U.S. patent application number 16/606377 was filed with the patent office on 2020-02-06 for p-ethoxy nucleic acids for igf-1r inhibition.
This patent application is currently assigned to Bio-Path Holdings, Inc.. The applicant listed for this patent is Bio-Path Holdings, Inc., Thomas Jefferson University. Invention is credited to David W. Andrews, Ana Ashizawa, Douglas Craig Hooper.
Application Number | 20200038429 16/606377 |
Document ID | / |
Family ID | 63856055 |
Filed Date | 2020-02-06 |
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United States Patent
Application |
20200038429 |
Kind Code |
A1 |
Ashizawa; Ana ; et
al. |
February 6, 2020 |
P-ETHOXY NUCLEIC ACIDS FOR IGF-1R INHIBITION
Abstract
Provided herein are methods of treating cancer or an autoimmune
disease comprising administering a liposome that comprises neutral
phospholipids and a P-ethoxy oligonucleotide that targets a
IGF-1R-encoding polynucleotide.
Inventors: |
Ashizawa; Ana; (Bellaire,
TX) ; Hooper; Douglas Craig; (Medford, NJ) ;
Andrews; David W.; (Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Path Holdings, Inc.
Thomas Jefferson University |
Bellaire
Philadephia |
TX
PA |
US
US |
|
|
Assignee: |
Bio-Path Holdings, Inc.
Bellaire
TX
Thomas Jefferson University
Philadephia
PA
|
Family ID: |
63856055 |
Appl. No.: |
16/606377 |
Filed: |
April 19, 2018 |
PCT Filed: |
April 19, 2018 |
PCT NO: |
PCT/US2018/028308 |
371 Date: |
October 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62487425 |
Apr 19, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7125 20130101;
A61K 45/06 20130101; A61K 9/19 20130101; A61P 35/00 20180101; A61K
9/0019 20130101; A61K 47/6911 20170801; A61K 9/127 20130101 |
International
Class: |
A61K 31/7125 20060101
A61K031/7125; A61K 47/69 20060101 A61K047/69; A61P 35/00 20060101
A61P035/00 |
Claims
1. A method of treating a subject with a cancer or an autoimmune
disease comprising administering to the subject a therapeutically
effective amount of a pharmaceutical composition comprising a
composition comprising a population of oligonucleotides, wherein
the oligonucleotides hybridize to an IGF-1R polynucleotide gene
product, wherein oligonucleotides of the population are composed of
nucleoside molecules linked together through phosphate backbone
linkages, wherein at least one of the phosphate backbone linkages
in each oligonucleotide is a P-ethoxy backbone linkage, and wherein
no more than 80% of the phosphate backbone linkages in each
oligonucleotide are P-ethoxy backbone linkages, phospholipids, and
a pharmaceutically acceptable carrier, wherein the oligonucleotides
and phospholipids form an oligonucleotide-lipid complex.
2. The method of claim 1, wherein the pharmaceutical composition
further comprises a chemotherapeutic agent.
3. The method of claim 1, wherein oligonucleotides of the
population comprise a sequence according to either of SEQ ID NOs: 1
or 2.
4. The method of claim 3, wherein oligonucleotides of the
population comprise a sequence according to SEQ ID NO: 1.
5. The method of claim 3, wherein oligonucleotides of the
population comprise a sequence according to SEQ ID NO: 2.
6. The method of claim 1, wherein 50% to 80% of the phosphate
backbone linkages are P-ethoxy backbone linkages.
7. The method of claim 6, wherein 60% to 75% of the phosphate
backbone linkages are P-ethoxy backbone linkages.
8. The method of claim 1, wherein 20% to 50% of the phosphate
backbone linkages are phosphodiester backbone linkages.
9. The method of claim 8, wherein 25% to 40% of the phosphate
backbone linkages are phosphodiester backbone linkages.
10. The method of claim 1, wherein the phosphodiester backbone
linkages are distributed throughout each oligonucleotide.
11. The method of claim 1, wherein the phosphodiester backbone
linkages are not clustered within a portion of each
oligonucleotide.
12. The method of claim 1, wherein the population of
oligonucleotides is heterogeneous as to the number of P-ethoxy
backbone linkages and phosphodiester backbone linkages present in
the oligonucleotides of the population.
13. The method of claim 1, wherein the oligonucleotides of the
population have a size ranging from 18 to 30 nucleotides.
14. The method of claim 13, wherein the oligonucleotides of the
population have an average size of 18 nucleotides, wherein no more
than 14 of the phosphate backbone linkages in each oligonucleotide
is a P-ethoxy backbone linkage.
15. The method of claim 13, wherein the oligonucleotides of the
population have an average size of 20 nucleotides, wherein no more
than 16 of the phosphate backbone linkages in each oligonucleotide
is a P-ethoxy backbone linkage.
16. The method of claim 13, wherein the oligonucleotides of the
population have an average size of 25 nucleotides, wherein no more
than 20 of the phosphate backbone linkages in each oligonucleotide
is a P-ethoxy backbone linkage.
17. The method of claim 13, wherein the oligonucleotides of the
population have an average size of 30 nucleotides, wherein no more
than 24 of the phosphate backbone linkages in each oligonucleotide
is a P-ethoxy backbone linkage.
18. The method of claim 1, wherein the population of
oligonucleotides comprises a single species of
oligonucleotides.
19. The method of claim 1, wherein the population of
oligonucleotides comprises at least two species of
oligonucleotides.
20. The method of claim 1, wherein the population of
oligonucleotides is heterogeneous as to the distribution of
phosphodiester backbone linkages among the oligonucleotides of the
population.
21. The method of claim 1, wherein the phospholipids are uncharged
or have a neutral charge at physiologic pH.
22. The method of claim 21, wherein the phospholipids are neutral
phospholipids.
23. The method of claim 22, wherein the neutral phospholipids are
phosphatidylcholines.
24. The method of claim 23, wherein the neutral phospholipids are
dioleoylphosphatidyl choline.
25. The method of claim 1, wherein the phospholipids are
essentially free of cholesterol.
26. The method of claim 1, wherein the phospholipids and
oligonucleotides are present at a molar ratio of from about 5:1 to
about 100:1.
27. The method of claim 1, wherein the oligonucleotide-lipid
complex is further defined as a population of liposomes.
28. The method of claim 27, wherein at least 90% of the liposomes
are less than 5 microns in diameter.
29. The method of claim 27, wherein at least 90% of the liposomes
are less than 4 microns in diameter.
30. The method of claim 27, wherein the population of
oligonucleotides is incorporated in the population of
liposomes.
31. The method of claim 15, wherein the subject is a human.
32. The method of claim 1, wherein the cancer is a non-small cell
lung cancer, pancreatic adenocarcinoma, breast cancer, prostate
cancer, melanoma, colon cancer, leukemia, lymphoma, glioma,
glioblastoma, astrocytoma, osteosarcoma, oral cavity cancer,
ovarian cancer, uterine cancer, bone cancer, brain cancer, prostate
cancer, kidney cancer, stomach cancer, esophageal cancer, rectal
cancer, bladder cancer, testicular cancer, or liver cancer.
33. The method of claim 32, wherein the astrocytoma is glioblastoma
multiforme.
34. The method of claim 1, wherein the composition is administered
subcutaneously, intravenously, or intraperitoneally.
35. The method of claim 1, further comprising administering at
least a second anticancer therapy to the subject.
36. The method of claim 24, wherein the second anticancer therapy
is a surgical therapy, chemotherapy, radiation therapy,
cryotherapy, hormone therapy, immunotherapy, anti-viral therapy,
immune suppression therapy, anti-bacterial therapy, anti-parasite
therapy, anti-fungal therapy, or cytokine therapy.
37. The method of claim 1, wherein the autoimmune disease is a Th2
dominant autoimmune disease.
38. The method of claim 37, wherein the Th2 dominant autoimmune
disease is lupus, allergic dermatitis, scleroderma, atopic eczema,
sinusitis, inflammatory bowel disease, asthma, ulcerative colitis,
or multiple chemical sensitivity.
39. The method of claim 1, wherein administration of the
composition reduces expression of IGF-1R protein in the
patient.
40. A method for reducing the expression level of IGF-1R protein in
a cell, comprising contacting the cell with a therapeutically
effective amount of a pharmaceutical composition comprising a
composition comprising a population of oligonucleotides, wherein
the oligonucleotides hybridize to an IGF-1R polynucleotide gene
product, wherein oligonucleotides of the population are composed of
nucleoside molecules linked together through phosphate backbone
linkages, wherein at least one of the phosphate backbone linkages
in each oligonucleotide is a P-ethoxy backbone linkage, and wherein
no more than 80% of the phosphate backbone linkages in each
oligonucleotide are P-ethoxy backbone linkages, phospholipids, and
a pharmaceutically acceptable carrier, wherein the oligonucleotides
and phospholipids form an oligonucleotide-lipid complex.
41. A method for delivering a therapeutically effective amount of
an oligonucleotide to a cell comprising contacting the cell with a
therapeutically effective amount of a pharmaceutical composition
comprising a composition comprising a population of
oligonucleotides, wherein the oligonucleotides hybridize to an
IGF-1R polynucleotide gene product, wherein oligonucleotides of the
population are composed of nucleoside molecules linked together
through phosphate backbone linkages, wherein at least one of the
phosphate backbone linkages in each oligonucleotide is a P-ethoxy
backbone linkage, and wherein no more than 80% of the phosphate
backbone linkages in each oligonucleotide are P-ethoxy backbone
linkages, phospholipids, and a pharmaceutically acceptable carrier,
wherein the oligonucleotides and phospholipids form an
oligonucleotide-lipid complex.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/487,425 filed Apr. 19, 2017, which is
incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The present application contains a sequence listing, which a
computer readable form (CRF) text file in compliance with 37 CFR
1.821 is submitted herewith and referenced herein in its
entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] The present invention relates generally to the field of
medicine. More particularly, it concerns liposomal formulations of
P-ethoxy oligonucleotides that hybridize to a IGF-1R polynucleotide
gene product and methods of making and using such formulations in
medicine, even more particularly in the treatment of cancers that
have high expression or increased activity of the IGF-1R gene.
2. Description of Related Art
[0004] The insulin-like growth factor 1 receptor (IGF-1R) is a
glycoprotein receptor with tyrosine kinase activity. It is a
hetero-tetrameric receptor of which each half--linked by disulfide
bridges--is composed of an extracellular .alpha.-subunit and of a
transmembrane (3-subunit. IGF-IR binds IGF I and IGF II with a very
high affinity. IGF-1R mediates mitogenic, differentiation, and
antiapoptosis effects. The cytoplasmic tyrosine kinase proteins are
activated by the binding of the ligand to the extracellular domain
of the receptor. The activation of the kinases in its turn involves
the stimulation of different intra-cellular substrates, including
IRS-1, IRS-2, Shc and Grb 10.
[0005] The role of the IGF system in carcinogenesis has become the
subject of intensive research. This interest followed the discovery
of the fact that in addition to its mitogenic and antiapoptosis
properties, IGF-1R seems to be required for the establishment and
the maintenance of a transformed phenotype. In fact, it has been
well established that an overexpression or a constitutive
activation of IGF-1R leads, in a great variety of cells, to a
growth of the cells independent of the support in media devoid of
fetal calf serum, and to the formation of tumors in nude mice.
IGF-IR is expressed in a great variety of tumors and of tumor lines
and the IGFs amplify the tumor growth via their attachment to
IGF-1R. Interestingly, murine monoclonal antibodies directed
against IGF-1R inhibit the proliferation of numerous cell lines in
culture and the growth of tumor cells in vivo (Arteaga et al.,
1989; Li et al., 1993; Zia et al., 1996; Scotlandi et al., 1998).
In addition, a negative dominant of IGF-IR is capable of inhibiting
tumor proliferation (Jiang et al., 1999). Thus, IGF-1R plays
important roles in carcinogenesis and tumor progression. As such,
compositions and methods for effectively inhibiting IGF-1R
expression are needed.
SUMMARY OF THE INVENTION
[0006] Provided herein are compositions for inhibiting IGF-1R
expression using a non-toxic nuclease resistant oligonucleotide
that targets IGF-1R-encoding polynucleotides in combination with a
neutral liposome that prevents IGF-1R protein expression, thus
eliminating the pool of available IGF-1R protein.
[0007] In one embodiment, compositions are provided comprising a
population of oligonucleotides that hybridize to a IGF-JR
polynucleotide gene product. In some aspects, the oligonucleotides
of the population are composed of nucleoside molecules linked
together through phosphate backbone linkages, wherein at least one
of the phosphate backbone linkages in each oligonucleotide is a
P-ethoxy backbone linkage, and wherein no more than 80% of the
phosphate backbone linkages in each oligonucleotide are P-ethoxy
backbone linkages. In some aspects, at least one of the phosphate
backbone linkages in each oligonucleotide is a phosphodiester
backbone linkage. In some aspects, the oligonucleotides of the
population comprise a sequence according to any one of SEQ ID NOs:
1 or 2. In some aspects, the oligonucleotides of the population
comprise a sequence according to SEQ ID NO: 1. In one aspect, the
oligonucleotides of the population comprise a sequence according to
SEQ ID NO: 1 and the phosphate backbone linkages at least between
nucleotides 5 and 6, between nucleotides 11 and 12, and between
nucleotides 16 and 17 of the oligonucleotides of the population are
phosphodiester backbone linkages. In some aspects, the
oligonucleotides of the population comprise a sequence according to
SEQ ID NO: 2. In one aspect, the oligonucleotides of the population
comprise a sequence according to SEQ ID NO: 2 and the phosphate
backbone linkages at least between nucleotides 5 and 6, between
nucleotides 11 and 12, and between nucleotides 17 and 18 of the
oligonucleotides of the population are phosphodiester backbone
linkages. In various aspects, the oligonucleotides of the
population inhibit the expression of IGF-1R protein. In some
aspects, the composition is lyophilized.
[0008] In some aspects, 10% to 80% of the phosphate backbone
linkages are P-ethoxy backbone linkages; 20% to 80% of the
phosphate backbone linkages are P-ethoxy backbone linkages; 30% to
80% of the phosphate backbone linkages are P-ethoxy backbone
linkages; 40% to 80% of the phosphate backbone linkages are
P-ethoxy backbone linkages; 50% to 80% of the phosphate backbone
linkages are P-ethoxy backbone linkages; or 60% to 70% of the
phosphate backbone linkages are P-ethoxy backbone linkages, or any
range derivable therein. In some aspects, 20% to 90% of the
phosphate backbone linkages are phosphodiester backbone linkages;
20% to 80% of the phosphate backbone linkages are phosphodiester
backbone linkages; 20% to 70% of the phosphate backbone linkages
are phosphodiester backbone linkages; 20% to 60% of the phosphate
backbone linkages are phosphodiester backbone linkages; 20% to 50%
of the phosphate backbone linkages are phosphodiester backbone
linkages; or 30% to 40% of the phosphate backbone linkages are
phosphodiester backbone linkages, or any range derivable therein.
In various aspects, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,
or any value therein, of the phosphate backbone linkages are
P-ethoxy backbone linkages. In various aspects, at most 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, or 95%, or any value therein, of the phosphate
backbone linkages are phosphodiester backbone linkages. In certain
aspects, the phosphodiester backbone linkages are distributed
throughout the oligonucleotides. As such, the oligonucleotides are
not chimeric molecules. In some aspects, the oligonucleotides do
not comprise a phosphorothioate backbone linkage.
[0009] In some aspects, the oligonucleotides of the population have
a size ranging from 7 to 30 nucleotides. In certain aspects, the
oligonucleotides of the population have a size ranging from 12 to
25 nucleotides. In various aspects, the oligonucleotides of the
population have a size of at least 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 nucleotides. The size range may be an average size of the
oligonucleotides in the population.
[0010] In some aspects, the oligonucleotides of the population have
an average size of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein
no more than 5, 6, 7, 8, 8, 9, 10, 11, 11, 12, 13, 14, 15, 15, 16,
17, 18, 19, 20, 20, 21, 22, 23, or 24, respectively, of the
phosphate backbone linkages in each oligonucleotide is a P-ethoxy
backbone linkage. In some aspects, the oligonucleotides of the
population have an average size of 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides and at least 2, 2, 2, 2, 3, 3, 3, 3, 4, 4, 4, 4, 4, 5,
5, 5, 5, 5, 5, 6, 6, 6, 6, or 6, respectively, of the phosphate
backbone linkages in each oligonucleotide is a phosphodiester
backbone linkage. By way of example, the oligonucleotides of the
population may have an average size of 18 nucleotides, wherein no
more than 14 of the phosphate backbone linkages in each
oligonucleotide is a P-ethoxy backbone linkage; the
oligonucleotides of the population may have an average size of 20
nucleotides, wherein no more than 16 of the phosphate backbone
linkages in each oligonucleotide is a P-ethoxy backbone linkage;
the oligonucleotides of the population may have an average size of
25 nucleotides, wherein no more than 20 of the phosphate backbone
linkages in each oligonucleotide is a P-ethoxy backbone linkage; or
the oligonucleotides of the population may have an average size of
30 nucleotides, wherein no more than 24 of the phosphate backbone
linkages in each oligonucleotide is a P-ethoxy backbone
linkage.
[0011] In some aspects, the population of oligonucleotides
comprises a single species of oligonucleotides. In other aspects,
the population of oligonucleotides comprises at least two species
of oligonucleotides. A single species of oligonucleotide may have
the same nucleotide sequence but either have or lack P-ethoxy
linkages in different positions within the molecule. As such, the
population may be homogeneous as to the nucleotide sequence and
heterogeneous as to the distribution of phosphodiester backbone
linkages among the oligonucleotides of the population. In addition,
the population may be heterogeneous as to the number of P-ethoxy
backbone linkages and phosphodiester backbone linkages among the
oligonucleotides of the population. As a non-limiting example, a
first portion of the oligonucleotides of the population may have
70% P-ethoxy linkages and 30% phosphodiester linkages while a
second portion of the oligonucleotides of the population may have
60% P-ethoxy linkages and 40% phosphodiester linkages. In some
aspects, the population of oligonucleotides comprises antisense
oligonucleotides, short interfering RNAs (siRNAs), microRNAs
(miRNAs), or piwiRNAs (piRNAs).
[0012] In various aspects, the composition further comprises
phospholipids. In some aspects, the phospholipids and
oligonucleotides are present at a molar ratio of from about 5:1 to
about 100:1. In some aspects, the oligonucleotides and
phospholipids form an oligonucleotide-lipid complex, such as, for
example, a liposome complex. In some aspects, at least 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the
liposomes are less than 5 microns in diameter. In some aspects, at
least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
of the liposomes are less than 4 microns in diameter. In some
aspects, the population of oligonucleotides are incorporated in the
population of liposomes.
[0013] In some aspects, the phospholipids are uncharged or have a
neutral charge at physiologic pH. In some aspects, the
phospholipids are neutral phospholipids. In certain aspects, the
neutral phospholipids are phosphatidylcholines. In certain aspects,
the neutral phospholipids are dioleoylphosphatidyl choline. In some
aspects, the phospholipids are essentially free of cholesterol.
[0014] In one embodiment, pharmaceutical compositions are provided
comprising a composition of oligonucleotides and phospholipids of
the present embodiments and a pharmaceutically acceptable carrier.
In some aspects, the composition further comprises a
chemotherapeutic agent.
