U.S. patent application number 14/268925 was filed with the patent office on 2014-08-21 for methods for treating a tumor using an antibody that specifically binds grp94.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is Elvira Favoino, Soldano Ferrone, Xinhui Wang, Yangyang Wang, Ling Yu. Invention is credited to Elvira Favoino, Soldano Ferrone, Xinhui Wang, Yangyang Wang, Ling Yu.
Application Number | 20140234324 14/268925 |
Document ID | / |
Family ID | 46172275 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234324 |
Kind Code |
A1 |
Ferrone; Soldano ; et
al. |
August 21, 2014 |
METHODS FOR TREATING A TUMOR USING AN ANTIBODY THAT SPECIFICALLY
BINDS GRP94
Abstract
Combinations of agents that have a synergistic effect for the
treatment of a tumor are disclosed herein. These combinations of
agents can be used to treat tumors, wherein the cells of the cancer
express a mutated BRAF. Methods are disclosed for treating a
subject diagnosed with a tumor that expresses a mutated BRAF. The
methods include administering to the subject (1) a therapeutically
effective amount of an antibody or antigen binding fragment thereof
that specifically binds glucose regulated protein (GRP) 94; and (2)
a therapeutically effective amount of a BRAF inhibitor. In some
embodiments, the tumor is melanoma. In some embodiments the method
includes selecting a subject with primary or secondary resistance
to a BRAF inhibitor. In further embodiments, treating the tumor
comprises decreasing the metastasis of the tumor. In additional
embodiments, the BRAF inhibitor comprises PLX4032 or PLX4720.
Inventors: |
Ferrone; Soldano; (Boston,
MA) ; Wang; Xinhui; (Boston, MA) ; Favoino;
Elvira; (Casamassima, IT) ; Yu; Ling;
(Pittsburgh, PA) ; Wang; Yangyang; (Pittsburgh,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ferrone; Soldano
Wang; Xinhui
Favoino; Elvira
Yu; Ling
Wang; Yangyang |
Boston
Boston
Casamassima
Pittsburgh
Pittsburgh |
MA
MA
PA
PA |
US
US
IT
US
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
46172275 |
Appl. No.: |
14/268925 |
Filed: |
May 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13309490 |
Dec 1, 2011 |
8771687 |
|
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14268925 |
|
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61419208 |
Dec 2, 2010 |
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
A61K 31/437 20130101;
A61K 45/06 20130101; A61K 39/39558 20130101; C07K 2317/21 20130101;
A61P 35/00 20180101; A61K 2039/505 20130101; C07K 16/3053 20130101;
A61K 39/3955 20130101; A61K 31/437 20130101; C07K 2317/73 20130101;
A61P 35/04 20180101; A61K 39/39558 20130101; C07K 2317/622
20130101; A61K 2300/00 20130101; C07K 16/18 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/139.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395; A61K 31/437 20060101 A61K031/437 |
Claims
1. A method of treating a subject diagnosed with a tumor that
expresses a BRAF mutation, comprising administering to the subject
(1) a therapeutically effective amount of an monoclonal antibody or
antigen binding fragment thereof that specifically binds glucose
regulated protein (GRP) 94; and (2) a therapeutically effective
amount of a BRAF inhibitor, thereby treating the tumor in the
subject.
2. The method of claim 1, wherein the tumor is a breast cancer,
prostate cancer, ovarian cancer, colon cancer, stomach cancer,
pancreatic cancer, glioma, chordoma, chondrosarcoma, thyroid
cancer, colon cancer, glioma, renal cancer, lung cancer, bladder
cancer, non-Hodgkin's lymphoma, or a squamous cell carcinoma,
wherein cells of the tumor express GRP94.
3. The method of claim 2, wherein the squamous cell carcinoma is
head and neck carcinoma, lung carcinoma, prostate carcinoma,
esophagus carcinoma, vagina carcinoma or cervix carcinoma.
4. The method of claim 1, wherein the subject has primary or
secondary resistance to the BRAF inhibitor.
5. The method of claim 1, wherein the BRAF inhibitor is PLX4032 or
PLX4720.
6. The method of claim 1, comprising administering to the subject a
therapeutically effective amount of an antigen binding fragment of
a monoclonal antibody that specifically binds GRP94.
7. The method of claim 1, wherein the monoclonal antibody or
antigen binding fragment comprises a heavy chain variable domain,
and wherein the heavy chain variable domain of the monoclonal
antibody comprises the amino acid sequence set forth as amino acids
26-33 of SEQ ID NO: 3, amino acids 51-58 of SEQ ID NO: 3, and amino
acids 97-103 of SEQ ID NO: 3.
8. The method of claim 5, wherein the monoclonal antibody or
antigen binding fragment comprises a light chain variable domain,
wherein the light chain variable domain of the antibody comprises
the amino acid sequence set forth as amino acids 27-32 of SEQ ID
NO: 4, amino acids 50-52 of SEQ ID NO: 4, and amino acids 89-97 of
SEQ ID NO: 4.
9. The method of claim 1, wherein treating the tumor comprises
decreasing the metastasis of the tumor.
10. The method of claim 1, wherein the subject is human.
11. The method of claim 1, further comprising administering one or
more additional chemotherapeutic agents.
12. The method of claim 11, wherein the one or more additional
chemotherapeutic agents comprises an alkylating agent, a
topoisomerase inhibitor, a platinum anti-cancer drug, or a
combination thereof.
13. The method of claim 12, wherein the alkylating agent comprises
a nitrosourea or a taxane.
14. The method of claim 1, wherein the therapeutically effective
amount of the BRAF inhibitor and the therapeutically effective
amount of the antibody or antigen binding fragment thereof that
specifically binds glucose regulated protein (GRP) 94 are
administered simultaneously.
15. The method of claim 1, wherein cells in the tumor comprise a
BRAF mutation.
16. The method of claim 15, wherein the BRAF mutation is a V600E
mutation.
17. The method of claim 15, wherein the BRAF mutation is a R462I,
I463S, G464E, G464V, G466A, G466E, G466V, G469A, G469E, N581S,
E585K, D594V, F595L, G596R, L597V, T599I, V600D, V600E, V600K,
V600R, K601E or A728V mutation.
18. The method of claim 15, further comprising detecting a V600E
BRAF mutation in a sample from the subject, wherein the sample
comprises cells from the tumor.
19. The method of claim 1, wherein: the BRAF inhibitor is PLX4032
or PLX4720; and the monoclonal antibody, or antigen binding
fragment thereof comprises a heavy chain variable domain and a
light chain variable domain, wherein the heavy chain variable
domain of the monoclonal antibody comprises the amino acid sequence
set forth as amino acids 26-33 of SEQ ID NO: 3, amino acids 51-58
of SEQ ID NO: 3, and amino acids 97-103 of SEQ ID NO: 3 and the
light chain variable domain of the antibody comprises the amino
acid sequence set forth as amino acids 27-32 of SEQ ID NO: 4, amino
acids 50-52 of SEQ ID NO: 4, and amino acids 89-97 of SEQ ID NO: 4.
Description
PRIORITY CLAIM
[0001] This is a continuation of U.S. patent application Ser. No.
13/309,490, filed on Dec. 1, 2011, which claims the benefit of U.S.
Provisional Application No. 61/419,208, filed Dec. 2, 2010. Both of
the prior applications are incorporated herein by reference in
their entirety.
FIELD
[0002] This application relates to the treatment of cancer,
specifically to the use of a combination of an antibody that
specifically binds glucose regulated protein (GRP) 94 and a BRAF
inhibitor.
BACKGROUND
[0003] Melanoma is a malignant tumor of melanocytes that are
predominately found in skin, but can also be found in the bowel and
eye. Although melanoma is not the most common form of skin cancer,
it causes the majority of skin cancer related deaths. Melanoma
incidence and mortality rates in fair-skinned populations are
increasing worldwide. Approximately 160,000 cases of malignant
melanoma are diagnosed in the world each year. Current treatments
include surgical removal of the tumor, adjuvant treatment,
chemotherapy, immunotherapy and radiation therapy.
[0004] RAF protein kinases are key components of signal
transduction pathways by which specific extracellular stimuli
elicit precise cellular responses in mammalian cells. Activated
cell surface receptors activate ras/rap proteins at the inner
aspect of the plasma-membrane which in turn recruit and activate
Raf proteins. Activated RAF proteins phosphorylate and activate the
intracellular protein kinases MEK1 and MEK2. In turn, activated
MEKs catalyze phosphorylation and activation of p42/p44
mitogen-activated protein kinase (MAPK). Several cytoplasmic and
nuclear substrates of activated MAPK are known that directly or
indirectly contribute to the cellular response to environmental
change. Three distinct genes have been identified in mammals that
encode Raf proteins: ARAF, BRAF and CRAF (also known as RAF-1).
[0005] Inhibitors of RAF kinases have been suggested for use in
disruption of tumor cell growth and in the treatment of cancers,
such as histiocytic lymphoma, adenocarcinoma, small cell lung
cancer, melanoma and pancreatic and breast carcinoma. Specific
inhibitors of BRAF mutants, such as V600E (BRAF.sup.V600E mutant)
are known and have been used for the treatment of cancer. However,
some subjects are refractory to treatment with BRAF inhibitors.
Furthermore, some subjects develop secondary resistance to BRAF
inhibitors, such that regression induced by a BRAF inhibitor is
only temporary. Thus, a need remains for agents that augment the
effect of BRAF inhibitors, such as a combination of agents of use
for treating cancer and for inhibiting secondary resistance to a
BRAF inhibitor.
SUMMARY
[0006] Combinations of agents that have a synergistic effect for
the treatment of cancer are disclosed herein. These combinations of
agents can be used to treat cancers, wherein the cells of the
cancer express a mutated BRAF. The compositions include a
therapeutically effective amount of a BRAF inhibitor.
[0007] Methods are disclosed for treating a subject diagnosed with
a tumor that expresses a mutated BRAF. The methods include
administering to the subject (1) a therapeutically effective amount
of an antibody or antigen binding fragment thereof that
specifically binds glucose regulated protein (GRP) 94; and (2) a
therapeutically effective amount of a BRAF inhibitor.
[0008] In some embodiments, the tumor is melanoma. In further
embodiments, treating the tumor comprises decreasing the metastasis
of the tumor. In additional embodiments, the BRAF inhibitor is
vemurafenib (PLX4032). The disclosed methods are of use to treat a
subject that has primary or secondary resistance to the BRAF
inhibitor. The cells in the tumor can have a BRAF V600E
mutation.
[0009] In some embodiments, the methods include administering to
the subject a therapeutically effective amount of an antibody or
antigen binding fragment thereof that specifically binds GRP94. For
example, the antibody can be a monoclonal antibody, wherein the
heavy chain of the antibody comprises the amino acid sequence set
forth as amino acids 26-33 of SEQ ID NO: 3 (CDR1), amino acids
51-58 of SEQ ID NO: 3 (CDR2), and amino acids 97-103 of SEQ ID NO:
3 (CDR3) and/or wherein the light chain of the antibody comprises
the amino acid sequence set forth as amino acids 27-32 of SEQ ID
NO: 4 (CDR1), amino acids 50-52 of SEQ ID NO: 4 (CDR2), and amino
acids 89-97 of SEQ ID NO: 4 (CDR3).
[0010] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Effect of BRAF-I on GRP94 Expression by M21 Melanoma
Cell Line.
[0012] Cells (2.times.10.sup.5/ml) were incubated in RPMI 1640
medium containing 10% FCS with 1 .mu.M of BRAF-I for 14 days. Cells
were harvested, stained with GRP94-specific fully human antibody
(mAb) W9, human IgG (HIg) as negative control, and analyzed by flow
cytometry. Untreated cells were used as control. Percentage of
stained cells and mean fluorescence intensity (MFI) are
indicated.
[0013] FIG. 2. GRP94 Expression in BRAF-I Resistant M21 Melanoma
Cell Line.
[0014] The melanoma cell line M21 acquired resistance to BRAF-I
PLX4720 following repeated exposures to this inhibitor namely M21R.
BRAF-I PLX4720 resistant M21R cells (red) and parental melanoma
cells M21 (black) were cell surface stained with GRP94-specific mAb
W9. Human immunoglobulin (HIg) was used as control. Stained cells
were analyzed with a flow cytometer. Percentage of stained cells
and mean fluorescence intensity (MFI) are indicated.
[0015] FIG. 3. Synergic Anti-Cell Growth Effect of GRP94-Specific
mAb W9 Combined with BRAF-I PLX 4720 on Melanoma Cells.
[0016] Human melanoma cells (MV3, M21R) were seeded
(2.5.times.10.sup.3 cells per well) in a 96-well plate (RPMI 1640
media plus 1% FCS) and treated with GRP94-specific mAb W9, HIg
(negative control) in presence of BRAF inhibitor PLX4720 (5 .mu.M)
for 1, 2, 3, 4, 5 days at 37.degree. C. in a 5% CO2 atmosphere.
Cells were then tested by MTT assay. The O.D. values at 540 nm
indicate the living cells. *p value<0.05; **p value<0.01 (W9
vs W9+BRAF-I).
[0017] FIG. 4. Synergic Anti-Cell Growth Effect of GRP94-Specific
mAb W9 Combined with BRAF-I PLX 4032 on Melanoma Cells.
[0018] Human melanoma cells (M21, M21R, Colo38, Colo38R) were
seeded (2.5.times.10.sup.3 cells per well) in a 96-well plate (RPMI
1640 media plus 1% FCS) and treated with GRP94-specific mAb W9, HIg
(negative control) in presence of BRAF inhibitor PLX4032 (500 nM)
for 1, 3, 5 days at 37.degree. C. in a 5% CO2 atmosphere. Cells
were then tested by MTT assay. The O.D. values at 540 nm indicate
the living cells. *p value<0.05; **p value<0.01 (W9 vs
W9+BRAF-I).
[0019] FIG. 5. Inhibition by GRP94-Specific Antibody W9 Combined
with BRAF Inhibitor PLX4720 of M21R Cells Migration.
[0020] M21R cells were incubated in RPMI 1640 medium containing 1%
FCS with W9 Ab, HIg, BRAF Inhibitor PLX4720 (BRAF-I) combined with
W9 Ab, BRAF-I combined with HIg, or BRAF-I in a 24-trans-well plate
(2.5.times.10.sup.4 per well) for 3 days in a migration assay.
Cells incubated in RPMI 1640 medium containing 1% FCS was used as a
reference for 100% cell migration. The results are expressed as %
inhibition of migration, utilizing the values obtained in RPMI 1640
medium containing 1% FCS without Ab as a reference. *p
value<0.05; **p value<0.01.
[0021] FIG. 6. Inhibition of Melanoma Cell M21R Signaling Pathways
RAS-MEK-ERK and FAK by BRAF-I in Combination with GRP94-Specific Ab
W9.
[0022] The human melanoma cell M21R was serum starved for 3 days
then seeded at the concentration of 1.0.times.10.sup.5 per well in
a 6-well plate in RPMI 1640 medium without serum and incubated with
W9 Ab, the human IgG (HIg), or untreated in presence of BRAF
inhibitor PLX4720 (5 .mu.M) for 72 hours at 37.degree. C. Cell
lysate were tested in Western blot with anti-RAS, c-RAF,
phosphorylated (p)-MEK, p-ERK1/2, ERK1/2, (p)-FAK (Tyr397), FAK,
and PKC.alpha. mAbs. Calnexin and .beta.-actin was used as the
loading control. The density of resultant bands was determined with
IMAGEJ.TM. software, normalized to that of Calnexin and
.beta.-actin, shown below the respective bands. Data are expressed
as the percentage of the expression in untreated control cells.
[0023] FIG. 7. Synergic Pro-Apoptosis of GRP94-Specific mAb W9
Combined With BRAF-I PLX 4032 on Melanoma Cells.
[0024] Human melanoma cells (M21 and M21R) were starved for 12
hours and seeded at a density of 2.times.105/ml in a 6-well plate
and treated for 6 hours with BRAF-I PLX4032 (500 nM) and
Grp94-specific mAb W9 (20 .mu.g/ml) in RPMI 1640 medium plus 1.5%
FCS. Cells were stained with Annexin V-FITC and PI, and evaluated
for apoptosis by flow cytometry according to the manufacturer's
protocol (BD PharMingen, San Diego, Calif., USA). The early
apoptotic cells (annexin V-positive, PI-negative) were determined
using a flow cytometer.
[0025] FIG. 8. Synergic Inhibition by Grp94-Specific mAb W9
Combined with BRAF-I of BRAFV600E Mutant and BRAF-I Resistant
Melanoma CICs Proliferation In Vitro.
[0026] Cells growing in the exponential phase were seeded at a
density of 2.times.105/ml. The cells were treated for 3 days with
BRAF-I PLX4032 (500 nM) and Grp94-specific mAb W9 (20 .mu.g/ml) in
RPMI 1640 medium plus with 1.5% FCS. Cells were stained with
ALDEFLUOR.RTM. according to the manufacturer's protocol (Stem Cell
Technologies). Incubation of cells with ALDEFLUOR.RTM. in the
presence the ALDH1-specific inhibitor diethylaminobenzaldehyde
(DEAB), was used as a negative staining control for the assay. Then
cells were stained with ABCB5-specific mAb RK1(1 .mu.g/ml) for 30
mM at 4.degree. C., and incubated with APC-conjugated secondary mAb
(1:200) (Jackson Immunoresearch).
[0027] FIG. 9. Synergic Targeting Multiple Signaling Pathways by
GRP94-Specific mAb W9 Combined with BRAF-I PLX 4032 on Melanoma
Cells.
[0028] Cells growing in the exponential phase were seeded at a
density of 2.times.10.sup.5/ml. The cells were treated for 3 days
with BRAF-I PLX4032 (500 nM) and Grp94-specific mAb W9 (5 .mu.g/ml)
in RPMI 1640 medium plus with 2% FCS. Then the cells were collected
and lysed in lysis buffer [10 mM Tris-HCl [pH 8.2], 1% NP40, 1 mM
EDTA, 0.1% BSA, 150 mM NaCl) containing 1/50 (vol/vol) of protease
inhibitor cocktail (Calbiochem, La Jolla, Calif.)]. Equal amount of
proteins (80 .mu.g per well) were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to polyvinylidene fluoride (PVDF) membrane of 0.45
.mu.m pore size (Millipore, Bedford, Mass.). After blocking the
membranes with 5% nonfat dry milk plus 2% BSA at room temperature
for 2 hrs, membranes were incubated overnight at 4.degree. C. with
anti-RAS, c-RAF, phosphorylated (p)-MEK (Ser217/221), p-ERK
(Thr202/Tyr204), p-AKT, PI3 Kp110.alpha., cleaved PARP, SHg, GLI
and .beta.-actin mAb. The appropriate peroxidase-conjugated
secondary mAb (Cell signaling technology) was added and incubation
was continued at room temperature for an additional 1 hr. After
washing the membrane, the bound antibodies were detected using ECL
PLUS.TM. Western Blotting Detection System (GE Healthcare,
Buckinghamshire, UK), and bands were visualized using the
FOTO/ANALYST.RTM. Investigator Eclipse System (Fotodyne
Incorporate, Hartland, Wis.). The .beta.-actin was used as the
protein loading control.
SEQUENCE LISTING
[0029] The Sequence Listing is submitted as an ASCII text file
[8123-86316-06_Sequence_Listing.txt, May 2, 2014, 21.8 KB], which
is incorporated by reference herein.
[0030] The nucleic and amino acid sequences listed are shown using
standard letter abbreviations for nucleotide bases, and three
letter code for amino acids, as defined in 37 C.F.R. 1.822. Only
one strand of each nucleic acid sequence is shown, but the
complementary strand is understood as included by any reference to
the displayed strand.
[0031] SEQ ID NO: 1 is the amino acid sequence of GRP94.
[0032] SEQ ID NO: 2 is an exemplary nucleic acid sequence encoding
GRP94.
[0033] SEQ ID NO: 3 is the amino acid sequence of a heavy chain of
an antibody that specifically binds GRP94.
[0034] SEQ ID NO: 4 is the amino acid sequence of a light chain of
an antibody that specifically binds GRP94.
[0035] SEQ ID NO: 5 is the amino acid sequence of a Pseudomonas
exotoxin.
[0036] SEQ ID NOs: 6-7 are the amino acid sequence of Pseudomonas
exotoxin motifs.
DETAILED DESCRIPTION
[0037] Disclosed herein are methods for treating a tumor in a
subject. The methods include selecting a subject with a BRAF
mutation, and administering to the subject a therapeutically
effective amount of 1 an antibody that specifically binds glucose
regulated protein (GRP) 94; and 2) a BRAF inhibitor. The use of a
combination of an antibody that specifically binds GRP94 and a BRAF
inhibitor for the treatment of cancer provides an unexpectedly
superior result for the treatment of a tumor, wherein cells in the
tumor comprise a BRAF mutation. In some embodiments, the tumor is a
melanoma. In other embodiments, the BRAF mutation is a V600E
mutation. In additional embodiments, the subject has resistance to
the BRAF inhibitor. In some specific non-limiting examples, the
BRAF inhibitor is PLX4032 or PLX4720.
[0038] Disclosed herein are methods to treat a subject diagnosed
with a tumor, such as a tumor that expresses GRP94. Methods are
also provided for treating a melanoma. Melanoma includes spreading
melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo
maligna (melanoma). However, the methods disclosed herein can also
be used to treat other cancers, such breast cancer, prostate
cancer, ovarian cancer, thyroid cancer, colon cancer, stomach
cancer, pancreatic cancer, glioma, chordoma, chondrosarcoma, glioma
or a squamous cell carcinoma. Squamous cell carcinomas include, but
are not limited to head and neck squamous cell carcinoma, and
squamous cell cancers of the skin, lung, prostate, esophagus,
vagina and cervix.
[0039] In some embodiments, the disclosed methods can also be used
to prevent metastasis or decrease the number of micrometastases,
such as micrometastases to regional lymph nodes.
[0040] Pharmaceutical compositions are also provided that include a
therapeutically effective amount of an antibody that specifically
binds GRP94 and a therapeutically effective amount of a BRAF
inhibitor. In specific, non-liming examples, the BRAF inhibitor is
PLX4032 or PLX4720.
Terms
[0041] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0042] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0043] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects.
[0044] Antibody: A polypeptide ligand comprising at least a light
chain or heavy chain immunoglobulin variable region which
specifically recognizes and specifically binds an epitope of an
antigen, such as GRP94, or a fragment thereof. Antibodies are
composed of a heavy and a light chain, each of which has a variable
region, termed the variable heavy (V.sub.H) region and the variable
light (V.sub.L) region. Together, the V.sub.H region and the
V.sub.L region are responsible for binding the antigen recognized
by the antibody.
[0045] Antibodies include intact immunoglobulins and the variants
and portions of antibodies well known in the art, such as Fab
fragments, Fab' fragments, F(ab)'2 fragments, single chain Fv
proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A
scFv protein is a fusion protein in which a light chain variable
region of an immunoglobulin and a heavy chain variable region of an
immunoglobulin are bound by a linker, while in dsFvs, the chains
have been mutated to introduce a disulfide bond to stabilize the
association of the chains. The term also includes genetically
engineered forms such as chimeric antibodies (for example,
humanized murine antibodies), heteroconjugate antibodies (such as,
bispecific antibodies). See also, Pierce Catalog and Handbook,
1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,
Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York,
1997.
[0046] Typically, a naturally occurring immunoglobulin has heavy
(H) chains and light (L) chains interconnected by disulfide bonds.
There are two types of light chain, lambda (.lamda.) and kappa (k).
There are five main heavy chain classes (or isotypes) which
determine the functional activity of an antibody molecule: IgM,
IgD, IgG, IgA and IgE.
