ser Asp Gly Arg Gly Phe Asp Phe 100 105 110
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 10112PRTHomo
sapiens 10Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val Ser
Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn
Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Asn Leu Leu Ile Tyr Lys Val
Ser Asn Arg Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Arg Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Tyr 85 90 95 Ser His
Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110
* * * * *