[0015] In one embodiment, methods are provided for reducing the
expression level of IGF-1R protein in a cell comprising contacting
the cell with an oligonucleotide composition of the present
embodiments. In some aspects, the expression of IGF-1R and genes
downstream of IGF-1R, such as, for example, hexokinase, are
downregulated in the cell. In some aspects, the cell is a mammalian
cell. In some aspects, the cell is a cancer cell. In some aspects,
the cell is a cell of the immune system, such as, for example, a
monocyte, neutrophil, eosinophil, basophil, leukocyte, natural
killer (NK) cell, lymphocyte, T cell, B cell, dendritic cell, mast
cell, or macrophage. In certain aspects, the macrophage is a M2
macrophage, which produces higher levels of IGF-1R than a M1
macrophage and expresses one or more of CD11b, CD14, CD15, CD23,
CD64, CD68, CD163, CD204, CD206 on its cell surface. In certain
aspects, the monocyte is a M2 monocyte, which expresses one or more
of CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, CD206 on its
cell surface.
[0016] In one embodiment, methods are provided for delivering a
therapeutically effective amount of an oligonucleotide to a cell
comprising contacting the cell with a pharmaceutical composition of
the present embodiments. In some aspects, the method is a method of
treating hyperplasia, cancer, an autoimmune disease, or an
infectious disease. In some aspects, the method is a method of
treating, preventing, or delaying Alzheimer's disease, inflammatory
bowel disease, insulin resistance in type 2 diabetes, and
psoriasis. In one embodiment, methods are provided for enhancing an
immune response induced by vaccination comprising administering to
the subject a therapeutically effective amount of a pharmaceutical
composition of the present embodiments.
[0017] In one embodiment, methods are provided for treating a
subject with cancer, an autoimmune disease, or an infectious
disease comprising administering to the subject a therapeutically
effective amount of a pharmaceutical composition of the present
embodiments. In some aspects, the subject is a human. In some
aspects, the cancer is a bladder, blood, lymphoma, pancreas, bone,
bone marrow, brain, breast, colon, esophagus, stomach, head and
neck, kidney, liver, lung, prostate, skin, testis, tongue, ovary,
or uterine cancer. Tumors treatable with the methods of the present
invention include, but are not limited to, melanoma, prostate
cancer, ovarian cancer, breast cancer, mammary cancer, head and
neck squamous cell cancer, papillary renal cell carcinoma, gall
bladder cancer, rectal cancer, pancreatic cancer, lung cancer,
colon cancer, glioma, astrocytoma, classical Hodgkin's lymphoma,
and smooth muscle tumors, as well as cells from glioblastoma, bone
marrow stem cells, hematopoietic cells, osteoblasts, epithelial
cells, fibroblasts, as well as any other tumor cells which undergo
apoptosis and induce resistance to or regression of tumor cells. In
some aspects, the autoimmune disease is a Th2 dominant autoimmune
disease, which means that the autoimmune disease is driven by the
activity of cells of the Th2 class of T helper cells. In some
aspects, the autoimmune disease is Lupus erythematosis, allergic
dermatitis, scleroderma, atopic eczema, sinusitis, inflammatory
bowel disease, asthma, allergies, ulcerative colitis, multiple
chemical sensitivity, Spondyloarthropathy, Sjogren's disease,
Crohn's disease, diabetes mellitus, multiple sclerosis, or
rheumatoid arthritis. In some aspects, the infectious disease is a
bacterial infection, fungal infection, viral infection, or
parasitic infection. In some aspects, the composition is
administering subcutaneously, intravenously, or intraperitoneally.
In some aspects, the method further comprises administering at
least a second anticancer therapy to the subject. In some aspects,
the second anticancer therapy is a surgical therapy, chemotherapy,
radiation therapy, cryotherapy, hormone therapy, immunotherapy, or
cytokine therapy. In some aspects, administration of the
composition reduces expression of IGF-1R protein in the patient. In
one embodiment, methods are provided for enhancing the immune
response induced by vaccination.
[0018] In one embodiment, methods are provided for reducing the
expression level of IGF-1R protein in a cell, comprising contacting
the cell with a therapeutically effective amount of a
pharmaceutical composition of the present embodiments comprising a
composition comprising a population of oligonucleotides, wherein
the oligonucleotides hybridize to an IGF-1R polynucleotide gene
product, wherein oligonucleotides of the population are composed of
nucleoside molecules linked together through phosphate backbone
linkages, wherein at least one of the phosphate backbone linkages
in each oligonucleotide is a P-ethoxy backbone linkage, and wherein
no more than 80% of the phosphate backbone linkages in each
oligonucleotide are P-ethoxy backbone linkages, phospholipids, and
a pharmaceutically acceptable carrier, wherein the oligonucleotides
and phospholipids form an oligonucleotide-lipid complex.
[0019] In one embodiment, methods are provided for delivering a
therapeutically effective amount of an oligonucleotide to a cell
comprising contacting the cell with a therapeutically effective
amount of a pharmaceutical composition of the present embodiments
comprising a composition comprising a population of
oligonucleotides, wherein the oligonucleotides hybridize to an
IGF-1R polynucleotide gene product, wherein oligonucleotides of the
population are composed of nucleoside molecules linked together
through phosphate backbone linkages, wherein at least one of the
phosphate backbone linkages in each oligonucleotide is a P-ethoxy
backbone linkage, and wherein no more than 80% of the phosphate
backbone linkages in each oligonucleotide are P-ethoxy backbone
linkages, phospholipids, and a pharmaceutically acceptable carrier,
wherein the oligonucleotides and phospholipids form an
oligonucleotide-lipid complex.
[0020] An oligonucleotide includes an antisense nucleic acid
molecule that specifically hybridizes to a nucleic acid molecule
encoding a target protein or regulating the expression of the
target protein. "Specific hybridization" (or variations of these
terms, including but not limited to "specifically hybridizes")
means that the antisense nucleic acid molecule hybridizes to the
targeted nucleic acid molecule and regulates its expression.
Preferably, "specific hybridization" also means that no other genes
or transcripts are affected. An oligonucleotide can be a
single-stranded nucleic acid and may comprise 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30 or more nucleobases. In particular aspects the oligonucleotide
can comprise 15 to 30, 19 to 25, 20 to 23, or 21 contiguous
nucleobases. In certain embodiments, the oligonucleotide inhibits
the translation of a gene that promotes growth of a cancerous or
pre-cancerous or hyperplastic mammalian cell (e.g., a human cell).
An oligonucleotide may induce apoptosis in the cell, and/or inhibit
the translation of an oncogene or other target gene. In certain
embodiments, the oligonucleotide component comprises a single
species of oligonucleotide. In other embodiments, the
oligonucleotide component comprises a 2, 3, 4 or more species of
oligonucleotide that target 1, 2, 3, 4, or more genes. The
composition may further comprise a chemotherapeutic or other
anti-cancer agent, which may or may not be incorporated in a lipid
component or liposome of the invention. In further embodiments, the
oligonucleotide component is incorporated within the liposome or
lipid component.
[0021] "Entrap," "encapsulate," and "incorporate" refer to the
lipid or liposome forming an impediment to free diffusion into
solution by an association with or around an agent of interest,
e.g., a liposome may encapsulate an agent within a lipid layer or
within an aqueous compartment inside or between lipid layers. In
certain embodiments, the composition is comprised in a
pharmaceutically acceptable carrier. The pharmaceutically
acceptable carrier may be formulated for administration to a human
subject or patient.
[0022] In certain embodiments, the lipid component has an
essentially neutral charge because it comprises a neutral
phospholipid or a net neutral charge. In certain aspects a neutral
phospholipid may be a phosphatidylcholine, such as DOPC, egg
phosphatidylcholine ("EPC"), dilauroylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"),
dilinoleoylphosphatidylcholine,
1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-di
eicosenoyl-sn-glycero-3-phosphocholine ("DEPC"),
1-myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"),
1-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"),
1-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"),
1-stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"),
1-palmitoyl-2-oleoyl phosphatidylcholine ("POPC"),
1-oleoyl-2-palmitoyl phosphatidylcholine ("OPPC"), or
lysophosphatidylcholine. In other aspects the neutral phospholipid
can be a phosphatidylethanolamine, such as
dioleoylphosphatidylethanolamine ("DOPE"),
distearoylphosphatidylethanolamine ("DSPE"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), palmitoyloleoyl
phosphatidylethanolamine ("POPE"), or lysophosphatidylethanolamine.
In certain embodiments, the phospholipid component can comprise 1,
2, 3, 4, 5, 6, 7, 8, or more kinds or types of neutral
phospholipid. In other embodiments, a phospholipid component can
comprise 2, 3, 4, 5, 6 or more kinds or type of neutral
phospholipids.
[0023] In certain embodiments, a lipid component can have an
essentially neutral charge because it comprises a positively
charged lipid and a negatively charged lipid. The lipid component
may further comprise a neutrally charged lipid(s) or
phospholipid(s). The positively charged lipid may be a positively
charged phospholipid. The negatively charged lipid may be a
negatively charged phospholipid. The negatively charged
phospholipid may be a phosphatidylserine, such as dimyristoyl
phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine
("DPPS"), or brain phosphatidylserine ("BPS"). The negatively
charged phospholipid may be a phosphatidylglycerol, such as
dilauroylphosphatidylglycerol ("DLPG"),
dimyristoylphosphatidylglycerol ("DMPG"),
dipalmitoylphosphatidylglycerol ("DPPG"), di
stearoylphosphatidylglycerol ("DSPG"), or diol
eoylphosphatidylglycerol ("DOPG"). In certain embodiments, the
composition further comprises cholesterol or polyethyleneglycol
(PEG). In other embodiments, the composition is essentially free of
cholesterol. In certain embodiments, a phospholipid is a
naturally-occurring phospholipid. In other embodiments, a
phospholipid is a synthetic phospholipid.
[0024] Liposomes can be made of one or more phospholipids, as long
as the lipid material is substantially uncharged. It is important
that the composition be substantially free of anionic and cationic
phospholipids and cholesterol. Suitable phospholipids include
phosphatidylcholines and others that are well known to persons that
are skilled in this field.
[0025] Another aspect of the present invention involves methods for
delivering oligonucleotide to a cell comprising contacting the cell
with a neutral lipid composition of the invention. The methods will
provide an inventive composition in an effective amount. An
effective amount is an amount of therapeutic component that
attenuates, slows, reduces or eliminates a cell, condition, or
disease state in a subject. The cell may be comprised in a subject
or patient, such as a human. The method may further comprise a
method of treating cancer or other hyperplastic condition. The
cancer may have originated in the bladder, blood, bone, bone
marrow, brain, breast, colon, esophagus, gastrointestine, gum,
head, kidney, liver, lymph node, lung, nasopharynx, neck, prostate,
skin, stomach, testis, tongue, ovary, or uterus. In certain
embodiments, the method further comprises a method of treating a
non-cancerous disease or hyperplastic condition. The cell may be a
pre-cancerous or a cancerous cell. In certain embodiments, the
compositions and methods inhibit the growth of the cell, induce
apoptosis in the cell, and/or inhibit the translation of an
oncogene. The oligonucleotide may inhibit the translation of a gene
that is overexpressed in the cancerous cell.
[0026] In certain embodiments, the methods of the invention further
comprise administering an additional therapy to the subject. The
additional therapy may comprise administering a chemotherapeutic
(e.g., paclitaxel or docetaxel), a surgery, a radiation therapy,
and/or a gene therapy. In certain aspects the chemotherapy is
docetaxel, paclitaxel, cisplatin (CDDP), carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase
inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin,
methotrexate, or combinations thereof. In certain embodiments the
chemotherapy is a taxane such as docetaxal or paclitaxel. The
chemotherapy can be delivered before, during, after, or
combinations thereof relative to a neutral lipid composition of the
invention. A chemotherapy can be delivered within 0, 1, 5, 10, 12,
20, 24, 30, 48, or 72 hours or more of the neutral lipid
composition. The neutral lipid composition, the second anti-cancer
therapy, or both the neutral lipid composition and the anti-cancer
therapy can be administered intratumorally, intravenously,
intraperitoneally, subcutaneously, orally or by various
combinations thereof.
[0027] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method or
composition of the invention, and vice versa. Furthermore,
compositions of the invention can be used to achieve the methods of
the invention.
[0028] As used herein, "essentially free," in terms of a specified
component, is used herein to mean that none of the specified
component has been purposefully formulated into a composition
and/or is present only as a contaminant or in trace amounts. The
total amount of the specified component resulting from any
unintended contamination of a composition is therefore well below
0.05%, preferably below 0.01%. Most preferred is a composition in
which no amount of the specified component can be detected with
standard analytical methods.
[0029] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising," the words "a" or "an" may mean one or
more than one.
[0030] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0031] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0032] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0034] FIG. 1--Liposomal IGF-1R antisense delays the formation of
GL261 cell tumors in mice. The ability of liposomal IGF-1R
antisense to prevent growth of GL261 cell tumors implanted in mice
was tested by administering liposomal IGF-1R antisense
corresponding to SEQ ID NO: 1 to mice 14 days after implantation of
GL261 cells.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0035] To inhibit the expression of IGF-1R protein, the present
invention provides compositions and methods for delivery of an
anti-IGF-1R oligonucleotide (e.g., an inhibitor of gene expression)
to a cell via a lipid composition, in certain aspects a lipid
composition with a net charge of about zero, i.e., a neutral lipid
composition, which allows it to be delivered systemically via
intravenous infusion. These methods may be effectively used to
treat a cancer, treat an autoimmune disease, or enhance an immune
response induced by vaccination.
I. LIPIDS AND LIPOSOMES
[0036] "Liposomes" is used herein to mean lipid-containing vesicles
having a lipid bilayer, as well as other lipid carrier particles
that can entrap or incorporate antisense oligonucleotides. As such,
liposome is a generic term encompassing a variety of unilamellar,
multilamellar, and multivesicular lipid vehicles formed by the
generation of enclosed lipid bilayers or aggregates. In addition,
liposomes may have an undefined lamellar structure. Liposomes may
be characterized as having vesicular structures with a phospholipid
bilayer membrane and an inner aqueous medium. Multilamellar
liposomes have multiple lipid layers separated by aqueous medium.
They form spontaneously when phospholipids are suspended in an
excess of aqueous solution. The lipid components undergo
self-rearrangement before the formation of closed structures and
entrap water and dissolved solutes between the lipid bilayers
(Ghosh and Bachhawat, 1991). However, the present invention also
encompasses compositions that have different structures in solution
than the normal vesicular structure. For example, the lipids may
assume a micellar structure or merely exist as non-uniform
aggregates of lipid molecules.
[0037] Liposomes are a form of nanoparticles that are carriers for
delivering a variety of drugs into a diseased tissue. Optimal
liposome size depends on the target tissue. In tumor tissue, the
vasculature is discontinuous, and pore sizes vary from 100 to 780
nm (Siwak et al., 2002). By comparison, pore size in normal
vascular endothelium is <2 nm in most tissues, and 6 nm in
post-capillary venules. Negatively charged liposomes are thought to
be more rapidly removed from circulation than neutral or positively
charged liposomes; however, recent studies have indicated that the
type of negatively charged lipid affects the rate of liposome
uptake by the reticulo-endothelial system (RES). For example,
liposomes containing negatively charged lipids that are not
sterically shielded (phosphatidylserine, phosphatidic acid, and
phosphatidylglycerol) are cleared more rapidly than neutral
liposomes. Interestingly, cationic liposomes
(1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) and
cationic-liposome-DNA complexes are more avidly bound and
internalized by endothelial cells of angiogenic blood vessels via
endocytosis than anionic, neutral, or sterically stabilized neutral
liposomes (Thurston et al., 1998; Krasnici et al., 2003). Cationic
liposomes may not be ideal delivery vehicles for tumor cells
because surface interactions with the tumor cells create an
electrostatically derived binding-site barrier effect, inhibiting
further association of the delivery systems with tumor spheroids
(Kostarelos et al., 2004). However, neutral liposomes appear to
have better intratumoral penetration. Toxicity with specific
liposomal preparations has also been a concern. Cationic liposomes
elicit dose-dependent toxicity and pulmonary inflammation by
promoting release of reactive oxygen intermediates, and this effect
is more pronounced with multivalent cationic liposomes than
monovalent cationic liposomes, such as DOTAP (Dokka et al., 2000).
Neutral and negative liposomes do not appear to exhibit lung
toxicity (Guitierrez-Puente et al., 1999). Cationic liposomes,
while efficiently taking up nucleic acids, have had limited success
for in vivo gene down-regulation, perhaps because of their stable
intracellular nature and resultant failure to release nucleic acid
contents. Lipids with neutral charge or lipid compositions with a
neutralized charge, e.g., 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC), are used herein because of the neutral properties and
success in delivering antisense oligonucleotides in vivo.
[0038] The present invention provides methods and compositions for
associating an oligonucleotide, such as an antisense
oligonucleotide, with a lipid and/or liposome. The oligonucleotide
may be incorporated in the aqueous interior of a liposome,
interspersed within the lipid bilayer of a liposome, attached to a
liposome via a linking molecule that is associated with both the
liposome and the oligonucleotide, entrapped in a liposome,
complexed with a liposome, dispersed in a solution containing a
lipid, mixed with a lipid, combined with a lipid, contained as a
suspension in a lipid, contained or complexed with a micelle, or
otherwise associated with a lipid. The liposome or
liposome/oligonucleotide-associated compositions provided herein
are not limited to any particular structure in solution. For
example, they may be present in a bilayer structure, as micelles,
or with a "collapsed" structure. They may also simply be
interspersed in a solution, possibly forming aggregates that are
not uniform in either size or shape.
[0039] A. Lipids
[0040] Lipids are fatty substances that may be naturally occurring
or synthetic. For example, lipids include the fatty droplets that
naturally occur in the cytoplasm as well as the class of compounds
that are well known to those of skill in the art that contain
long-chain aliphatic hydrocarbons and their derivatives, such as
fatty acids, alcohols, amines, amino alcohols, and aldehydes. An
example is the lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC).
[0041] Lipid compositions of the present invention may comprise
phospholipids. In certain embodiments, a single kind or type of
phospholipid may be used in the creation of lipid compositions,
such as liposomes. In other embodiments, more than one kind or type
of phospholipid may be used.
[0042] Phospholipids include glycerophospholipids and certain
sphingolipids. Phospholipids include, but are not limited to,
dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine
("EPC"), dilauryloylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"),
dilinoleoylphosphatidylcholine,
1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DAPC"),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"),
1-myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"),
1-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"),
1-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"),
1-stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), palmitoyloeoyl
phosphatidylcholine ("POPC"), 1-oleoyl-2-palmitoyl
phosphatidylcholine ("OPPC"), dilauryloylphosphatidylglycerol
("DLPG"), dimyristoylphosphatidylglycerol ("DWG"),
dipalmitoylphosphatidylglycerol ("DPPG"),
distearoylphosphatidylglycerol ("DSPG"),
dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic
acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), distearoyl
phosphatidic acid ("DSPA"), dioleoyl phosphatidic acid ("DOPA"),
dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"),
distearoylphophatidylethanolamine ("DSPE"), dioleoylphosphatidyl
ethanolamine ("DOPE"), palmitoyloeoyl phosphatidyletlianolamine
("POPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl
phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"),
distearoyl sphingomyelin ("DS SP"), brain sphingomyelin ("BSP"),
dipalmitoyl sphingomyelin ("DPSP"), lysophosphatidylcholine, and
lysophosphatidylethanolamine.
[0043] Phospholipids include, for example, phosphatidylcholines,
phosphatidylglycerols, and phosphatidylethanolamines; because
phosphatidylethanolamines and phosphatidylcholines are non-charged
under physiological conditions (i.e., at about pH 7), these
compounds may be particularly useful for generating neutral
liposomes. In certain embodiments, the phospholipid DOPC is used to
produce non-charged liposomes or lipid compositions. In certain
embodiments, a lipid that is not a phospholipid (e.g., a
cholesterol) can also be used
[0044] Phospholipids may be from natural or synthetic sources.
However, phospholipids from natural sources, such as egg or soybean
phosphatidylcholine, brain phosphatidic acid, brain or plant
phosphatidylinositol, heart cardiolipin, and plant or bacterial
phosphatidylethanolamine, are not used in certain embodiments as
the primary phosphatide (i.e., constituting 50% or more of the
total phosphatide composition) because this may result in
instability and leakiness of the resulting liposomes.