[0047] Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). In
combination, the heavy and the light chain variable regions
specifically bind the antigen. Light and heavy chain variable
regions contain a "framework" region interrupted by three
hypervariable regions, also called "complementarity-determining
regions" or "CDRs." The extent of the framework region and CDRs has
been defined (see, Kabat et al., Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human
Services, 1991, which is hereby incorporated by reference). The
Kabat database is now maintained online. The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species, such as humans. The framework region of
an antibody, that is the combined framework regions of the
constituent light and heavy chains, serves to position and align
the CDRs in three-dimensional space.
[0048] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found. An
antibody that binds GRP94 will have a specific V.sub.H region and
the V.sub.L region sequence, and thus specific CDR sequences.
Antibodies with different specificities (i.e. different combining
sites for different antigens) have different CDRs. Although it is
the CDRs that vary from antibody to antibody, only a limited number
of amino acid positions within the CDRs are directly involved in
antigen binding. These positions within the CDRs are called
specificity determining residues (SDRs).
[0049] References to "V.sub.H" or "VH" refer to the variable region
of an immunoglobulin heavy chain, including that of an Fv, scFv,
dsFv or Fab. References to "V.sub.L" or "VL" refer to the variable
region of an immunoglobulin light chain, including that of an Fv,
scFv, dsFv or Fab.
[0050] A "monoclonal antibody" is an antibody produced by a single
clone of B-lymphocytes or by a cell into which the light and heavy
chain genes of a single antibody have been transfected. Monoclonal
antibodies are produced by methods known to those of skill in the
art, for instance by making hybrid antibody-forming cells from a
fusion of myeloma cells with immune spleen cells. Monoclonal
antibodies include humanized monoclonal antibodies.
[0051] A "chimeric antibody" has framework residues from one
species, such as human, and CDRs (which generally confer antigen
binding) from another species, such as a murine antibody that
specifically binds GRP94.
[0052] A "human" antibody (also called a "fully human" antibody) is
an antibody that includes human framework regions and all of the
CDRs from a human immunoglobulin. In one example, the framework and
the CDRs are from the same originating human heavy and/or light
chain amino acid sequence. However, frameworks from one human
antibody can be engineered to include CDRs from a different human
antibody. A "humanized" immunoglobulin is an immunoglobulin
including a human framework region and one or more CDRs from a
non-human (for example a mouse, rat, or synthetic) immunoglobulin.
The non-human immunoglobulin providing the CDRs is termed a
"donor," and the human immunoglobulin providing the framework is
termed an "acceptor." In one embodiment, all the CDRs are from the
donor immunoglobulin in a humanized immunoglobulin. Constant
regions need not be present, but if they are, they must be
substantially identical to human immunoglobulin constant regions,
i.e., at least about 85-90%, such as about 95% or more identical.
Hence, all parts of a humanized immunoglobulin, except possibly the
CDRs, are substantially identical to corresponding parts of natural
human immunoglobulin sequences. A "humanized antibody" is an
antibody comprising a humanized light chain and a humanized heavy
chain immunoglobulin. A humanized antibody binds to the same
antigen as the donor antibody that provides the CDRs. The acceptor
framework of a humanized immunoglobulin or antibody may have a
limited number of substitutions by amino acids taken from the donor
framework. Humanized or other monoclonal antibodies can have
additional conservative amino acid substitutions which have
substantially no effect on antigen binding or other immunoglobulin
functions. Humanized immunoglobulins can be constructed by means of
genetic engineering (see for example, U.S. Pat. No. 5,585,089).
[0053] Binding affinity: Affinity of an antibody for an antigen. In
one embodiment, affinity is calculated by a modification of the
Scatchard method described by Frankel et al., Mol. Immunol.,
16:101-106, 1979. In another embodiment, binding affinity is
measured by an antigen/antibody dissociation rate.
[0054] In another embodiment, a high binding affinity is measured
by a competition radioimmunoassay. In another embodiment, binding
affinity is measured by ELISA. An antibody that "specifically
binds" an antigen, such as GRP94 with a high affinity and does not
significantly bind other unrelated antigens.
[0055] BRAF: A member of the Raf kinase family of
serine/threonine-specific protein kinases. This protein plays a
role in regulating the MAP kinase/ERKs signaling pathway, which
affects cell division, differentiation, and secretion. BRAF
transduces cellular regulatory signals from Ras to MEK in vivo.
BRAF is also referred to as v-raf murine sarcoma viral oncogene
homolog B1.
[0056] BRAF Mutant: A mutated form of BRAF that has increased basal
kinase activity relative to the basal kinase activity of wild type
BRAF is also an activated form of BRAF. More than 30 mutations of
the BRAF gene that are associated with human cancers have been
identified. The frequency of BRAF mutations in melanomas and nevi
are 80%. In 90% of the cases, a Glu for Val substitution at
position 600 (referred to as V600E) in the activation segment has
been found in human cancers. This mutation is observed in papillary
thyroid cancer, colorectal cancer and melanoma. Other mutations
which have been found are R462I, I463S, G464E, G464V, G466A, G466E,
G466V, G469A, G469E, N581S, E585K, D594V, F595L, G596R, L597V,
T599I, V600D, V600K, V600R, K601E or A728V. Most of these mutations
are clustered to two regions: the glycine-rich P loop of the N lobe
and the activation segment and flanking regions. A mutated form of
BRAF that induces focus formation more efficiently than wild type
BRAF is also an activated form of BRAF.
[0057] Breast cancer: A neoplastic condition of breast tissue that
can be benign or malignant. The most common type of breast cancer
is ductal carcinoma. Ductal carcinoma in situ is a non-invasive
neoplastic condition of the ducts. Lobular carcinoma is not an
invasive disease but is an indicator that a carcinoma may develop.
Infiltrating (malignant) carcinoma of the breast can be divided
into stages (I, IIA, IIB, IIIA, IIIB, and IV).
[0058] Chemotherapeutic agents: Any chemical agent with therapeutic
usefulness in the treatment of diseases characterized by abnormal
cell growth. Such diseases include tumors, neoplasms, and cancer as
well as diseases characterized by hyperplastic growth such as
psoriasis. In one embodiment, a chemotherapeutic agent is an agent
of use in treating a melanoma or another tumor. In one embodiment,
a chemotherapeutic agent is a radioactive compound. One of skill in
the art can readily identify a chemotherapeutic agent (see for
example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86
in Harrison's Principles of Internal Medicine, 14th edition; Perry
et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed.,
.COPYRGT. 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R.
(eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis,
Mosby-Year Book, 1995; Fischer, D. S., Knobf, M. F., Durivage, H.
J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis,
Mosby-Year Book, 1993). Combination chemotherapy is the
administration of more than one agent to treat cancer. One example
is the administration of an antibody that binds GRP94, used in
combination with a BRAF inhibitor, such as a chemical compound.
[0059] Decrease in survival: As used herein, "decrease in survival"
refers to a decrease in the length of time before death of a
patient, or an increase in the risk of death for the patient. A
decrease in survival also can refer to a decrease in the average
time to death in a group, such as a group of patients diagnosed
with a cancer, such as melanoma.
[0060] Diagnosing: Refers to the process of identifying the nature
or cause of a disease or disorder.
[0061] Effector molecule: The portion of a chimeric molecule that
is intended to have a desired effect on a cell to which the
chimeric molecule is targeted. Effector molecule is also known as
an effector moiety (EM), therapeutic agent, or diagnostic agent, or
similar terms.
[0062] Therapeutic agents include such compounds as nucleic acids,
proteins, peptides, amino acids or derivatives, glycoproteins,
radioisotopes, lipids, carbohydrates, or recombinant viruses.
Nucleic acid therapeutic and diagnostic moieties include antisense
nucleic acids, derivatized oligonucleotides for covalent
cross-linking with single or duplex DNA, and triplex forming
oligonucleotides. Alternatively, the molecule linked to a targeting
moiety, such as an anti-GRP94 antibody, may be an encapsulation
system, such as a liposome or micelle that contains a therapeutic
composition such as a drug, a nucleic acid (such as an antisense
nucleic acid), or another therapeutic moiety that can be shielded
from direct exposure to the circulatory system. Means of preparing
liposomes attached to antibodies are well known to those of skill
in the art (see, for example, U.S. Pat. No. 4,957,735; and Connor
et al., Pharm. Ther. 28:341-365, 1985). Diagnostic agents or
moieties include radioisotopes and other detectable labels.
Detectable labels useful for such purposes are also well known in
the art, and include radioactive isotopes such as .sup.35S,
.sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.19F, .sup.99mTc,
.sup.131I, .sup.3H, .sup.14C, .sup.15N, .sup.90Y, .sup.99Tc,
.sup.111In and .sup.125I, fluorophores, chemiluminescent agents,
and enzymes.
[0063] Epitope: An antigenic determinant. These are particular
chemical groups or peptide sequences on a molecule that are
antigenic, i.e. that elicit a specific immune response. An antibody
specifically binds a particular antigenic epitope on a polypeptide,
such as GRP94.
[0064] Glucose regulated protein (GRP).sub.94 (also known as
Endoplasmin): A protein which is the endoplasmic reticulum
(ER)-resident member of the heat-shock-protein 90 (Hsp90) family.
In vivo, hsp90 and GRP94 interact with client proteins and function
to protect them from ubiquitin-dependent proteasomal degradation.
Although the GRP94 protein is expressed constitutively in all cell
types, its expression is up-regulated under various stress
conditions including low glucose levels, low extracellular pH,
expression of mutated proteins, and viral infections. Heat-shock
proteins have a cytoprotective function and modulate apoptosis
directly or indirectly.
[0065] It has been shown that expression of GRP94 is increased in
tumor cells, including hepatocellular carcinoma, colorectal
carcinoma and lung cancer cells, and that GRP94 has an
anti-apoptotic effect on some tumor cells. Moreover, increased
levels of GRP94 were observed when a chronic hepatitis B virus
(HBV) infection progressed to cirrhosis and hepatocellular
carcinoma (HCC) Inhibitors of Hsp90 and GRP94 (such as geldanamycin
(GA) and its less toxic derivative 17-AAG) have been investigated
for efficacy in cancer treatment.
[0066] GRP94 (endoplasmin) may be encoded by the following genes,
but not limited thereto: GENBANK.RTM. Accession Nos.
NM.sub.--003299, BC066656 (Homo sapiens); NM.sub.--011631 (Mus
musculus); NM.sub.--001045763: (Xenopus (Silurana) tropicalis);
NM.sub.--214103 (Sus scrofa) NM.sub.--98210 (Danio rerio);
NM.sub.--001012197 (Rattus norvegicus); NM.sub.--001134101: Pongo
abelii; NM.sub.--001003327 (Canis lupus familiaris) heat shock
protein 90 kDa beta (GRP94); NM.sub.--204289 (Gallus gallus).
[0067] Expression Control Sequences: Nucleic acid sequences that
regulate the expression of a heterologous nucleic acid sequence to
which it is operatively linked. Expression control sequences are
operatively linked to a nucleic acid sequence when the expression
control sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signal for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of mRNA, and stop codons. The term "control
sequences" is intended to include, at a minimum, components whose
presence can influence expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0068] A promoter is a minimal sequence sufficient to direct
transcription. Also included are those promoter elements which are
sufficient to render promoter-dependent gene expression
controllable for cell-type specific, tissue-specific, or inducible
by external signals or agents; such elements may be located in the
5' or 3' regions of the gene. Both constitutive and inducible
promoters are included (see e.g., Bitter et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
lambda, plac, pap, ptac (ptrp-lac hybrid promoter) and the like may
be used. In one embodiment, when cloning in mammalian cell systems,
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
retrovirus long terminal repeat; the adenovirus late promoter; the
vaccinia virus 7.5K promoter) can be used. Promoters produced by
recombinant DNA or synthetic techniques may also be used to provide
for transcription of the nucleic acid sequences.
[0069] Inhibitor: As used herein, an "inhibitor" refers to any
compound that is capable of reducing or altering the expression or
activity of a target molecule. In some embodiments, the inhibitor
is an inhibitor of BRAF.
[0070] Isolated: An "isolated" biological component, such as a
nucleic acid, protein (including antibodies) or organelle, has been
substantially separated or purified away from other biological
components in the environment (such as a cell) in which the
component naturally occurs, i.e., other chromosomal and
extra-chromosomal DNA and RNA, proteins and organelles. Nucleic
acids and proteins that have been "isolated" include nucleic acids
and proteins purified by standard purification methods. The term
also embraces nucleic acids and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acids.
[0071] Melanoma: A form of cancer that originates in melanocytes
(cells that make the pigment melanin). Melanocytes are found
primarily in the skin, but are also present in the bowel and eye.
As used herein, "melanoma" refers to any stage of melanoma, or any
subtype of melanoma, such as superficial spreading melanoma,
nodular melanoma, acral lentiginous melanoma, lentigo maligna,
melanoma-in-situ, mucosal melanoma and uveal melanoma. Melanoma in
the skin includes superficial spreading melanoma, nodular melanoma,
acral lentiginous melanoma, and lentigo maligna (melanoma). Any of
the above types may produce melanin or can be amelanotic.
Similarly, any subtype may show desmoplasia (dense fibrous reaction
with neurotropism) which is a marker of aggressive behavior and a
tendency to local recurrence. Other melanomas include clear cell
sarcoma, mucosal melanoma and uveal melanoma.
[0072] Features that affect prognosis are tumor thickness in
millimeters (Breslow's depth), depth related to skin structures
(Clark level), type of melanoma, presence of ulceration, presence
of lymphatic/perineural invasion, presence of tumor infiltrating
lymphocytes (if present, prognosis is better), location of lesion,
presence of satellite lesions, and presence of regional or distant
metastasis. When melanomas have spread to the lymph nodes, one of
the most important factors is the number of nodes with malignancy.
The extent of malignancy within a node is also important;
micrometastases in which malignancy is only microscopic have a more
favorable prognosis than macrometastases. When there is distant
metastasis, the five year survival rate is less than 10 percent;
the median survival is 6 to 12 months. Metastases to skin and lungs
have a better prognosis. Metastases to brain, bone and liver are
associated with a worse prognosis.
[0073] Melanoma can be staged as follows: [0074] Stage 0: Melanoma
in Situ (Clark Level I), 100% Survival [0075] Stage I/II: Invasive
Melanoma, 85-95% Survival [0076] T1a: Less than 1.00 mm primary,
w/o Ulceration, Clark Level II-III [0077] T1b: Less than 1.00 mm
primary, w/Ulceration or Clark Level IV-V [0078] T2a: 1.00-2.00 mm
primary, w/o Ulceration [0079] Stage II: High Risk Melanoma, 40-85%
Survival [0080] T2b: 1.00-2.00 mm primary, w/Ulceration [0081] T3a:
2.00-4.00 mm primary, w/o Ulceration [0082] T3b: 2.00-4.00 mm
primary, w/Ulceration [0083] T4a: 4.00 mm or greater primary w/o
Ulceration [0084] T4b: 4.00 mm or greater primary w/Ulceration
[0085] Stage III: Regional Metastasis, 25-60% Survival [0086] N1:
Single Positive Lymph Node [0087] N2: 2-3 Positive Lymph Nodes OR
Regional Skin/In-Transit Metastasis [0088] N3: 4 Positive Lymph
Nodes OR Lymph Node and Regional Skin/In Transit Metastases [0089]
Stage IV: Distant Metastasis, 9-15% Survival [0090] M1a: Distant
Skin Metastasis, Normal lactate dehydrogenase (LDH) [0091] M1b:
Lung Metastasis, Normal LDH [0092] M1c: Other Distant Metastasis OR
Any Distant Metastasis with Elevated LDH
[0093] Metastasis: Refers to the spread of cancer cells from the
original tumor to other sites in the body.
[0094] Monoclonal antibody: An antibody produced by a single clone
of B-lymphocytes or by a cell into which the light and heavy chain
genes of a single antibody have been transfected. Monoclonal
antibodies are produced by methods known to those of skill in the
art, for instance by making hybrid antibody-forming cells from a
fusion of myeloma cells with immune spleen cells. Monoclonal
antibodies include humanized and fully human monoclonal antibodies.
As used herein a monoclonal antibody includes antibody fragments,
such as, but not limited to scFv, Fv, dsRv, or Fab.
[0095] Mutation: Any change of the DNA sequence within a gene or
chromosome. In some instances, a mutation will alter a
characteristic or trait (phenotype), but this is not always the
case. Types of mutations include base substitution point mutations
(e.g., transitions or transversions), deletions and insertions.
Missense mutations are those that introduce a different amino acid
into the sequence of the encoded protein; nonsense mutations are
those that introduce a new stop codon. In the case of insertions or
deletions, mutations can be in-frame (not changing the frame of the
overall sequence) or frame shift mutations, which may result in the
misreading of a large number of codons (and often leads to abnormal
termination of the encoded product due to the presence of a stop
codon in the alternative frame).
[0096] This term specifically encompasses variations that arise
through somatic mutation, for instance those that are found only in
disease cells (such as cancer cells), but not constitutionally, in
a given individual. Examples of such somatically-acquired
variations include the point mutations that frequently result in
altered function of various genes that are involved in development
of cancers. This term also encompasses DNA alterations that are
present constitutionally, that alter the function of the encoded
protein in a readily demonstrable manner, and that can be inherited
by the children of an affected individual. In this respect, the
term overlaps with "polymorphism," as discussed below, but
generally refers to the subset of constitutional alterations that
have arisen within the past few generations in a kindred and that
are not widely disseminated in a population group. In some
embodiments, a mutation in BRAF refers to a nucleotide substitution
in the BRAF gene or cDNA, or an amino acid substitution in the BRAF
protein.
[0097] Neoplasia, malignancy, cancer or tumor: The result of
abnormal and uncontrolled growth of cells. A neoplasm is an
abnormal growth of tissue or cells that results from excessive cell
division. Neoplastic growth can produce a tumor.
[0098] malignancy, cancer and tumor are often used interchangeably.
The amount of a tumor in an individual is the "tumor burden" which
can be measured as the number, volume, or weight of the tumor. A
tumor that does not metastasize is referred to as "benign." A tumor
that invades the surrounding tissue and/or can metastasize is
referred to as "malignant." A "non-cancerous tissue" is a tissue
from the same organ wherein the malignant neoplasm formed, but does
not have the characteristic pathology of the neoplasm. Generally,
noncancerous tissue appears histologically normal. A "normal
tissue" is tissue from an organ, wherein the organ is not affected
by cancer or another disease or disorder of that organ. A
"cancer-free" subject has not been diagnosed with a cancer of that
organ and does not have detectable cancer.
[0099] Examples of hematological tumors include leukemias,
including acute leukemias (such as 11q23-positive acute leukemia,
acute lymphocytic leukemia, acute myelocytic leukemia, acute
myelogenous leukemia and myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia), chronic leukemias
(such as chronic myelocytic (granulocytic) leukemia, chronic
myelogenous leukemia, and chronic lymphocytic leukemia),
polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's
lymphoma (indolent and high grade forms), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease,
myelodysplastic syndrome, hairy cell leukemia and
myelodysplasia.
[0100] Examples of solid tumors, such as sarcomas and carcinomas,
include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
lymphoid malignancy, pancreatic cancer, breast cancer (including
basal breast carcinoma, ductal carcinoma and lobular breast
carcinoma), lung cancers, ovarian cancer, prostate cancer,
hepatocellular carcinoma, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid
carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,
cervical cancer, testicular tumor, seminoma, bladder carcinoma, and
CNS tumors (such as a glioma, astrocytoma, medulloblastoma,
craniopharyogioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, menangioma, melanoma,
neuroblastoma and retinoblastoma).
[0101] In several examples, a tumor is melanoma, breast cancer,
prostate cancer, glioma or a squamous cell carcinoma, such as head
and neck cancer.
[0102] Nevi: Melanocytic lesions that can be considered regional
melanocytic hyperplasias. The term "nevi" includes all types of
nevi, such as congenital nevi, acquired nevi, intradermal nevi,
compound nevi, dysplastic nevi, atypical nevi, and junctional
nevi.
[0103] Nevus: The term "nevus" encompasses one or more nevi,
including one or more in vivo nevi cells and one or more in vitro
nevi cells. A "nevus" also encompasses one or more melanocytic
lesions that can be considered regional melanocytic hyperplasias.
The term "nevus" as used herein includes all types of nevi, such as
congenital nevi, acquired nevi, intradermal nevi, compound nevi,
dysplastic nevi, atypical nevi, and junctional nevi.
[0104] Nucleic acid: A polymer composed of nucleotide units
(ribonucleotides, deoxyribonucleotides, related naturally occurring
structural variants, and synthetic non-naturally occurring analogs
thereof) linked via phosphodiester bonds, related naturally
occurring structural variants, and synthetic non-naturally
occurring analogs thereof. Thus, the term includes nucleotide
polymers in which the nucleotides and the linkages between them
include non-naturally occurring synthetic analogs, such as, for
example and without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,
2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the
like. Such polynucleotides can be synthesized, for example, using
an automated DNA synthesizer. The term "oligonucleotide" typically
refers to short polynucleotides, generally no greater than about 50
nucleotides. It will be understood that when a nucleotide sequence
is represented by a DNA sequence (i.e., A, T, G, C), this also
includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces
"T."
[0105] Conventional notation is used herein to describe nucleotide
sequences: the left-hand end of a single-stranded nucleotide
sequence is the 5'-end; the left-hand direction of a
double-stranded nucleotide sequence is referred to as the
5'-direction. The direction of 5' to 3' addition of nucleotides to
nascent RNA transcripts is referred to as the transcription
direction. The DNA strand having the same sequence as an mRNA is
referred to as the "coding strand;" sequences on the DNA strand
having the same sequence as an mRNA transcribed from that DNA and
which are located 5' to the 5'-end of the RNA transcript are
referred to as "upstream sequences;" sequences on the DNA strand
having the same sequence as the RNA and which are 3' to the 3' end
of the coding RNA transcript are referred to as "downstream
sequences."
[0106] "cDNA" refers to a DNA that is complementary or identical to
an mRNA, in either single stranded or double stranded form.
[0107] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA produced by that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and
non-coding strand, used as the template for transcription, of a
gene or cDNA can be referred to as encoding the protein or other
product of that gene or cDNA. Unless otherwise specified, a
"nucleotide sequence encoding an amino acid sequence" includes all
nucleotide sequences that are degenerate versions of each other and
that encode the same amino acid sequence. Nucleotide sequences that
encode proteins and RNA may include introns.
[0108] "Recombinant nucleic acid" refers to a nucleic acid having
nucleotide sequences that are not naturally joined together. This
includes nucleic acid vectors comprising an amplified or assembled
nucleic acid which can be used to transform a suitable host cell. A
host cell that comprises the recombinant nucleic acid is referred
to as a "recombinant host cell." The gene is then expressed in the
recombinant host cell to produce, such as a "recombinant
polypeptide." A recombinant nucleic acid may serve a non-coding
function (such as a promoter, origin of replication,
ribosome-binding site, etc.) as well.
[0109] A first sequence is an "antisense" with respect to a second
sequence if a polynucleotide whose sequence is the first sequence
specifically hybridizes with a polynucleotide whose sequence is the
second sequence.
[0110] Terms used to describe sequence relationships between two or
more nucleotide sequences or amino acid sequences include
"reference sequence," "selected from," "comparison window,"
"identical," "percentage of sequence identity," "substantially
identical," "complementary," and "substantially complementary."
[0111] For sequence comparison of nucleic acid sequences, typically
one sequence acts as a reference sequence, to which test sequences
are compared. When using a sequence comparison algorithm, test and
reference sequences are entered into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. Default program parameters are
used. Methods of alignment of sequences for comparison are well
known in the art. Optimal alignment of sequences for comparison can
be conducted, for example, by the local homology algorithm of Smith
& Waterman, Adv. Appl. Math. 2:482, 1981, by the homology
alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
48:443, 1970, by the search for similarity method of Pearson &
Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see for example, Current Protocols
in Molecular Biology (Ausubel et al., eds 1995 supplement)).
[0112] One example of a useful algorithm is PILEUP. PILEUP uses a
simplification of the progressive alignment method of Feng &
Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is
similar to the method described by Higgins & Sharp, CABIOS
5:151-153, 1989. Using PILEUP, a reference sequence is compared to
other test sequences to determine the percent sequence identity
relationship using the following parameters: default gap weight
(3.00), default gap length weight (0.10), and weighted end gaps.
PILEUP can be obtained from the GCG sequence analysis software
package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.
12:387-395, 1984.
[0113] Another example of algorithms that are suitable for
determining percent sequence identity and sequence similarity are
the BLAST and the BLAST 2.0 algorithm, which are described in
Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et
al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information (http://www.ncbi nlm nih.gov/). The
BLASTN program (for nucleotide sequences) uses as defaults a word
length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5,
N=-4, and a comparison of both strands. The BLASTP program (for
amino acid sequences) uses as defaults a word length (W) of 3, and
expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,
1989).
[0114] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter, such as the
CMV promoter, is operably linked to a coding sequence if the
promoter affects the transcription or expression of the coding
sequence. Generally, operably linked DNA sequences are contiguous
and, where necessary to join two protein-coding regions, in the
same reading frame.
[0115] Patient: As used herein, the term "patient" includes human
and non-human animals. The preferred patient for treatment is a
human.
[0116] Pharmaceutically acceptable vehicles: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of one or more therapeutic compounds, molecules or agents.
[0117] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0118] Pharmaceutical agent: A chemical compound or composition
capable of inducing a desired therapeutic or prophylactic effect
when properly administered to a subject or a cell.
[0119] Polymorphism: Variant in a sequence of a gene, or any
genomic sequence, usually carried from one generation to another in
a population. Polymorphisms can be those variations (nucleotide
sequence differences) that, while having a different nucleotide
sequence, produce functionally equivalent gene products, such as
those variations generally found between individuals, different
ethnic groups, and geographic locations. The term polymorphism also
encompasses variations that produce gene products with altered
function, i.e., variants in the gene sequence that lead to gene
products that are not functionally equivalent. This term also
encompasses variations that produce no gene product, an inactive
gene product, a truncated gene product, or increased or increased
activity gene product.
[0120] Polymorphisms can be referred to, for instance, by the
nucleotide position at which the variation exists, by the change in
amino acid sequence caused by the nucleotide variation, or by a
change in some other characteristic of the nucleic acid molecule or
protein that is linked to the variation (e.g., an alteration of a
secondary structure such as a stem-loop, or an alteration of the
binding affinity of the nucleic acid for associated molecules, such
as polymerases, RNAses, a change in the availability of a site for
cleavage by a restriction endonuclease, either the formation of a
new site, or lose of a site, and so forth).
[0121] Polypeptide: A polymer in which the monomers are amino acid
residues which are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used. The terms "polypeptide" or
"protein" as used herein are intended to encompass any amino acid
sequence and include modified sequences such as glycoproteins. The
term "polypeptide" is specifically intended to cover naturally
occurring proteins, as well as those which are recombinantly or
synthetically produced.
[0122] The term "residue" or "amino acid residue" includes
reference to an amino acid that is incorporated into a protein,
polypeptide, or peptide.
[0123] Conservative amino acid substitutions are those
substitutions that, when made, least interfere with the properties
of the original protein, that is, the structure and especially the
function of the protein is conserved and not significantly changed
by such substitutions. Examples of conservative substitutions are
shown in the following table:
TABLE-US-00001 Original Residue Conservative Substitutions Ala Ser
Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln
Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met;
Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu
[0124] Conservative substitutions generally maintain (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain.
[0125] The substitutions which in general are expected to produce
the greatest changes in protein properties will be
non-conservative, for instance changes in which (a) a hydrophilic
residue, for example, seryl or threonyl, is substituted for (or by)
a hydrophobic residue, for example, leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, for example, lysyl, arginyl, or
histadyl, is substituted for (or by) an electronegative residue,
for example, glutamyl or aspartyl; or (d) a residue having a bulky
side chain, for example, phenylalanine, is substituted for (or by)
one not having a side chain, for example, glycine.
[0126] Preventing, treating or ameliorating a disease: "Preventing"
a disease (such as metastatic melanoma) refers to inhibiting the
full development of a disease. "Treating" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition after it has begun to develop.
"Ameliorating" refers to the reduction in the number or severity of
signs or symptoms of a disease.
[0127] Prognosis: The likelihood of the clinical outcome for a
subject afflicted with a specific disease or disorder. With regard
to cancer, the prognosis is a representation of the likelihood
(probability) that the subject will survive (such as for one, two,
three, four or five years) and/or the likelihood (probability) that
the tumor will metastasize. A "poor prognosis" indicates a greater
than 50% chance that the subject will not survive to a specified
time point (such as one, two, three, four or five years), and/or a
greater than 50% chance that the tumor will metastasize. In several
examples, a poor prognosis indicates that there is a greater than
60%, 70%, 80%, or 90% chance that the subject will not survive
and/or a greater than 60%, 70%, 80% or 90% chance that the tumor
will metastasize. Conversely, a "good prognosis" indicates a
greater than 50% chance that the subject will survive to a
specified time point (such as one, two, three, four or five years),
and/or a greater than 50% chance that the tumor will not
metastasize. In several examples, a good prognosis indicates that
there is a greater than 60%, 70%, 80%, or 90% chance that the
subject will survive and/or a greater than 60%, 70%, 80% or 90%
chance that the tumor will not metastasize.
[0128] Proliferation: One or more cellular events that result in
cell growth. Proliferation includes any of a number of growth
activities including increase in the number of cells, increase in
the rate of cell division, increase in the number of cell
divisions, increase in the size of a cell, change in cellular
differentiation, transformation to a malignant state, metastatic
transformation, change in cell cycle phase to a more mitotically
active cell cycle phase (e.g., S phase), or a combination of two or
more of those activities. Cell growth (either in vitro or in vivo)
can be a hyper-proliferative condition, such as is characteristic
of certain disorders or diseases, for instance neoplasia or tumor
formation.
[0129] Inhibiting proliferation includes any of a number of
anti-growth activities that reduce or even eliminate the ability of
a cell to proliferate Inhibiting proliferation includes, for
instance, decreasing cell number, decreasing colony forming
ability, decreasing the rate of cell division, decreasing the
number of cell divisions, stopping cell division, inducing
apoptosis, inducing senescence, inducing quiescence, changing cell
cycle phase to a less mitotically active cell cycle phase,
decreasing cellular de-differentiation, preventing transformation
to a malignancy, decreasing malignant potential, decreasing
metastatic ability or potential or a combination of two or more of
those activities.
[0130] Resistance: The lack of response of a disease, such as a
cancer, to a therapeutic agent. In some embodiment, the agent is a
BRAF inhibitor. Primary resistance is the lack of a response to a
therapeutic agent upon initial treatment with the agent. Secondary
resistance is the lack of a response to a therapeutic agent,
wherein the cancer in the subject is initially susceptible to
treatment with the agent. Resistance of a cancer to an agent can be
measured by an increase in tumor burden, an increase in the number
of metastases, or an increase in the amount of a tumor marker
present in the subject.
[0131] Sample: A biological specimen containing genomic DNA, RNA,
protein, or combinations thereof, obtained from a subject. Examples
include, but are not limited to, peripheral blood, urine, saliva,
tissue biopsy (such as skin tissue), surgical specimen, and autopsy
material. In one example, a sample includes a biopsy of a melanoma
tumor or a sample of normal tissue, such as skin tissue (from a
subject not afflicted with a known disease or disorder, such as a
cancer-free subject).
[0132] Somatic mutation: An acquired mutation that occurs in a
somatic cell (as opposed to a germ cell).
[0133] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and non-human mammals. In some
embodiments, the subject is a human subject.
[0134] Therapeutic agent: A chemical compound, small molecule, or
other composition, such as an antisense compound, antibody, peptide
or nucleic acid molecule capable of inducing a desired therapeutic
or prophylactic effect when properly administered to a subject. For
example, therapeutic agents for melanoma include agents that
prevent or inhibit development or metastasis of melanoma. In some
embodiments, the therapeutic agent is an inhibitor of BRAF.
[0135] Therapy: The mode of treatment or care of a patient. In some
cases, therapy refers to administration of a therapeutic agent. In
some embodiments herein, therapy includes administration of a BRAF
inhibitor. In other examples, therapy includes surgery, such as
surgical resection of a melanoma tumor, chemotherapy, radiation
therapy, or any combination thereof.
[0136] Therapeutically effective amount: A quantity of an agent
sufficient to achieve a desired effect in a subject or a cell being
treated. For instance, this can be the amount necessary to inhibit
or to measurably reduce B-Raf activity in a nevi or to inhibit
melanoma proliferation. A therapeutically effective amount of an
agent may be administered in a single dose, or in several doses,
for example daily or more often, during a course of treatment.
However, the effective amount will be dependent on the particular
agent applied, the subject being treated, the severity and type of
the affliction, and the manner of administration.
[0137] Treating: Includes inhibiting or preventing the partial or
full development or progression of a disease or medical condition
or abnormal biological state in a subject, for example in a person
who is known to have a predisposition to or to be at risk for the
disease or medical condition or abnormal biological state, or a
cell or a lesion, for instance a nevus or a melanoma. Furthermore,
"treating" refers to a therapeutic intervention that ameliorates at
least one sign or symptom of a disease or pathological condition,
or interferes with a pathophysiological process, after the disease
or pathological condition has begun to develop. The therapeutic
intervention can be prophylactic inhibition of a disease or medical
condition or biological state, and therapeutic interventions to
alter the natural course of an untreated disease process or medical
condition or a biological state, such as a tumor growth. The
therapeutic intervention can be surgical, including but not limited
to cryosurgery or ablation, such as laser ablation, and
administration of agents, systemically, regionally, or topically or
locally.
[0138] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including explanations of terms, will control. In addition, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
Antibodies that Specifically Bind GRP94, Antigen Binding Fragments
and Immunotoxins
[0139] Antibodies have been produced that specifically bind GRP94
(also known as endoplasmin) including monoclonal antibodies, such
as fully human monoclonal antibodies. These antibodies and/or
antigen binding fragments thereof can be used in the methods
disclosed herein. These antibodies can be conjugated to labels or
effector molecules. The antibodies used in the disclosed methods
can be fully human monoclonal antibodies and functional fragments
thereof that specifically bind GRP 94 (endoplasmin)
[0140] Exemplary GRP94 antibodies of use in the methods and
compositions disclosed herein include those listed in the following
table:
TABLE-US-00002 Name Source Clonality/Source HSP90B1 antibody (Cat.
# 60012-1-Ig) Proteintech Group monoclonal, mouse HSP90B1 antibody
(Cat. # 14700-1-AP) Proteintech Group polyclonal, rabbit HSP90B1
antibody (Cat. # 10979-1-AP) Proteintech Group polyclonal, rabbit
Human TRA-1-60 MAb (clone TRA- R & D Systems monoclonal, mouse
1-60) Human TRA-1-85 MAb (clone TRA- R & D Systems monoclonal,
mouse 1-85) Human TRA-1-85 phycoerythrin R & D Systems
monoclonal, mouse MAb (clone TRA-1-85) GRP94 polyclonal antibody
Enzo Life Sciences polyclonal, rabbit GRP94 monoclonal antibody
(9G10), Enzo Life Sciences monoclonal, mouse PE GRP94 monoclonal
antibody (9G10), Enzo Life Sciences monoclonal, rat DyLight 488
Anti-human TRA1 antibody (clone ATGen monoclonal, mouse 2H3)
HSP90B1 antibody Epitomics polyclonal, rabbit HSP90B antibody
Epitomics polyclonal, rabbit GRP94 antibody Epitomics monoclonal,
rabbit HSP90B1 antibody OriGene polyclonal, goat Anti-GRP94/TRA1
antibody Everest Biotech polyclonal, goat Anti-HSP90B1 antibody
AbDSerotec polyclonal, rabbit Anti-human GRP94 antibody AbDSerotec
polyclonal, rabbit Anti-human TRA1 antibody MyBioSource polyclonal,
goat Anti-human TRA1 antibody (clone MyBioSource monoclonal, mouse
2H3) Anti-GRP94 antibody MyBioSource monoclonal, rat HSP90B1
antibody GeneTex polyclonal, rabbit GRP94 antibody Abcam
monoclonal, mouse Alexa Fluor .TM. 488 anti-human TRA- BioLegend
monoclonal, mouse 1-60-R PE anti-human TRA-1-60-R BioLegend
monoclonal, mouse Heat Shock Protein 94 antibody Thermo Fisher
polyclonal, rabbit Scientific Glucose-Regulated Protein 94 Thermo
Fisher polyclonal, rabbit Scientific Anti-human Heat Shock Protein
ProSpec monoclonal, mouse 90 kDa Beta (GRP94) Member 1 antibody
Anti-GRP94 StressMarq monoclonal, rat Biosciences GRP94 antibody
Abbiotec polyclonal, rabbit HSP90B1 antibody (N-term) Abgent
polyclonal, rabbit GRP94 antibody US Biological polyclonal, rabbit
TRA-1-60 antibody US Biological monoclonal, mouse GRP94 antibody US
Biological polyclonal, goat GRP94 antibody Novus Biologicals
polyclonal, rabbit TRA-1-60 antibody (MG38) Novus Biologicals
monoclonal, mouse TRA-1-60(S) antibody Cell Signaling monoclonal,
mouse Technology TRA-1-85 mAb Cell Signaling monoclonal, mouse
Technology HSP90B1 anti-mouse polyclonal LifeSpan Biosciences
polyclonal, rabbit antibody HSP90B1 anti-human polyclonal LifeSpan
Biosciences polyclonal, goat antibody HSP90B1 anti-chicken
monoclonal LifeSpan Biosciences monoclonal, rat antibody (9G10) GRP
94 (H-212) Santa Cruz polyclonal, rabbit Biotechnology GRP 94
(C-19) Santa Cruz polyclonal, goat Biotechnology GRP 94 (4E89)
Santa Cruz monoclonal, rabbit Biotechnology Glucose Regulated
Protein 94 Biotrend polyclonal, rat antibody Anti-TRA-1-60, clone
TRA-1-60 Millipore monoclonal, mouse
[0141] Humanized forms and antigen binding fragments of these
antibodies are also of use in the presently disclosed methods.
[0142] In one example, human GRP94 (also known as endoplasmin) has
an amino acid sequence set forth as:
TABLE-US-00003 SEQ ID NO: 1
MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRT
DDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLR
ELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELV
KNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNND
TQHINESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFIN
FPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWE
LMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTS
APRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRET
LQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAK
LLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVI
YLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKD
KALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQK
KTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIE
RMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTA EKDEL,
See also GENBANK .RTM. Accession No. NM 003299 incorporated herein
by reference.
[0143] In another example, the GRP94 is encoded by the nucleic acid
sequence set forth as:
TABLE-US-00004 SEQ ID NO: 2 gtgggcggac cgcgcggctg gaggtgtgag
gatccgaacc caggggtggg gggtggaggc ggctcctgcg atcgaagggg acttgagact
caccggccgc acgccatgag ggccctgtgg gtgctgggcc tctgctgcgt cctgctgacc
ttcgggtcgg tcagagctga cgatgaagtt gatgtggatg gtacagtaga agaggatctg
ggtaaaagta gagaaggatc aaggacggat gatgaagtag tacagagaga ggaagaagct
attcagttgg atggattaaa tgcatcacaa ataagagaac ttagagagaa gtcggaaaag
tttgccttcc aagccgaagt taacagaatg atgaaactta tcatcaattc attgtataaa
aataaagaga ttttcctgag agaactgatt tcaaatgctt ctgatgcttt agataagata
aggctaatat cactgactga tgaaaatgct ctttctggaa atgaggaact aacagtcaaa
attaagtgtg ataaggagaa gaacctgctg catgtcacag acaccggtgt aggaatgacc
agagaagagt tggttaaaaa ccttggtacc atagccaaat ctgggacaag cgagttttta
aacaaaatga ctgaagcaca ggaagatggc cagtcaactt ctgaattgat tggccagttt
ggtgtcggtt tctattccgc cttccttgta gcagataagg ttattgtcac ttcaaaacac
aacaacgata cccagcacat ctgggagtct gactccaatg aattttctgt aattgctgac
ccaagaggaa acactctagg acggggaacg acaattaccc ttgtcttaaa agaagaagca
tctgattacc ttgaattgga tacaattaaa aatctcgtca aaaaatattc acagttcata
aactttccta tttatgtatg gagcagcaag actgaaactg ttgaggagcc catggaggaa
gaagaagcag ccaaagaaga gaaagaagaa tctgatgatg aagctgcagt agaggaagaa
gaagaagaaa agaaaccaaa gactaaaaaa gttgaaaaaa ctgtctggga ctgggaactt
atgaatgata tcaaaccaat atggcagaga ccatcaaaag aagtagaaga agatgaatac
aaagctttct acaaatcatt ttcaaaggaa agtgatgacc ccatggctta tattcacttt
actgctgaag gggaagttac cttcaaatca attttatttg tacccacatc tgctccacgt
ggtctgtttg acgaatatgg atctaaaaag agcgattaca ttaagctcta tgtgcgccgt
gtattcatca cagacgactt ccatgatatg atgcctaaat acctcaattt tgtcaagggt
gtggtggact cagatgatct ccccttgaat gtttcccgcg agactcttca gcaacataaa
ctgcttaagg tgattaggaa gaagcttgtt cgtaaaacgc tggacatgat caagaagatt
gctgatgata aatacaatga tactttttgg aaagaatttg gtaccaacat caagcttggt
gtgattgaag accactcgaa tcgaacacgt cttgctaaac ttcttaggtt ccagtcttct
catcatccaa ctgacattac tagcctagac cagtatgtgg aaagaatgaa ggaaaaacaa
gacaaaatct acttcatggc tgggtccagc agaaaagagg ctgaatcttc tccatttgtt
gagcgacttc tgaaaaaggg ctatgaagtt atttacctca cagaacctgt ggatgaatac
tgtattcagg cccttcccga atttgatggg aagaggttcc agaatgttgc caaggaagga
gtgaagttcg atgaaagtga gaaaactaag gagagtcgtg aagcagttga gaaagaattt
gagcctctgc tgaattggat gaaagataaa gcccttaagg acaagattga aaaggctgtg
gtgtctcagc gcctgacaga atctccgtgt gctttggtgg ccagccagta cggatggtct
ggcaacatgg agagaatcat gaaagcacaa gcgtaccaaa cgggcaagga catctctaca
aattactatg cgagtcagaa gaaaacattt gaaattaatc ccagacaccc gctgatcaga
gacatgcttc gacgaattaa ggaagatgaa gatgataaaa cagttttgga tcttgctgtg
gttttgtttg aaacagcaac gcttcggtca gggtatcttt taccagacac taaagcatat
ggagatagaa tagaaagaat gcttcgcctc agtttgaaca ttgaccctga tgcaaaggtg
gaagaagagc ccgaagaaga acctgaagag acagcagaag acacaacaga agacacagag
caagacgaag atgaagaaat ggatgtggga acagatgaag aagaagaaac agcaaaggaa
tctacagctg aaaaagatga attgtaaatt atactctcac catttggatc ctgtgtggag
agggaatgtg aaatttacat catttctttt tgggagagac ttgttttgga tgccccctaa
tccccttctc ccctgcactg taaaatgtgg gattatgggt cacaggaaaa agtgggtttt
ttagttgaat tttttttaac attcctcatg aatgtaaatt tgtactattt aactgactat
tcttgatgta aaatcttgtc atgtgtataa aaataaaaaa gatcccaaat, see also
GENBANK .RTM. Accession No. NM 003299, incorporated herein by
reference.
[0144] Once of skill in the art can readily use a nucleic acid
sequence to produce a polypeptide, such as GRP94 using standard
method in molecular biology (see, for example, Molecular Cloning: A
Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0145] Described herein are methods for using isolated human
monoclonal antibodies and fragments thereof that specifically bind
human GRP94 (endoplasmin) for the treatment of cancer, such as, but
not limited to, melanoma. In some embodiments, the human monoclonal
antibody functional fragment is a scFv. Also described are
compositions including a monoclonal antibody or antigen fragment
thereof, a BRAF inhibitor, and a pharmaceutically acceptable
carrier. Nucleic acids encoding these antibodies, expression
vectors comprising these nucleic acids, and isolated host cells
that express the nucleic acids are also provided. Thus, in other
embodiments, compositions are provided that include a nucleic acid
encoding a monoclonal antibody or antigen fragment thereof, a BRAF
inhibitor, and a pharmaceutically acceptable carrier
[0146] In some embodiments, the human monoclonal antibody or
functional fragment thereof comprises at least a portion of the
variable chain of the heavy chain amino acid sequence set forth as
SEQ ID NO: 3 and specifically binds GRP94. For example, the human
monoclonal antibody can include the SDRs (specificity determining
residues), the CDRs, or the variable region of the amino acid
sequence set forth as SEQ ID NO: 3. In the amino acid sequence
shown below, the constant region is in bold, and the CDRs are
underlined:
TABLE-US-00005 (SEQ ID NO: 3) ##STR00001## ##STR00002##
##STR00003## S V F P L A P S S K S T S G G T A A L G C L V K D Y F
P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S
V V T V P S S S L G T Q T Y I C N V N H K P S N T K V D K K V E P K
S C D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M
I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N
A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K
C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S
R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E
N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V
F S C S V M H E A L H N H Y T Q K S L S L S P G K
[0147] In some embodiments, the monoclonal antibody or functional
fragment thereof comprises at least a portion of the heavy chain
amino acid sequence set forth as SEQ ID NO: 3 and specifically
binds GRP94. The monoclonal antibody can be a human monoclonal
antibody. In some examples, at least one of the CDRs of the light
chain of the antibody comprises one or more of the amino acid
sequences set forth as amino acids 26-33 of SEQ ID NO: 3 (CDR1),
amino acids 51-58 of SEQ ID NO: 3 (CDR2), and amino acids 97-103 of
SEQ ID NO: 3 (CDR3). In additional examples, the heavy chain of the
antibody comprises the amino acid sequence set forth as amino acids
26-33 of SEQ ID NO: 3 (CDR1), amino acids 51-58 of SEQ ID NO: 3
(CDR2), and amino acids 97-103 of SEQ ID NO: 3 (CDR3). In some
examples, the variable region of the heavy chain of the antibody
can include, or consist of, amino acids 1-113 of SEQ ID NO: 3. The
heavy chain of the antibody can include, or consist of, SEQ ID NO:
3.