[0045] B. Neutral Liposomes
[0046] "Neutral liposomes or lipid composition" or "non-charged
liposomes or lipid composition," as used herein, are defined as
liposomes or lipid compositions having one or more lipids that
yield an essentially-neutral net charge (substantially
non-charged). In certain embodiments, neutral liposomes or lipid
compositions may include mostly lipids and/or phospholipids that
are themselves neutral. In certain embodiments, amphipathic lipids
may be incorporated into or used to generate neutral liposomes or
lipid compositions. For example, a neutral liposome may be
generated by combining positively and negatively charged lipids so
that those charges substantially cancel one another, thereby
yielding an essentially-neutral net charge. By "essentially
neutral" or "essentially non-charged," it is meant that few, if
any, lipids within a given population (e.g., a population of
liposomes) include a charge that is not canceled by an opposite
charge of another component (e.g., fewer than 10% of components
include a non-canceled charge, more preferably fewer than 5%, and
most preferably fewer than 1%). In certain embodiments of the
present invention, a composition may be prepared wherein the lipid
component of the composition is essentially neutral but is not in
the form of liposomes.
[0047] The size of the liposomes varies depending on the method of
synthesis. A liposome suspended in an aqueous solution is generally
in the shape of a spherical vesicle, and may have one or more
concentric layers of lipid bilayer molecules. Each layer consists
of a parallel array of molecules represented by the formula XY,
wherein X is a hydrophilic moiety and Y is a hydrophobic moiety. In
aqueous suspension, the concentric layers are arranged such that
the hydrophilic moieties tend to remain in contact with an aqueous
phase and the hydrophobic regions tend to self-associate. For
example, when aqueous phases are present within the liposome, the
lipid molecules may form a bilayer, known as a lamella, of the
arrangement XY-YX. Aggregates of lipids may form when the
hydrophilic and hydrophobic parts of more than one lipid molecule
become associated with each other. The size and shape of these
aggregates will depend upon many different variables, such as the
nature of the solvent and the presence of other compounds in the
solution.
[0048] Liposomes within the scope of the present invention can be
prepared in accordance with known laboratory techniques, such as,
for example, the method of Bangham et al. (1965), the contents of
which are incorporated herein by reference; the method of
Gregoriadis (1979), the contents of which are incorporated herein
by reference; the method of Deamer and Uster (1983), the contents
of which are incorporated by reference; and the reverse-phase
evaporation method as described by Szoka and Papahadjopoulos
(1978). The aforementioned methods differ in their respective
abilities to entrap aqueous material and their respective aqueous
space-to-lipid ratios.
[0049] In certain embodiments, a neutral liposome may be used to
deliver an oligonucleotide, such as an antisense oligonucleotide.
The neutral liposome may contain a single species of
oligonucleotide directed to the suppression of translation of a
single gene, or the neutral liposome may contain multiple species
of oligonucleotides that are directed to the suppression of
translation of multiple genes. Further, the neutral liposome may
also contain a chemotherapeutic in addition to the oligonucleotide;
thus, in certain embodiments, a chemotherapeutic and an
oligonucleotide may be delivered to a cell (e.g., a cancerous cell
in a human subject) in the same or separate compositions.
[0050] Dried lipids or lyophilized liposomes may be dehydrated and
reconstituted at an appropriate concentration with a suitable
solvent (e.g., DPBS or Hepes buffer). The mixture may then be
vigorously shaken in a vortex mixer. The liposomes may be
resuspended at an appropriate total phospholipid concentration
(e.g., about 10-200 mM). Unencapsulated oligonucleotide may be
removed by centrifugation at 29,000 g and the liposomal pellets
washed. Alternatively, the unencapsulated oligonucleotides may be
removed by dialyzing against an excess of solvent. The amount of
oligonucleotide encapsulated can be determined in accordance with
standard methods.
II. INHIBITION OF GENE EXPRESSION
[0051] An inhibitory oligonucleotide can inhibit the transcription
or translation of a gene in a cell. An oligonucleotide may be from
5 to 50 or more nucleotides long, and in certain embodiments from 7
to 30 nucleotides long. In certain embodiments, the oligonucleotide
maybe 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. The
oligonucleotide may comprise a nucleic acid and/or a nucleic acid
analog. Typically, an inhibitory oligonucleotide will inhibit the
translation of a single gene within a cell; however, in certain
embodiments, an inhibitory oligonucleotide may inhibit the
translation of more than one gene within a cell.
[0052] Within an oligonucleotide, the components of the
oligonucleotide need not be of the same type or homogenous
throughout (e.g., an oligonucleotide may comprise a nucleotide and
a nucleic acid or nucleotide analog). In certain embodiments of the
present invention, the oligonucleotide may comprise only a single
nucleic acid or nucleic acid analog. The inhibitory oligonucleotide
may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30 or more contiguous nucleobases, including all ranges
therebetween, that hybridize with a complementary nucleic acid to
form a double-stranded structure.
III. NUCLEIC ACIDS
[0053] The present invention provides methods and compositions for
the delivery of an oligonucleotide via neutral liposomes. Because
an oligonucleotide is composed of a nucleic acid, methods relating
to nucleic acids (e.g., production of a nucleic acid, modification
of a nucleic acid, etc.) may also be used with regard to an
oligonucleotide.
[0054] The term "nucleic acid" is well known in the art. A "nucleic
acid" as used herein generally refers to a molecule (i.e., a
strand) of DNA, RNA, or a derivative or analog thereof, comprising
a nucleobase. These definitions refer to a single-stranded or
double-stranded nucleic acid. Double-stranded nucleic acids may be
formed by fully complementary binding; however, in some
embodiments, a double-stranded nucleic acid may be formed by
partial or substantial complementary binding. As used herein, a
single-stranded nucleic acid may be denoted by the prefix "ss" and
a double-stranded nucleic acid by the prefix "ds."
[0055] A. Nucleobases
[0056] As used herein a "nucleobase" refers to a heterocyclic base,
such as, for example, a naturally occurring nucleobase (i.e., an A,
T, G, C or U) found in at least one naturally occurring nucleic
acid (i.e., DNA and RNA), and naturally or non-naturally occurring
derivative(s) and analogs of such a nucleobase. A nucleobase
generally can form one or more hydrogen bonds (i.e., "anneal" or
"hybridize") with at least one naturally occurring nucleobase in a
manner that may substitute for naturally occurring nucleobase
pairing (e.g., the hydrogen bonding between A and T, G and C, and A
and U). A nucleobase may be comprised in a nucleoside or
nucleotide, using any chemical or natural synthesis method
described herein or known to one of ordinary skill in the art.
[0057] "Purine" and/or "pyrimidine" nucleobase(s) encompass
naturally occurring purine and/or pyrimidine nucleobases and also
derivative(s) and analog(s) thereof, including but not limited to,
a purine or pyrimidine substituted by one or more of an alkyl,
carboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro,
bromo, or iodo), thiol, or alkylthiol moiety. Preferred alkyl
(e.g., alkyl, caboxyalkyl, etc.) moieties comprise of from about 1,
about 2, about 3, about 4, about 5, to about 6 carbon atoms. Other
non-limiting examples of a purine or pyrimidine include a
deazapurine, a 2,6-diaminopurine, a 5-fluorouracil, a xanthine, a
hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, a bromothyline,
a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a
8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a
5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil,
a 5-chlorouracil, a 5-propyluracil, a thiouracil, a
2-methyladenine, a methylthioadenine, a N,N-diemethyladenine, an
azaadenines, a 8-bromoadenine, a 8-hydroxyadenine, a
6-hydroxyaminopurine, a 6-thiopurine, a 4-(6-aminohexyl/cytosine),
and the like. Purine and pyrimidine derivatives or analogs include,
but are not limited to (abbreviation/modified base description):
ac4c/4-acetylcytidine, Mam5 s2u/5-methoxyaminomethyl-2-thiouridine,
Chm5u/5-(carboxyhydroxylmethyl) uridine, Man q/Beta,
D-mannosylqueosine, Cm/2'-O-methylcytidine,
Mcm5s2u/5-methoxycarbonylmethyl-2-thiouridine,
Cmnm5s2u/5-carboxymethylamino-methyl-2-thioridine,
Mcm5u/5-methoxycarbonylmethyluridine, Cmnm5u/5-carboxymethyl
aminomethyluridine, Mo5u/5-methoxyuridine, D/Dihydrouridine,
Ms2i6a, 2-methylthio-N6-isopentenyladenosine,
Fm/2'-O-methylpseudouridine,
Ms2t6a/N-((9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threo-
nine, Gal q/Beta,D-galactosylqueosine,
Mt6a/N-((9-beta-D-ribofuranosylpurine-6-yl)N-methyl-carbamoyl)threonine,
Gm/2'-O-methylguanosine, Mv/Uridine-5-oxyacetic acid methylester,
I/Inosine, o5u/Uridine-5-oxyacetic acid (v),
I6a/N6-isopentenyladenosine, Osyw/Wybutoxosine,
mla/1-methyladenosine, P/Pseudouridine, mlf/1-methylpseudouridine,
Q/Queosine, mlg/1-methylguanosine, s2c/2-thiocytidine,
mlIl1-methylinosine, s2t/5-methyl-2-thiouridine,
m22g/2,2-dimethylguanosine, s2u/2-thiouridine,
m2a/2-methyladenosine, s4u/4-thiouridine, m2g/2-methylguanosine,
T/5-methyluridine, m3c/3-methylcytidine, t6a/N-((9-b
eta-D-ribofuranosylpurine-6-yl)carbamoyl)threonine,
m5c/5-methylcytidine, Tm/2'-O-methyl-5-methyluridine,
m6a/N6-methyladenosine, Um/2'-O-methyluridine,
m7g/7-methylguanosine, Yw/Wybutosine,
Mam5u/5-methylaminomethyluridine, or
X/3-(3-amino-3-carboxypropyl)uridine, (acp3)u.
[0058] B. Nucleosides
[0059] As used herein, a "nucleoside" refers to an individual
chemical unit comprising a nucleobase covalently attached to a
nucleobase linker moiety. A non-limiting example of a "nucleobase
linker moiety" is a sugar comprising 5-carbon atoms (i.e., a
"5-carbon sugar"), including but not limited to a deoxyribose, a
ribose, an arabinose, or a derivative or an analog of a 5-carbon
sugar. Non-limiting examples of a derivative or an analog of a
5-carbon sugar include a 2'-fluoro-2'-deoxyribose or a carbocyclic
sugar where a carbon is substituted for an oxygen atom, in the
sugar ring. As used herein, a "moiety" generally refers to a
smaller chemical or molecular component of a larger chemical or
molecular structure.
[0060] Different types of covalent attachment(s) of a nucleobase to
a nucleobase linker moiety are known in the art. By way of
non-limiting example, a nucleoside comprising a purine (i.e., A or
G) or a 7-deazapurine nucleobase typically comprises a covalent
attachment of the 9 position of the purine or 7-deazapurine to a
1'-position of a 5-carbon sugar. In another non-limiting example, a
nucleoside comprising a pyrimidine nucleobase (i.e., C, T, or U)
typically comprises a covalent attachment of the 1 position of the
pyrimidine to a 1'-position of a 5-carbon sugar (Kornberg and
Baker, 1992).
[0061] C. Nucleotides
[0062] As used herein, a "nucleotide" refers to a nucleoside
further comprising a "backbone linkage." A backbone linkage
generally covalently attaches a nucleotide to another molecule
comprising a nucleotide, or to another nucleotide to form a nucleic
acid. The "backbone linkage" in naturally occurring nucleotides
typically comprises a phosphate moiety (e.g., a phosphodiester
backbone linkage), which is covalently attached to a 5-carbon
sugar. The attachment of the backbone moiety typically occurs at
either the 3'- or 5'-position of the 5-carbon sugar. However, other
types of attachments are known in the art, particularly when a
nucleotide comprises derivatives or analogs of a naturally
occurring 5-carbon sugar or phosphate moiety.
[0063] D. Nucleic Acid Analogs
[0064] A nucleic acid may comprise, or be composed entirely of, a
derivative or analog of a nucleobase, a nucleobase linker moiety,
and/or backbone linkage that may be present in a naturally
occurring nucleic acid. As used herein a "derivative" refers to a
chemically modified or altered form of a naturally occurring
molecule, while the terms "mimic" or "analog" refer to a molecule
that may or may not structurally resemble a naturally occurring
molecule or moiety, but possesses similar functions. Nucleobase,
nucleoside, and nucleotide analogs or derivatives are well known in
the art.
[0065] Non-limiting examples of nucleosides, nucleotides, or
nucleic acids comprising 5-carbon sugar and/or backbone linkage
derivatives or analogs, include those in U.S. Pat. No. 5,681,947
which describes oligonucleotides comprising purine derivatives that
form triple helixes with and/or prevent expression of dsDNA; U.S.
Pat. Nos. 5,652,099 and 5,763,167 which describe nucleic acids
incorporating fluorescent analogs of nucleosides found in DNA or
RNA, particularly for use as fluorescent nucleic acids probes; U.S.
Pat. No. 5,614,617 which describes oligonucleotide analogs with
substitutions on pyrimidine rings that possess enhanced nuclease
stability; U.S. Pat. Nos. 5,670,663, 5,872,232 and 5,859,221 which
describe oligonucleotide analogs with modified 5-carbon sugars
(i.e., modified 2'-deoxyfuranosyl moieties) used in nucleic acid
detection; U.S. Pat. No. 5,446,137 which describes oligonucleotides
comprising at least one 5-carbon sugar moiety substituted at the 4'
position with a substituent other than hydrogen that can be used in
hybridization assays; U.S. Pat. No. 5,886,165 which describes
oligonucleotides with both deoxyribonucleotides with 3'-5' backbone
linkages and ribonucleotides with 2'-5' backbone linkages; U.S.
Pat. No. 5,714,606 which describes a modified backbone linkage
wherein a 3'-position oxygen of the backbone linkage is replaced by
a carbon to enhance the nuclease resistance of nucleic acids; U.S.
Pat. No. 5,672,697 which describes oligonucleotides containing one
or more 5' methylene phosphonate backbone linkages that enhance
nuclease resistance; U.S. Pat. Nos. 5,466,786 and 5,792,847 which
describe the linkage of a substituent moiety that may comprise a
drug or label to the 2' carbon of an oligonucleotide to provide
enhanced nuclease stability and ability to deliver drugs or
detection moieties; U.S. Pat. No. 5,223,618 which describes
oligonucleotide analogs with a 2 or 3 carbon backbone linkage
attaching the 4' position and 3' position of adjacent 5-carbon
sugar moiety to enhanced cellular uptake, resistance to nucleases,
and hybridization to target RNA; U.S. Pat. No. 5,470,967 which
describes oligonucleotides comprising at least one sulfamate or
sulfamide backbone linkage that are useful as nucleic acid
hybridization probes; U.S. Pat. Nos. 5,378,825, 5,777,092,
5,623,070, 5,610,289 and 5,602,240 which describe oligonucleotides
with a three or four atom backbone linkage moiety replacing the
phosphodiester backbone linkage used for improved nuclease
resistance, cellular uptake, and regulating RNA expression; U.S.
Pat. No. 5,858,988 which describes hydrophobic carrier agent
attached to the 2'-O position of oligonucleotides to enhance their
membrane permeability and stability; U.S. Pat. No. 5,214,136 which
describes oligonucleotides conjugated to anthraquinone at the 5'
terminus that possess enhanced hybridization to DNA or RNA;
enhanced stability to nucleases; U.S. Pat. No. 5,700,922 which
describes PNA-DNA-PNA chimeras wherein the DNA comprises
2'-deoxy-erythro-pentofaranosyl nucleotides for enhanced nuclease
resistance, binding affinity, and ability to activate RNase H; U.S.
Pat. No. 5,708,154 which describes RNA linked to a DNA to form a
DNA-RNA hybrid; U.S. Pat. No. 5,908,845 which describes polyether
nucleic acids wherein one or more nucleobases are linked to chiral
carbon atoms in a polyether backbone; U.S. Pat. Nos. 5,786,461,
5,891,625, 5,786,461, 5,773,571, 5,766,855, 5,736,336, 5,719,262,
5,714,331, 5,539,082, and WO 92/20702 which describe peptide
nucleic acids (PNA or peptide-based nucleic acid analog; or PENAM)
that generally comprise one or more nucleotides or nucleosides that
comprise a nucleobase moiety, a nucleobase linker moiety that is
not a 5-carbon sugar (e.g., aza nitrogen atoms, amido and/or ureido
tethers), and/or a backbone linkage that is not a phosphate
backbone linkage (e.g., aminoethylglycine, polyamide, polyethyl,
polythioamide, polysulfinamide, or polysulfonamide backbone
linkage); and U.S. Pat. No. 5,855,911 which describes the
hydrophobic, nuclease resistant P-ethoxy backbone linkage.
[0066] Other modifications and uses of nucleic acid analogs are
known in the art, and it is anticipated that these techniques and
types of nucleic acid analogs may be used with the present
invention.
[0067] E. Preparation of Nucleic Acids
[0068] A nucleic acid may be made by any technique known to one of
ordinary skill in the art, such as chemical synthesis, enzymatic
production or biological production. Non-limiting examples of a
synthetic nucleic acid (e.g., a synthetic oligonucleotide) include
a nucleic acid made by in vitro chemical synthesis using
phosphotriester, phosphite, or phosphoramidite chemistry and solid
phase techniques, such as described in EP 266,032, incorporated
herein by reference, or by deoxynucleoside H-phosphonate
intermediates as described by Froehler et al. (1986) and U.S. Pat.
No. 5,705,629, each incorporated herein by reference. In the
methods of the present invention, one or more species of
oligonucleotide may be used. Various mechanisms of oligonucleotide
synthesis have been disclosed in, for example, U.S. Pat. Nos.
4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148,
5,554,744, 5,574,146, 5,602,244, each of which is incorporated
herein by reference.
[0069] F. Purification of Nucleic Acids
[0070] A nucleic acid may be purified on polyacrylamide gels,
cesium chloride centrifugation gradients, or by any other means
known to one of ordinary skill in the art (see for example,
Sambrook et al. (2001), incorporated herein by reference).
[0071] In certain embodiments, the present invention concerns a
nucleic acid that is an isolated nucleic acid. As used herein, the
term "isolated nucleic acid" refers to a nucleic acid molecule
(e.g., an RNA or DNA molecule) that has been isolated free of, or
is otherwise free of, the bulk of the total genomic and transcribed
nucleic acids of one or more cells. In certain embodiments,
"isolated nucleic acid" refers to a nucleic acid that has been
isolated free of, or is otherwise free of, the bulk of cellular
components or in vitro reaction components, such as, for example,
macromolecules, such as lipids or proteins, small biological
molecules, and the like.
[0072] G. Hybridization
[0073] As used herein, "hybridization," "hybridize(s)," or "capable
of hybridizing" is understood to mean the forming of a double or
triple stranded molecule or a molecule with partial double or
triple stranded nature. The term "anneal" as used herein is
synonymous with "hybridize."
[0074] As used herein "stringent condition(s)" or "high stringency"
are those conditions that allow hybridization between or within one
or more nucleic acid strand(s) containing complementary
sequence(s), but precludes hybridization of random sequences.
Stringent conditions tolerate little, if any, mismatch between a
nucleic acid and a target strand. Such conditions are well known to
those of ordinary skill in the art, and are preferred for
applications requiring high selectivity.
[0075] Stringent conditions may comprise low salt and/or high
temperature conditions, such as provided by about 0.02 M to about
0.15 M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. It is understood that the temperature and ionic
strength of a desired stringency are determined in part by the
length of the particular nucleic acid(s), the length and nucleobase
content of the target sequence(s), the charge composition of the
nucleic acid(s), and to the presence or concentration of formamide,
tetramethylammonium chloride, or other solvent(s) in a
hybridization mixture.