[0148] In some embodiments, the monoclonal antibody or functional
fragment thereof comprises at least a portion of the variable
region of the light chain amino acid sequence set forth as SEQ ID
NO: 4 and specifically binds GRP94. The monoclonal antibody can be
a human monoclonal antibody. In the amino acid sequence shown
below, the constant region is in bold, and the CDRs are
underlined:
TABLE-US-00006 (SEQ ID NO: 4) ##STR00004## ##STR00005##
##STR00006## S G T A S V V C L L N N F Y P R E A K V Q W K V D N A
L Q S G N S Q E S V T E Q D S K D S T Y S L S S T L T L S K A D Y E
K H K V Y A C E V T H Q G L S S P V T K S F N R G E C
In some examples, at least one of the CDRs of the light chain of
the antibody comprises one or more of the amino acid sequences set
forth as amino acids 27-32 of SEQ ID NO: 8 (CDR1), amino acids
50-52 of SEQ ID NO: 4 (CDR2), and amino acids 89-97 of SEQ ID NO: 4
(CDR3). In additional examples, the light chain of the antibody
comprises amino acids amino acids 27-32 of SEQ ID NO: 4 (CDR1),
amino acids 50-52 of SEQ ID NO: 4 (CDR2), and amino acids 89-97 of
SEQ ID NO: 4 (CDR3). The variable region of the light chain of the
antibody can include, or consist of, amino acids 1-107 of SEQ ID
NO: 4. The light chain of the antibody can include, or consist of,
SEQ ID NO: 4.
[0149] Fully human monoclonal antibodies include human framework
regions. The human framework regions can include the framework
regions disclosed in one or both of SEQ ID NO: 3 or SEQ ID NO: 4
(these sequences include CDR sequences as well as framework
sequences). However, the framework regions can be from another
source. Additional examples of framework sequences that can be used
include the amino acid framework sequences of the heavy and light
chains disclosed in PCT Publication No. WO 2006/074071 (see, for
example, SEQ ID NOs: 1-16), which is herein incorporated by
reference.
[0150] Chimeric antibodies include CDRs from one species, and
framework regions (and/or a constant domain), from another species.
In some embodiments, the monoclonal antibody, or antigen binding
fragment, is chimeric. In one specific non-limiting example, the
monoclonal antibody includes the CDRs from a murine antibody, and a
human framework region. In another specific non-liming example, the
monoclonal antibody includes the CDRs from a rabbit antibody, and a
human framework region.
[0151] The monoclonal antibody of use in the disclosed methods can
be of any isotype. The monoclonal antibody can be, for example, an
IgA, IgM or an IgG antibody, such as IgG.sub.1 or an IgG.sub.2. The
class of an antibody that specifically binds GRP94 can be switched
with another. In one aspect, a nucleic acid molecule encoding
V.sub.L or V.sub.H is isolated using methods well-known in the art,
such that it does not include any nucleic acid sequences encoding
the constant region of the light or heavy chain, respectively. The
nucleic acid molecule encoding V.sub.L or V.sub.H is then
operatively linked to a nucleic acid sequence encoding a C.sub.L or
C.sub.H from a different class of immunoglobulin molecule. This can
be achieved using a vector or nucleic acid molecule that comprises
a C.sub.L or C.sub.H chain, as known in the art. For example, an
antibody that specifically binds GRP94 that was originally IgM may
be class switched to an IgG. Class switching can be used to convert
one IgG subclass to another, such as from IgG.sub.1 to
IgG.sub.2.
[0152] The method disclosed herein can utilize immunoconjugates
comprising the monoclonal antibodies or functional fragment thereof
that specifically binds human GRP94, such as human monoclonal
antibodies. The immunoconjugates can comprise any therapeutic
agent, toxin or other moiety. In one example, the toxin is PE or a
variant or fragment thereof.
[0153] Antibody fragments that specifically bind GRP94 are
encompassed by the present disclosure, such as Fab, F(ab).sub.2,
and Fv which include a heavy chain and light chain variable region
and are capable of binding the epitopic determinant on GRP94. These
antibody fragments retain the ability to specifically bind with the
antigen. These fragments include:
[0154] (1) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule, can be produced
by digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain;
[0155] (2) Fab', the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody
molecule;
[0156] (3) (Fab).sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds;
[0157] (4) Fv, a genetically engineered fragment containing the
variable region of the light chain and the variable region of the
heavy chain expressed as two chains; and
[0158] (5) Single chain antibody (such as scFv), defined as a
genetically engineered molecule containing the variable region of
the light chain, the variable region of the heavy chain, linked by
a suitable polypeptide linker as a genetically fused single chain
molecule.
[0159] (6) A dimer of a single chain antibody (scFV.sub.2), defined
as a dimer of a scFV. This has also been termed a
"miniantibody."
[0160] Methods of making these fragments are known in the art (see
for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988). In several examples, the
variable region included in the antibody is the variable region of
M912.
[0161] In a further group of embodiments, the antibodies are Fv
antibodies, which are typically about 25 kDa and contain a complete
antigen-binding site with three CDRs per each heavy chain and each
light chain. To produce these antibodies, the V.sub.H and the
V.sub.L can be expressed from two individual nucleic acid
constructs in a host cell. If the V.sub.H and the V.sub.L are
expressed non-contiguously, the chains of the Fv antibody are
typically held together by noncovalent interactions. However, these
chains tend to dissociate upon dilution, so methods have been
developed to crosslink the chains through glutaraldehyde,
intermolecular disulfides, or a peptide linker. Thus, in one
example, the Fv can be a disulfide stabilized Fv (dsFv), wherein
the heavy chain variable region and the light chain variable region
are chemically linked by disulfide bonds.
[0162] In an additional example, the Fv fragments comprise V.sub.H
and V.sub.L chains connected by a peptide linker. These
single-chain antigen binding proteins (scFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the V.sub.H and V.sub.L domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
scFvs are known in the art (see Whitlow et al., Methods: a
Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et
al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al.,
Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of a
single chain antibody (scFV2), are also contemplated.
[0163] The antibody can also be included in a bi-specific antibody.
In further embodiments, an engineered antibody domain, including
the heavy chain CDRs or the light chain CDRs can be utilized,
provided the engineered antibody domain specifically binds
GRP94.
[0164] Antibody fragments can be prepared by proteolytic hydrolysis
of the antibody or by expression in E. coli of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No.
4,331,647, and references contained therein; Nisonhoff et al.,
Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119,
1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422,
Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10
and 2.10.1-2.10.4).
[0165] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0166] One of skill will realize that conservative variants of the
antibodies can be produced. Such conservative variants employed in
antibody fragments, such as dsFv fragments or in scFv fragments,
will retain critical amino acid residues necessary for correct
folding and stabilizing between the V.sub.H and the V.sub.L
regions, and will retain the charge characteristics of the residues
in order to preserve the low pI and low toxicity of the molecules
Amino acid substitutions (such as at most one, at most two, at most
three, at most four, or at most five amino acid substitutions) can
be made in the V.sub.H and the V.sub.L regions to increase yield.
Conservative amino acid substitution tables providing functionally
similar amino acids are well known to one of ordinary skill in the
art.
[0167] In some embodiments, the disclosed methods utilize
immunoconjugates. Immunoconjugates include, but are not limited to,
molecules in which there is a covalent linkage of a diagnostic or
therapeutic agent with an antibody. A therapeutic agent is an agent
with a particular biological activity directed against a particular
target molecule or a cell bearing a target molecule. Therapeutic
agents include various drugs such as vinblastine, daunomycin and
the like, and effector molecules such as cytotoxins such as native
or modified Pseudomonas exotoxin or Diphtheria toxin, encapsulating
agents (e.g., liposomes), which themselves contain pharmacological
compositions, target moieties and ligands.
[0168] The choice of a particular therapeutic agent depends on the
particular target molecule or cell and the biological effect
desired. Thus, for example, the therapeutic agent may be an
effector molecule that is cytotoxin which is used to bring about
the death of a particular target cell. Conversely, where it is
merely desired to invoke a non-lethal biological response, a
therapeutic agent can be conjugated to a non-lethal pharmacological
agent or a liposome containing a non-lethal pharmacological
agent.
[0169] Toxins can be employed with antibodies that bind GRP94
polypeptide and antigen biding antibody fragments, such as a svFv
or a dsFv, to yield chimeric molecules, which are of use as
immunotoxins. Exemplary toxins include Pseudomonas exotoxin (PE),
ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin,
ribonuclease, saporin, and calicheamicin, as well as botulinum
toxins A through F. These toxins are well known in the art and many
are readily available from commercial sources (for example, Sigma
Chemical Company, St. Louis, Mo.).
[0170] Diphtheria toxin is isolated from Corynebacterium
diphtheriae. Typically, diphtheria toxin for use in immunotoxins is
mutated to reduce or to eliminate non-specific toxicity. A mutant
known as CRM107, which has full enzymatic activity but markedly
reduced non-specific toxicity, has been known since the 1970's
(Laird and Groman, J. Virol. 19:220, 1976), and has been used in
human clinical trials. See, U.S. Pat. No. 5,792,458 and U.S. Pat.
No. 5,208,021. As used herein, the term "diphtheria toxin" refers
as appropriate to native diphtheria toxin or to diphtheria toxin
that retains enzymatic activity but which has been modified to
reduce non-specific toxicity.
[0171] Ricin is the lectin RCA60 from Ricinus communis (Castor
bean). The term "ricin" also references toxic variants thereof. For
example, see U.S. Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401.
Ricinus communis agglutinin (RCA) occurs in two forms designated
RCA.sub.60 and RCA.sub.120 according to their molecular weights of
approximately 65 and 120 kD, respectively (Nicholson &
Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is
responsible for inactivating protein synthesis and killing cells.
The B chain binds ricin to cell-surface galactose residues and
facilitates transport of the A chain into the cytosol (Olsnes et
al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).
[0172] Ribonucleases have also been conjugated to targeting
molecules for use as immunotoxins (see Suzuki et al., Nat Biotech
17:265-270, 1999). Exemplary ribotoxins such as .alpha.-sarcin and
restrictocin are discussed in, e.g., Rathore et al., Gene
190:31-35, 1997; and Goyal and Batra, Biochem 345 Pt 2:247-254,
2000. Calicheamicins were first isolated from Micromonospora
echinospora and are members of the enediyne antitumor antibiotic
family that cause double strand breaks in DNA that lead to
apoptosis (see, e.g., Lee et al., J. Antibiot 42:1070-1087.
1989).
[0173] The drug is the toxic moiety of an immunotoxin in clinical
trials (see, e.g., Gillespie et al., Ann Oncol 11:735-741,
2000).
[0174] Abrin includes toxic lectins from Abrus precatorius. The
toxic principles, abrin a, b, c, and d, have a molecular weight of
from about 63 and 67 kD and are composed of two disulfide-linked
polypeptide chains A and B. The A chain inhibits protein synthesis;
the B-chain (abrin-b) binds to D-galactose residues (see, Funatsu
et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods
Enzymol. 50:330-335, 1978).
[0175] In one embodiment, the toxin is Pseudomonas exotoxin (PE).
Native Pseudomonas exotoxin A ("PE") is an extremely active
monomeric protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells.
The native PE sequence and the sequence of modified PE are provided
in U.S. Pat. No. 5,602,095, incorporated herein by reference. In
one embodiment, native PE has a sequence set forth as:
TABLE-US-00007 (SEQ ID NO: 5) AEEAFDLWNE CAKACVLDLK DGVRSSRMSV
DPAIADTNGQ GVLHYSMVLE GGNDALKLAI DNALSITSDG LTIRLEGGVE PNKPVRYSYT
RQARGSWSLN WLVPIGHEKP SNIKVFIHEL NAGNQLSHMS PIYTIEMGDE LLAKLARDAT
FFVRAHESNE MQPTLAISHA GVSVVMAQTQ PRREKRWSEW ASGKVLCLLD PLDGVYNYLA
QQRCNLDDTW EGKIYRVLAG NPAKHDLDIK PTVISHRLHF PEGGSLAALT AHQACHLPLE
TFTRHRQPRG WEQLEQCGYP VQRLVALYLAARLSWNQVDQ VIRNALASPG SGGDLGEAIR
EQPEQARLAL TLAAAESERF VRQGTGNDEA GAANADVVSL TCPVAAGECA GPADSGDALL
ERNYPTGAEF LGDGGDVSFS TRGTQNWTVE RLLQAHRQLE ERGYVFVGYH GTFLEAAQSI
VFGGVRARSQ DLDAIWRGFY IAGDPALAYG YAQDQEPDAR GRIRNGALLR VYVPRSSLPG
FYRTSLTLAA PEAAGEVERL IGHPLPLRLD AITGPEEEGG RLETILGWPL AERTVVIPSA
IPTDPRNVGG DLDPSSIPDK EQAISALPDYASQPGKPPRE DLK
[0176] The method of action of PE is inactivation of the
ADP-ribosylation of elongation factor 2 (EF-2). The exotoxin
contains three structural domains that act in concert to cause
cytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.
Domain II (amino acids 253-364) is responsible for translocation
into the cytosol and domain III (amino acids 400-613) mediates ADP
ribosylation of elongation factor 2. The function of domain Ib
(amino acids 365-399) remains undefined, although a large part of
it, amino acids 365-380, can be deleted without loss of
cytotoxicity. See Siegall et al., J. Biol. Chem. 264:14256-14261,
1989.
[0177] The term "Pseudomonas exotoxin" ("PE") as used herein refers
as appropriate to a full-length native (naturally occurring) PE or
to a PE that has been modified. Such modifications may include, but
are not limited to, elimination of domain Ia, various amino acid
deletions in domains Ib, II and III, single amino acid
substitutions and the addition of one or more sequences at the
carboxyl terminus, such as KDEL (SEQ ID NO: 6) and REDL (SEQ ID NO:
7) (see Siegall et al., supra). In several examples, the cytotoxic
fragment of PE retains at least 50%, such as about 75%, about 90%,
or about 95% of the cytotoxicity of native PE. In one embodiment,
the cytotoxic fragment is more toxic than native PE.
[0178] Thus, the PE used in the immunotoxins disclosed herein
includes the native sequence, cytotoxic fragments of the native
sequence, and conservatively modified variants of native PE and its
cytotoxic fragments. Cytotoxic fragments of PE include those which
are cytotoxic with or without subsequent proteolytic or other
processing in the target cell (e.g., as a protein or pre-protein).
Cytotoxic fragments of PE known in the art include PE40, PE38, and
PE35.
[0179] In several embodiments, the PE has been modified to reduce
or eliminate non-specific cell binding, typically by deleting
domain Ia, as taught in U.S. Pat. No. 4,892,827, although this can
also be achieved, for example, by mutating certain residues of
domain Ia. U.S. Pat. No. 5,512,658, for instance, discloses that a
mutated PE in which Domain Ia is present but in which the basic
residues of domain Ia at positions 57, 246, 247, and 249 are
replaced with acidic residues (glutamic acid, or "E") exhibits
greatly diminished non-specific cytotoxicity. This mutant form of
PE is sometimes referred to as PE4E. PE40 is a truncated derivative
of PE (see, Pai et al., Proc. Nat'l Acad. Sci. U.S.A. 88:3358-3362,
1991; and Kondo et al., J. Biol. Chem. 263:9470-9475, 1988). PE35
is a 35 kD carboxyl-terminal fragment of PE in which amino acid
residues 1-279 have deleted and the molecule commences with a met
at position 280 followed by amino acids 281-364 and 381-613 of
native PE. PE35 and PE40 are disclosed, for example, in U.S. Pat.
No. 5,602,095 and U.S. Pat. No. 4,892,827.
[0180] In some embodiments, the cytotoxic fragment PE38 is
employed. PE38 is a truncated PE pro-protein composed of amino
acids 253-364 and 381-613 of SEQ ID NO: 3 which is activated to its
cytotoxic form upon processing within a cell (see e.g., U.S. Pat.
No. 5,608,039, and Pastan et al., Biochim. Biophys. Acta
1333:C1-C6, 1997).
[0181] While in some embodiments, the PE is PE4E, PE40, or PE38,
any form of PE in which non-specific cytotoxicity has been
eliminated or reduced to levels in which significant toxicity to
non-targeted cells does not occur can be used in the immunotoxins
disclosed herein so long as it remains capable of translocation and
EF-2 ribosylation in a targeted cell.
[0182] Conservatively modified variants of PE or cytotoxic
fragments thereof have at least about 80% sequence identity, such
as at least about 85% sequence similarity, at least about 90%
sequence identity, or at least about 95% sequence similarity at the
amino acid level, with the PE of interest, such as PE38.
Nucleic Acids
[0183] Nucleic acids encoding antibodies that specifically bind
GRP94, and conjugates and fusion thereof, are provided herein.
These nucleic acids can be used in conjunction with a BRAF
inhibitor. With the antibodies and immunotoxins herein provided,
one of skill can readily construct a variety of clones containing
functionally equivalent antibodies, and nucleic acids encoding
these antibodies, such as nucleic acids which differ in sequence
but which encode the same effector molecule ("EM") or antibody
sequence. Thus, nucleic acids encoding antibodies and conjugates
and fusion proteins are provided herein.
[0184] Nucleic acid sequences encoding the antibodies and/or
immunotoxins can be prepared by any suitable method including, for
example, cloning of appropriate sequences or by direct chemical
synthesis by methods such as the phosphotriester method of Narang,
et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of
Brown et al., Meth. Enzymol. 68:109-151, 1979; the
diethylphosphoramidite method of Beaucage et al., Tetra. Lett.
22:1859-1862, 1981; the solid phase phosphoramidite triester method
described by Beaucage & Caruthers, Tetra. Letts.
22(20):1859-1862, 1981, e.g., using an automated synthesizer as
described in, for example, Needham-VanDevanter et al. Nucl. Acids
Res. 12:6159-6168, 1984; and, the solid support method of U.S. Pat.
No. 4,458,066. Chemical synthesis produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. One of
skill would recognize that while chemical synthesis of DNA is
limited to sequences of about 100 bases, longer sequences may be
obtained by the ligation of shorter sequences.
[0185] In one embodiment, the nucleic acid sequences encoding the
antibody or immunotoxin are prepared by cloning techniques.
Examples of appropriate cloning and sequencing techniques, and
instructions sufficient to direct persons of skill through many
cloning exercises are found in Sambrook et al., supra, Berger and
Kimmel (eds.), supra, and Ausubel, supra. Product information from
manufacturers of biological reagents and experimental equipment
also provide useful information. Such manufacturers include the
SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems
(Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.),
CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp.,
Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,
GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
Invitrogen (San Diego, Calif.), and Applied Biosystems (Foster
City, Calif.), as well as many other commercial sources known to
one of skill.
[0186] Nucleic acids can also be prepared by amplification methods.
Amplification methods include polymerase chain reaction (PCR), the
ligase chain reaction (LCR), the transcription-based amplification
system (TAS), the self-sustained sequence replication system (3SR).
A wide variety of cloning methods, host cells, and in vitro
amplification methodologies are well known to persons of skill.
[0187] In one example, an immunotoxin of use is prepared by
inserting the cDNA which encodes a variable region into a vector
which comprises the cDNA encoding the EM. The insertion is made so
that the variable region and the EM are read in frame so that one
continuous polypeptide is produced. The polypeptide contains a
functional Fv region and a functional EM region. In one embodiment,
cDNA encoding a cytotoxin is ligated to a scFv so that the
cytotoxin is located at the carboxyl terminus of the scFv. In one
example, cDNA encoding a Pseudomonas exotoxin ("PE"), mutated to
eliminate or to reduce non-specific binding, is ligated to a scFv
so that the toxin is located at the amino terminus of the scFv. In
another example, PE38 is located at the amino terminus of the scFv.
In a further example, cDNA encoding a cytotoxin is ligated to a
heavy chain variable region of an antibody that binds the antigen
of interest so that the cytoxin is located at the carboxyl terminus
of the heavy chain variable region. The heavy chain-variable region
can subsequently be ligated to a light chain variable region of the
antibody using disulfide bonds. In yet another example, cDNA
encoding a cytotoxin is ligated to a light chain variable region of
an antibody that binds the antigen (for example, GRP94), so that
the cytotoxin is located at the carboxyl terminus of the light
chain variable region. The light chain-variable region can
subsequently be ligated to a heavy chain variable region of the
antibody using disulfide bonds.
[0188] Once the nucleic acids encoding the antibody immunotoxin is
isolated and cloned, the protein can be expressed in a
recombinantly engineered cell such as bacteria, plant, yeast,
insect and mammalian cells. One or more DNA sequences encoding an
antibody immunotoxin can be expressed in vitro by DNA transfer into
a suitable host cell. The cell may be prokaryotic or eukaryotic.
The term also includes any progeny of the subject host cell. It is
understood that all progeny may not be identical to the parental
cell since there may be mutations that occur during replication.
Methods of stable transfer, meaning that the foreign DNA is
continuously maintained in the host, are known in the art.
[0189] Polynucleotide sequences encoding the antibody or
immunotoxin can be operatively linked to expression control
sequences. An expression control sequence operatively linked to a
coding sequence is ligated such that expression of the coding
sequence is achieved under conditions compatible with the
expression control sequences. The expression control sequences
include, but are not limited to appropriate promoters, enhancers,
transcription terminators, a start codon (i.e., ATG) in front of a
protein-encoding gene, splicing signal for introns, maintenance of
the correct reading frame of that gene to permit proper translation
of mRNA, and stop codons.
[0190] The polynucleotide sequences encoding the antibody or
immunotoxin can be inserted into an expression vector including,
but not limited to a plasmid, virus or other vehicle that can be
manipulated to allow insertion or incorporation of sequences and
can be expressed in either prokaryotes or eukaryotes. Hosts can
include microbial, yeast, insect and mammalian organisms. Methods
of expressing DNA sequences having eukaryotic or viral sequences in
prokaryotes are well known in the art. Biologically functional
viral and plasmid DNA vectors capable of expression and replication
in a host are known in the art.
[0191] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired, or by electroporation.
[0192] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors may be used.
Eukaryotic cells can also be cotransformed with polynucleotide
sequences encoding the immunotoxin, and a second foreign DNA
molecule encoding a selectable phenotype, such as the herpes
simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein (see for example, Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One of
skill in the art can readily use an expression systems such as
plasmids and vectors of use in producing proteins in cells
including higher eukaryotic cells such as the COS, CHO, HeLa and
myeloma cell lines.
[0193] Isolation and purification of recombinantly expressed
polypeptide may be carried out by conventional means including
preparative chromatography and immunological separations. Once
expressed, the recombinant immunotoxins can be purified according
to standard procedures of the art, including ammonium sulfate
precipitation, affinity columns, column chromatography, and the
like (see, generally, R. Scopes, Protein Purification,
Springer-Verlag, N.Y., 1982). Substantially pure compositions of at
least about 90 to 95% homogeneity are disclosed herein, and 98 to
99% or more homogeneity can be used for pharmaceutical purposes.
Once purified, partially or to homogeneity as desired, if to be
used therapeutically, the polypeptides should be substantially free
of endotoxin.
[0194] Methods for expression of single chain antibodies and/or
refolding to an appropriate active form, including single chain
antibodies, from bacteria such as E. coli have been described and
are well-known and are applicable to the antibodies disclosed
herein. See, Buchner et al., Anal. Biochem. 205:263-270, 1992;
Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science
246:1275, 1989 and Ward et al., Nature 341:544, 1989, all
incorporated by reference herein.
[0195] Often, functional heterologous proteins from E. coli or
other bacteria are isolated from inclusion bodies and require
solubilization using strong denaturants, and subsequent refolding.
During the solubilization step, as is well known in the art, a
reducing agent must be present to separate disulfide bonds. An
exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M
guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of
the disulfide bonds can occur in the presence of low molecular
weight thiol reagents in reduced and oxidized form, as described in
Saxena et al., Biochemistry 9: 5015-5021, 1970, incorporated by
reference herein, and especially as described by Buchner et al.,
supra.
[0196] Renaturation is typically accomplished by dilution (e.g.,
100-fold) of the denatured and reduced protein into refolding
buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M
1-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
[0197] As a modification to the two chain antibody purification
protocol, the heavy and light chain regions are separately
solubilized and reduced and then combined in the refolding
solution. An exemplary yield is obtained when these two proteins
are mixed in a molar ratio such that a 5-fold molar excess of one
protein over the other is not exceeded. It is desirable to add
excess oxidized glutathione or other oxidizing low molecular weight
compounds to the refolding solution after the redox-shuffling is
completed.
[0198] In addition to recombinant methods, the immunoconjugates,
EM, and antibodies disclosed herein can also be constructed in
whole or in part using standard peptide synthesis. Solid phase
synthesis of the polypeptides of less than about 50 amino acids in
length can be accomplished by attaching the C-terminal amino acid
of the sequence to an insoluble support followed by sequential
addition of the remaining amino acids in the sequence. Techniques
for solid phase synthesis are described by Barany & Merrifield,
The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods
in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am.
Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase
Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984.
Proteins of greater length may be synthesized by condensation of
the amino and carboxyl termini of shorter fragments. Methods of
forming peptide bonds by activation of a carboxyl terminal end
(e.g., by the use of the coupling reagent
N,N'-dicycylohexylcarbodiimide) are well known in the art.
BRAF Inhibitors
[0199] The methods disclosed herein utilize BRAF inhibitors. One
exemplary amino acid sequence for human BRAF is provided below.
TABLE-US-00008 MAALSGGGGG GAEPGQALFN GDMEPEAGAG AGAAASSAAD
PAIPEEVWNI KQMIKLTQEHIEALLDKFGG EHNPPSIYLE AYEEYTSKLD ALQQREQQLL
ESLGNGTDFS VSSSASMDTVTSSSSSSLSV LPSSLSVFQN PTDVARSNPK SPQKPIVRVF
LPNKQRTVVP ARCGVTVRDSLKKALMMRGL IPECCAVYRI QDGEKKPIGW DTDISWLTGE
ELHVEVLENV PLTTHNFVRKTFFTLAFCDF CRKLLFQGFR CQTCGYKFHQ RCSTEVPLMC
VNYDQLDLLF VSKFFEHHPI PQEEASLAET ALTSGSSPSA PASDSIGPQI LTSPSPSKSI
PIPQPFRPAD EDHRNQFGQRDRSSSAPNVH INTIEPVNID DLIRDQGFRG DGGSTTGLSA
TPPASLPGSL TNVKALQKSPGPQRERKSSS SSEDRNRMKT LGRRDSSDDW EIPDGQITVG
QRIGSGSFGT VYKGKWHGDVAVKMLNVTAP TPQQLQAFKN EVGVLRKTRH VNILLFMGYS
TKPQLAIVTQ WCEGSSLYHHLHIIETKFEM IKLIDIARQT AQGMDYLHAK SIIHRDLKSN
NIFLHEDLTV KIGDFGLATV KSRWSGSHQF EQLSGSILWM APEVIRMQDK NPYSFQSDVY
AFGIVLYELM TGQLPYSNINNRDQIIFMVG RGYLSPDLSK VRSNCPKAMK RLMAECLKKK
RDERPLFPQI LASIELLARSLPKIHRSASE PSLNRAGFQT EDFSLYACAS PKTPIQAGGY
GAFPVH (SEQ ID NO: 10, see GENBANK .RTM. Accession No. ACD11489.1,
incorporated herein by reference)
[0200] A number of BRAF inhibitors have been previously described
(see, for example, PCT Publication Nos. WO 2007/002325, WO
2007/002433, WO 2009/047505, WO 03/086467; WO 2009/143024, WO
2010/104945, WO 2010/104973, WO 2010/111527 and WO 2009/152087;
U.S. Pat. Nos. 6,187,799 and 7,329,670; and U.S. Patent Application
Publication Nos. 2005/0176740 and 2009/0286783, each of which is
herein incorporated by reference).
[0201] PLX 4032 (also known as RG7204, RO5185426, and Vemurafenib,
C.sub.23H.sub.18ClF.sub.2N.sub.3O.sub.3S) is a BRAF small molecule
inhibitor being developed by Plexxikon and Roche (Genentech) for
the treatment of melanoma. Phase I clinical trials in patients with
advanced melanoma demonstrated that PLX 4032 was effective in
promoting tumor regression and increasing overall survival in
patients with the V600E BRAF mutation. In particular embodiments of
the present disclosure, the BRAF inhibitor is PLX 4032:
##STR00007##
or a salt, solvate or functional derivative thereof. Thus, a
therapeutically effective amount of PLX4032 can be used in
combination with a therapeutically effective amount of an antibody
that specifically binds GRP94 (or a nucleic acid encoding this
antibody) for the treatment of tumors.
[0202] In other embodiments, the BRAF inhibitor is PLX 4720
(C.sub.17H.sub.14ClF.sub.2N.sub.3O.sub.3S):
##STR00008##
or a salt, solvate or functional derivative thereof. Thus, a
therapeutically effective amount of PLX4720 can be used in
combination with a therapeutically effective amount of an antibody
that specifically binds GRP94 (or a nucleic acid encoding this
antibody) for the treatment of tumors.
[0203] In further embodiments, the BRAF inhibitor is sofafenib
(C.sub.21H.sub.16ClF.sub.3N.sub.4O.sub.3):
##STR00009##
or a salt, solvate or functional derivative thereof. Sofafenib
(Nexavar) is used for the treatment of renal cancer, liver cancer,
thyroid cancer, lung cancer, glioblastoma and kidney cancer. Thus a
therapeutically effective amount of sofafenib can be used in
combination with a therapeutically effective amount of an antibody
that specifically binds GRP94 (or a nucleic acid encoding this
antibody) for the treatment of these tumors.
[0204] In some embodiments, the BRAF inhibitors have the structure
of Formula III, shown below, or any salts, prodrugs, tautomers or
isomers thereof, as described in PCT Publication Nos. WO
2007/002325 and WO 2007/002433 (which are incorporated herein by
reference):
##STR00010##
[0205] wherein: Q has a structure selected from the group
consisting of
##STR00011##
in which
##STR00012##
indicates the attachment point of Q to A of Formula III; [0206]
Z.sub.2 is N or CR.sup.12; Z.sub.4 is N or CR.sup.14; Z.sub.5 is N
or CR.sup.15; Z.sub.6 is N or CR.sup.16; [0207] L.sub.2 is selected
from the group consisting of
--(CR.sup.10R.sup.11).sub.p--NR.sup.25--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--O--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--S--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--C(O)--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--C(S)--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--S--(O)--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--S(O).sub.2--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--C(O)NR.sup.25--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--C(S)NR.sup.25--(CR.sup.10R.sup.11).sub.q--,
--(CR.sup.10R.sup.11).sub.p--S(O).sub.3NR.sup.25--(CR.sup.10R.sup.11).sub-
.q--,
--(CR.sup.10R.sup.11).sub.p--NR.sup.25C(O)--(CR.sup.10R.sup.11).sub.-
q--,
--(CR.sup.10R.sup.11).sub.p--NR.sup.25C(S)--(CR.sup.10R.sup.11).sub.q-
--, and
--(CR.sup.10R.sup.11).sub.p--NR.sup.25S(O).sub.2--(CR.sup.10R.sup.-
11).sub.q--, [0208] p and q are independently 0, 1, or 2 provided,
however, that at least one of p and q is 0; [0209] s is 1 or 2;
[0210] X is O or S; [0211] A is selected from the group consisting
of --O--, --S--, --CR.sup.aR.sup.b--, --NR.sup.1--, --C(O)--,
--C(S)--, --S(O)--, and --S(O).sub.2--; [0212] R.sup.a and R.sup.b
at each occurrence are independently selected from the group
consisting of hydrogen, fluoro, --OH, --NH.sub.2, lower alkyl,
lower alkoxy, lower alkylthio, mono-alkylamino, di-alkylamino, and
--NR.sup.8R.sup.9, wherein the alkyl chain(s) of lower alkyl, lower
alkoxy, lower alkylthio, mono-alkylamino, or di-alkylamino are
optionally substituted with one or more substituents selected from
the group consisting of fluoro, --OH, --NH.sub.2, lower alkoxy,
fluoro substituted lower alkoxy, lower alkylthio, fluoro
substituted lower alkylthio, mono-alkylamino, di-alkylamino, and
cycloalkylamino, provided, however, that any substitution of the
alkyl chain carbon bound to O of alkoxy, S of thioalkyl or N of
mono- or di-alkylamino is fluoro; or [0213] R.sup.a and R.sup.b
combine to form a 3-7 membered monocyclic cycloalkyl or 5-7
membered monocyclic heterocycloalkyl, wherein the monocyclic
cycloalkyl or monocyclic heterocycloalkyl are optionally
substituted with one or more substituents selected from the group
consisting of halogen, --OH, --NH.sub.2, lower alkyl, fluoro
substituted lower alkyl, lower alkoxy, fluoro substituted lower
alkoxy, lower alkylthio, fluoro substituted lower alkylthio,
mono-alkylamino, di-alkylamino, and cycloalkylamino; [0214] R.sup.1
is selected from the group consisting of hydrogen, lower alkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, --C(O)R.sup.7,
--C(S)R.sup.7, --S(O).sub.2R.sup.7, --C(O)NHR.sup.7,
--C(S)NHR.sup.7, and --S(O).sub.2NHR.sup.7, wherein lower alkyl is
optionally substituted with one or more substituents selected from
the group consisting of fluoro, --OH, --NH.sub.2, lower alkoxy,
lower alkylthio, mono-alkylamino, di-alkylamino, and
--NR.sup.8R.sup.9, wherein the alkyl chain(s) of lower alkoxy,
lower alkylthio, mono-alkylamino, or di-alkylamino are optionally
substituted with one or more substituents selected from the group
consisting of fluoro, --OH, --NH.sub.2, lower alkoxy, fluoro
substituted lower alkoxy, lower alkylthio, fluoro substituted lower
alkylthio, mono-alkylamino, di-alkylamino, and cycloalkylamino,
provided, however, that any substitution of the alkyl chain carbon
bound to O of alkoxy, S of thioalkyl or N of mono- or di-alkylamino
is fluoro, further provided that when R.sup.1 is lower alkyl, any
substitution on the lower alkyl carbon bound to the N of
--NR.sup.1-- is fluoro, and wherein cycloalkyl, heterocycloalkyl,
aryl or heteroaryl are optionally substituted with one or more
substituents selected from the group consisting of halogen, --OH,
--NH.sub.2, lower alkyl, fluoro substituted lower alkyl, lower
alkoxy, fluoro substituted lower alkoxy, lower alkylthio, fluoro
substituted lower alkylthio, mono-alkylamino, di-alkylamino, and
cycloalkylamino; [0215] R.sup.7 is selected from the group
consisting of lower alkyl, cycloalkyl, heterocycloalkyl, aryl, and
heteroaryl, wherein lower alkyl is optionally substituted with one
or more substituents selected from the group consisting of fluoro,
--OH, --NH.sub.2, lower alkoxy, lower alkylthio, mono-alkylamino,
di-alkylamino, and --NR.sup.8R.sup.9, provided, however, that any
substitution of the alkyl carbon bound to the N of --C(O)NHR.sup.7,
--C(S)NHR.sup.7 or --S(O).sub.2NHR.sup.7 is fluoro, wherein the
alkyl chain(s) of lower alkoxy, lower alkylthio, mono-alkylamino,
or di-alkylamino are optionally substituted with one or more
substituents selected from the group consisting of fluoro, --OH,
--NH.sub.2, lower alkoxy, fluoro substituted lower alkoxy, lower
alkylthio, fluoro substituted lower alkylthio, mono-alkylamino,
di-alkylamino, and cycloalkylamino, provided, however, that any
substitution of the alkyl chain carbon bound to O of alkoxy, S of
thioalkyl or N of mono- or di-alkylamino is fluoro, and wherein
cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally
substituted with one or more substituents selected from the group
consisting of halogen, --OH, --NH.sub.2, lower alkyl, fluoro
substituted lower alkyl, lower alkoxy, fluoro substituted lower
alkoxy, lower alkylthio, fluoro substituted lower alkylthio,
mono-alkylamino, di-alkylamino, and cycloalkylamino; [0216]
R.sup.4, R.sup.5, R.sup.6, R.sup.12, R.sup.14, R.sup.15, R.sup.16,
R.sup.42, R.sup.43, R.sup.45, R.sup.46, and R.sup.47 are
independently selected from the group consisting of hydrogen,
halogen, optionally substituted lower alkyl, optionally substituted
lower alkenyl, optionally substituted lower alkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl,
--CN, --NO.sub.2, --CR.sup.aR.sup.bR.sup.26, and -LR.sup.26; [0217]
L at each occurrence is independently selected from the group
consisting of -(alk).sub.a-S-(alk).sub.b-,
-(alk).sub.a-O-(alk).sub.b-, -(alk).sub.a-NR.sup.25-(alk).sub.b-,
-(alk).sub.a-C(O)-(alk).sub.b-, -(alk).sub.a-C(S)-(alk).sub.b-,
-(alk).sub.a-S(O)-(alk).sub.b-,
-(alk).sub.a-S(O).sub.2-(alk).sub.b-,
-(alk).sub.a-OC(O)-(alk).sub.b-, -(alk).sub.a-C(O)O-(alk).sub.b-,
-(alk).sub.a-OC(S)-(alk).sub.b-, -(alk).sub.a-C(S)O-(alk).sub.b-,
-(alk).sub.a-C(O)NR.sup.25-(alk).sub.b-,
-(alk).sub.a-C(S)NR.sup.25-(alk).sub.b-,
-(alk).sub.a-S(O).sub.2NR.sup.25-(alk).sub.b-,
-(alk).sub.a-NR.sup.25C(O)-(alk).sub.b-,
-(alk).sub.a-NR.sup.25C(S)-(alk).sub.b-,
-(alk).sub.a-NR.sup.25S(O).sub.2-(alk).sub.b-,
-(alk).sub.a-NR.sup.25C(O)O-(alk).sub.b-,
-(alk).sub.a-NR.sup.25C(S)O-(alk).sub.b-,
-(alk).sub.a-OC(O)NR.sup.25-(alk).sub.b-,
-(alk).sub.a-OC(S)NR.sup.25-(alk).sub.b-,
-(alk).sub.a-NR.sup.25C(O)NR.sup.25-(alk).sub.b-,
-(alk).sub.a-NR.sup.25C(S)NR.sup.25-(alk).sub.b-, and
-(alk).sub.a-NR.sup.25S(O).sub.2NR.sup.25-(alk).sub.b-; [0218] a
and b are independently 0 or 1; [0219] alk is C.sub.1-3 alkylene or
C.sub.1-3 alkylene substituted with one or more substituents
selected from the group consisting of fluoro, --OH, --NH.sub.2,
lower alkyl, lower alkoxy, lower alkylthio, mono-alkylamino,
di-alkylamino, and --NR.sup.8R.sup.9, wherein lower alkyl or the
alkyl chain(s) of lower alkoxy, lower alkylthio, mono-alkylamino or
di-alkylamino are optionally substituted with one or more
substituents selected from the group consisting of fluoro, --OH,
--NH.sub.2, lower alkoxy, fluoro substituted lower alkoxy, lower
alkylthio, fluoro substituted lower alkylthio, mono-alkylamino,
di-alkylamino and cycloalkylamino, provided, however, that any
substitution of the alkyl chain carbon bound to O of alkoxy, S of
thioalkyl or N of mono- or di-alkylamino is fluoro; [0220] R.sup.25
at each occurrence is independently selected from the group
consisting of hydrogen, optionally substituted lower alkyl,
optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl, and optionally
substituted heteroaryl; [0221] R.sup.26 at each occurrence is
independently selected from the group consisting of hydrogen,
provided, however, that hydrogen is not bound to any of S(O),
S(O).sub.2, C(O) or C(S) of L, optionally substituted lower alkyl,
optionally substituted lower alkenyl, provided, however, that when
R.sup.26 is optionally substituted lower alkenyl, no alkene carbon
thereof is bound to N, S, O, S(O), S(O).sub.2, C(O) or C(S) of L,
optionally substituted lower alkynyl, provided, however, that when
R.sup.26 is optionally substituted lower alkynyl, no alkyne carbon
thereof is bound to N, S, O, S(O), S(O).sub.2, C(O) or C(S) of L,
optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl, and optionally
substituted heteroaryl; [0222] R.sup.10 and R.sup.11 at each
occurrence are independently selected from the group consisting of
hydrogen, fluoro, lower alkyl, and lower alkyl optionally
substituted with one or more substituents selected from the group
consisting of fluoro, --OH, --NH.sub.2, lower alkoxy, fluoro
substituted lower alkoxy, lower alkylthio, fluoro substituted lower
alkylthio, mono-alkylamino, di-alkylamino, and cycloalkylamino; or
[0223] any two of R.sup.10 and R.sup.11 on the same or adjacent
carbon atoms combine to form a 3-7 membered monocyclic cycloalkyl
or 5-7 membered monocyclic heterocycloalkyl, and any others of
R.sup.10 and R.sup.11 are independently selected from the group
consisting of hydrogen, fluoro, lower alkyl, and lower alkyl
optionally substituted with one or more substituents selected from
the group consisting of fluoro, --OH, --NH.sub.2, lower alkoxy,
fluoro substituted lower alkoxy, lower alkylthio, fluoro
substituted lower alkylthio, mono-alkylamino, di-alkylamino, and
cycloalkylamino, and wherein the monocyclic cycloalkyl or
monocyclic heterocycloalkyl are optionally substituted with one or
more substituents selected from the group consisting of halogen,
--OH, --NH.sub.2, lower alkyl, fluoro substituted lower alkyl,
lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,
fluoro substituted lower alkylthio, mono-alkylamino, di-alkylamino,
and cycloalkylamino; [0224] R.sup.8 and R.sup.9 combine with the
nitrogen to which they are attached to form a 5-7 membered
heterocycloalkyl optionally substituted with one or more
substituents selected from the group consisting of fluoro, --OH,
--NH.sub.2, lower alkyl, fluoro substituted lower alkyl, lower
alkoxy, fluoro substituted lower alkoxy, lower alkylthio, and
fluoro substituted lower alkylthio; [0225] R.sup.17 is selected
from the group consisting of hydrogen, halogen, optionally
substituted lower alkyl and --OR.sup.18; [0226] R.sup.31 and
R.sup.33 are independently selected from the group consisting of
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted cycloalkyl, and optionally substituted
heterocycloalkyl; [0227] R.sup.36 is selected from the group
consisting of substituted methyl, optionally substituted C.sub.2-6
alkyl, optionally substituted lower alkenyl, provided, however,
that when R.sup.36 is optionally substituted lower alkenyl, no
alkene carbon thereof is bound to the S(O).sub.2 of
S(O).sub.2R.sup.36, optionally substituted lower alkynyl, provided,
however, that when R.sup.36 is optionally substituted lower
alkynyl, no alkyne carbon thereof is bound to the S(O).sub.2 of
S(O).sub.2R.sup.36, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl,
optionally substituted heteroaryl, and --NR.sup.19R.sup.20; [0228]
R.sup.19, R.sup.20, R.sup.34, R.sup.35, R.sup.37, and R.sup.38 are
independently selected from the group consisting of hydrogen,
optionally substituted lower alkyl, optionally substituted lower
alkenyl, provided, however, that when R.sup.19, R.sup.20, R.sup.34,
R.sup.35, R.sup.37, or R.sup.38 is optionally substituted lower
alkenyl, no alkene carbon thereof is bound to the N of
NR.sup.19R.sup.20, NR.sup.34R.sup.35 or NR.sup.37R.sup.38,
optionally substituted lower alkynyl, provided, however, that when
R.sup.19, R.sup.20, R.sup.34, R.sup.35, R.sup.37, or R.sup.38 is
optionally substituted lower alkynyl, no alkyne carbon thereof is
bound to the N of NR.sup.19R.sup.20, NR.sup.34R.sup.35 or
NR.sup.37R.sup.38, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted aryl and
optionally substituted heteroaryl; or [0229] R.sup.34 and R.sup.35
together with the nitrogen to which they are attached form
optionally substituted 5-7 membered heterocycloalkyl or optionally
substituted 5 or 7 membered nitrogen containing heteroaryl; or
[0230] R.sup.37 and R.sup.38 together with the nitrogen to which
they are attached form optionally substituted 5-7 membered
heterocycloalkyl or optionally substituted 5 or 7 membered nitrogen
containing heteroaryl; [0231] R.sup.32 is selected from the group
consisting of hydrogen, optionally substituted lower alkyl,
optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl, optionally
substituted heteroaryl, and --OR.sup.18; [0232] R.sup.82 is
selected from hydrogen or lower alkyl; and [0233] R.sup.18 is
hydrogen or optionally substituted lower alkyl;
[0234] In other embodiments of the present disclosure, the BRAF
inhibitor comprises a formula as described in PCT Publication No.
WO 03/086467 (incorporated herein by reference) as Formula I,
Formula II or Formula III:
Formula I--
##STR00013##
[0235] or a salt, solvate, physiologically functional derivative
thereof; wherein
Y is CR.sup.1 and V is N;
or Y is CR.sup.1 and V is CR.sup.2;
[0236] R.sup.1 represents a group
CH.sub.3SO.sub.2CH.sub.2CH.sub.2NHCH.sub.2--Ar--, wherein Ar is
selected from phenyl, furan, thiophene, pyrrole and thiazole, each
of which may optionally be substituted by one or two halo,
C.sub.1-4 alkyl or C.sub.1-4 alkoxy groups; R.sup.2 is selected
from the group comprising hydrogen, halo, hydroxy, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, C.sub.1-4 alkylamino and di[C.sub.1-4
alkyl]amino; U represents a phenyl, pyridyl, 3H-imidazolyl,
indolyl, isoindolyl, indolinyl, isoindolinyl, 1H-indazolyl,
2,3-dihydro-1H-indazolyl, 1H-benzimidazolyl,
2,3-dihydro-1H-benzimidazolyl or 1H-benzotriazolyl group,
substituted by an R.sup.3 group and optionally substituted by at
least one independently selected R.sup.4 group; R.sup.3 is selected
from a group comprising benzyl, halo-, dihalo- and trihalobenzyl,
benzoyl, pyridylmethyl, pyridylmethoxy, phenoxy, benzyloxy, halo-,
dihalo- and trihalobenzyloxy and benzenesulphonyl; or R.sup.3
represents trihalomethylbenzyl or trihalomethylbenzyloxy; or
R.sup.3 represents a group of formula
##STR00014##
wherein each R.sup.5 is independently selected from halogen,
C.sub.1-4 alkyl and C.sub.1-4 alkoxy; and n is 0 to 3; each R.sup.4
is independently hydroxy, halogen, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, C.sub.1-4 alkoxy, amino, C.sub.1-4
alkylamino, di[C.sub.1-4 alkyl]amino, C.sub.1-4 alkylthio,
C.sub.1-4 alkylsulphinyl, C.sub.1-4 alkylsulphonyl, C.sub.1-4
alkylcarbonyl, carboxy, carbamoyl, C.sub.1-4 alkoxycarbonyl,
C.sub.1-4 alkanoylamino, N--(C.sub.1-4 alkyl)carbamoyl,
N,N-di(C.sub.1-4 alkyl) carbamoyl, cyano, nitro and
trifluoromethyl.
[0237] Additional BRAF inhibitors are shown below:
##STR00015##
wherein R is --Cl or --Br, X is CH, N, or CF, and Z is thiazole or
furan.