[0076] It is also understood that these ranges, compositions and
conditions for hybridization are mentioned by way of non-limiting
examples only, and that the desired stringency for a particular
hybridization reaction is often determined empirically by
comparison to one or more positive or negative controls. Depending
on the application envisioned it is preferred to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of a nucleic acid towards a target sequence. In a
non-limiting example, identification or isolation of a related
target nucleic acid that does not hybridize to a nucleic acid under
stringent conditions may be achieved by hybridization at low
temperature and/or high ionic strength. Such conditions are termed
"low stringency" or "low stringency conditions," and non-limiting
examples of low stringency include hybridization performed at about
0.15 M to about 0.9 M NaCl at a temperature range of about
20.degree. C. to about 50.degree. C. Of course, it is within the
skill of one in the art to further modify the low or high
stringency conditions to suit a particular application.
IV. METHOD OF MANUFACTURING LIPOSOMAL P-ETHOXY ANTISENSE DRUG
PRODUCT
[0077] Antisense oligonucleotides (oligos) complementary to
specific regions of a target mRNA have been used to inhibit the
expression of endogenous genes. When the antisense oligonucleotides
bind to a target mRNA, a DNA-RNA hybrid is formed. This hybrid
formation inhibits the translation of the mRNA and, thus, the
expression of the encoded protein. If the protein is essential for
the survival of the cell, the inhibition of its expression may lead
to cell death. Therefore, antisense oligonucleotides can be useful
tools in anticancer and antiviral therapies.
[0078] The main obstacles in using antisense oligonucleotides to
inhibit gene expression are cellular instability, low cellular
uptake, and poor intercellular delivery. Natural phosphodiesters
are not resistant to nuclease hydrolysis; thus high concentrations
of antisense oligonucleotides are needed before any inhibitory
effect is observed. Modified phosphodiester analogs, such as
P-ethoxy, have been made to overcome this nuclease hydrolysis
problem, but they have not provided a satisfactory solution to the
problem.
[0079] The cellular uptake of antisense oligonucleotides is low. To
solve this problem, physical techniques, such as calcium-phosphate
precipitation, DEAE-dextran mediation, or electroporation, have
been used to increase the cellular uptake of oligonucleotides.
These techniques are difficult to reproduce and are inapplicable in
vivo. Cationic lipids, such as Lipofectin, have also been used to
deliver oligonucleotides. An electrostatic interaction is formed
between the cationic lipids and the negatively charged
oligonucleotides, which results in a complex that is then taken up
by the target cells. Since these cationic lipids do not protect the
oligonucleotides from nuclease digestion and are harmful to the
cell membrane, they are only useful in delivering the
nuclease-resistant phosphorothioates, but not the
nuclease-cleavable phosphodiesters.
[0080] Another modified phosphodiester analog that has been
prepared is P-ethoxy. The P-ethoxy antisense backbone does not have
an adverse effect on bleeding and complement activation, which are
some of the toxicities that have been reported for other antisense
analogs. The modifications of P-ethoxy oligonucleotides are made in
the phosphate backbone so that the modification will not interfere
with the binding of these oligonucleotides to a target mRNA.
P-ethoxy oligonucleotides are made by adding an ethyl group to the
non-bridging oxygen atom of the phosphate backbone, thus rendering
these oligonucleotides uncharged compounds. In spite of their
resistance to nucleases, the cellular uptake and intracellular
delivery of P-ethoxy oligonucleotides is poor because upon
internalization, these oligonucleotides remain sequestered inside
the endosomal/lysosomal vacuoles, impeding their access to target
mRNA.
[0081] A. P-Ethoxy Antisense Drug Product
[0082] The liposomal P-ethoxy antisense drug product is composed of
two cGMP products, both of which have a FDA-required Certificate of
Analysis with FDA-approved release criteria. The raw materials,
solvents, and final drug product are described herein. When
manufactured, the drug product is a lyophilized crystal or powder
of amber or white color that comprises the following materials:
oligonucleotide (e.g., P-ethoxy antisense drug substance), neutral
lipids (e.g., DOPC), and surfactant (e.g., polysorbate 20). In
preparation for administration to a patient, normal saline is added
to the vial, at which time liposomes are formed with the P-ethoxy
antisense incorporated into the interior.
[0083] B. P-Ethoxy Antisense Drug Substance
[0084] Specific physical properties (e.g., solubility and
hydrophobicity, which then affect drug product solubility in
saline, incorporation of oligo into liposomes, and liposome
particle size) of the finished product can be defined using a
pre-determined P-ethoxy and phosphodiester amidite raw material mix
during production of the P-ethoxy antisense drug substance. While
loss of the P-ethoxy backbone group randomly occurs during
oligonucleotide manufacturing resulting in phosphodiester bonds at
those linkages, that loss may not generate the preferred ratio of
P-ethoxy:phosphodiester backbone linkage within the
oligonucleotide. In this case, the mix of P-ethoxy and
phosphodiester amidite raw material supplements the expected value
of P-ethoxy backbone deletions, thus generating an oligonucleotide
with the desired ratio. Increasing the number of P-ethoxy molecules
in the backbone of the oligonucleotide causes the molecule to be
more hydrophobic (which results in larger liposome particles; Table
1), less polar, and less soluble (Table 2). Methods of testing the
charge-neutral, hydrophobic P-ethoxy drug substance include mass
spectrometry to determine the distribution of oligonucleotide
lengths and assays to determine the solubility of drug substance,
which for practical purposes for solubility is a visual inspection
of the drug product reconstituted in saline. As the oligonucleotide
becomes less soluble due to a greater number of P-ethoxy backbone
linkages the reconstituted solution becomes whiter until
particulates form as hydrophobicity becomes too high.
[0085] Formulation must use a particle size, wherein the 90% value
is less than 5000 nm in size and is soluble, which is a function of
the nucleotide composition. By way of example, if an
oligonucleotide is 18-20 nucleotides in length, then at least five
of the phosphate backbone linkages should be phosphodiester
backbone linkages. This is supported clearly by the Experiments
7-10 below in Table 1, which provides data from 18mer
oligonucleotides. Wherein if an oligonucleotide is 25 nucleotides
in length, then at least six of the phosphate backbone linkages
should be phosphodiester backbone linkages.
TABLE-US-00001 TABLE 1 Liposome Particle Size Variability with
Antisense Backbone Composition of oligonucleotides of 18 mer
Post-Manufacturing Particle Size Characteristics: Backbone
Cumulative Distribution Function Engineered Ethyl Deletion 90% 50%
Antisense Principal Composite Value Value 300 nm Experiment
Backbone Peak.sup.d Deletion.sup.e (nm)** (nm) Value (%) 1 3
amidite -6 -5.67 2130 911 15.30 substitution 2 3 amidite -6 -5.67
2420 1004 15.50 substitution 3 3 amidite -6 -6.12 3682 943 15.50
substitution 4 3 amidite -7 -6.66 3805 978 14.60 substitution 5
100% P- -5 -5.66 3924 976 16.00 ethoxy 6 2 amidite -5 -5.32 4387
1888 11.60 substitution 7.sup.a 100% P- -4 -4.22 5057 1131 17.70
ethoxy 8 100% P- -4 -4.52 5659 1359 10.00 ethoxy 9.sup.b 100% P- -4
-4.38 7571 1909 2.60 ethoxy 10.sup.c 100% P- -4 -4.38 7994 1653
14.40 ethoxy **Drug product release criteria is for 90% of the
liposome particles to be less than or equal to 5000 nm. .sup.aThis
lot was discarded due to poor solubility; specifically, antisense
particles in the reconstituted solution. .sup.bThis lot had lower
DMSO and tBA volume with 2 mg antisense in a 20 mL vial, which
added an additional component to liposome enlargement. .sup.cThis
lot was not released because it failed the particle size release
spec. .sup.dThe principal peak represents the most common number of
p-ethoxy deletions in the oligonucleotides of the population.
.sup.eThe composite deletion represents the average number of
p-ethoxy deletions in the population of oligonucleotides.
TABLE-US-00002 TABLE 2 Liposome Particle Solubility with Antisense
Backbone Composition Post-Manufacturing Engineered Backbone Ethyl
Deletion Drug Solubility Antisense Composite Visual Solubility
Experiment Backbone Principal Peak Deletion Observation**
Assessment 1 3 amidite -6 -5.67 skim milk good substitution
solution 2 3 amidite -6 -5.67 skim milk good substitution solution
3 3 amidite -6 -6.12 skim milk good substitution solution 4 3
amidite -7 -6.66 skim milk good substitution solution 5 100% P- -5
-5.66 skim milk good ethoxy solution 6 2 amidite -5 -5.32 skim milk
good substitution solution 7 100% P- -4 -4.52 white pass ethoxy
solution 8.sup.b 100% P- -4 -4.38 white pass ethoxy solution
9.sup.c 100% P- -4 -4.38 white pass ethoxy solution 10.sup.a 100%
P- -4 -4.22 white fail ethoxy solution particles **If the drug
product sample has particles the lot will be rejected .sup.aThis
lot was discarded due to poor solubility; specifically, antisense
particles in the reconstituted solution. .sup.bThis lot had lower
DMSO and tBA volume with 2 mg antisense in a 20 mL vial, which
added an additional component to liposome enlargement. .sup.cThis
lot was not released because it failed the particle size release
spec.
[0086] C. Formulation, Filtration, and Lyophilization of Liposomal
P-Ethoxy Antisense Drug Product
[0087] One gram (1 g) of pE oligos is dissolved in DMSO at a ratio
of 10 mg oligonucleotide per 1 mL DMSO. Next, DOPC is added to
tert-butyl alcohol at a ratio of 1 g DOPC per 1719 mL of tert-butyl
alcohol. The oligo and DOPC are combined and mixed at a ratio of 1
g oligonucleotide per 2.67 g DOPC. Then, 20 mL of a 0.835% (v/v)
solution of polysorbate 20 is added to the mixture resulting in a
final concentration of 0.039 mg/mL. The solution is passed through
a sterile filter prior to dispensing into glass vials for
lyophilization.
[0088] The effect of the surfactant on liposome particle size was
determined by titrating the amount of surfactant (Table 3). In the
absence of polysorbate 20, only 2.8% of the particles had a
diameter of 300 nm or less. In the presence of 1.times. polysorbate
20, 12.5% of the particles had a diameter of 300 nm or less. With
the addition of 3.times.-10.times. polysorbate 20, around 20% of
the particles had a diameter of 300 nm or less. Thus an increase in
surfactant from 1.times. to 3.times. results in a decrease in
particle size.
TABLE-US-00003 TABLE 3 Liposome Particle Size Variability with
Surfactant Particle Size Characteristics: Cumulative Distribution
Function Amount of 50% 90% 300 nm Experiment Surfactant Value
Value** Value 1 0x 5301 nm 10719 nm 2.8% 2 1x 1053 nm 4054 nm 12.5%
3 3x 785 nm 2926 nm 19.1% 4 5x 721 nm 2691 nm 21.9% 5 10x 734 nm
2937 nm 21.4% **Drug product release criteria is for 90% of the
liposome particles to be less than or equal to 5000 nm.
[0089] D. Preparation of Liposomal P-Ethoxy Antisense Drug Product
for Administration
[0090] The lyophilized preparation was hydrated with normal saline
(0.9%/10 mM NaCl) at a final oligo concentration of 10-5000 .mu.M.
The liposomal-P-ethoxy oligos were mixed by hand shaking.
[0091] E. Methods of Testing Liposomal P-Ethoxy Antisense Drug
Product
[0092] Visual Inspection of Manufactured Drug Product:
[0093] After manufacturing, a sample vial containing drug product
is selected and visually inspected. The absence of liquid is
mandatory, and then amber crystals at the bottom of the vial are
acceptable, and increasing in acceptance to a white, flocculated
powder or appearance, the best result. The white appearance
indicates a better drying process, with a high surface area to mass
ratio, which is very conducive to reconstitution for use.
[0094] Visual Inspection of Reconstituted Drug Ready for Patient
IV:
[0095] Normal saline is added to a vial containing the manufactured
Liposomal P-ethoxy Antisense Drug Product and shaken to
reconstitute into a solution with the drug crystal or powder
completely dissolved. Three main observations are made: 1) that the
crystal or powder is completely dissolved, 2) there are no white
clumps of undissolved material, and 3) the appearance is a milky
white or skim milk appearance. The bluer the appearance of the
reconstituted liquid, the better, as this signals a smaller
liposome particle size that reflects light in the blue
spectrum.
[0096] Mass Spectrometry:
[0097] Mass spectrometry (mass spec) is used to display the profile
of the various masses in a sample. When P-ethoxy antisense material
is produced, a mass spec is run on the sample. The result shows
peaks of material present on a grid that has increasing mass on the
"x" axis to the right, and relative mass abundance on the "y" axis
increasing upward. The profile from a sample is analyzed to
determine the relative quantity of P-ethoxy backbones in the
P-ethoxy sample, recognizing that the profile of peaks represents
(starting farthest to the right), full length material with all
backbones comprised of the P-ethoxy linkage, the next peak moving
left a full length with one backbone with a P-ethoxy deletion (and
therefore, the ethyl being knocked off and the result being a
normal phosphodiester backbone linkage), and continuing. The mass
spec pattern shifted to the right represents a P-ethoxy sample
having more P-ethoxy backbones, and therefore having the properties
of being more hydrophobic and less soluble; and likewise, shifted
to the left having the opposite effects. Inspection of the mass
spec chart of a sample also can be used to determine if filtration
during manufacturing produces any adverse effects on
oligonucleotide composition present in the filtered drug
product.
[0098] UV Testing:
[0099] Ultraviolent light testing is used to determine the mass of
oligonucleotide present in a sample. Oligonucleotides absorb light
in the 260 nanometer range. As a result, UV testing of the finished
reconstituted drug product has come to be used as a method in
determining the quantity of oligonucleotide drug substance in a
vial of drug product. In terms of manufacturing development and
innovations, UV testing was used to determine if there were
problems experienced during filtration in manufacturing or poor
solubility of the P-ethoxy antisense drug substance, resulting in
less oligonucleotide in solution and therefore a lower UV reading.
The method will be validated and likely become part of the final
product release testing.
[0100] Liposome Particle Size:
[0101] A vial of finished drug product is reconstituted and tested
for liposome particle size. The result is often a roughly normal
distribution, having a central point, tails and average values or a
roughly normal distribution of the majority of the particles and
smaller, secondary peaks of the smaller liposomes particles
resulting from second-order particle formation effects. It is
important that liposome particles not be too large, as they may
create adverse effects in patients (for example, create blood flow
problems in smaller blood vessels in the lungs). As a result, the
drug product release criteria include that particle size testing
show that 90% of liposomes be 5 microns or less in size. In
addition, smaller liposomes are preferred because they will have
better uptake into cells, and secondly, smaller liposomes can
penetrate vascular pores, thereby allowing the liposomes to
penetrate inside tumors, increasing treatment effectiveness of a
Liposomal P-ethoxy Antisense Drug Product.
V. METHODS OF TREATMENT
[0102] Certain aspects of the present invention provide an
oligonucleotide-lipid complex (e.g., an oligonucleotide
incorporated into a non-charged liposome) for treating diseases,
such as cancer, autoimmune disease, or infectious disease. Certain
aspects of the present invention provide an oligonucleotide-lipid
complex (e.g., an oligonucleotide incorporated into a non-charged
liposome) for enhancing an immune response, such as an immune
response induced by vaccination, in a subject, thereby enhancing
therapeutic immunity. Particularly, the oligonucleotide may have a
sequence that allows for base pairing with a human nucleotide
sequence (e.g., IGF-1R) and thus may inhibit the expression of a
protein encoded by the human nucleotide sequence.
[0103] The expression of IGF-1R, and potentially genes downstream
of IGF-1R, such as, for example, hexokinase, may be downregulated
in a cell exposed to the oligonucleotide. The cell may be a
mammalian cell. The cell may be a cancer cell. The cell may be a
cell of the immune system, such as, for example, a monocyte,
neutrophil, eosiophil, basophil, leukocyte, natural killer (NK)
cell, lymphocyte, T cell, B cell, dendritic cell, mast cell, or
macrophage. The functions of macrophages include phagocytosis,
antigen presentation, and cytokine presentation. The macrophage may
be a M2 macrophage, which produces higher levels of IGF-1R than a
M1 macrophage and expresses one or more of CD11b, CD14, CD15, CD23,
CD64, CD68, CD163, CD204, CD206 on its cell surface. The monocyte
may be a M2 monocyte, which expresses one or more of CD11b, CD14,
CD15, CD23, CD64, CD68, CD163, CD204, CD206 on its cell surface.
Inhibiting the expression of IGF-1R in an undifferentiated monocyte
or macrophage may prevent the undifferentiated monocyte or
macrophage from being polarized to be a M2 monocyte or macrophage.
Inhibiting the expression of IGF-1R in a M2 monocyte or macrophage
may cause the M2 monocyte or macrophage to lose its M2 phenotype
and function, and/or undergo cell cycle arrest, and/or undergo cell
death, such as, for example, apoptosis or necrosis. Inhibiting the
expression of IGF-1R in macrophages may selectively affect M2
macrophages over M1 macrophages because M2 macrophages produce
higher levels of IGF-1R than M1 macrophages.
[0104] "Treatment" and "treating" refer to administration or
application of a therapeutic agent to a subject or performance of a
procedure or modality on a subject for the purpose of obtaining a
therapeutic benefit of a disease or health-related condition. For
example, a treatment may include administration of a
pharmaceutically effective amount of an IGF-1R
oligonucleotide-lipid complex.
[0105] "Subject" and "patient" refer to either a human or
non-human, such as primates, mammals, and vertebrates. In
particular embodiments, the subject is a human.
[0106] The term "therapeutic benefit" or "therapeutically
effective" as used throughout this application refers to anything
that promotes or enhances the well-being of the subject with
respect to the medical treatment of this condition. This includes,
but is not limited to, a reduction in the frequency or severity of
the signs or symptoms of a disease. For example, treatment of
cancer may involve, for example, a regression of a tumor, a
reduction in the size of a tumor, a reduction in the invasiveness
of a tumor, reduction in the growth rate of the cancer, prevention
of metastasis, or elimination of a tumor. Treatment of cancer may
also refer to prolonging survival of a subject with cancer.
Treatment of an autoimmune disease may involve, for example,
reducing the expression of a self-antigen against which there is an
undesired immune response, inducing tolerance of a self-antigen
against which there is an undesired immune response, or inhibiting
the immune response towards the self-antigen. Treatment of an
infectious disease may involve, for example, eliminate the
infectious agent, reduce the level of the infectious agent, or
maintain the level of the infectious agent at a certain level.
[0107] Tumors for which the present treatment methods are useful
include any malignant cell type, such as those found in a solid
tumor, a hematological tumor, metastatic cancer, or non-metastatic
cancer. Exemplary solid tumors can include, but are not limited to,
a tumor of an organ selected from the group consisting of pancreas,
colon, cecum, esophagus, gastrointestine, gum, liver, skin,
stomach, testis, tongue, uterus, stomach, brain, head, neck, ovary,
kidney, larynx, sarcoma, bone, lung, bladder, melanoma, prostate,
and breast. Exemplary hematological tumors include tumors of the
bone marrow, T or B cell malignancies, leukemias, lymphomas, such
as, for example, diffuse large B-cell lymphoma, blastomas,
myelomas, and the like. Further examples of cancers that may be
treated using the methods provided herein include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia,
squamous cell cancer, lung cancer (including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung, and
squamous carcinoma of the lung), cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer (including
gastrointestinal cancer and gastrointestinal stromal cancer),
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, breast cancer, colon cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland
carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,
thyroid cancer, various types of head and neck cancer, melanoma,
superficial spreading melanoma, lentigo malignant melanoma, acral
lentiginous melanomas, nodular melanomas, as well as B-cell
lymphoma (including low grade/follicular non-Hodgkin's lymphoma
(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular
NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL;
high grade lymphoblastic NHL; high grade small non-cleaved cell
NHL; bulky disease NHL; diffuse large B-cell lymphoma; mantle cell
lymphoma; AIDS-related lymphoma; and Waldenstrom's
macroglobulinemia), chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), Hairy cell leukemia, multiple
myeloma, acute myeloid leukemia (AML) and chronic myeloblastic
leukemia.