##STR00016##
[0238] In some embodiments, the BRAF inhibitor is selected from a
compound as described in PCT Publication No. WO 2010/104973 which
is incorporated by reference herein. These include the
following:
[0239] In a first aspect, a compound selected from the group
consisting of
N-[3-(4-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]--
4-trifluorometh)/1-benzenesulfonamide (P-0001),
N-[3-(4-ethynyl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl-
]-4-trifluoromethyl-benzenesulfonamide (P-0002), any salt thereof,
any formulation thereof, any conjugate thereof, any derivative
thereof, and any form thereof is provided. In certain embodiments
P-0001, P-0002, or a salt thereof, formulation thereof, conjugate
thereof, derivative thereof, or form thereof is an inhibitor of one
or more Raf protein kinases, including A-Raf, B-Raf, and c-Raf-1
(including any mutations of these kinases),
[0240] In a second aspect the compound
N-[3-(4-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]--
4-trifluoromethyl-benzenesulfonamide (P-0001), or a salt thereof,
formulation thereof, conjugate thereof, derivative thereof, or form
thereof is provided. In certain embodiments P-0001, or a salt
thereof, formulation thereof, conjugate thereof, derivative
thereof, or form thereof is an inhibitor of one or more Raf protein
kinases, including
A-Raf, B-Raf, and c-Raf-1 (including any mutations of these
kinases).
[0241] In a third aspect the compound
N-[3-(4-ethynyl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl-
]-4-trifluoromethyl-benzenesulfonamide (P-0002), or a salt thereof,
formulation thereof, conjugate thereof, derivative thereof, or form
thereof is provided. In certain embodiments P-0002, or a salt
thereof, formulation thereof, conjugate thereof, derivative
thereof, or form thereof is an inhibitor of one or more Raf protein
kinases, including A-Raf, B-Raf, and c-Raf-1 (including any
mutations of these kinases).
[0242] In some embodiments, the BRAF inhibitor is selected from a
compound as described in PCT Publication No. WO 2010/104945, which
is incorporated by reference herein. These include the
following:
[0243] In a first aspect, a compound selected from the group
consisting of propane-1-sulfonic acid
{2,4-difluoro-3-[5-(2-methoxy-pyrimidin-5-yl)-1H-pyr[tau]olo[2,3-bJpyridi-
nc-3-carbonyl]-phenyl}-amide (P-0001), propane-1-sulfonic acid
[3-(5-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenylj-am-
ide (P-0002), propane-1-sulfonic acid
[3-(5-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2-fluoro-phenyl]-amide
(P-0003),
N-[3-(5-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluor-
o-phenyl]-2,5-diflu[upsilon]r[upsilon]-ben/enesulfonamide (P-0004),
N-[3-(5-cyano-1H-pyrrolo[2,3-b[pyridine-3-carbonyl)-2,4-difluoro-phenylJ--
3-fluoro-benzenesulfonamide (P-0005), pyrrolidine-1-sulfonic acid
[3-(5-cyano-1H-pyi[tau]olo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl-
]-amide (P-0006), N,N-dimethylamino-sulfonic acid
[3-(5-cyano-1H-pyrrolo[2,3-bJpyridine-3-carbonyl)-2.4-difluoro-phenylJ-am-
ide (P-0007), any salt thereof, any formulation thereof, any
conjugate thereof, any derivative thereof, and any form thereof is
provided. In certain embodiments P-0001, P-0002, P-0003, P-0004,
P-0005, P-0006, P-0007. or a salt thereof, formulation thereof,
conjugate thereof, derivative thereof, or form thereof is an
inhibitor of one or more Raf prolei[pi]kinases, including
[Lambda]-Raf, R-Raf. and c-Raf-1 (including any mutations of these
kinases).
[0244] In some embodiments, the BRAF inhibitor is a compound having
a formula from U.S. Pat. No. 6,187,799, which is incorporated by
reference herein:
##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021##
[0245] In some embodiments, the BRAF inhibitor is a compound as
disclosed in PCT Publication No. WO 2010/111527, which is
incorporated by reference herein. For example, the BRAF inhibitor
can have a structure according to Formula I of PCT Publication No.
WO 2010/111527.
##STR00022##
or a salt, a prodrug, a tautomer or an isomer thereof,
[0246] wherein:
[0247] Ar is selected from the group consisting of:
##STR00023## ##STR00024##
wherein
##STR00025##
indicates the point of attachment of Ar to Lj of Formula I and
##STR00026##
indicates the point of attachment of Ar to L of Formula I;
[0248] L, is selected from the group consisting Of
--C(R.sup.5R.sup.6)--, --C(O)--, --C(S)--, --N(R.sup.7)--, --O--,
--S--, --S(O)--, and --S(O)2-;
[0249] L.sub.2 is selected from the group consisting of
--N(R.sup.8)--C(O)--, --N(R.sup.8)--C(S)--,
--N(R.sup.8)--S(O)--N(R.sup.8)--S(O)2-,
--N(R.sup.8)--C(O)--N(R.sup.8)--, --N(R.sup.8)--C(S)--N(R.sup.8)--,
and --N(R.sup.8)--S(O)2-N(R.sup.8)--;
[0250] R.sup.1 is selected from the group consisting of optionally
substituted lower alkyl, optionally substituted lower alkenyl,
optionally substituted lower alkynyl, optionally substituted
cycloalkyl, optionally substituted hetcrocycloalkyl, optionally
substituted aryl, and optionally substituted heteroaryl;
[0251] R.sup.2 is selected from the group consisting of hydrogen,
halogen, optionally substituted lower alkyl, optionally substituted
lower alkenyl, optionally substituted lower alkynyl, optionally
substituted cycloalkyl, optionally substituted hetcrocycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl,
--CN, --NO.sub.2, --O--R.sup.9, --S--R.sup.11,
--N(R.sup.9)--R.sup.10, --C(O)--R.sup.11, --C(S)--R.sup.11,
--C(O)--N(R.sup.9R.sup.10, --C(S)--N(R.sup.9)--R.sup.10,
--C(O)--N(R.sup.13)--OR.sup.9, --C(S)--N(R.sup.13)--OR.sup.9,
--C(O)--N(R.sup.13)--S(O).sub.2--R.sup.11,
--C(S)--N(R.sup.13)--S(O).sub.2--R.sup.11, --C(O)--O--R.sup.9,
--S(O)--R.sup.11, --S(O).sub.2--R.sup.11,
--S(O)--N(R.sup.9)--R.sup.10, --S(O).sub.2--N(R.sup.9)--R.sup.10,
--S(O).sub.2--N(R.sup.13)--C(O)R.sup.11,
--S(O).sub.2--N(R.sup.13)--C(S)R.sup.11,
--N(R.sup.13)--C(O)--R.sup.11, --N(R.sup.13)--C(S)--R.sup.11,
--N(R.sup.13)--S(O)--R.sup.11, --N(R.sup.13)--S(O).sub.2--R.sup.11,
--N(R.sup.13)--C(O)--N(R.sup.9)--R.sup.10,
--N(R.sup.13)--C(S)--N(R.sup.9)--R.sup.10, and
--N(R.sup.13)--S(O).sub.2--N(R.sup.9)--R.sup.10;
[0252] R.sup.3 is selected from the group consisting of hydrogen,
halogen, optionally substituted lower alkyl, optionally substituted
lower alkenyl, optionally substituted lower alkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl,
--CN, --NO.sub.2, --O--R.sup.17, --S--R.sup.19,
--N(R.sup.17)--R.sup.18, --C(O)--R.sup.19, --C(S)--R.sup.19,
--C(O)--N(R.sup.17R.sup.18, --C(S)--N(R.sup.17)--R.sup.18,
--C(O)--N(R.sup.20)--OR.sup.17, --C(S)--N(R.sup.20)--OR.sup.17,
--C(O)--N(R.sup.20)--S(O).sub.2--R.sup.19,
--C(S)--N(R.sup.20)--S(O).sub.2--R.sup.19, --C(O)--O--R.sup.17,
--S(O)--R.sup.19, --S(O).sub.2--R.sup.19,
--S(O)--N(R.sup.17)--R.sup.18, --S(O).sub.2--N(R.sup.17)--R.sup.18,
--S(O).sub.2--N(R.sup.20)--C(O)R.sup.19,
--S(O).sub.2--N(R.sup.20)--C(S)R.sup.19,
--N(R.sup.20)--C(O)--R.sup.19, --N(R.sup.20)--C(S)--R.sup.19,
--N(R.sup.20S(O)--R.sup.19, --N(R.sup.20S(O).sub.2--R.sup.19,
--N(R.sup.20C(O)--N(R.sup.17)--R.sup.18,
--N(R.sup.20)--C(S)--N(R.sup.17)--R.sup.18, and
--N(R.sup.20)--S(O).sub.2--N(R.sup.17)--R.sup.18;
[0253] R.sup.4 is selected from the group consisting of hydrogen,
halogen, optionally substituted lower alkyl, optionally substituted
lower alkenyl, optionally substituted lower alkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl,
--CN, NO.sub.2, --O--R.sup.21, --S--R.sup.23,
--N(R.sup.21)--R.sup.22, --C(O)--R.sup.23, --C(S)--R.sup.23,
--C(O)--N(R.sup.21)--R.sup.22, --C(S)--N(R.sup.21)--R.sup.22,
--C(O)--N(R.sup.24)--OR.sup.21, --C(S)--N(R.sup.24)--OR.sup.21,
--C(O)--N(R.sup.24)--S(O).sub.2--R.sup.23,
--C(S)--N(R.sup.24)--S(O).sub.2--R.sup.23, --C(O)--O--R.sup.21,
--S(O).sub.2--R.sup.23, --S(O).sub.2--R.sup.23,
--S(O)--N(R.sup.21)--R.sup.22, --S(O)2-N(R.sup.21)--R.sup.22,
--S(O).sub.2--N(R.sup.24)--C(O)R.sup.23,
--S(O.sub.2--N(R.sup.24)--C(S)R.sup.23,
--N(R.sup.24)--C(O)--R.sup.23, --N(R.sup.24)--C(S)--R.sup.23,
--N(R.sup.24)--S(O)--R.sup.23, --N(R.sup.24)--S(O).sub.2--R.sup.23,
--N(R.sup.24)--C(O)--N(R.sup.21)--R.sup.22,
--N(R.sup.24)--C(S)--N(R.sup.21)--R.sup.22, and
--N(R.sup.24)--S(O).sub.2--N(R.sup.21)--R.sup.22;
[0254] R.sup.5 and R.sup.6 are independently selected from the
group consisting of hydrogen, fluoro, --OH, --NH.sub.2, lower
alkyl, lower alkoxy, lower alklylthio, mono-alkylamino,
di-alkylamino, and --N(R.sup.25)--R.sup.26, wherein the alkyl
chain(s) of lower alkyl, lower alkoxy, lower alkylthio,
mono-alkylamino, or di-alkylamino are optionally substituted with
one or more substituents selected from the group consisting of
fluoro, --OH, --NH.sub.2, lower alkoxy, fluoro substituted lower
alkoxy, lower alkylthio, fluoro substituted lower alkylthio,
mono-alkylamino, di-alkylamino, and cycloalkylamino; or
[0255] R.sup.5 and R.sup.6 combine to form a 3-7 membered
monocyclic cycloalkyl or 5-7 membered monocyclic heterocycloalkyl,
wherein the 3-7 membered monocyclic cycloalkyl or 5-7 membered
monocyclic heterocycloalkyl are optionally substituted with one or
more substituents selected from the group consisting of halogen,
--OH, --NH.sub.2, lower alkyl, fluoro substituted lower alkyl,
lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,
fluoro substituted lower alkylthio, mono-alkylamino, di-alkylamino,
and cycloalkylamino,
[0256] R.sup.7, R.sup.13, R.sup.20, and R.sup.24 are independently
selected from the group consisting of hydrogen, optionally
substituted lower alkyl, optionally substituted cycloalkyl,
optionally substituted heterocycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, --C(O)--R.sup.27,
--C(S)--R.sup.27, --S(O)--R.sup.27, --S(O).sub.2--R.sup.27,
--C(O)--N(H)--R.sup.27, --C(S)--N(H)--R.sup.27, and
--S(O).sub.2--N(H)--R.sup.27,
[0257] R.sup.8 at each occurrence is independently hydrogen, lower
alkyl, or lower alkyl substituted with one or more substituents
selected from the group consisting of fluoro, --OH, --NH.sub.2,
lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,
fluoro substituted lower alkylthio, mono-alkylamino, fluoro
substituted mono-alkylamino, di-alkylamino, fluoro substituted
di-alkylamino, and --N(R.sup.25)--R.sup.26,
[0258] R.sup.12, R.sup.14, R.sup.15, and R.sup.16 are independently
selected from the group consisting of hydrogen, halogen, optionally
substituted lower alkyl, --N(R.sup.28)--R.sup.29, --O--R.sup.28,
and --S--R.sup.30,
[0259] R.sup.11, R.sup.19 and R.sup.23 are independently selected
from the group consisting of optionally substituted lower alkyl,
optionally substituted lower alkenyl, optionally substituted lower
alkynyl, optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl, and optionally
substituted heteroaryl,
[0260] R.sup.9, R.sup.10, R.sup.17, R.sup.18, R.sup.21 and R.sup.22
are independently selected from the group consisting of hydrogen,
optionally substituted lower alkyl, optionally substituted lower
alkenyl, optionally substituted lower alkynyl, optionally
substituted cycloalkyl, optionally substituted heterocycloalkyl,
optionally substituted aryl, and optionally substituted
heteroaryl;
[0261] R.sup.25 and R.sup.26 at each occurrence combine with the
nitrogen to which they are attached to form a 5-7 membered
heterocycloalkyl optionally substituted with one or more
substituents selected from the group consisting of fluoro, --OH,
--NH.sub.2, lower alkyl, fluoro substituted lower alkyl, lower
alkoxy, fluoro substituted lower alkoxy, lower alkylthio, and
fluoro substituted lower alkylthio;
[0262] R.sup.27 at each occurrence is independently selected from
the group consisting of optionally substituted lower alkyl,
optionally substituted cycloalkyl, optionally substituted
heterocycloalkyl, optionally substituted aryl, and optionally
substituted heteroaryl;
[0263] R.sup.28 and R.sup.29 at each occurrence are independently
hydrogen or optionally substituted lower alkyl; and
[0264] R.sup.30 at each occurrence is optionally substituted lower
alkyl.
[0265] In some embodiments, the BRAF inhibitor is a compound having
the formula disclosed in U.S. Pat. No. 7,329,670, which is
incorporated by reference herein. For example, the BRAF inhibitor
can have the following structure
##STR00027##
wherein B is generally an unsubstituted or substituted, up to
tricyclic, aryl or heteroaryl moiety with up 30 carbon atoms with
at least one 5 or 6 member aromatic structure containing 0-4
members of the group consisting of nitrogen, oxygen and sulfur. A
is a heteroaryl moiety.
[0266] In some examples of the presence disclosure, the BRAF
inhibitor is GDC 0789 (Genentech):
##STR00028##
In other examples, the inhibitor is PD-325901, XL518, PD-184352,
PD-318088, AZD6244 or CI-1040 (see PCT Publication No. WO
2009/047505, incorporated herein by reference).
[0267] In further examples, the BRAF inhibitor can be an antibody
that specifically binds BRAF, or an antigen binding fragment
thereof. This includes monoclonal antibodies and antigen binding
fragments that specifically bind BRAF. The BRAF inhibitor can also
be an inhibitory RNA, such as, but not limited to, anti-sense RNA,
small inhibitor RNA, and shRNA.
Methods of Treatment
[0268] Methods are provided herein for treating a subject diagnosed
with a tumor that expresses a mutated BRAF. The methods include
administering to the subject (1) a therapeutically effective amount
of an antibody or antigen binding fragment thereof that
specifically binds glucose regulated protein (GRP) 94, or a nucleic
acid encoding the antibody or antigen binding fragment; and (2) a
therapeutically effective amount of a BRAF inhibitor, thereby
treating the cancer in the subject.
[0269] These use of a BRAF inhibitor in combination with an
antibody or antigen binding fragment thereof that specifically
binds GRP94 provide an unexpectedly superior result for the
treatment of any tumor, wherein cells in the tumor comprise a BRAF
mutation. In other embodiments, the BRAF mutation is a V600E
mutation. In other embodiments, the BRAF mutation is R462I, I463S,
G464E, G464V, G466A, G466E, G466V, G469A, G469E, N581S, E585K,
D594V, F595L, G596R, L597V, T599I, V600D, V600E, V600K, V600R,
K601E, and A728V. In further embodiments the BRAF mutation is in
one of two regions: the glycine-rich P loop of the N lobe and the
activation segment and flanking regions. The cells in the tumor can
have more than one BRAF mutation.
[0270] In additional embodiments, the subject has a primary or
secondary resistance to the BRAF inhibitor. A subject that does not
respond initially to a BRAF inhibitor has primary resistance. A
subject that initially responds to the BRAF inhibitor, but then
ceases to respond, has secondary resistance. In some examples, the
subject responds for one, two, three, four, five, six, seven,
eight, nine, ten, eleven or twelve months, but then does not
respond to the BRAF inhibitor. Thus, the methods can include
selecting a subject with a resistance to a BRAF inhibitor, such as
selecting a subject with primary resistance to the BRAF inhibitor
or secondary resistance to the BRAF inhibitor.
[0271] The subject can have a tumor. Thus, in some embodiments, the
method includes selecting a subject that has secondary resistance
to a BRAF inhibitor, such as a subject with secondary resistance to
a BRAF inhibitor that has a tumor, such as melanoma. In other
embodiments, the method includes selecting a subject with primary
resistance to a BRAF inhibitor, such as a subject with primary
resistance to a BRAF inhibitor that has a tumor, such as
melanoma.
[0272] The disclosed methods can also be used to prevent metastasis
or decrease the number of micrometastases, such as micrometastases
to regional lymph nodes. Disclosed herein are methods to treat a
subject diagnosed with a tumor, such as a melanoma. Melanoma
includes spreading melanoma, nodular melanoma, acral lentiginous
melanoma, and lentigo maligna (melanoma). However, the methods
disclosed herein can also be used to treat other cancers, such
breast cancer, prostate cancer, ovarian cancer, colon cancer,
stomach cancer, pancreatic cancer, glioma, chordoma,
chondrosarcoma, thyroid cancer, colon cancer, glioma or a squamous
cell carcinoma. Squamous cells carcinomas include, but are not
limited to head and neck squamous cell carcinoma, and squamous cell
cancers of the skin, lung, prostate, esophagus, vagina and cervix.
The methods disclosed herein can also be used to breast cancer,
head and neck squamous cell carcinoma, renal cancer, lung cancer,
glioma, bladder cancer, ovarian cancer, colon cancer or pancreatic
cancer, wherein cells of the cancer express GRP94. The methods also
can be used to treat non-Hodgkin lymphoma, colorectal cancer,
papillary thyroid carcinoma, non-small cell lung carcinoma, and
adenocarcinoma of lung. Thus, the methods can include selecting a
subject with a tumor.
[0273] A variety of different types of melanoma may be treated in
accordance with the present methods including, such as superficial
spreading melanoma, nodular malignant melanoma, acral lentiginous
melanoma, lentiginous malignant melanoma, and mucosal lentiginous
melanoma. The primary melanoma may also be cutaneous or
extracutaneous. Extracutaneous primary malignant melanomas include
ocular melanoma and clear-cell sarcoma of the soft tissues.
Additional indications include rare melanomas or precancerous
lesions where relevance of RTK targets may be implicated. The
present methods are also useful in the treatment of melanoma that
has metastasized.
[0274] A therapeutically effective amount of the agents will depend
upon the severity of the disease and the general state of the
patient's health. A therapeutically effective amount of the
antibodies or antigen binding fragments thereof (or nucleic acids
encoding the antibody or antigen binding fragment) and BRAF
inhibitor is that which provides either a reduction in tumor
burden, decreased metastatic lesions and/or subjective relief of a
symptom(s) or an objectively identifiable improvement as noted by
the clinician or other qualified observer.
[0275] The antibody or antigen binding fragment can be administered
in the same formulation or separately. They can be given at the
same time or a different time (simultaneously or sequentially), but
sufficiently concurrently to have the beneficial effect disclosed
herein. Compositions are provided herein that include a carrier and
one or more of the antibodies that specifically bind GRP94 and/or
antigen binding fragments thereof, in combination with a BRAF
inhibitor. Compositions comprising immunoconjugates or immunotoxins
of these antibodies are also provided. The compositions can be
prepared in unit dosage forms for administration to a subject. The
amount and timing of administration are at the discretion of the
treating physician to achieve the desired purposes.
[0276] The antibody (or a nucleic acid encoding the antibody)
and/or BRAF inhibitor can be formulated for systemic or local (such
as intra-tumor) administration. In one example, the antibodies
and/or BRAF inhibitor is formulated for parenteral administration,
such as intravenous administration. In another example, the
antibodies, antigen binding fragments, nucleic acid and/or one or
more BRAF inhibitors are formulated for topical administration,
subcutaneous or intradermal administration. A BRAF inhibitor and/or
and antibody or antigen binding fragment described above (or a
nucleic acid encoding the antibody or antigen binding fragment) can
be delivered transdermally via, for example, a transdermal delivery
device or a suitable vehicle or, such in an ointment base, which
may be incorporated into a patch for controlled delivery. Such
devices are advantageous, as they may allow a prolonged period of
treatment relative to, for example, an oral or intravenous
medicament. Examples of transdermal delivery devices may include,
for example, a patch, dressing, bandage or plaster adapted to
release a compound or substance through the skin of a patient.
[0277] The compositions for administration can include a solution
of the antibodies that specifically bind GRP94 (or nucleic acids
encoding these antibodies) and/or BRAF inhibitor(s) dissolved or
suspended in a pharmaceutically acceptable carrier, such as an
aqueous carrier. A variety of aqueous carriers can be used, for
example, buffered saline and the like. These solutions are sterile
and generally free of undesirable matter. These compositions may be
sterilized by conventional, well known sterilization techniques.
The compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, toxicity adjusting agents and
the like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of antibody in these formulations can vary widely,
and will be selected primarily based on fluid volumes, viscosities,
body weight and the like in accordance with the particular mode of
administration selected and the subject's needs.
[0278] A typical pharmaceutical composition for intravenous
administration includes about 0.1 to 10 mg of antibody per subject
per day. Dosages from 0.1 up to about 100 mg per subject per day
may be used, particularly if the agent is administered to a
secluded site and not into the circulatory or lymph system, such as
into a body cavity or into a lumen of an organ. Actual methods for
preparing administrable compositions will be known or apparent to
those skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 19th ed., Mack
Publishing Company, Easton, Pa. (1995).
[0279] Antibodies can be provided in lyophilized form and
rehydrated with sterile water before administration, although they
are also provided in sterile solutions of known concentration. The
antibody solution is then added to an infusion bag containing 0.9%
sodium chloride, USP, and typically administered at a dosage of
from 0.5 to 15 mg/kg of body weight. Considerable experience is
available in the art in the administration of antibody drugs, which
have been marketed in the U.S. since the approval of RITUXAN.RTM.
in 1997. Antibodies can be administered by slow infusion, rather
than in an intravenous push or bolus. In one example, a higher
loading dose is administered, with subsequent, maintenance doses
being administered at a lower level. For example, an initial
loading dose of 4 mg/kg may be infused over a period of some 90
minutes, followed by weekly maintenance doses for 4-8 weeks of 2
mg/kg infused over a 30 minute period if the previous dose was well
tolerated.
[0280] Compositions for administering BRAF inhibitors are known in
the art. Suitable BRAF inhibitors and pharmaceutical formations
including these inhibitors are disclosed for example, in PCT
Publication No. WO2009047505A2, PCT Publication No. WO03086467A1,
U.S. Published Patent Application US20050176740A1, U.S. Pat. No.