[0108] The cancer may specifically be of the following histological
type, though it is not limited to these: neoplasm, malignant;
carcinoma; carcinoma, undifferentiated; giant and spindle cell
carcinoma; small cell carcinoma; papillary carcinoma; squamous cell
carcinoma; lymphoepithelial carcinoma; basal cell carcinoma;
pilomatrix carcinoma; transitional cell carcinoma; papillary
transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;
cholangiocarcinoma; hepatocellular carcinoma; combined
hepatocellular carcinoma and cholangiocarcinoma; trabecular
adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in
adenomatous polyp; adenocarcinoma, familial polyposis coli; solid
carcinoma; carcinoid tumor, malignant; branchiolo-alveolar
adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma;
clear cell adenocarcinoma; granular cell carcinoma; follicular
adenocarcinoma; papillary and follicular adenocarcinoma;
nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;
endometroid carcinoma; skin appendage carcinoma; apocrine
adenocarcinoma; sebaceous adenocarcinoma; ceruminous
adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;
papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;
mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring
cell carcinoma; infiltrating duct carcinoma; medullary carcinoma;
lobular carcinoma; inflammatory carcinoma; paget's disease,
mammary; acinar cell carcinoma; adenosquamous carcinoma;
adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian
stromal tumor, malignant; thecoma, malignant; granulosa cell tumor,
malignant; androblastoma, malignant; sertoli cell carcinoma; leydig
cell tumor, malignant; lipid cell tumor, malignant; paraganglioma,
malignant; extra-mammary paraganglioma, malignant;
pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic
melanoma; superficial spreading melanoma; malignant melanoma in
giant pigmented nevus; epithelioid cell melanoma; blue nevus,
malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant;
myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;
embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal
sarcoma; mixed tumor, malignant; mullerian mixed tumor;
nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma,
malignant; brenner tumor, malignant; phyllodes tumor, malignant;
synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal
carcinoma; teratoma, malignant; struma ovarii, malignant;
choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;
hemangioendothelioma, malignant; kaposi's sarcoma;
hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;
juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma,
malignant; mesenchymal chondrosarcoma; giant cell tumor of bone;
ewing's sarcoma; odontogenic tumor, malignant; ameloblastic
odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma;
pinealoma, malignant; chordoma; glioma, malignant; ependymoma;
astrocytoma (grade I, grade II, grade III, or grade IV);
protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma;
glioblastoma; glioblastoma multiforme; oligodendroglioma;
oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;
ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory
neurogenic tumor; meningioma, malignant; neurofibrosarcoma;
neurilemmoma, malignant; granular cell tumor, malignant; malignant
lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant
lymphoma, small lymphocytic; malignant lymphoma, large cell,
diffuse; malignant lymphoma, follicular; mycosis fungoides; other
specified non-hodgkin's lymphomas; malignant histiocytosis;
multiple myeloma; mast cell sarcoma; immunoproliferative small
intestinal disease; leukemia; lymphoid leukemia; plasma cell
leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid
leukemia; basophilic leukemia; eosinophilic leukemia; monocytic
leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid
sarcoma; and hairy cell leukemia.
[0109] Autoimmune diseases for which the present treatment methods
are useful include, without limitation, lupus, scleroderma, atopic
eczema, sinusitis, asthma, allergies, multiple chemical
sensitivity, type 1 diabetes, Hashimoto's thyroiditis, Grave's
disease, lichen planus, spondyloarthropathy, ankylosing
spondylitis, psoriatic arthritis, reactive arthritis, enteropathic
arthritis, diabetes mellitus, celiac disease, autoimmune thyroid
disease, autoimmune liver disease, Addison's disease, transplant
rejection, graft vs. host disease, host vs. graft disease,
ulcerative colitis, Crohn's disease, irritable bowel disease,
inflammatory bowel disease, rheumatoid arthritis, juvenile
rheumatoid arthritis, familial Mediterranean fever, amyotrophic
lateral sclerosis, Sjogren's syndrome, early arthritis, viral
arthritis, multiple sclerosis, or psoriasis. The diagnosis and
treatment of these diseases are well documented in the
literature.
[0110] Infectious diseases for which the present treatment methods
are useful include, without limitation, bacterial infections, viral
infections, fungal infections, and parasitic infections. Exemplary
viral infections include hepatitis B virus, hepatitis C virus,
human immunodeficiency virus 1, human immunodeficiency virus 2,
human papilloma virus, herpes simplex virus 1, herpes simplex virus
2, herpes zoster, varicella zoster, coxsackievirus A16,
cytomegalovirus, ebola virus, enterovirus, Epstein-Barr virus,
hanta virus, hendra virus, viral meningitis, respiratory syncytial
virus, rotavirus, west nile virus, adenovirus, and influenza virus
infections. Exemplary bacterial infections include Chlamydia
trachomatis, Listeria monocytogenes, Helicobacter pylori,
Escherichia coli, Borelia burgdorferi, Legionella pneumophilia,
Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intraceliuiar
e, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria
gonorrhoeae, Neisseria meningitides, Streptococcus pyogenes (Group
A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans group), Streptococcus faecalis,
Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus
pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus influenzae, Bacillus anthracis, Corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasteurella multocida,
Bacteroides sp., Fusobacterium nucleatum, Streptobacillus
moniliformis, Treponema pallidium, Treponema pertenue, Leptospira,
Rickettsia, Actinomyces israelli, Shigella sps (e.g., S.flexneri,
S. sonnei, S. dysenteriae), and Salmonella spp infections.
Exemplary fungal infections include Candida albicans, Candida
glabrata, Aspergillus fumigatus, Aspergillus terreus, Cryptococcus
neoformans, Histoplasma capsulatum, Coccidioides immitis,
Blastomyces dermatitidis, and Chlamydia irachomatis infections.
[0111] The oligonucleotide-lipid complex may be used herein as an
antitumor, antiviral, antibacterial, antifungal, antiparasite, or
anti-autoimmune agent in a variety of modalities. In a particular
embodiment, the invention contemplates methods of using an
oligonucleotide-lipid complex comprises contacting a population of
diseased cells with a therapeutically effective amount of an
oligonucleotide-lipid complex for a time period sufficient to
inhibit or reverse disease.
[0112] In one embodiment, the contacting in vivo is accomplished by
administering, by intravenous, intraperitoneal, subcutaneous, or
intratumoral injection, a therapeutically effective amount of a
physiologically tolerable composition comprising an
oligonucleotide-lipid complex of this invention to a patient. The
oligonucleotide-lipid complex can be administered parenterally by
injection or by gradual infusion over time.
[0113] Therapeutic compositions comprising oligonucleotide-lipid
complex are conventionally administered intravenously or
subcutaneously, such as by injection of a unit dose, for example.
The term "unit dose" when used in reference to a therapeutic
composition refers to physically discrete units suitable as unitary
dosage for the subject, each unit containing a predetermined
quantity of active material calculated to produce the desired
therapeutic effect in association with the required diluent, i.e.,
carrier, or vehicle.
[0114] The compositions are administered in a manner compatible
with the dosage formulation, and in a therapeutically effective
amount. The quantity to be administered depends on the subject to
be treated, capacity of the subject's system to utilize the active
ingredient, and degree of therapeutic effect desired. Precise
amounts of active ingredient required to be administered depend on
the judgment of the practitioner and are peculiar to each
individual. However, suitable dosage ranges for systemic
application are disclosed herein and depend on the route of
administration. Suitable regimes for initial and booster
administration are also contemplated and are typified by an initial
administration followed by repeated doses at one or more hour
intervals by a subsequent injection or other administration.
Exemplary multiple administrations are described herein and are
particularly preferred to maintain continuously high serum and
tissue levels of polypeptide. Alternatively, continuous intravenous
infusion sufficient to maintain concentrations in the blood in the
ranges specified for in vivo therapies are contemplated.
[0115] It is contemplated that an oligonucleotide of the invention
can be administered systemically or locally to treat disease, such
as to inhibit tumor cell growth or to kill cancer cells in cancer
patients with locally advanced or metastatic cancers. They can be
administered intravenously, intrathecally, subcutaneously, and/or
intraperitoneally. They can be administered alone or in combination
with anti-proliferative drugs. In one embodiment, they are
administered to reduce the cancer load in the patient prior to
surgery or other procedures. Alternatively, they can be
administered after surgery to ensure that any remaining cancer
(e.g., cancer that the surgery failed to eliminate) does not
survive.
[0116] A therapeutically effective amount of an oligonucleotide is
a predetermined amount calculated to achieve the desired effect,
i.e., to inhibit the expression of a target protein. Thus, the
dosage ranges for the administration of oligonucleotides of the
invention are those large enough to produce the desired effect. The
dosage should not be so large as to cause adverse side effects,
such as hyperviscosity syndromes, pulmonary edema, congestive heart
failure, neurological effects, and the like. Generally, the dosage
will vary with age of, condition of, sex of, and extent of the
disease in the patient and can be determined by one of skill in the
art. The dosage can be adjusted by the individual physician in the
event of any complication.
[0117] A composition of the present invention is preferably
administered to a patient parenterally, for example by intravenous,
intraarterial, intramuscular, intralymphatic, intraperitoneal,
subcutaneous, intrapleural, or intrathecal injection, or may be
used ex vivo. Preferred dosages are between 5-25 mg/kg. The
administration is preferably repeated on a timed schedule until the
cancer disappears or regresses, and may be in conjunction with
other forms of therapy.
VI. PHARMACEUTICAL PREPARATIONS
[0118] A pharmaceutical composition comprising the liposomes will
usually include a sterile, pharmaceutically acceptable carrier or
diluent, such as dextrose or saline solution.
[0119] Where clinical application of non-charged lipid component
(e.g., in the form of a liposome) containing an oligonucleotide is
undertaken, it will generally be beneficial to prepare the lipid
complex as a pharmaceutical composition appropriate for the
intended application. This will typically entail preparing a
pharmaceutical composition that is essentially free of pyrogens, as
well as any other impurities that could be harmful to humans or
animals. One may also employ appropriate buffers to render the
complex stable and allow for uptake by target cells.
[0120] The phrases "pharmaceutical or pharmacologically acceptable"
refers to molecular entities and compositions that do not produce
an adverse, allergic or other untoward reaction when administered
to an animal, such as a human, as appropriate. The preparation of a
pharmaceutical composition that contains at least one non-charged
lipid component comprising an oligonucleotide or additional active
ingredient will be known to those of skill in the art in light of
the present disclosure, as exemplified by Remington: The Science
and Practice of Pharmacy, 21st, 2005, incorporated herein by
reference. Moreover, for animal (e.g., human) administration, it
will be understood that preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards.
[0121] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art. A pharmaceutically acceptable carrier is preferably
formulated for administration to a human, although in certain
embodiments it may be desirable to use a pharmaceutically
acceptable carrier that is formulated for administration to a
non-human animal but which would not be acceptable (e.g., due to
governmental regulations) for administration to a human. Except
insofar as any conventional carrier is incompatible with the active
ingredient, its use in the therapeutic or pharmaceutical
compositions is contemplated.
[0122] The actual dosage amount of a composition of the present
invention administered to a patient or subject can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject.
[0123] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 0.1% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein. In
other non-limiting examples, a dose may also comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about
10 microgram/kg/body weight, about 50 microgram/kg/body weight,
about 100 microgram/kg/body weight, about 200 microgram/kg/body
weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5
milligram/kg/body weight, about 10 milligram/kg/body weight, about
50 milligram/kg/body weight, about 100 milligram/kg/body weight,
about 200 milligram/kg/body weight, about 350 milligram/kg/body
weight, about 500 milligram/kg/body weight, to about 1000
mg/kg/body weight or more per administration, and any range
derivable therein. In non-limiting examples of a derivable range
from the numbers listed herein, a range of about 5 jag/kg/body
weight to about 1000 mg/kg/body weight, about 5 microgram/kg/body
weight to about 500 milligram/kg/body weight, etc., can be
administered.
[0124] An oligonucleotide of the present embodiments may be
administered in a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 40, 50, 60, 70, 80, 90, 100 or more .mu.g of nucleic acid
per dose. Each dose may be in a volume of 1, 10, 50, 100, 200, 500,
1000 or more .mu.l or ml.
[0125] Solutions of therapeutic compositions can be prepared in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0126] The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain 10
mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per
milliliter of phosphate buffered saline. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the
like.
[0127] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti-oxidants, chelating agents and
inert gases. The pH and exact concentration of the various
components the pharmaceutical composition are adjusted according to
well-known parameters.
[0128] The therapeutic compositions of the present invention may
include classic pharmaceutical preparations. Administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Topical administration may be particularly advantageous
for the treatment of skin cancers, to prevent chemotherapy-induced
alopecia or other dermal hyperproliferative disorder.
Alternatively, administration may be by orthotopic, intradermal,
subcutaneous, intramuscular, intraperitoneal or intravenous
injection. Such compositions would normally be administered as
pharmaceutically acceptable compositions that include
physiologically acceptable carriers, buffers or other excipients.
For treatment of conditions of the lungs, aerosol delivery can be
used. Volume of the aerosol is between about 0.01 ml and 0.5
ml.
[0129] An effective amount of the therapeutic composition is
determined based on the intended goal. The term "unit dose" or
"dosage" refers to physically discrete units suitable for use in a
subject, each unit containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired responses
discussed above in association with its administration, i.e., the
appropriate route and treatment regimen. The quantity to be
administered, both according to number of treatments and unit dose,
depends on the protection or effect desired.
[0130] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting the dose include the physical and
clinical state of the patient, the route of administration, the
intended goal of treatment (e.g., alleviation of symptoms versus
cure) and the potency, stability and toxicity of the particular
therapeutic substance.
VII. COMBINATION TREATMENTS
[0131] In certain embodiments, the compositions and methods of the
present invention involve an inhibitory oligonucleotide, or
oligonucleotide capable of expressing an inhibitor of gene
expression, in combination with a second or additional therapy. The
methods and compositions including combination therapies enhance
the therapeutic or protective effect, and/or increase the
therapeutic effect of another anti-cancer or
anti-hyperproliferative therapy. Therapeutic and prophylactic
methods and compositions can be provided in a combined amount
effective to achieve the desired effect, such as the killing of a
cancer cell and/or the inhibition of cellular hyperproliferation.
This process may involve contacting the cells with both an
inhibitor of gene expression and a second therapy. A tissue, tumor,
or cell can be contacted with one or more compositions or
pharmacological formulation(s) including one or more of the agents
(i.e., inhibitor of gene expression or an anti-cancer agent), or by
contacting the tissue, tumor, and/or cell with two or more distinct
compositions or formulations, wherein one composition provides 1)
an inhibitory oligonucleotide; 2) an anti-cancer agent, or 3) both
an inhibitory oligonucleotide and an anti-cancer agent. Also, it is
contemplated that such a combination therapy can be used in
conjunction with a chemotherapy, radiotherapy, surgical therapy, or
immunotherapy.
[0132] An inhibitory oligonucleotide may be administered before,
during, after or in various combinations relative to an anti-cancer
treatment. The administrations may be in intervals ranging from
concurrently to minutes to days to weeks. In embodiments where the
inhibitory oligonucleotide is provided to a patient separately from
an anti-cancer agent, one would generally ensure that a significant
period of time did not expire between the time of each delivery,
such that the two compounds would still be able to exert an
advantageously combined effect on the patient. In such instances,
it is contemplated that one may provide a patient with the
inhibitory oligonucleotide therapy and the anti-cancer therapy
within about 12 to 24 or 72 h of each other and, more preferably,
within about 6-12 h of each other. In some situations it may be
desirable to extend the time period for treatment significantly
where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3,
4, 5, 6, 7 or 8) lapse between respective administrations.
[0133] In certain embodiments, a course of treatment will last 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90 days or more. It is contemplated that one agent may be given
on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, and/or 90, any combination thereof, and another
agent is given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, and/or 90, or any combination
thereof. Within a single day (24-hour period), the patient may be
given one or multiple administrations of the agent(s). Moreover,
after a course of treatment, it is contemplated that there is a
period of time at which no anti-cancer treatment is administered.
This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3,
4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or
more, depending on the condition of the patient, such as their
prognosis, strength, health, etc.
[0134] Various combinations may be employed. For the example below
an inhibitory oligonucleotide therapy is "A" and an anti-cancer
therapy is "B":
TABLE-US-00004 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0135] Administration of any compound or therapy of the present
invention to a patient will follow general protocols for the
administration of such compounds, taking into account the toxicity,
if any, of the agents. Therefore, in some embodiments there is a
step of monitoring toxicity that is attributable to combination
therapy. It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the described therapy.
[0136] In specific aspects, it is contemplated that a standard
therapy will include chemotherapy, radiotherapy, immunotherapy,
surgical therapy or gene therapy and may be employed in combination
with the inhibitor of gene expression therapy, anticancer therapy,
or both the inhibitor of gene expression therapy and the
anti-cancer therapy, as described herein.
[0137] A. Chemotherapy
[0138] A wide variety of chemotherapeutic agents may be used in
accordance with the present embodiments. The term "chemotherapy"
refers to the use of drugs to treat cancer. A "chemotherapeutic
agent" is used to connote a compound or composition that is
administered in the treatment of cancer. These agents or drugs are
categorized by their mode of activity within a cell, for example,
whether and at what stage they affect the cell cycle.
Alternatively, an agent may be characterized based on its ability
to directly cross-link DNA, to intercalate into DNA, or to induce
chromosomal and mitotic aberrations by affecting nucleic acid
synthesis.
[0139] Examples of chemotherapeutic agents include alkylating
agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates,
such as busulfan, improsulfan, and piposulfan; aziridines, such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines, including altretamine, tri ethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide, and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards, such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, and uracil
mustard; nitrosureas, such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics,
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gammalI and calicheamicin omegaI1); dynemicin,
including dynemicin A; bisphosphonates, such as clodronate; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores, aclacinomysins,
actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins, such as
mitomycin C, mycophenolic acid, nogalarnycin, olivomycins,
peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and
zorubicin; anti-metabolites, such as methotrexate and
5-fluorouracil (5-FU); folic acid analogues, such as denopterin,
pteropterin, and trimetrexate; purine analogs, such as fludarabine,
6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs,
such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, and
floxuridine; androgens, such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, and testolactone;
anti-adrenals, such as mitotane and trilostane; folic acid
replenisher, such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids,
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;
PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g.,
paclitaxel and docetaxel gemcitabine; 6-thioguanine;
mercaptopurine; platinum coordination complexes, such as cisplatin,
oxaliplatin, and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine;
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase
inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids,
such as retinoic acid; capecitabine; carboplatin,
procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein
tansferase inhibitors, transplatinum, and pharmaceutically
acceptable salts, acids, or derivatives of any of the above.
[0140] B. Radiotherapy
[0141] Other factors that cause DNA damage and have been used
extensively include what are commonly known as y-rays, X-rays,
and/or the directed delivery of radioisotopes to tumor cells. Other
forms of DNA damaging factors are also contemplated such as
microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and
4,870,287) and UV-irradiation. It is most likely that all of these
factors affect a broad range of damage on DNA, on the precursors of
DNA, on the replication and repair of DNA, and on the assembly and
maintenance of chromosomes. Dosage ranges for X-rays range from
daily doses of 50 to 200 roentgens for prolonged periods of time (3
to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges
for radioisotopes vary widely, and depend on the half-life of the
isotope, the strength and type of radiation emitted, and the uptake
by the neoplastic cells.