6,187,799, U.S. Pat. No. 7,329,670, PCT Publication No.
WO2009143024A2, PCT Publication No. WO2010104945A1, PCT Publication
No. WO2010104973A1, PCT Publication No. WO2010111527A1, U.S.
Published Patent Application No. 20090286783A1 and PCT Publication
No. WO2009152087A1, all of which are incorporated by reference
herein.
The antibody or antigen binding fragment thereof that specifically
binds GRP94 (or a nucleic acid encoding the antibody or antigen
binding fragment) and the BRAF inhibitors can be administered to
slow or inhibit the growth of cells, such as cancer cells. In these
applications, a therapeutically effective amount of an antibody is
administered to a subject in an amount sufficient to inhibit
growth, replication or metastasis of cancer cells, or to inhibit a
sign or a symptom of the cancer. In some embodiments, the
compositions are administered to a subject to inhibit or prevent
the development of metastasis, or to decrease the size or number of
metasases, such as micrometastases, for example micrometastases to
the regional lymph nodes (Goto et al., Clin. Cancer Res.
14(11):3401-3407, 2008).
[0281] These compositions disclosed herein can be administered in
conjunction with another chemotherapeutic agent, either
simultaneously or sequentially. Many chemotherapeutic agents are
presently known in the art. In one embodiment, the chemotherapeutic
agents is selected from the group consisting of mitotic inhibitors,
alkylating agents, anti-metabolites, intercalating antibiotics,
growth factor inhibitors, cell cycle inhibitors, enzymes,
topoisomerase inhibitors, anti-survival agents, biological response
modifiers, anti-hormones, e.g. anti-androgens, and
anti-angiogenesis agents.
[0282] Methods of treating melanoma can further include
administering one or more additional anti-cancer drugs for the
treatment of melanoma with a compound as defined herein. For
example, anti-cancer drugs for the treatment of melanoma,
especially metastatic melanoma, may be selected from alkylating
anti-cancer drugs such as dacarbazine, temozolomide,
mechlorethamine, and nitrosoureas such as carmustine, lomustine,
and fotemustine; taxanes, such as paclitaxel and docetaxel; vinca
alkaloids, such as vinblastine; topoisomerase inhibitors such as
irinotecan; thalidomide; anti-cancer antibiotics such as
streptozocin and dactinomycin; or platinum anti-cancer drugs, such
as cisplatin and carboplatin. Compounds of the invention may be
added to polychemotherapeutic regimes such as the Dartmouth regime,
CVD (cisplatin, vinblastine, and dacarbazine) and BOLD (bleomycin,
vincristine, lomustine, and dacarbazine). In some embodiments, the
anti-cancer drugs are selected from interferons such as, but not
limited to, interferon alpha-2a, interferon alpha-2b, pegylated
interferons such as pegylated interferon alpha-2b. Interleukins
such as interleukin-2 may also be used in combination with
compounds disclosed herein.
[0283] In the methods of treating melanoma described herein, the
therapeutically effective amount of the compound can range from
about 0.25 mg/kg to about 30 mg/kg body weight of the subject. In
some embodiments, the therapeutically effective amount of the
compound can range from about 0.5 mg/kg to about 30 mg/kg, from
about 1 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 25
mg/kg, from about 1 mg/kg to about 15 mg/kg, or from about 1 or 2
mg/kg to about 10 mg/kg. In other embodiments, the amount of the
compound administered to the subject ranges from about 25 to about
1500 mg/day and, preferably, from about 100 or 200 mg/day to about
500 or 600 mg/day.
[0284] Treatment may also include administering the pharmaceutical
formulations of the present methods in combination with other
therapies. For example, a pharmaceutical formulations including the
antibody and BRAF inhibitor may be administered before, during, or
after a surgical procedure and/or radiation therapy.
[0285] Anti-angiogenesis agents, such as MMP-2
(matrix-metalloproteinase 2) inhibitors, MMP-9
(matrix-metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase
II) inhibitors, can be used in conjunction with the antibody and
the BRAF inhibitor. Examples of useful COX-II inhibitors include
CELEBREX.TM. (alecoxib), valdecoxib, and rofecoxib. Examples of
useful matrix metalloproteinase inhibitors are described in PCT
Publication No. WO 96/33172 (published Oct. 24, 1996), PCT
Publication No. WO 96/27583 (published Mar. 7, 1996), European
Patent Application No. 97304971.1 (filed Jul. 8, 1997), European
Patent Application No. 99308617.2 (filed Oct. 29, 1999), PCT
Publication No. WO 98/07697 (published Feb. 26, 1998), PCT
Publication No WO 98/03516 (published Jan. 29, 1998), PCT
Publication No WO 98/34918 (published Aug. 13, 1998), PCT
Publication No WO 98/34915 (published Aug. 13, 1998), PCT
Publication No WO 98/33768 (published Aug. 6, 1998), PCT
Publication No WO 98/30566 (published Jul. 16, 1998), European
Patent Publication 606,046 (published Jul. 13, 1994), European
Patent Publication 931,788 (published Jul. 28, 1999), PCT
Publication No WO 90/05719 (published May 31, 1990), PCT
Publication No WO 99/52910 (published Oct. 21, 1999), PCT
Publication No WO 99/52889 (published Oct. 21, 1999), PCT
Publication No WO 99/29667 (published Jun. 17, 1999), PCT
International Application No. PCT/IB98/01113 (filed Jul. 21, 1998),
European Patent Application No. 99302232.1 (filed Mar. 25, 1999),
U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No.
5,861,510 (issued Jan. 19, 1999), and European Patent Publication
780,386 (published Jun. 25, 1997). In one example, the MMP
inhibitors do not induce arthralgia upon administration. In another
example, the MMP inhibitor selectively inhibits MMP-2 and/or MMP-9
relative to the other matrix-metalloproteinases (such as MMP-1,
MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12,
and MMP-13). Some specific examples of MMP inhibitors of use are
AG-3340, RO 32-3555, RS 13-0830,
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl-
)-amino]-propionic acid;
3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]o-
ctane-3-carboxylic acid hydroxyamide; (2R,3R)
1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-pi-
peridine-2-carboxylic acid hydroxyamide;
4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxyl-
ic acid hydroxyamide;
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-
-amino-]-propionic acid;
4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxyl-
ic acid hydroxyamide; (R)
3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxyl-
ic acid hydroxyamide; (2R,3R)
1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-pi-
peridine-2-carboxylic acid hydroxyamide;
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-et-
hyl)-amino-1-propionic acid;
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro--
pyran-4-yl)-amino]-propionic acid;
3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxaicyclo[3.2.1]oct-
ane-3-carboxylic acid hydroxyamide;
3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]o-
ctane-3-carboxylic acid hydroxyamide; and (R)
3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino-1-tetrahydro-furan-3-carboxy-
lic acid hydroxyamide; and pharmaceutically acceptable salts and
solvates of said compounds.
[0286] The antibody or antigen binding fragment that specifically
bind GRP94 (or nucleic acid encoding the antibody or antigen
binding fragment) and the BRAF inhibitor can also be used with
signal transduction inhibitors, such as agents that can inhibit
EGF-R (epidermal growth factor receptor) responses, such as EGF-R
antibodies, EGF antibodies, and molecules that are EGF-R
inhibitors; VEGF (vascular endothelial growth factor) inhibitors,
such as VEGF receptors and molecules that can inhibit VEGF; and
erbB2 receptor inhibitors, such as organic molecules or antibodies
that bind to the erbB2 receptor, for example, HERCEPTIN.TM.
(Genentech, Inc.). EGF-R inhibitors are described in, for example
in PCT Publication Nos. WO 95/19970 (published Jul. 27, 1995), WO
98/14451 (published Apr. 9, 1998), WO 98/02434 (published Jan. 22,
1998), and U.S. Pat. No. 5,747,498 (issued May 5, 1998).
EGFR-inhibiting agents also include, but are not limited to, the
monoclonal antibodies C225 and anti-EGFR 22Mab (ImClone Systems
Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck
KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. and Merck
KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839
(AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis),
PTK 787 (Novartis), CP 701 (Cephalon), leflunomide
(Pharmacia/Sugen), CI-1033 (Warner Lambert Parke Davis), CI-1033/PD
183,805 (Warner Lambert Parke Davis), CL-387,785 (Wyeth-Ayerst),
BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A (Bristol
Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (Boehringer
Ingelheim), OLX-103 (Merck & Co.), VRCTC-310 (Ventech
Research), EGF fusion toxin (Seragen Inc.), DAB-389
(Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund),
RG-50864 (INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97
(Parker Hughes Cancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa
Hakko) and EGF-R Vaccine (York Medical/Centro de Immunologia
Molecular (CIM)).
[0287] VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen
Inc.), SH-268 (Schering), and NX-1838 (NeXstar) can also be used in
conjunction with an antibody that specifically binds endoplasmin.
VEGF inhibitors are described in, for example in PCT Publication
No. WO 99/24440 (published May 20, 1999), PCT International
Application PCT/IB99/00797 (filed May 3, 1999), PCT Publication No.
WO 95/21613 (published Aug. 17, 1995), PCT Publication No. WO
99/61422 (published Dec. 2, 1999), U.S. Pat. No. 5,834,504 (issued
Nov. 10, 1998), PCT Publication No. WO 98/50356 (published Nov. 12,
1998), U.S. Pat. No. 5,883,113 (issued Mar. 16, 1999), U.S. Pat.
No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No. 5,792,783
(issued Aug. 11, 1998), PCT Publication No. WO 99/10349 (published
Mar. 4, 1999), PCT Publication No. WO 97/32856 (published Sep. 12,
1997), PCT Publication No. WO 97/22596 (published Jun. 26, 1997),
PCT Publication No. WO 98/54093 (published Dec. 3, 1998), PCT
Publication No. WO 98/02438 (published Jan. 22, 1998), WO 99/16755
(published Apr. 8, 1999), and PCT Publication No. WO 98/02437
(published Jan. 22, 1998). Other examples of some specific VEGF
inhibitors are IM862 (Cytran Inc.); anti-VEGF monoclonal antibody
of Genentech, Inc.; and angiozyme, a synthetic ribozyme from
Ribozyme and Chiron. These and other VEGF inhibitors can be used in
conjunction with the antibodies, antigen binding fragments, and the
BRAF inhibitor.
[0288] ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome
plc), and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals
Inc.) and 2B-1 (Chiron), can furthermore be combined with the
antibodies, antigen binding fragments and BRAF inhibitor, for
example those indicated in PCT Publication No. WO 98/02434
(published Jan. 22, 1998), PCT Publication No. WO 99/35146
(published Jul. 15, 1999), PCT Publication No. WO 99/35132
(published Jul. 15, 1999), PCT Publication No. WO 98/02437
(published Jan. 22, 1998), PCT Publication No. WO 97/13760
(published Apr. 17, 1997), PCT Publication No. WO 95/19970
(published Jul. 27, 1995), U.S. Pat. No. 5,587,458 (issued Dec. 24,
1996), and U.S. Pat. No. 5,877,305 (issued Mar. 2, 1999). ErbB2
receptor inhibitors of use are also described in U.S. Provisional
Application No. 60/117,341, filed Jan. 27, 1999, and in U.S.
Provisional Application No. 60/117,346, filed Jan. 27, 1999.
[0289] For the treatment of cancer, such as melanoma, the antibody
or antigen binding fragment that specifically bind GRP94 (or
nucleic acid encoding the antibody or antigen binding fragment) and
a BRAF inhibitor can be used with surgical treatment, or with
another therapeutic including dacarbazine (also termed DTIC), or
interleukin-2 (IL-2) or interferon, such as interferon (IFN). For
the treatment of a superficial melanoma, the antibody or antigen
binding fragment that specifically bind GRP94 (or nucleic acid
encoding the antibody or antigen binding fragment) and a BRAF
inhibitor can be used in conjunction with Imiquimod. However, for
the treatment of another cancer, such as head and neck squamous
cell carcinoma, the antibody or antigen binding fragment that
specifically bind GRP94 (or nucleic acid encoding the antibody or
antigen binding fragment) and a BRAF inhibitor can be used in
conjunction with surgery, radiation therapy, chemotherapy, other
antibodies (such as cetuximab and bevacizumab) or small-molecule
therapeutics (such as erlotinib). One of skill in the art can
readily determine surgical procedures and additional
chemotherapeutic agents of use.
[0290] Single or multiple administrations of the compositions are
administered depending on the dosage and frequency as required and
tolerated by the patient. In any event, the composition should
provide a sufficient quantity of at least one of the antibodies (or
antigen binding fragments thereof) and BRAF inhibitor to
effectively treat the patient. The dosage can be administered once
but may be applied periodically until either a therapeutic result
is achieved or until side effects warrant discontinuation of
therapy. The subject can be treated at regular intervals, such as
daily, weekly, biweekly, or monthly, until a desired therapeutic
result is achieved.
[0291] Generally, the dose is sufficient to treat or ameliorate
symptoms or signs of disease without producing unacceptable
toxicity to the patient.
[0292] Controlled release parenteral formulations can be made as
implants, oily injections, or as particulate systems. For a broad
overview of protein delivery systems see, Banga, A. J., Therapeutic
Peptides and Proteins: Formulation, Processing, and Delivery
Systems, Technomic Publishing Company, Inc., Lancaster, Pa., (1995)
incorporated herein by reference. Particulate systems include
microspheres, microparticles, microcapsules, nanocapsules,
nanospheres, and nanoparticles. Microcapsules contain the
therapeutic protein, such as a cytotoxin or a drug, as a central
core. In microspheres the therapeutic is dispersed throughout the
particle. Particles, microspheres, and microcapsules smaller than
about 1 .mu.m are generally referred to as nanoparticles,
nanospheres, and nanocapsules, respectively. Capillaries have a
diameter of approximately 5 .mu.m so that only nanoparticles are
administered intravenously. Microparticles are typically around 100
.mu.m in diameter and are administered subcutaneously or
intramuscularly. See, for example, Kreuter, J., Colloidal Drug
Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York,
N.Y., pp. 219-342 (1994); and Tice & Tabibi, Treatise on
Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc.
New York, N.Y., pp. 315-339, (1992) both of which are incorporated
herein by reference.
[0293] Polymers can be used for ion-controlled release of the
antibody compositions disclosed herein. Various degradable and
non-degradable polymeric matrices for use in controlled drug
delivery are known in the art (Langer, Accounts Chem. Res.
26:537-542, 1993). For example, the block copolymer, polaxamer 407,
exists as a viscous yet mobile liquid at low temperatures but forms
a semisolid gel at body temperature. It has been shown to be an
effective vehicle for formulation and sustained delivery of
recombinant interleukin-2 and urease (Johnston et al., Pharm.
Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.
44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as
a microcarrier for controlled release of proteins (Ijntema et al.,
Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes
are used for controlled release as well as drug targeting of the
lipid-capsulated drug (Betageri et al., Liposome Drug Delivery
Systems, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)).
Numerous additional systems for controlled delivery of therapeutic
molecules are known (see U.S. Pat. No. 5,055,303; U.S. Pat. No.
5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S.
Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; U.S. Pat. No.
5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S.
Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No.
5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.
Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S. Pat. No.
5,534,496).
[0294] The disclosure is illustrated by the following non-limiting
Examples.
EXAMPLES
[0295] The treatment of tumors, such as metastatic melanoma is
changing rapidly due to the great success in translational research
from bench to bedside. PLX4032 (RG7204, Plexxikon,) is a selective
V600E mutated BRAF inhibitor. A response rate of 70% in 32 BRAF
mutated advanced melanoma patients treated by PLX4032 was reported
in the phase II study (Flaherty et al, N Engl J Med 363:809-819,
2010). Although the significant clinical activity of BRAF selective
inhibitors is a major breakthrough in the treatment of this
disease, there are still hurdles to overcome to optimize this
targeted therapy approach (Flaherty and McArthur, Cancer
116:4902-4913, 2010). Some patients did not respond (primary
resistance). Most patients had partial responses. Moreover, the
median duration of response is approximately only 8 months
(secondary resistance). The next urgent clinical goal is to
identify rational therapy to obtain complete response and to
improve the longevity of initial response to BRAF-inhibitor
(BRAF-I). It is disclosed herein that antibodies that specifically
bind GRP94 have a synergistic effect with BRAF inhibitors to lower
viability of tumor cells and decrease metastasis.
Example 1
Material and Methods
[0296] Generation of BRAFinhibitor (BRAF-I) PLX4720 M21 Resistant
(M21R) Melanoma Cell Line.
[0297] The cell line M21 (harboring the V600E BRAF missense
mutation) (4.times.10.sup.5/well) was cultured in a 6-well plate
containing RPMI 1640 medium supplemented with 10% FBS and 2.5 .mu.M
of PLX4720. Medium was changed every 4 days. On the 8.sup.th day
the concentration of PLX4720 was increased to 5 .mu.M. Cells were
cultured under these conditions for 12 days changing the medium
every 4 days. Then the concentration of PLX4720 in the medium was
increased to 10 .mu.M, and the medium has been changed every 3
days. Cells were cultured until when resistant colonies appeared
(around 3 weeks). Cells were tested for resistance to BRAF-I
PLX4720 by analyzing their growth in the presence of 10 .mu.M of
PLX4720, using the MTT assay.
[0298] Effect of BRAF-I PLX4720 on GRP94 Expression by M21 Cell
Line:
[0299] M21 cells were cultured in RPMI 1640 medium supplemented
with 10% FBS and BIM of PLX4720. Cells were harvested after 14 days
of culture, and washed twice with PBS-BSA. Cells
(1.times.10.sup.5/sample), and were stained with 1 .mu.g of
anti-GRP94 mAb W9. Human IgG (HIg) and untreated cells were used as
controls. Following an incubation of 30 min at 4.degree. C., cells
were washed three times with PBS containing 2% of BSA (PBS-BSA).
Cells were then incubated with an appropriate dilution of
PE-conjugated anti-human IgG for 30 min at 4.degree. C. Cells were
washed and fixed with 2% PFA. Immunofluorescence was measured using
a Cyan cytofluorimeter.
[0300] GRP94 Expression by M21R Melanoma Cell Line:
[0301] M21 acquired resistance to BRAF-I PLX4720 following repeated
exposures to this inhibitor. BRAF-I PLX4720 resistant melanoma
cells M21R and parental melanoma cells M21 were cell surface
stained with the GRP94-specific mAb W9. HIg was used as negative
control. The staining was performed as previously described
Immunofluorescence was measured using a Cyan cytofluorimeter.
[0302] Anti-Cell Growth Effect of GRP94-Specific mAb W9 Combined
with BRAF-I:
[0303] Cells (1.times.10.sup.4 cells/ml) were seeded in
quadruplicate into 96-well plates and treated with W9 mAb combined
with 5 .mu.M of BRAF-I. HIg was used as a control. Cells growth was
analyzed at different time points (1, 2, 3, 4, and 5 days) using
the MTT assay.
[0304] Migration:
[0305] BRAF-I PLX4720 resistant melanoma cells M21R
(2.5.times.10.sup.4/well) were seeded in a 24-transwell plate
(24-well insert, pore size 8 .mu.m; BD Biosciences) in RPMI 1640
medium containing 1% FCS with W9 antibody (Ab), HIg, BRAF-I
combined with W9 Ab, BRAF-I combined with HIg, or BRAF-I only.
Cells migrated toward RPMI1640 medium containing 1% FCS and 10
.mu.g/ml fibronectin. After 72 hours, migrated cells were stained
with HEMA 3 stain set, taken picture and counted under a Zeiss
Inverted Fluorescence Microscope (AxioVision Software). Mean of six
independent high power field (100.times.) were shown as columns.
The experiments were performed in triplicates.
[0306] Immunoblot:
[0307] Human melanoma cell line M21R was serum starved for 3 days
then seeded at the concentration of 1.0.times.10.sup.5 per well in
a 6-well plate in RPMI 1640 medium without serum and incubated with
the GRP94 mAb, HIg, untreated, BRAF-I PLX4720 (5 .mu.M) combined
with W9 mAb, BRAF-I combined with HIg, or BRAF-I only, at
37.degree. C. for an additional 3 days. Cells were lysed in lysis
buffer (10 mM Tris-HCl [pH 8.2], 1% NP40, 1 mM EDTA, 0.1% BSA, 150
mM NaCl) containing 1/50 (vol/vol) of protease inhibitor cocktail
(Calbiochem). Protein concentrations in the lysates were measured
utilizing the Bradford reagent (Bio-Rad, Laboratories, Hercules,
Calif.). Equal amount of proteins (60 .mu.g per well) from the
clarified lysates were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to polyvinylidene fluoride (PVDF) membrane of 0.45
.mu.m pore size (Millipore). After blocking the membranes with 5%
nonfat dry milk plus 2% BSA at room temperature for 2 hours,
membranes were incubated overnight at 4.degree. C. with rabbit
anti-RAS, C-RAF, phosphorylated (p)-MEK(Ser217/221), ERK, p-ERK1/2
(Thr202/Tyr204), PKC.alpha. and .beta.-actin mAb (Cell Signaling
Technology), mouse anti-FAK, p-FAK (Tyr397) mAb (BD Transduction
Laboratories), mouse anti-calnexin mAb TO-5. Then
peroxidase-conjugated secondary antibodies (anti-mouse IgG
antibody, or anti-rabbit IgG antibody) were added and incubation
was continued at room temperature for an additional 45 minutes.
Between the incubations, membranes were washed five times, 5
minutes each, with PBS (pH 7.4) containing 0.1% Tween. Then bound
antibodies were detected using ECL Plus Western Blotting Detection
System (GE Healthcare), and bands were visualized using the
FOTO/Analyst Investigator Eclipse System (Fotodyne Incorporate).
The calnexin and .beta.-actin were used as the protein loading
controls. The densities of resultant bands were determined with
ImageJ software (NIH), normalized to that of Calnexin and
.beta.-actin, and are shown below the respective bands. Data are
expressed as the percentage of the expression in untreated control
cells.
[0308] Statistical Analysis:
[0309] Statistical analysis was performed using the t-test.
Statistical significance was indicated by p<0.05.
Example 2
Effect of BRAF-I PLX4720 on the Expression of GRP94 by M21 Cell
Line
[0310] M21 cells were cultured in RPMI 1640 medium supplemented
with 10% FBS and 1 .mu.M of PLX4720. Cells were harvested after 14
days of culture, and washed twice with PBS-BSA. Cells
(1.times.10.sup.5/sample), and were stained with 1 .mu.g of
anti-GRP94 mAb W9. Human IgG (HIg) and untreated cells were used as
controls. Following an incubation of 30 min at 4.degree. C., cells
were washed three times with PBS containing 2% of BSA (PBS-BSA).
Cells were then incubated with an appropriate dilution of
PE-conjugated anti-human IgG for 30 min at 4.degree. C. Cells were
washed and fixed with 2% PFA Immunofluorescence was measured using
a Cyan cytofluorimeter and the results show treatment of BRAF-I
sensitive melanoma cell line M21 with the BRAF-I PLX4720 results in
increased expression of GRP94. No change in human IgG binding was
detected (FIG. 1). These data suggest that BRAF-I treatment
enhances the anti-tumor effect of GRP94-specific W9 mAb.
Example 3
GRP94 Expression by M21R Melanoma Cell Line M21
[0311] BRAF-I PLX4720 resistant melanoma cells M21R and parental
melanoma cells M21 were cell surface stained with the
GRP94-specific mAb W9. HIg was used as negative control. The
staining was performed as previously described. Immunofluorescence
was measured using a CYAN.TM. cytofluorimeter. The results show
that BRAF-I resistant melanoma cells display an increased
expression of GRP94 when compared to the parental M21 cells (FIG.