[0142] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing, for example, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0143] C. Immunotherapy
[0144] In the context of cancer treatment, immunotherapeutics,
generally, rely on the use of immune effector cells and molecules
to target and destroy cancer cells. Trastuzumab (Herceptin.TM.) is
such an example. The immune effector may be, for example, an
antibody specific for some marker on the surface of a tumor cell.
The antibody alone may serve as an effector of therapy or it may
recruit other cells to actually affect cell killing. The antibody
also may be conjugated to a drug or toxin (chemotherapeutic,
radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.)
and serve merely as a targeting agent. Alternatively, the effector
may be a lymphocyte carrying a surface molecule that interacts,
either directly or indirectly, with a tumor cell target. Various
effector cells include cytotoxic T cells and NK cells. The
combination of therapeutic modalities, i.e., direct cytotoxic
activity and inhibition or reduction of ErbB2 would provide
therapeutic benefit in the treatment of ErbB2 overexpressing
cancers.
[0145] Another immunotherapy could also be used as part of a
combined therapy with gen silencing therapy discussed above. In one
aspect of immunotherapy, the tumor cell must bear some marker that
is amenable to targeting, i.e., is not present on the majority of
other cells. Many tumor markers exist and any of these may be
suitable for targeting in the context of the present invention.
Common tumor markers include carcinoembryonic antigen, prostate
specific antigen, urinary tumor associated antigen, fetal antigen,
tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA,
MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. An
alternative aspect of immunotherapy is to combine anticancer
effects with immune stimulatory effects. Immune stimulating
molecules also exist including: cytokines such as IL-2, IL-4,
IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and
growth factors such as FLT3 ligand. Combining immune stimulating
molecules, either as proteins or using gene delivery in combination
with a tumor suppressor has been shown to enhance anti-tumor
effects. Moreover, antibodies against any of these compounds can be
used to target the anti-cancer agents discussed herein.
[0146] Examples of immunotherapies currently under investigation or
in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium
falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat.
Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998;
Christodoulides et al., 1998), cytokine therapy, e.g., interferons
.alpha., .beta. and .gamma.; IL-1, GM-CSF and TNF (Bukowski et al.,
1998; Davidson et al., 1998; Hellstrand et al., 1998) gene therapy,
e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and
Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) and
monoclonal antibodies, e.g., anti-ganglioside GM2, anti-HER-2,
anti-p185 (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat.
No. 5,824,311). It is contemplated that one or more anti-cancer
therapies may be employed with the gene silencing therapies
described herein.
[0147] In active immunotherapy, an antigenic peptide, polypeptide
or protein, or an autologous or allogenic tumor cell composition or
"vaccine" is administered, generally with a distinct bacterial
adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992;
Mitchell et al., 1990; Mitchell et al., 1993).
[0148] In adoptive immunotherapy, the patient's circulating
lymphocytes, or tumor infiltrated lymphocytes, are isolated in
vitro, activated by lymphokines such as IL-2 or transduced with
genes for tumor necrosis, and readministered (Rosenberg et al.,
1988; 1989).
[0149] In some embodiments, the immunotherapy may be an immune
checkpoint inhibitor. Immune checkpoints either turn up a signal
(e.g., co-stimulatory molecules) or turn down a signal. Inhibitory
immune checkpoints that may be targeted by immune checkpoint
blockade include adenosine A2A receptor (A2AR), B7-H3 (also known
as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic
T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152),
indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin
(KIR), lymphocyte activation gene-3 (LAG3), programmed death 1
(PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and
V-domain Ig suppressor of T cell activation (VISTA). In particular,
the immune checkpoint inhibitors target the PD-1 axis and/or
CTLA-4.
[0150] The immune checkpoint inhibitors may be drugs such as small
molecules, recombinant forms of ligand or receptors, or, in
particular, are antibodies, such as human antibodies (e.g.,
International Patent Publication WO2015016718; Pardoll, Nat Rev
Cancer, 12(4): 252-64, 2012; both incorporated herein by
reference). Known inhibitors of the immune checkpoint proteins or
analogs thereof may be used, in particular chimerized, humanized or
human forms of antibodies may be used. As the skilled person will
know, alternative and/or equivalent names may be in use for certain
antibodies mentioned in the present disclosure. Such alternative
and/or equivalent names are interchangeable in the context of the
present disclosure. For example, it is known that lambrolizumab is
also known under the alternative and equivalent names MK-3475 and
pembrolizumab.
[0151] In some embodiments, the PD-1 binding antagonist is a
molecule that inhibits the binding of PD-1 to its ligand binding
partners. In a specific aspect, the PD-1 ligand binding partners
are PDL1 and/or PDL2. In another embodiment, a PDL1 binding
antagonist is a molecule that inhibits the binding of PDL1 to its
binding partners. In a specific aspect, PDL1 binding partners are
PD-1 and/or B7-1. In another embodiment, the PDL2 binding
antagonist is a molecule that inhibits the binding of PDL2 to its
binding partners. In a specific aspect, a PDL2 binding partner is
PD-1. The antagonist may be an antibody, an antigen binding
fragment thereof, an immunoadhesin, a fusion protein, or
oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos.
8,735,553, 8,354,509, and 8,008,449, all incorporated herein by
reference. Other PD-1 axis antagonists for use in the methods
provided herein are known in the art such as described in U.S.
Patent Publication Nos. 20140294898, 2014022021, and 20110008369,
all incorporated herein by reference.
[0152] In some embodiments, the PD-1 binding antagonist is an
anti-PD-1 antibody (e.g., a human antibody, a humanized antibody,
or a chimeric antibody). In some embodiments, the anti-PD-1
antibody is selected from the group consisting of nivolumab,
pembrolizumab, and CT-011. In some embodiments, the PD-1 binding
antagonist is an immunoadhesin (e.g., an immunoadhesin comprising
an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a
constant region (e.g., an Fc region of an immunoglobulin sequence).
In some embodiments, the PD-1 binding antagonist is AMP-224.
Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538,
BMS-936558, and OPDIVO.RTM., is an anti-PD-1 antibody described in
WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475,
lambrolizumab, KEYTRUDA.RTM., and SCH-900475, is an anti-PD-1
antibody described in WO2009/114335. CT-011, also known as hBAT or
hBAT-1, is an anti-PD-1 antibody described in WO2009/101611.
AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble
receptor described in WO2010/027827 and WO2011/066342.
[0153] Another immune checkpoint that can be targeted in the
methods provided herein is the cytotoxic T-lymphocyte-associated
protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence
of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is
found on the surface of T cells and acts as an "off" switch when
bound to CD80 or CD86 on the surface of antigen-presenting cells.
CTLA4 is a member of the immunoglobulin superfamily that is
expressed on the surface of Helper T cells and transmits an
inhibitory signal to T cells. CTLA4 is similar to the T-cell
co-stimulatory protein, CD28, and both molecules bind to CD80 and
CD86, also called B7-1 and B7-2 respectively, on antigen-presenting
cells. CTLA4 transmits an inhibitory signal to T cells, whereas
CD28 transmits a stimulatory signal. Intracellular CTLA4 is also
found in regulatory T cells and may be important to their function.
T cell activation through the T cell receptor and CD28 leads to
increased expression of CTLA-4, an inhibitory receptor for B7
molecules.
[0154] In some embodiments, the immune checkpoint inhibitor is an
anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody,
or a chimeric antibody), an antigen binding fragment thereof, an
immunoadhesin, a fusion protein, or oligopeptide.
[0155] Anti-human-CTLA-4 antibodies (or VH and/or VL domains
derived therefrom) suitable for use in the present methods can be
generated using methods well known in the art. Alternatively, art
recognized anti-CTLA-4 antibodies can be used. For example, the
anti-CTLA-4 antibodies disclosed in: U.S. Pat. No. 8,119,129, WO
01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as
tremelimumab; formerly ticilimumab), U.S. Pat. No. 6,207,156;
Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071;
Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505
(antibody CP-675206); and Mokyr et al. (1998) Cancer Res
58:5301-5304 can be used in the methods disclosed herein. The
teachings of each of the aforementioned publications are hereby
incorporated by reference. Antibodies that compete with any of
these art-recognized antibodies for binding to CTLA-4 also can be
used. For example, a humanized CTLA-4 antibody is described in
International Patent Application No. WO2001014424, WO2000037504,
and U.S. Pat. No. 8,017,114; all incorporated herein by
reference.
[0156] An exemplary anti-CTLA-4 antibody is ipilimumab (also known
as 10D1, MDX-010, MDX-101, and Yervoy.RTM.) or antigen binding
fragments and variants thereof (see, e.g., WO 01/14424). In other
embodiments, the antibody comprises the heavy and light chain CDRs
or VRs of ipilimumab. Accordingly, in one embodiment, the antibody
comprises the CDR1, CDR2, and CDR3 domains of the VH region of
ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of
ipilimumab. In another embodiment, the antibody competes for
binding with and/or binds to the same epitope on CTLA-4 as the
above-mentioned antibodies. In another embodiment, the antibody has
at least about 90% variable region amino acid sequence identity
with the above-mentioned antibodies (e.g., at least about 90%, 95%,
or 99% variable region identity with ipilimumab).
[0157] Other molecules for modulating CTLA-4 include CTLA-4 ligands
and receptors such as described in U.S. Pat. Nos. 5,844,905,
5,885,796 and International Patent Application Nos. WO1995001994
and WO1998042752; all incorporated herein by reference, and
immunoadhesins such as described in U.S. Pat. No. 8,329,867,
incorporated herein by reference.
[0158] In some embodiment, the immune therapy could be adoptive
immunotherapy, which involves the transfer of autologous
antigen-specific T cells generated ex vivo. The T cells used for
adoptive immunotherapy can be generated either by expansion of
antigen-specific T cells or redirection of T cells through genetic
engineering (Park, Rosenberg et al. 2011). Isolation and transfer
of tumor specific T cells has been shown to be successful in
treating melanoma. Novel specificities in T cells have been
successfully generated through the genetic transfer of transgenic T
cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et
al. 2010). CARs are synthetic receptors consisting of a targeting
moiety that is associated with one or more signaling domains in a
single fusion molecule. In general, the binding moiety of a CAR
consists of an antigen-binding domain of a single-chain antibody
(scFv), comprising the light and variable fragments of a monoclonal
antibody joined by a flexible linker. Binding moieties based on
receptor or ligand domains have also been used successfully. The
signaling domains for first generation CARs are derived from the
cytoplasmic region of the CD3zeta or the Fc receptor gamma chains.
CARs have successfully allowed T cells to be redirected against
antigens expressed at the surface of tumor cells from various
malignancies including lymphomas and solid tumors (Jena, Dotti et
al. 2010).
[0159] In one embodiment, the present application provides for a
combination therapy for the treatment of cancer wherein the
combination therapy comprises adoptive T-cell therapy and a
checkpoint inhibitor. In one aspect, the adoptive T-cell therapy
comprises autologous and/or allogenic T cells. In another aspect,
the autologous and/or allogenic T cells are targeted against tumor
antigens.
[0160] D. Surgery
[0161] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative, and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0162] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
microscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0163] Upon excision of part or all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0164] E. Other Agents
[0165] It is contemplated that other agents may be used in
combination with certain aspects of the present embodiments to
improve the therapeutic efficacy of treatment. These additional
agents include agents that affect the upregulation of cell surface
receptors and GAP junctions, cytostatic and differentiation agents,
inhibitors of cell adhesion, agents that increase the sensitivity
of the hyperproliferative cells to apoptotic inducers, or other
biological agents. Increases in intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with certain aspects of the present embodiments to improve the
anti-hyperproliferative efficacy of the treatments. Inhibitors of
cell adhesion are contemplated to improve the efficacy of the
present embodiments. Examples of cell adhesion inhibitors are focal
adhesion kinase (FAKs) inhibitors and Lovastatin. It is further
contemplated that other agents that increase the sensitivity of a
hyperproliferative cell to apoptosis, such as the antibody c225,
could be used in combination with certain aspects of the present
embodiments to improve the treatment efficacy.
VIII. KITS AND DIAGNOSTICS
[0166] In various aspects of the invention, a kit is envisioned
containing therapeutic agents and/or other therapeutic and delivery
agents. In some embodiments, the present invention contemplates a
kit for preparing and/or administering a therapy of the invention.
The kit may comprise reagents capable of use in administering an
active or effective agent(s) of the invention. Reagents of the kit
may include at least one inhibitor of gene expression (e.g., a
IGF-1R oligonucleotide), one or more lipid component, one or more
anti-cancer component of a combination therapy, as well as reagents
to prepare, formulate, and/or administer the components of the
invention or perform one or more steps of the inventive
methods.
[0167] In some embodiments, the kit may also comprise a suitable
container means, which is a container that will not react with
components of the kit, such as an eppendorf tube, an assay plate, a
syringe, a bottle, or a tube. The container may be made from
sterilizable materials such as plastic or glass.
[0168] The kit may further include an instruction sheet that
outlines the procedural steps of the methods, and will follow
substantially the same procedures as described herein or are known
to those of ordinary skill.
IX. EXAMPLES
[0169] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--IGF-1R-Targeted P-Ethoxy Oligonucleotides
[0170] Oligonucleotides targeting IGF-1R were designed for use in a
liposomal IGF-1R antisense drug product to inhibit the expression
of IGF-1R protein. The contiguous cDNA sequence of IGF-1R is
provided in SEQ ID NO: 3 and the protein sequence of IGF-1R is
provided in SEQ ID NO: 4. The sequence of each of the
oligonucleotides is provided in Table 4.
TABLE-US-00005 TABLE 4 IGF-1R antisense sequences Antisense name
Sequence SEQ ID NO: IGF-1R_AS1 5'-TCC TCC GGA GCC AGA CTT-3' 1
IGF-1R_AS2 5'-GGA CCC TCC TCC GGA GCC-3' 2
[0171] The liposomal IGF-1R antisense drug product was manufactured
according to the methods described herein. Mass spectrometry
testing for the IGF-1R_AS1 base oligonucleotide showed that over
80% of the oligonucleotide drug substance had between three and
seven phosphodiester backbone linkages and that over 70% of the
oligonucleotide drug substance had between 4 and seven
phosphodiester backbone linkages.
Example 2--Effects of Liposomal IGF-1R Antisense on GL261 Tumor
Growth in Mice
[0172] The ability of liposomal IGF-1R_AS1 antisense to prevent
growth of GL261 cell tumors implanted in mice was tested. GL261
cells (10.sup.5) were implanted in the flanks of C57BL/6 mice on
day 0. Fourteen days later, liposomal IGF-1R_AS1 antisense (0.75
mg, 0.25 mg, or 0.075 mg) was administered intraperitoneally. Mice
were followed to track tumor development. Administration of
liposomal IGF-1R_AS1 antisense delayed formation of tumors (FIG.
1).
[0173] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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Sequence CWU 1
1
4118DNAArtificial SequenceSynthetic polynucleotide 1tcctccggag
ccagactt 18218DNAArtificial SequenceSynthetic polynucleotide
2ggaccctcct ccggagcc 18312262DNAHomo sapiensCDS(1044)..(5147)
3agtgtgtggc agcggcggcg gcggcgcggc gaggctgggg ctcttgttta ccagcattaa
60ctccgctgag cggaaaaaaa aagggaaaaa acccgaggag gagcgagcgc accaggcgaa
120ctcgagagag gcgggagagc gagagggacg ccgccagcga gcctgcccac
ggccggcgct 180cgcagaccct cggccccgct ccccggatcc ccccgcgccc
tccacgcccc tcccgcgcgg 240gggcagctcc acggcgcgcc tcgcctcggc
tgtgaccttc agcgagccgg agcccccgcg 300cagagcaggc ggcggcgggc
gggggccggg cgggggccgg cgcggggcgg gcggcggcgc 360agagccgggc
ggcgcggcgg gagtgctgag cgcggcgcgg ccggcccgcc gctttgtgtg
420tgtcctggat ttgggaagga gctcgccgcg gcggcggcgg cgctgaggga
ggaggcggcg 480gcgagcggag ccaggaggag gaggaggagg gggagccgct
cattcatttt gactccgcgt 540ttctgcccct cgccggcctc gcctgtgacc
cggacttcgg ggcgatcttg cgaactgcgt 600cgcgccctcc cgcggcggaa
gctcgggcgt ccggccgcct cccgcgcggc cagggccggg 660cttgtttttc
ctcgcctagg cagatttggg ctttgccccc tttctttgca gttttccccc
720cttcctgcct ctccgggttt gaaaatggag gccgacgacg ccgacagccc
gccccggcgc 780gcctcgggtt cccgactccg ccgagccctg ggccgctgct
gccggcgctg aggggccgcc 840ccgcgccgcc cgccccgtcc gcgcacccgg
agggccccgg cggcgccgcc ttcggagtat 900tgtttccttc gcccttgttt
ttggaggggg agcgaagact gagtttgaga cttgtttcct 960ttcatttcct
ttttttcttt tcttttcttt tttttttttt tttttttttt tgagaaaggg
1020gaatttcatc ccaaataaaa gga atg aag tct ggc tcc gga gga ggg tcc
ccg 1073 Met Lys Ser Gly Ser Gly Gly Gly Ser Pro 1 5 10acc tcg ctg
tgg ggg ctc ctg ttt ctc tcc gcc gcg ctc tcg ctc tgg 1121Thr Ser Leu
Trp Gly Leu Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp 15 20 25ccg acg
agt gga gaa atc tgc ggg cca ggc atc gac atc cgc aac gac 1169Pro Thr
Ser Gly Glu Ile Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp 30 35 40tat
cag cag ctg aag cgc ctg gag aac tgc acg gtg atc gag ggc tac 1217Tyr
Gln Gln Leu Lys Arg Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr 45 50
55ctc cac atc ctg ctc atc tcc aag gcc gag gac tac cgc agc tac cgc
1265Leu His Ile Leu Leu Ile Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg
60 65 70ttc ccc aag ctc acg gtc att acc gag tac ttg ctg ctg ttc cga
gtg 1313Phe Pro Lys Leu Thr Val Ile Thr Glu Tyr Leu Leu Leu Phe Arg
Val75 80 85 90gct ggc ctc gag agc ctc gga gac ctc ttc ccc aac ctc
acg gtc atc 1361Ala Gly Leu Glu Ser Leu Gly Asp Leu Phe Pro Asn Leu
Thr Val Ile 95 100 105cgc ggc tgg aaa ctc ttc tac aac tac gcc ctg
gtc atc ttc gag atg 1409Arg Gly Trp Lys Leu Phe Tyr Asn Tyr Ala Leu
Val Ile Phe Glu Met 110 115 120acc aat ctc aag gat att ggg ctt tac
aac ctg agg aac att act cgg 1457Thr Asn Leu Lys Asp Ile Gly Leu Tyr
Asn Leu Arg Asn Ile Thr Arg 125 130 135ggg gcc atc agg att gag aaa
aat gct gac ctc tgt tac ctc tcc act 1505Gly Ala Ile Arg Ile Glu Lys
Asn Ala Asp Leu Cys Tyr Leu Ser Thr 140 145 150gtg gac tgg tcc ctg
atc ctg gat gcg gtg tcc aat aac tac att gtg 1553Val Asp Trp Ser Leu
Ile Leu Asp Ala Val Ser Asn Asn Tyr Ile Val155 160 165 170ggg aat
aag ccc cca aag gaa tgt ggg gac ctg tgt cca ggg acc atg 1601Gly Asn
Lys Pro Pro Lys Glu Cys Gly Asp Leu Cys Pro Gly Thr Met 175 180
185gag gag aag ccg atg tgt gag aag acc acc atc aac aat gag tac aac
1649Glu Glu Lys Pro Met Cys Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn
190 195 200tac cgc tgc tgg acc aca aac cgc tgc cag aaa atg tgc cca
agc acg 1697Tyr Arg Cys Trp Thr Thr Asn Arg Cys Gln Lys Met Cys Pro
Ser Thr 205 210 215tgt ggg aag cgg gcg tgc acc gag aac aat gag tgc
tgc cac ccc gag 1745Cys Gly Lys Arg Ala Cys Thr Glu Asn Asn Glu Cys
Cys His Pro Glu 220 225 230tgc ctg ggc agc tgc agc gcg cct gac aac
gac acg gcc tgt gta gct 1793Cys Leu Gly Ser Cys Ser Ala Pro Asp Asn
Asp Thr Ala Cys Val Ala235 240 245 250tgc cgc cac tac tac tat gcc
ggt gtc tgt gtg cct gcc tgc ccg ccc 1841Cys Arg His Tyr Tyr Tyr Ala
Gly Val Cys Val Pro Ala Cys Pro Pro 255 260 265aac acc tac agg ttt
gag ggc tgg cgc tgt gtg gac cgt gac ttc tgc 1889Asn Thr Tyr Arg Phe
Glu Gly Trp Arg Cys Val Asp Arg Asp Phe Cys 270 275 280gcc aac atc
ctc agc gcc gag agc agc gac tcc gag ggg ttt gtg atc 1937Ala Asn Ile
Leu Ser Ala Glu Ser Ser Asp Ser Glu Gly Phe Val Ile 285 290 295cac
gac ggc gag tgc atg cag gag tgc ccc tcg ggc ttc atc cgc aac 1985His
Asp Gly Glu Cys Met Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn 300 305
310ggc agc cag agc atg tac tgc atc cct tgt gaa ggt cct tgc ccg aag
2033Gly Ser Gln Ser Met Tyr Cys Ile Pro Cys Glu Gly Pro Cys Pro
Lys315 320 325 330gtc tgt gag gaa gaa aag aaa aca aag acc att gat
tct gtt act tct 2081Val Cys Glu Glu Glu Lys Lys Thr Lys Thr Ile Asp
Ser Val Thr Ser 335 340 345gct cag atg ctc caa gga tgc acc atc ttc
aag ggc aat ttg ctc att 2129Ala Gln Met Leu Gln Gly Cys Thr Ile Phe
Lys Gly Asn Leu Leu Ile 350 355 360aac atc cga cgg ggg aat aac att
gct tca gag ctg gag aac ttc atg 2177Asn Ile Arg Arg Gly Asn Asn Ile
Ala Ser Glu Leu Glu Asn Phe Met 365 370 375ggg ctc atc gag gtg gtg
acg ggc tac gtg aag atc cgc cat tct cat 2225Gly Leu Ile Glu Val Val
Thr Gly Tyr Val Lys Ile Arg His Ser His 380 385 390gcc ttg gtc tcc
ttg tcc ttc cta aaa aac ctt cgc ctc atc cta gga 2273Ala Leu Val Ser
Leu Ser Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly395 400 405 410gag
gag cag cta gaa ggg aat tac tcc ttc tac gtc ctc gac aac cag 2321Glu
Glu Gln Leu Glu Gly Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln 415 420
425aac ttg cag caa ctg tgg gac tgg gac cac cgc aac ctg acc atc aaa
2369Asn Leu Gln Gln Leu Trp Asp Trp Asp His Arg Asn Leu Thr Ile Lys
430 435 440gca ggg aaa atg tac ttt gct ttc aat ccc aaa tta tgt gtt
tcc gaa 2417Ala Gly Lys Met Tyr Phe Ala Phe Asn Pro Lys Leu Cys Val
Ser Glu 445 450 455att tac cgc atg gag gaa gtg acg ggg act aaa ggg
cgc caa agc aaa 2465Ile Tyr Arg Met Glu Glu Val Thr Gly Thr Lys Gly
Arg Gln Ser Lys 460 465 470ggg gac ata aac acc agg aac aac ggg gag
aga gcc tcc tgt gaa agt 2513Gly Asp Ile Asn Thr Arg Asn Asn Gly Glu
Arg Ala Ser Cys Glu Ser475 480 485 490gac gtc ctg cat ttc acc tcc
acc acc acg tcg aag aat cgc atc atc 2561Asp Val Leu His Phe Thr Ser
Thr Thr Thr Ser Lys Asn Arg Ile Ile 495 500 505ata acc tgg cac cgg
tac cgg ccc cct gac tac agg gat ctc atc agc 2609Ile Thr Trp His Arg
Tyr Arg Pro Pro Asp Tyr Arg Asp Leu Ile Ser 510 515 520ttc acc gtt
tac tac aag gaa gca ccc ttt aag aat gtc aca gag tat 2657Phe Thr Val
Tyr Tyr Lys Glu Ala Pro Phe Lys Asn Val Thr Glu Tyr 525 530 535gat
ggg cag gat gcc tgc ggc tcc aac agc tgg aac atg gtg gac gtg 2705Asp
Gly Gln Asp Ala Cys Gly Ser Asn Ser Trp Asn Met Val Asp Val 540 545
550gac ctc ccg ccc aac aag gac gtg gag ccc ggc atc tta cta cat ggg
2753Asp Leu Pro Pro Asn Lys Asp Val Glu Pro Gly Ile Leu Leu His
Gly555 560 565 570ctg aag ccc tgg act cag tac gcc gtt tac gtc aag
gct gtg acc ctc 2801Leu Lys Pro Trp Thr Gln Tyr Ala Val Tyr Val Lys
Ala Val Thr Leu 575 580 585acc atg gtg gag aac gac cat atc cgt ggg
gcc aag agt gag atc ttg 2849Thr Met Val Glu Asn Asp His Ile Arg Gly
Ala Lys Ser Glu Ile Leu 590 595 600tac att cgc acc aat gct tca gtt
cct tcc att ccc ttg gac gtt ctt 2897Tyr Ile Arg Thr Asn Ala Ser Val
Pro Ser Ile Pro Leu Asp Val Leu 605 610 615tca gca tcg aac tcc tct
tct cag tta atc gtg aag tgg aac cct ccc 2945Ser Ala Ser Asn Ser Ser
Ser Gln Leu Ile Val Lys Trp Asn Pro Pro 620 625 630tct ctg ccc aac
ggc aac ctg agt tac tac att gtg cgc tgg cag cgg 2993Ser Leu Pro Asn
Gly Asn Leu Ser Tyr Tyr Ile Val Arg Trp Gln Arg635 640 645 650cag
cct cag gac ggc tac ctt tac cgg cac aat tac tgc tcc aaa gac 3041Gln
Pro Gln Asp Gly Tyr Leu Tyr Arg His Asn Tyr Cys Ser Lys Asp 655 660
665aaa atc ccc atc agg aag tat gcc gac ggc acc atc gac att gag gag
3089Lys Ile Pro Ile Arg Lys Tyr Ala Asp Gly Thr Ile Asp Ile Glu Glu
670 675 680gtc aca gag aac ccc aag act gag gtg tgt ggt ggg gag aaa
ggg cct 3137Val Thr Glu Asn Pro Lys Thr Glu Val Cys Gly Gly Glu Lys
Gly Pro 685 690 695tgc tgc gcc tgc ccc aaa act gaa gcc gag aag cag
gcc gag aag gag 3185Cys Cys Ala Cys Pro Lys Thr Glu Ala Glu Lys Gln
Ala Glu Lys Glu 700 705 710gag gct gaa tac cgc aaa gtc ttt gag aat
ttc ctg cac aac tcc atc 3233Glu Ala Glu Tyr Arg Lys Val Phe Glu Asn
Phe Leu His Asn Ser Ile715 720 725 730ttc gtg ccc aga cct gaa agg
aag cgg aga gat gtc atg caa gtg gcc 3281Phe Val Pro Arg Pro Glu Arg
Lys Arg Arg Asp Val Met Gln Val Ala 735 740 745aac acc acc atg tcc
agc cga agc agg aac acc acg gcc gca gac acc 3329Asn Thr Thr Met Ser
Ser Arg Ser Arg Asn Thr Thr Ala Ala Asp Thr 750 755 760tac aac atc
acc gac ccg gaa gag ctg gag aca gag tac cct ttc ttt 3377Tyr Asn Ile
Thr Asp Pro Glu Glu Leu Glu Thr Glu Tyr Pro Phe Phe 765 770 775gag
agc aga gtg gat aac aag gag aga act gtc att tct aac ctt cgg 3425Glu
Ser Arg Val Asp Asn Lys Glu Arg Thr Val Ile Ser Asn Leu Arg 780 785
790cct ttc aca ttg tac cgc atc gat atc cac agc tgc aac cac gag gct
3473Pro Phe Thr Leu Tyr Arg Ile Asp Ile His Ser Cys Asn His Glu
Ala795 800 805 810gag aag ctg ggc tgc agc gcc tcc aac ttc gtc ttt
gca agg act atg 3521Glu Lys Leu Gly Cys Ser Ala Ser Asn Phe Val Phe
Ala Arg Thr Met 815 820 825ccc gca gaa gga gca gat gac att cct ggg
cca gtg acc tgg gag cca 3569Pro Ala Glu Gly Ala Asp Asp Ile Pro Gly
Pro Val Thr Trp Glu Pro 830 835 840agg cct gaa aac tcc atc ttt tta
aag tgg ccg gaa cct gag aat ccc 3617Arg Pro Glu Asn Ser Ile Phe Leu
Lys Trp Pro Glu Pro Glu Asn Pro 845 850 855aat gga ttg att cta atg
tat gaa ata aaa tac gga tca caa gtt gag 3665Asn Gly Leu Ile Leu Met
Tyr Glu Ile Lys Tyr Gly Ser Gln Val Glu 860 865 870gat cag cga gaa
tgt gtg tcc aga cag gaa tac agg aag tat gga ggg 3713Asp Gln Arg Glu
Cys Val Ser Arg Gln Glu Tyr Arg Lys Tyr Gly Gly875 880 885 890gcc
aag cta aac cgg cta aac ccg ggg aac tac aca gcc cgg att cag 3761Ala
Lys Leu Asn Arg Leu Asn Pro Gly Asn Tyr Thr Ala Arg Ile Gln 895 900
905gcc aca tct ctc tct ggg aat ggg tcg tgg aca gat cct gtg ttc ttc
3809Ala Thr Ser Leu Ser Gly Asn Gly Ser Trp Thr Asp Pro Val Phe Phe
910 915 920tat gtc cag gcc aaa aca gga tat gaa aac ttc atc cat ctg
atc atc 3857Tyr Val Gln Ala Lys Thr Gly Tyr Glu Asn Phe Ile His Leu
Ile Ile 925 930 935gct ctg ccc gtc gct gtc ctg ttg atc gtg gga ggg
ttg gtg att atg 3905Ala Leu Pro Val Ala Val Leu Leu Ile Val Gly Gly
Leu Val Ile Met 940 945 950ctg tac gtc ttc cat aga aag aga aat aac
agc agg ctg ggg aat gga 3953Leu Tyr Val Phe His Arg Lys Arg Asn Asn
Ser Arg Leu Gly Asn Gly955 960 965 970gtg ctg tat gcc tct gtg aac
ccg gag tac ttc agc gct gct gat gtg 4001Val Leu Tyr Ala Ser Val Asn
Pro Glu Tyr Phe Ser Ala Ala Asp Val 975 980 985tac gtt cct gat gag
tgg gag gtg gct cgg gag aag atc acc atg agc 4049Tyr Val Pro Asp Glu
Trp Glu Val Ala Arg Glu Lys Ile Thr Met Ser 990 995 1000cgg gaa ctt
ggg cag ggg tcg ttt ggg atg gtc tat gaa gga gtt 4094Arg Glu Leu Gly
Gln Gly Ser Phe Gly Met Val Tyr Glu Gly Val 1005 1010 1015gcc aag
ggt gtg gtg aaa gat gaa cct gaa acc aga gtg gcc att 4139Ala Lys Gly
Val Val Lys Asp Glu Pro Glu Thr Arg Val Ala Ile 1020 1025 1030aaa
aca gtg aac gag gcc gca agc atg cgt gag agg att gag ttt 4184Lys Thr
Val Asn Glu Ala Ala Ser Met Arg Glu Arg Ile Glu Phe 1035 1040
1045ctc aac gaa gct tct gtg atg aag gag ttc aat tgt cac cat gtg
4229Leu Asn Glu Ala Ser Val Met Lys Glu Phe Asn Cys His His Val
1050 1055 1060gtg cga ttg ctg ggt gtg gtg tcc caa ggc cag cca aca
ctg gtc 4274Val Arg Leu Leu Gly Val Val Ser Gln Gly Gln Pro Thr Leu
Val 1065 1070 1075atc atg gaa ctg atg aca cgg ggc gat ctc aaa agt
tat ctc cgg 4319Ile Met Glu Leu Met Thr Arg Gly Asp Leu Lys Ser Tyr
Leu Arg 1080 1085 1090tct ctg agg cca gaa atg gag aat aat cca gtc
cta gca cct cca 4364Ser Leu Arg Pro Glu Met Glu Asn Asn Pro Val Leu
Ala Pro Pro 1095 1100 1105agc ctg agc aag atg att cag atg gcc gga
gag att gca gac ggc 4409Ser Leu Ser Lys Met Ile Gln Met Ala Gly Glu
Ile Ala Asp Gly 1110 1115 1120atg gca tac ctc aac gcc aat aag ttc
gtc cac aga gac ctt gct 4454Met Ala Tyr Leu Asn Ala Asn Lys Phe Val
His Arg Asp Leu Ala 1125 1130 1135gcc cgg aat tgc atg gta gcc gaa
gat ttc aca gtc aaa atc gga 4499Ala Arg Asn Cys Met Val Ala Glu Asp
Phe Thr Val Lys Ile Gly 1140 1145 1150gat ttt ggt atg acg cga gat
atc tat gag aca gac tat tac cgg 4544Asp Phe Gly Met Thr Arg Asp Ile
Tyr Glu Thr Asp Tyr Tyr Arg 1155 1160 1165aaa gga ggg aaa ggg ctg
ctg ccc gtg cgc tgg atg tct cct gag 4589Lys Gly Gly Lys Gly Leu Leu
Pro Val Arg Trp Met Ser Pro Glu 1170 1175 1180tcc ctc aag gat gga
gtc ttc acc act tac tcg gac gtc tgg tcc 4634Ser Leu Lys Asp Gly Val
Phe Thr Thr Tyr Ser Asp Val Trp Ser 1185 1190 1195ttc ggg gtc gtc
ctc tgg gag atc gcc aca ctg gcc gag cag ccc 4679Phe Gly Val Val Leu
Trp Glu Ile Ala Thr Leu Ala Glu Gln Pro 1200 1205 1210tac cag ggc
ttg tcc aac gag caa gtc ctt cgc ttc gtc atg gag 4724Tyr Gln Gly Leu
Ser Asn Glu Gln Val Leu Arg Phe Val Met Glu 1215 1220 1225ggc ggc
ctt ctg gac aag cca gac aac tgt cct gac atg ctg ttt 4769Gly Gly Leu
Leu Asp Lys Pro Asp Asn Cys Pro Asp Met Leu Phe 1230 1235 1240gaa
ctg atg cgc atg tgc tgg cag tat aac ccc aag atg agg cct 4814Glu Leu
Met Arg Met Cys Trp Gln Tyr Asn Pro Lys Met Arg Pro 1245 1250
1255tcc ttc ctg gag atc atc agc agc atc aaa gag gag atg gag cct
4859Ser Phe Leu Glu Ile Ile Ser Ser Ile Lys Glu Glu Met Glu Pro
1260 1265 1270ggc ttc cgg gag gtc tcc ttc tac tac agc gag gag aac
aag ctg 4904Gly Phe Arg Glu Val Ser Phe Tyr Tyr Ser Glu Glu Asn Lys
Leu 1275 1280 1285ccc gag ccg gag gag ctg gac ctg gag cca gag aac
atg gag agc 4949Pro Glu Pro Glu Glu Leu Asp Leu Glu Pro Glu Asn Met
Glu Ser 1290 1295 1300gtc ccc ctg gac ccc tcg gcc tcc tcg tcc tcc
ctg cca ctg ccc 4994Val Pro Leu Asp Pro Ser Ala Ser Ser Ser Ser Leu
Pro Leu Pro 1305 1310 1315gac aga cac tca gga cac aag gcc gag aac
ggc ccc ggc cct ggg 5039Asp Arg His Ser Gly His Lys Ala Glu Asn Gly
Pro Gly Pro Gly 1320 1325 1330gtg ctg gtc ctc cgc gcc agc ttc gac
gag aga cag cct tac gcc 5084Val Leu Val Leu Arg Ala Ser Phe Asp Glu
Arg Gln Pro Tyr Ala 1335 1340 1345cac atg aac ggg ggc cgc aag aac
gag cgg gcc ttg ccg ctg ccc 5129His Met Asn Gly Gly Arg Lys Asn Glu
Arg Ala Leu Pro Leu Pro 1350 1355 1360cag tct tcg acc tgc tga
tccttggatc ctgaatctgt gcaaacagta 5177Gln Ser Ser Thr Cys
1365acgtgtgcgc acgcgcagcg gggtgggggg ggagagagag ttttaacaat
ccattcacaa 5237gcctcctgta cctcagtgga tcttcagaac tgcccttgct
gcccgcggga gacagcttct
5297ctgcagtaaa acacatttgg gatgttcctt ttttcaatat gcaagcagct
ttttattccc 5357tgcccaaacc cttaactgac atgggccttt aagaacctta
atgacaacac ttaatagcaa 5417cagagcactt gagaaccagt ctcctcactc
tgtccctgtc cttccctgtt ctccctttct 5477ctctcctctc tgcttcataa
cggaaaaata attgccacaa gtccagctgg gaagcccttt 5537ttatcagttt
gaggaagtgg ctgtccctgt ggccccatcc aaccactgta cacacccgcc