2). These results suggest that BRAF-I resistant melanoma cells are
more susceptible to the anti-tumor effect of GRP94-specific
mAb.
Example 4
Anti-Cell Growth Effect of GRP94-Specific mAb W9 Combined with
BRAF-I
[0312] Cells (1.times.10.sup.4 cells/ml) were seeded in
quadruplicate into 96-well plates and treated with W9 mAb combined
with 5 .mu.M of BRAF-I PLX4720. HIg was used as a control. Cells
growth was analyzed at different time points (1, 2, 3, 4, and 5
days) using the MTT assay. The results show that treatment of
BRAF-I resistant M21R cell line with GRP94-specific mAb in
combination with BRAF-I results in lower viability than cells
treated with mAb W9 alone. No changes are detectable in the
viability of BRAF wild type MV3 cells treated with W9 mAb alone or
combined with BRAF-I (FIG. 3). This data shows the synergic
anti-proliferative effect of W9 mAb and BRAF-I on BRAF-I resistant
cell line.
Example 5
Anti-Cell Growth Effect of GRP94-Specific mAb W9 Combined with
BRAF-I
[0313] Cells (1.times.10.sup.4 cells/ml) were seeded in
quadruplicate into 96-well plates and treated with W9 mAb combined
with 500 nM of BRAF-I PLX4032. HIg was used as a control. Cells
growth was analyzed at different time points (1, 3 and 5 days)
using the MTT assay. The results show that treatment of M21, M21R,
Colo38 and Colo38R cell line with GRP94-specific mAb in combination
with BRAF-I results in lower viability than cells treated with mAb
W9 alone (FIG. 4). This data shows the synergic anti-proliferative
effect of W9 mAb and BRAF-I on BRAF-I sensitive and resistant cell
line.
Example 6
Migration
[0314] BRAF-I PLX4720 resistant melanoma cells M21R
(2.5.times.10.sup.4/well) were seeded in a 24-transwell plate
(24-well insert, pore size 8 .mu.m; BD Biosciences) in RPMI 1640
medium containing 1% FCS with W9 antibody (Ab), HIg, BRAF-I
combined with W9 Ab, BRAF-I combined with HIg, or BRAF-I only.
Cells migrated toward RPMI1640 medium containing 1% FCS and 10
.mu.g/ml fibronectin. After 72 hrs incubation, as show in FIG. 5,
GRP94-specific Ab W9 inhibited around 20% of the motility of M21R
cells towards fibronectin in a Boyden chamber assay. When cells
incubated with GRP94-specific Ab W9 combined with BRAF-I, the
inhibition of migration was increased to 45%. This data shows the
synergic anti-migration effect of W9 mAb and BRAF-I on BRAF-I
resistant cell line.
Example 7
Immunoblot
[0315] In vitro incubation with the GRP94-specific W9 mAb showed a
decrease in the level of phosphorylated (p)-MEK (Ser217/221), and
p-ERK1/2 (Thr202/Tyr204) and p-FAK (Tyr397), in the M21R cell line
(FIG. 6). In addition, the total protein levels of RAS, c-RAF, and
PKC.alpha. were also decreased. Treatment of BRAF-I resistant M21R
cell line with W9 in combination with BRAF-I results in lower level
of RAS, c-RAF, p-MEK (Ser217/221) and p-ERK1/2(Thr202/Tyr204) than
those in cells treated with W9 or BRAF-I alone.
Example 8
Combination Therapy with BRAF-I PLX4720 (or PLX4320) and Anti-GRP94
Antibody is More Effective than Either Agent Alone in Inducing
Melanoma Cell Apoptosis
[0316] To measure cell apoptosis in vitro, cells are seeded
(5.0.times.10.sup.4 cells per well) in a 96-well plate and treated
with 5 different regimens as described above for 2, 6 and 24 hours.
Cells are then subjected to staining simultaneously with
FITC-Annexin V (green fluorescence) (BD Biosciences) and the
non-vital dye propidium iodide (red fluorescence) (BD Biosciences),
which allows bivariate analysis to discriminate intact cells
(FITC.sup.-PI.sup.-), early apoptotic (FITC.sup.+PI.sup.-) and late
apoptotic or necrotic cells (FITC.sup.+PI.sup.+). All experiments
are performed three independent times.
[0317] To investigate the mechanisms of action underlying the
effects on cell growth, migration and apoptosis, cells treated as
described above are lysed for examining the changes in the level of
total and activated signaling protein molecules, such as FAK, RAS,
BRAF, ERK1/2, PI3, AKT, PKC.alpha. by Western blot. The
significance of the difference in cell growth, migration, apoptosis
and the level of signaling molecules are analyzed using the Student
T test.
[0318] The primary resistance of melanoma cells to BRAF-I can be
reversed and the secondary resistance of melanoma cells to BRAF-I
can be delayed/prevented by targeting multiple signaling pathways
with BRAF-I and anti-GRP94 antibody. The M233.sup.V600E cell line
is naturally, i.e., primarily resistant to BRAF-I (Sondergaard et
al., J Transl Med 8:39, 2010). In addition to M233 cell line, in
order to obtain at least one more BRAF-I primary resistant cell
line, additional V600E mutated human melanoma cell lines are
screened using a cell growth MTT assay. It is determined that the
combination therapy can reverse their resistance to BRAF-I in these
two primary resistant cell lines in cell growth assays.
Furthermore, the cell lines M21.sup.V600E and SK-MEL-5.sup.V600E
are used, both of which are BRAF-I sensitive to confirm that
secondary resistance can be delayed or prevented in the presence of
anti-GPR94 in cell growth assays.
[0319] Cells were starved for 12 hours and seeded at a density of
2.times.10.sup.5/ml in a 6-well plate and treated for 6 hours with
BRAF-I PLX4032 (500 nM) and Grp94-specific mAb W9 (20 .mu.g/ml) in
RPMI 1640 medium plus 1.5% FCS. Cells were stained with Annexin
V-FITC and PI, and evaluated for apoptosis by flow cytometry
according to the manufacturer's protocol (BD PharMingen, San Diego,
Calif., USA). The early apoptotic cells (annexin V-positive,
PI-negative) were determined using a flow cytometer. The results
show that treatment of M21 and M21R cell lines with GRP94-specific
mAb in combination with BRAF-I PLX4032 induced more cell apoptosis
than cells treated with mAb W9 and BRAF-I PLX4032 alone (FIG. 7).
This data shows the synergic pro-apoptpsis effect of W9 mAb and
BRAF-I on BRAF-I sensitive and resistant cell line.
Example 9
Combination Therapy with BRAF-I PLX4032 and Anti-GRP94 Antibody is
More Effective than Either Agent Alone in Inhibition of Cancer Stem
Cell Proliferation in Vitro
[0320] Cells growing in the exponential phase were seeded at a
density of 2.times.10.sup.5/ml. The cells were treated for 3 days
with BRAF-I PLX4032 (500 nM) and Grp94-specific mAb W9 (20
.mu.g/ml) in RPMI 1640 medium plus with 1.5% FCS. Cells were
stained with ALDEFLUOR.RTM. according to the manufacturer's
protocol (Stem Cell Technologies). Incubation of cells with
ALDEFLUOR.RTM. in the presence the ALDH1-specific inhibitor
diethylaminobenzaldehyde (DEAB) was used as a negative staining
control for the assay. Then cells were stained with ABCB5-specific
mAb RK1(1 .mu.g/ml) for 30 min at 4.degree. C., and incubated with
APC-conjugated secondary mAb (1:200) (Jackson Immunoresearch). The
results show that treatment of M21 and M21R cell lines with
GRP94-specific mAb in combination with BRAF-I PLX 4032 results in
lower viability than cells treated with mAb W9 and PLX4032 alone
(FIG. 8). This data shows the synergic inhibition of growth of
cancer stem cell effect of W9 mAb and BRAF-I on BRAF-I sensitive
and resistant cell line.
Example 10
Inhibition by Grp94-Specific mAb W9 and BRAF-I PLX 4032 of
Signaling Pathways in BRAFV600E Mutant and BRAF-I Resistant
Melanoma Cells
[0321] Cells growing in the exponential phase were seeded at a
density of 2.times.10.sup.5/ml. The cells were treated for 3 days
with BRAF-I PLX4032 (500 nM) and Grp94-specific mAb W9 (5 .mu.g/ml)
in RPMI 1640 medium plus with 2% FCS. Then the cells were collected
and lysed in lysis buffer. The expression and activation of
multiple signaling molecules were analyzed by immunoblot. The
results show that the combination of GRP94-specific mAb W9 and
BRAF-I PLX4032 inhibited the expression and activation of signaling
molecules important for cell proliferation (RAS, MER, ERK1/2), for
hedgehog signaling pathway (GLI1 and SHh) as compared to the
protein level in cells treated by GRP94-specific mAb W9 and BRAF-I
PLX4032 alone (FIG. 9).
Example 11
V600E Mutated Human Melanoma Cell Lines and Resistance to PLX4720
and/or PLX4320
[0322] Several V600E mutated human melanoma cell lines are tested
to identify the cell lines which maintain the same growth rate at
days 1, 3 and 7 in the presence and absence of BRAF-I PLX4720 (or
PLX4320) at 5 .mu.M in MTT assays. The identified cell lines, which
are primary resistant to BRAF-I are confirmed with a higher dose
(10 .mu.M) of PLX4720 (or PLX4320) in cell growth assays.
[0323] The cells are treated with treatment with BRAF-I PLX4720 and
an antibody that specifically binds GRP94 (anti-GRP94) to determine
that primary resistance of human melanoma cell lines to BRAF-I
PLX4720 (or PLX4320) is affected by the combination therapy. In
addition to cell line M233.sup.V600E, the additional primary
resistant cell lines are treated in several different regimens and
then cell growth, migration and apoptosis is measured. Combination
therapy affects the growth, migration and apoptosis of the cell
lines.
[0324] It is also determined that combination treatment with BRAF-I
PLX4720 (or PLX4320) and anti-GRP94 delays and/or prevents
secondary resistance of human melanoma cell lines to BRAF-I PLX4720
(or PLX4320). It has been determined that V600E mutated melanoma
cell lines, which are sensitive to BRAF-I in vitro treatment,
became resistant, i.e., secondary resistant, to PLX4720 (or
PLX4320) after 3-4 week of in vitro treatment with increasing doses
(2.5-10 .mu.M) of PLX4720 (or PLX4320).
[0325] The cells (4.times.10.sup.5/well) are cultured in a 6-well
plate containing 2 ml of RPMI 1640 medium supplemented with 10% FBS
and 2.5 .mu.M of PLX-4720 (or PLX4320). Medium is changed every 4
days. On day 8, the dose of PLX4720 (or PLX4320) is increased to 5
.mu.M. Cells are cultured under these conditions for 12 days
changing the medium every 4 days. Then the dose of PLX4720 (or
PLX4320) is increased to 10 .mu.M, changing the medium every 3
days. Cells are cultured until resistant colonies appear (around
3-4 weeks). Cells are tested for resistance to BRAF-I by testing
their growth in the presence of 10 .mu.M of PLX4720 (or PLX4320),
using the MTT assay. Simultaneously, cells in one well will be set
up in the exact same way except adding anti-GRP94 every 3-4 days at
its optimal dose identified as described above. In the control
well, everything is not changed but only anti-GRP94 is replaced
with the isotype control mAb. The cells, in the presence of both
PLX4720 (or PLX4320) and anti-GRP94, are kept in culture as long as
needed for monitoring whether and/or when secondary resistance
occurs.
[0326] To analyze the mechanisms of action of combination treatment
in primary and secondary resistances, cells are lysed for examining
the changes in the level of total and activated signaling protein
molecules by western blot as above. The significance of the
difference in cell growth and the level of signaling molecules is
analyzed using the Student T test.
[0327] It will be apparent that the precise details of the methods
or compositions described may be varied or modified without
departing from the spirit of the described invention. We claim all
such modifications and variations that fall within the scope and
spirit of the claims below.
Sequence CWU 1
1
71803PRTHomo sapiens 1Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys
Val Leu Leu Thr Phe 1 5 10 15 Gly Ser Val Arg Ala Asp Asp Glu Val
Asp Val Asp Gly Thr Val Glu 20 25 30 Glu Asp Leu Gly Lys Ser Arg
Glu Gly Ser Arg Thr Asp Asp Glu Val 35 40 45 Val Gln Arg Glu Glu
Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser 50 55 60 Gln Ile Arg
Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala 65 70 75 80 Glu
Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn 85 90
95 Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu
100 105 110 Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu
Ser Gly 115 120 125 Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys
Glu Lys Asn Leu 130 135 140 Leu His Val Thr Asp Thr Gly Val Gly Met
Thr Arg Glu Glu Leu Val 145 150 155 160 Lys Asn Leu Gly Thr Ile Ala
Lys Ser Gly Thr Ser Glu Phe Leu Asn 165 170 175 Lys Met Thr Glu Ala
Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile 180 185 190 Gly Gln Phe
Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys 195 200 205 Val
Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu 210 215
220 Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr
225 230 235 240 Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu
Glu Ala Ser 245 250 255 Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu
Val Lys Lys Tyr Ser 260 265 270 Gln Phe Ile Asn Phe Pro Ile Tyr Val
Trp Ser Ser Lys Thr Glu Thr 275 280 285 Val Glu Glu Pro Met Glu Glu
Glu Glu Ala Ala Lys Glu Glu Lys Glu 290 295 300 Glu Ser Asp Asp Glu
Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys 305 310 315 320 Pro Lys
Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met 325 330 335
Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu 340
345 350 Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp
Asp 355 360 365 Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val
Thr Phe Lys 370 375 380 Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg
Gly Leu Phe Asp Glu 385 390 395 400 Tyr Gly Ser Lys Lys Ser Asp Tyr
Ile Lys Leu Tyr Val Arg Arg Val 405 410 415 Phe Ile Thr Asp Asp Phe
His Asp Met Met Pro Lys Tyr Leu Asn Phe 420 425 430 Val Lys Gly Val
Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg 435 440 445 Glu Thr
Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu 450 455 460
Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr 465
470 475 480 Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu
Gly Val 485 490 495 Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys
Leu Leu Arg Phe 500 505 510 Gln Ser Ser His His Pro Thr Asp Ile Thr
Ser Leu Asp Gln Tyr Val 515 520 525 Glu Arg Met Lys Glu Lys Gln Asp
Lys Ile Tyr Phe Met Ala Gly Ser 530 535 540 Ser Arg Lys Glu Ala Glu
Ser Ser Pro Phe Val Glu Arg Leu Leu Lys 545 550 555 560 Lys Gly Tyr
Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys 565 570 575 Ile
Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala 580 585
590 Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg
595 600 605 Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met
Lys Asp 610 615 620 Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val
Ser Gln Arg Leu 625 630 635 640 Thr Glu Ser Pro Cys Ala Leu Val Ala
Ser Gln Tyr Gly Trp Ser Gly 645 650 655 Asn Met Glu Arg Ile Met Lys
Ala Gln Ala Tyr Gln Thr Gly Lys Asp 660 665 670 Ile Ser Thr Asn Tyr
Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn 675 680 685 Pro Arg His
Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp 690 695 700 Glu
Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr 705 710
715 720 Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr
Gly 725 730 735 Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile
Asp Pro Asp 740 745 750 Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro
Glu Glu Thr Ala Glu 755 760 765 Asp Thr Thr Glu Asp Thr Glu Gln Asp
Glu Asp Glu Glu Met Asp Val 770 775 780 Gly Thr Asp Glu Glu Glu Glu
Thr Ala Lys Glu Ser Thr Ala Glu Lys 785 790 795 800 Asp Glu Leu
22780DNAHomo sapiens 2gtgggcggac cgcgcggctg gaggtgtgag gatccgaacc
caggggtggg gggtggaggc 60ggctcctgcg atcgaagggg acttgagact caccggccgc
acgccatgag ggccctgtgg 120gtgctgggcc tctgctgcgt cctgctgacc
ttcgggtcgg tcagagctga cgatgaagtt 180gatgtggatg gtacagtaga
agaggatctg ggtaaaagta gagaaggatc aaggacggat 240gatgaagtag
tacagagaga ggaagaagct attcagttgg atggattaaa tgcatcacaa
300ataagagaac ttagagagaa gtcggaaaag tttgccttcc aagccgaagt
taacagaatg 360atgaaactta tcatcaattc attgtataaa aataaagaga
ttttcctgag agaactgatt 420tcaaatgctt ctgatgcttt agataagata
aggctaatat cactgactga tgaaaatgct 480ctttctggaa atgaggaact
aacagtcaaa attaagtgtg ataaggagaa gaacctgctg 540catgtcacag
acaccggtgt aggaatgacc agagaagagt tggttaaaaa ccttggtacc
600atagccaaat ctgggacaag cgagttttta aacaaaatga ctgaagcaca
ggaagatggc 660cagtcaactt ctgaattgat tggccagttt ggtgtcggtt
tctattccgc cttccttgta 720gcagataagg ttattgtcac ttcaaaacac
aacaacgata cccagcacat ctgggagtct 780gactccaatg aattttctgt
aattgctgac ccaagaggaa acactctagg acggggaacg 840acaattaccc
ttgtcttaaa agaagaagca tctgattacc ttgaattgga tacaattaaa
900aatctcgtca aaaaatattc acagttcata aactttccta tttatgtatg
gagcagcaag 960actgaaactg ttgaggagcc catggaggaa gaagaagcag
ccaaagaaga gaaagaagaa 1020tctgatgatg aagctgcagt agaggaagaa
gaagaagaaa agaaaccaaa gactaaaaaa 1080gttgaaaaaa ctgtctggga
ctgggaactt atgaatgata tcaaaccaat atggcagaga 1140ccatcaaaag
aagtagaaga agatgaatac aaagctttct acaaatcatt ttcaaaggaa
1200agtgatgacc ccatggctta tattcacttt actgctgaag gggaagttac
cttcaaatca 1260attttatttg tacccacatc tgctccacgt ggtctgtttg
acgaatatgg atctaaaaag 1320agcgattaca ttaagctcta tgtgcgccgt
gtattcatca cagacgactt ccatgatatg 1380atgcctaaat acctcaattt
tgtcaagggt gtggtggact cagatgatct ccccttgaat 1440gtttcccgcg
agactcttca gcaacataaa ctgcttaagg tgattaggaa gaagcttgtt
1500cgtaaaacgc tggacatgat caagaagatt gctgatgata aatacaatga
tactttttgg 1560aaagaatttg gtaccaacat caagcttggt gtgattgaag
accactcgaa tcgaacacgt 1620cttgctaaac ttcttaggtt ccagtcttct
catcatccaa ctgacattac tagcctagac 1680cagtatgtgg aaagaatgaa
ggaaaaacaa gacaaaatct acttcatggc tgggtccagc 1740agaaaagagg
ctgaatcttc tccatttgtt gagcgacttc tgaaaaaggg ctatgaagtt
1800atttacctca cagaacctgt ggatgaatac tgtattcagg cccttcccga
atttgatggg 1860aagaggttcc agaatgttgc caaggaagga gtgaagttcg
atgaaagtga gaaaactaag 1920gagagtcgtg aagcagttga gaaagaattt
gagcctctgc tgaattggat gaaagataaa 1980gcccttaagg acaagattga
aaaggctgtg gtgtctcagc gcctgacaga atctccgtgt 2040gctttggtgg
ccagccagta cggatggtct ggcaacatgg agagaatcat gaaagcacaa
2100gcgtaccaaa cgggcaagga catctctaca aattactatg cgagtcagaa
gaaaacattt 2160gaaattaatc ccagacaccc gctgatcaga gacatgcttc
gacgaattaa ggaagatgaa 2220gatgataaaa cagttttgga tcttgctgtg
gttttgtttg aaacagcaac gcttcggtca 2280gggtatcttt taccagacac
taaagcatat ggagatagaa tagaaagaat gcttcgcctc 2340agtttgaaca
ttgaccctga tgcaaaggtg gaagaagagc ccgaagaaga acctgaagag
2400acagcagaag acacaacaga agacacagag caagacgaag atgaagaaat
ggatgtggga 2460acagatgaag aagaagaaac agcaaaggaa tctacagctg
aaaaagatga attgtaaatt 2520atactctcac catttggatc ctgtgtggag
agggaatgtg aaatttacat catttctttt 2580tgggagagac ttgttttgga
tgccccctaa tccccttctc ccctgcactg taaaatgtgg 2640gattatgggt
cacaggaaaa agtgggtttt ttagttgaat tttttttaac attcctcatg
2700aatgtaaatt tgtactattt aactgactat tcttgatgta aaatcttgtc
atgtgtataa 2760aaataaaaaa gatcccaaat 27803443PRTHomo sapiens 3Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu
Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr
Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr
Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala His Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145
150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp 195 200 205 Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265
270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
275 280 285 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390
395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 4 213PRTHomo sapiens 4Glu Ile Glu 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 Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly 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 Ser Tyr Ser Thr Pro
Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Val
Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205 Asn Arg Gly Glu Cys 210 5613PRTPseudomonas aeruginosa 5Ala
Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val 1 5 10
15 Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro
20 25 30 Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser
Met Val 35 40 45 Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile
Asp Asn Ala Leu 50 55 60 Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg
Leu Glu Gly Gly Val Glu 65 70 75 80 Pro Asn Lys Pro Val Arg Tyr Ser
Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95 Trp Ser Leu Asn Trp Leu
Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105 110 Ile Lys Val Phe
Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His 115 120 125 Met Ser
Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140
Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu 145
150 155 160 Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val
Val Met 165 170 175 Ala Gln Thr Gln Pro Arg Arg Glu Lys Arg Trp Ser
Glu Trp Ala Ser 180 185 190 Gly Lys Val Leu Cys Leu Leu Asp Pro Leu
Asp Gly Val Tyr Asn Tyr 195 200 205 Leu Ala Gln Gln Arg Cys Asn Leu
Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220 Tyr Arg Val Leu Ala Gly
Asn Pro Ala Lys His Asp Leu Asp Ile Lys 225 230 235 240 Pro Thr Val
Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu 245 250 255 Ala
Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe 260 265
270 Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly
275 280 285 Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg
Leu Ser 290 295 300 Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu
Ala Ser Pro Gly 305 310 315 320 Ser Gly Gly Asp Leu Gly Glu Ala Ile
Arg Glu Gln Pro Glu Gln Ala 325 330 335 Arg Leu Ala Leu Thr Leu Ala
Ala Ala Glu Ser Glu Arg Phe Val Arg 340 345 350 Gln Gly Thr Gly Asn
Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val 355 360 365 Ser Leu Thr
Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp 370 375 380 Ser
Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe 385 390
395
400 Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn
405 410 415 Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu
Glu Arg 420 425 430 Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu
Glu Ala Ala Gln 435 440 445 Ser Ile Val Phe Gly Gly Val Arg Ala Arg
Ser Gln Asp Leu Asp Ala 450 455 460 Ile Trp Arg Gly Phe Tyr Ile Ala
Gly Asp Pro Ala Leu Ala Tyr Gly 465 470 475 480 Tyr Ala Gln Asp Gln
Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly 485 490 495 Ala Leu Leu
Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr 500 505 510 Arg
Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu 515 520
525 Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly
530 535 540 Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp
Pro Leu 545 550 555 560 Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile
Pro Thr Asp Pro Arg 565 570 575 Asn Val Gly Gly Asp Leu Asp Pro Ser
Ser Ile Pro Asp Lys Glu Gln 580 585 590 Ala Ile Ser Ala Leu Pro Asp
Tyr Ala Ser Gln Pro Gly Lys Pro Pro 595 600 605 Arg Glu Asp Leu Lys
610 64PRTPseudomonas aeruginosa 6Lys Asp Glu Leu 1 74PRTPseudomonas
aeruginosa 7Arg Glu Asp Leu 1
* * * * *
References