5597tgacaccgtg ggtcattaca aaaaaacacg tggagatgga aatttttacc
tttatctttc 5657acctttctag ggacatgaaa tttacaaagg gccatcgttc
atccaaggct gttaccattt 5717taacgctgcc taattttgcc aaaatcctga
actttctccc tcatcggccc ggcgctgatt 5777cctcgtgtcc ggaggcatgg
gtgagcatgg cagctggttg ctccatttga gagacacgct 5837ggcgacacac
tccgtccatc cgactgcccc tgctgtgctg ctcaaggcca caggcacaca
5897ggtctcattg cttctgacta gattattatt tgggggaact ggacacaata
ggtctttctc 5957tcagtgaagg tggggagaag ctgaaccggc ttccctgccc
tgcctcccca gccccctgcc 6017caacccccaa gaatctggtg gccatgggcc
ccgaagcagc ctggcggaca ggcttggagt 6077caaggggccc catgcctgct
tctctcccag ccccagctcc cccgcccgcc cccaaggaca 6137cagatgggaa
ggggtttcca gggactcagc cccactgttg atgcaggttt gcaaggaaag
6197aaattcaaac accacaacag cagtaagaag aaaagcagtc aatggattca
agcattctaa 6257gctttgttga cattttctct gttcctagga cttcttcatg
ggtcttacag ttctatgtta 6317gaccatgaaa catttgcata cacatcgtct
ttaatgtcac ttttataact tttttacggt 6377tcagatattc atctatacgt
ctgtacagaa aaaaaaaagc tgctattttt tttgttcttg 6437atctttgtgg
atttaatcta tgaaaacctt caggtccacc ctctcccctt tctgctcact
6497ccaagaaact tcttatgctt tgtactagag tgcgtgactt tcttcctctt
ttcccggtaa 6557tggatacttc tatcacataa tttgccatga actgttggat
gcctttttat aaatacatcc 6617cccatccctg ctcccacctg cccctttagt
tgttttctaa cccgtaggct ctctgggcac 6677gaggcagaaa gcaggccggg
cacccatcct gagagggccg cgctcctctc cccagcctgc 6737cctcacagca
ttggagcctg ttacagtgca agacatgata caaactcagg tcagaaaaac
6797aaaggttaaa tatttcacac gtctttgttc agtgtttcca ctcaccgtgg
ttgagaagcc 6857tcaccctctc tttcccttgc ctttgcttag gttgtgacac
acatatatat atattttttt 6917aattcttggg tacaacagca gtgttaaccg
cagacactag gcatttggat tactattttt 6977cttaatggct atttaatcct
tccatcccac gaaaaacagc tgctgagtcc aagggagcag 7037cagagcgtgg
tccggcaggg cctgttgtgg ccctcgccac ccccctcacc ggaccgactg
7097acctgtcttt ggaaccagaa catcccaagg gaactccttc gcactggcgt
tgagtgggac 7157cccgggatcc aggctggccc agggcggcac cctcagggct
gtgcccgctg gagtgctagg 7217tggaggcagc acagacgcca cggtggccca
agagcccctt tgcttcttgc tgggggacca 7277gggctgtggt gctggcccac
tttccctcgg ccaggaatcc aggtccttgg ggcccagggg 7337tcttgtcttg
tttcattttt agcacttctc accagagaga tgacagcaca agagttgctt
7397ctgggataga aatgtttagg agtaagaaca aagctgggat acggtgattg
ctagttgtga 7457ctgaagattc aacacagaaa agaaagttta tacggctttt
ttgctggtca gcagtttgtc 7517ccactgcttt ctctagtctc tatcccatag
cgtgttccct ttaaaaaaaa aaaaaaggta 7577ttatatgtag gagttttctt
ttaatttatt ttgtgataaa ttaccagttt caatcactgt 7637agaaaagccc
cattatgaat ttaaatttca aggaaagggt gtgtgtgtgt gtatgtgtgg
7697ggtgtgtgtg tgtgagagtg atgggacagt tcttgatttt ttgggttttt
tttcccccaa 7757acatttatct acctcactct tattttttat atgtgtatat
agacaaaaga atacatctca 7817cctttctcag cacctgacaa taggccgttg
atactggtaa cctcatccac gccacaggcg 7877ccacacccag gtgatgcagg
gggaagccag gctgtattcc ggggtcaaag caacactaac 7937tcacctctct
gctcatttca gacagcttgc ctttttctga gatgtcctgt tttgtgttgc
7997tttttttgtt ttgttttcta tcttggtttc caccaaggtg ttagatttct
cctcctccta 8057gccaggtggc cctgtgaggc caacgagggc accagagcac
acctggggga gccaccaggc 8117tgtccctggc tggttgtctt tggaacaaac
tgcttctgtg cagatggaat gaccaacaca 8177tttcgtcctt aagagagcag
tggttcctca ggttctgagg agaggaaggt gtccaggcag 8237caccatctct
gtgcgaatcc ccagggtaaa ggcgtggggc attgggtttg ctccccttgc
8297tgctgctcca tccctgcagg aggctcgcgc tgaggcagga ccgtgcggcc
atggctgctg 8357cattcattga gcacaaaggt gcagctgcag cagcagctgg
agagcaagag tcacccagcc 8417tgtgcgccag aatgcagagg ctcctgacct
cacagccagt ccctgataga acacacgcag 8477gagcagagtc ccctccccct
ccaggctgcc ctctcaactt ctccctcacc tccttcccta 8537ggggtagaca
gagatgtacc aaaccttccg gctggaaagc ccagtggccg gcgccgaggc
8597tcgtggcgtc acgccccccc cgccagggct gtacctccgt ctccctggtc
ctgctgctca 8657caggacagac ggctcgctcc cctcttccag cagctgctct
tacaggcact gatgatttcg 8717ctgggaagtg tggcgggcag ctttgcctaa
gcgtggatgg ctcctcggca attccagcct 8777aagtgaaggc gctcaggagc
ctcctgctgg aacgcgaccc atctctccca ggaccccggg 8837gatcttaagg
tcattgagaa atactgttgg atcagggttt tgttcttcca cactgtaggt
8897gaccccttgg aataacggcc tctcctctcg tgcacatacc taccggtttc
cacaactgga 8957tttctacaga tcattcagct ggttataagg gttttgttta
aactgtccga gttactgatg 9017tcattttgtt tttgttttat gtaggtagct
tttaagtaga aaacactaac agtgtagtgc 9077ccatcatagc aaatgcttca
gaaacacctc aataaaagag aaaacttggc ttgtgtgatg 9137gtgcagtcac
tttactggac caacccaccc accttgacta taccaaggca tcatctatcc
9197acagttctag cctaacttca tgctgatttc tctgcctctt gatttttctc
tgtgtgttcc 9257aaataatctt aagctgagtt gtggcatttt ccatgcaacc
tccttctgcc agcagctcac 9317actgcttgaa gtcatatgaa ccactgaggc
acatcatgga attgatgtga gcattaagac 9377gttctcccac acagcccttc
cctgaggcag caggagctgg tgtgtactgg agacactgtt 9437gaacttgatc
aagacccaga ccaccccagg tctccttcgt gggatgtcat gacgtttgac
9497atacctttgg aacgagcctc ctccttggaa gatggaagac cgtgttcgtg
gccgacctgg 9557cctctcctgg cctgtttctt aagatgcgga gtcacatttc
aatggtacga aaagtggctt 9617cgtaaaatag aagagcagtc actgtggaac
taccaaatgg cgagatgctc ggtgcacatt 9677ggggtgcttt gggataaaag
atttatgagc caactattct ctggcaccag attctaggcc 9737agtttgttcc
actgaagctt ttcccacagc agtccacctc tgcaggctgg cagccgaatg
9797gcttgccagt ggctctgtgg caagatcaca ctgagatcga tgggtgagaa
ggctaggatg 9857cttgtctagt gttcttagct gtcacgttgg ctccttccag
ggtggccaga cggtgttggc 9917cactcccttc taaaacacag gcgccctcct
ggtgacagtg acccgccgtg gtatgccttg 9977gcccattcca gcagtcccag
ttatgcattt caagtttggg gtttgttctt ttcgttaatg 10037ttcctctgtg
ttgtcagctg tcttcatttc ctgggctaag cagcattggg agatgtggac
10097cagagatcca ctccttaaga accagtggcg aaagacactt tctttcttca
ctctgaagta 10157gctggtggta caaatgagaa cttcaagaga ggatgttatt
tagactgaac ctctgttgcc 10217agagatgctg aagatacaga ccttggacag
gtcagagggt ttcatttttg gccttcatct 10277tagatgactg gttgcgtcat
ttggagaagt gagtgctcct tgatggtgga atgaccgggt 10337ggtgggtaca
gaaccattgt cacagggatc ctggcacaga gaagagttac gagcagcagg
10397gtgcagggct tggaaggaat gtgggcaagg ttttgaactt gattgttctt
gaagctatca 10457gaccacatcg aggctcagca gtcatccgtg ggcatttggt
ttcaacaaag aaacctaaca 10517tcctactctg gaaactgatc tcggagttaa
ggcgaattgt tcaagaacac aaactacatc 10577gcactcgtca gttgtcagtt
ctggggcatg actttagcgt tttgtttctg cgagaacata 10637acgatcactc
atttttatgt cccacgtgtg tgtgtccgca tctttctggt caacattgtt
10697ttaactagtc actcattagc gttttcaata gggctcttaa gtccagtaga
ttacgggtag 10757tcagttgacg aagatctggt ttacaagaac taattaaatg
tttcattgca tttttgtaag 10817aacagaataa ttttataaaa tgtttgtagt
ttataattgc cgaaaataat ttaaagacac 10877tttttttttc tctgtgtgtg
caaatgtgtg tttgtgatcc attttttttt ttttttttta 10937ggacacctgt
ttactagcta gctttacaat atgccaaaaa aggatttctc cctgacccca
10997tccgtggttc accctctttt ccccccatgc tttttgccct agtttataac
aaaggaatga 11057tgatgattta aaaagtagtt ctgtatcttc agtatcttgg
tcttccagaa ccctctggtt 11117gggaagggga tcatttttta ctggtcattt
ccctttggag tgtagctact ttaacagatg 11177gaaagaacct cattggccat
ggaaacagcc gaggtgttgg agcccagcag tgcatggcac 11237cgttcggcat
ctggcttgat tggtctggct gccgtcattg tcagcacagt gccatggaca
11297tgggaagact tgactgcaca gccaatggtt ttcatgatga ttacagcata
cacagtgatc 11357acataaacga tgacagctat ggggcacaca ggccatttgc
ttacatgcct cgtatcatga 11417ctgattactg ctttgttaga acacagaaga
gaccctattt tatttaaggc agaaccccga 11477agatacgtat ttccaataca
gaaaagaatt tttaataaaa actataacat acacaaaaat 11537tggttttaaa
gttgactcca cttcctctaa ctccagtgga ttgttggcca tgtctcccca
11597actccacaat atctctatca tgggaaacac ctggggtttt tgcgctacat
aggagaaaga 11657tctggaaact atttgggttt tgttttcaac ttttcatttg
gatgtttggc gttgcacaca 11717cacatccacc ggtggaagag acgcccggtg
aaaacacctg tctgctttct aagccagtga 11777ggttgaggtg agaggtttgc
cagagtttgt ctacctctgg gtatcccttt gtctgggata 11837aaaaaaatca
aaccagaagg cgggatggaa tggatgcacc gcaaataatg cattttctga
11897gttttcttgt taaaaaaaaa tttttttaag taagaaaaaa aaaggtaata
acatggccaa 11957tttgttacat aaaatgactt tctgtgtata aattattcct
aaaaaatcct gtttatataa 12017aaaatcagta gatgaaaaaa atttcaaaat
gtttttgtat attctgttgt aagaatttat 12077tcctgttatt gcgatatact
ctggattctt tacataatgg aaaaaagaaa ctgtctattt 12137tgaatggctg
aagctaaggc aacgttagtt tctcttactc tgcttttttc tagtaaagta
12197ctacatggtt taagttaaat aaaataattc tgtatgcaaa aaaaaaaaaa
aaaaaaaaaa 12257aaaaa 1226241367PRTHomo sapiens 4Met Lys Ser Gly
Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu1 5 10 15Leu Phe Leu
Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile 20 25 30Cys Gly
Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg 35 40 45Leu
Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu His Ile Leu Leu Ile 50 55
60Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val65
70 75 80Ile Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser
Leu 85 90 95Gly Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Trp Lys
Leu Phe 100 105 110Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn
Leu Lys Asp Ile 115 120 125Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg
Gly Ala Ile Arg Ile Glu 130 135 140Lys Asn Ala Asp Leu Cys Tyr Leu
Ser Thr Val Asp Trp Ser Leu Ile145 150 155 160Leu Asp Ala Val Ser
Asn Asn Tyr Ile Val Gly Asn Lys Pro Pro Lys 165 170 175Glu Cys Gly
Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys 180 185 190Glu
Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr 195 200
205Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys
210 215 220Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser
Cys Ser225 230 235 240Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys
Arg His Tyr Tyr Tyr 245 250 255Ala Gly Val Cys Val Pro Ala Cys Pro
Pro Asn Thr Tyr Arg Phe Glu 260 265 270Gly Trp Arg Cys Val Asp Arg
Asp Phe Cys Ala Asn Ile Leu Ser Ala 275 280 285Glu Ser Ser Asp Ser
Glu Gly Phe Val Ile His Asp Gly Glu Cys Met 290 295 300Gln Glu Cys
Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met Tyr305 310 315
320Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys
325 330 335Lys Thr Lys Thr Ile Asp Ser Val Thr Ser Ala Gln Met Leu
Gln Gly 340 345 350Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile
Arg Arg Gly Asn 355 360 365Asn Ile Ala Ser Glu Leu Glu Asn Phe Met
Gly Leu Ile Glu Val Val 370 375 380Thr Gly Tyr Val Lys Ile Arg His
Ser His Ala Leu Val Ser Leu Ser385 390 395 400Phe Leu Lys Asn Leu
Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly 405 410 415Asn Tyr Ser
Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp 420 425 430Asp
Trp Asp His Arg Asn Leu Thr Ile Lys Ala Gly Lys Met Tyr Phe 435 440
445Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met Glu Glu
450 455 460Val Thr Gly Thr Lys Gly Arg Gln Ser Lys Gly Asp Ile Asn
Thr Arg465 470 475 480Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp
Val Leu His Phe Thr 485 490 495Ser Thr Thr Thr Ser Lys Asn Arg Ile
Ile Ile Thr Trp His Arg Tyr 500 505 510Arg Pro Pro Asp Tyr Arg Asp
Leu Ile Ser Phe Thr Val Tyr Tyr Lys 515 520 525Glu Ala Pro Phe Lys
Asn Val Thr Glu Tyr Asp Gly Gln Asp Ala Cys 530 535 540Gly Ser Asn
Ser Trp Asn Met Val Asp Val Asp Leu Pro Pro Asn Lys545 550 555
560Asp Val Glu Pro Gly Ile Leu Leu His Gly Leu Lys Pro Trp Thr Gln
565 570 575Tyr Ala Val Tyr Val Lys Ala Val Thr Leu Thr Met Val Glu
Asn Asp 580 585 590His Ile Arg Gly Ala Lys Ser Glu Ile Leu Tyr Ile
Arg Thr Asn Ala 595 600 605Ser Val Pro Ser Ile Pro Leu Asp Val Leu
Ser Ala Ser Asn Ser Ser 610 615 620Ser Gln Leu Ile Val Lys Trp Asn
Pro Pro Ser Leu Pro Asn Gly Asn625 630 635 640Leu Ser Tyr Tyr Ile
Val Arg Trp Gln Arg Gln Pro Gln Asp Gly Tyr 645 650 655Leu Tyr Arg
His Asn Tyr Cys Ser Lys Asp Lys Ile Pro Ile Arg Lys 660 665 670Tyr
Ala Asp Gly Thr Ile Asp Ile Glu Glu Val Thr Glu Asn Pro Lys 675 680
685Thr Glu Val Cys Gly Gly Glu Lys Gly Pro Cys Cys Ala Cys Pro Lys
690 695 700Thr Glu Ala Glu Lys Gln Ala Glu Lys Glu Glu Ala Glu Tyr
Arg Lys705 710 715 720Val Phe Glu Asn Phe Leu His Asn Ser Ile Phe
Val Pro Arg Pro Glu 725 730 735Arg Lys Arg Arg Asp Val Met Gln Val
Ala Asn Thr Thr Met Ser Ser 740 745 750Arg Ser Arg Asn Thr Thr Ala
Ala Asp Thr Tyr Asn Ile Thr Asp Pro 755 760 765Glu Glu Leu Glu Thr
Glu Tyr Pro Phe Phe Glu Ser Arg Val Asp Asn 770 775 780Lys Glu Arg
Thr Val Ile Ser Asn Leu Arg Pro Phe Thr Leu Tyr Arg785 790 795
800Ile Asp Ile His Ser Cys Asn His Glu Ala Glu Lys Leu Gly Cys Ser
805 810 815Ala Ser Asn Phe Val Phe Ala Arg Thr Met Pro Ala Glu Gly
Ala Asp 820 825 830Asp Ile Pro Gly Pro Val Thr Trp Glu Pro Arg Pro
Glu Asn Ser Ile 835 840 845Phe Leu Lys Trp Pro Glu Pro Glu Asn Pro
Asn Gly Leu Ile Leu Met 850 855 860Tyr Glu Ile Lys Tyr Gly Ser Gln
Val Glu Asp Gln Arg Glu Cys Val865 870 875 880Ser Arg Gln Glu Tyr
Arg Lys Tyr Gly Gly Ala Lys Leu Asn Arg Leu 885 890 895Asn Pro Gly
Asn Tyr Thr Ala Arg Ile Gln Ala Thr Ser Leu Ser Gly 900 905 910Asn
Gly Ser Trp Thr Asp Pro Val Phe Phe Tyr Val Gln Ala Lys Thr 915 920
925Gly Tyr Glu Asn Phe Ile His Leu Ile Ile Ala Leu Pro Val Ala Val
930 935 940Leu Leu Ile Val Gly Gly Leu Val Ile Met Leu Tyr Val Phe
His Arg945 950 955 960Lys Arg Asn Asn Ser Arg Leu Gly Asn Gly Val
Leu Tyr Ala Ser Val 965 970 975Asn Pro Glu Tyr Phe Ser Ala Ala Asp
Val Tyr Val Pro Asp Glu Trp 980 985 990Glu Val Ala Arg Glu Lys Ile
Thr Met Ser Arg Glu Leu Gly Gln Gly 995 1000 1005Ser Phe Gly Met
Val Tyr Glu Gly Val Ala Lys Gly Val Val Lys 1010 1015 1020Asp Glu
Pro Glu Thr Arg Val Ala Ile Lys Thr Val Asn Glu Ala 1025 1030
1035Ala Ser Met Arg Glu Arg Ile Glu Phe Leu Asn Glu Ala Ser Val
1040 1045 1050Met Lys Glu Phe Asn Cys His His Val Val Arg Leu Leu
Gly Val 1055 1060 1065Val Ser Gln Gly Gln Pro Thr Leu Val Ile Met
Glu Leu Met Thr 1070 1075 1080Arg Gly Asp Leu Lys Ser Tyr Leu Arg
Ser Leu Arg Pro Glu Met 1085 1090 1095Glu Asn Asn Pro Val Leu Ala
Pro Pro Ser Leu Ser Lys Met Ile 1100 1105 1110Gln Met Ala Gly Glu
Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala 1115 1120 1125Asn Lys Phe
Val His Arg Asp Leu Ala Ala Arg Asn Cys Met Val 1130 1135 1140Ala
Glu Asp Phe Thr Val Lys Ile Gly Asp Phe Gly Met Thr Arg 1145 1150
1155Asp Ile Tyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Lys Gly Leu
1160 1165 1170Leu Pro Val Arg Trp Met Ser Pro Glu Ser Leu Lys Asp
Gly Val 1175 1180 1185Phe Thr Thr Tyr Ser Asp Val Trp Ser Phe Gly
Val Val Leu Trp 1190 1195 1200Glu Ile Ala Thr Leu Ala Glu Gln Pro
Tyr Gln Gly Leu Ser Asn 1205 1210 1215Glu Gln Val Leu Arg Phe Val
Met Glu Gly Gly Leu Leu Asp Lys 1220 1225 1230Pro Asp Asn Cys Pro
Asp Met Leu Phe Glu Leu Met Arg Met Cys 1235 1240 1245Trp Gln Tyr
Asn Pro Lys Met Arg Pro Ser Phe Leu Glu Ile Ile 1250 1255 1260Ser
Ser Ile Lys Glu Glu Met Glu Pro Gly Phe Arg Glu Val Ser 1265 1270
1275Phe Tyr Tyr Ser Glu Glu Asn Lys Leu Pro Glu Pro Glu Glu Leu
1280 1285 1290Asp Leu Glu Pro Glu Asn Met Glu Ser Val Pro Leu Asp
Pro Ser 1295 1300 1305Ala Ser Ser Ser Ser Leu Pro Leu Pro
Asp Arg His Ser Gly His 1310 1315 1320Lys Ala Glu Asn Gly Pro Gly
Pro Gly Val Leu Val Leu Arg Ala 1325 1330 1335Ser Phe Asp Glu Arg
Gln Pro Tyr Ala His Met Asn Gly Gly Arg 1340 1345 1350Lys Asn Glu
Arg Ala Leu Pro Leu Pro Gln Ser Ser Thr Cys 1355 1360 1365
* * * * *