U.S. patent application number 15/058116 was filed with the patent office on 2016-06-23 for filip1l compositions and methods for treating cancer.
The applicant listed for this patent is The United States of America as Represented by the Secretary of the Department of Health and Human S. Invention is credited to Mijung Kwon, Steven K. Libutti, Anita Tandle.
Application Number | 20160176935 15/058116 |
Document ID | / |
Family ID | 42245592 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160176935 |
Kind Code |
A1 |
Libutti; Steven K. ; et
al. |
June 23, 2016 |
FILIP1L compositions and methods for treating cancer
Abstract
A purified DOC1 polypeptide comprising a fragment of SEQ ID NO:
1 is provided, wherein the DOC1 polypeptide is not the full-length
DOC1 polypeptide sequence. A method of inhibiting angiogenesis in a
subject is provided comprising administering to a subject a nucleic
acid encoding a DOC1 polypeptide, whereby a cell in the subject
produces the DOC1 polypeptide, thus inhibiting angiogenesis. A
method of inhibiting tumor growth in a subject is provided
comprising administering to a subject a nucleic acid encoding a
DOC1 polypeptide, whereby a cell in the subject produces the DOC1
polypeptide, thus inhibiting tumor growth.
Inventors: |
Libutti; Steven K.; (North
Potomac, MD) ; Kwon; Mijung; (Frederick, MD) ;
Tandle; Anita; (Towson, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America as Represented by the Secretary of the
Department of Health and Human S |
Rockville |
MD |
US |
|
|
Family ID: |
42245592 |
Appl. No.: |
15/058116 |
Filed: |
March 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13952192 |
Jul 26, 2013 |
9279009 |
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15058116 |
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12745279 |
Oct 8, 2010 |
8501912 |
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PCT/IB2008/005015 |
Dec 8, 2008 |
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13952192 |
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61005363 |
Dec 3, 2007 |
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Current U.S.
Class: |
514/44R ;
435/254.11; 435/254.2; 435/320.1; 435/325; 435/348; 435/366;
435/419; 435/6.12; 435/7.1; 435/7.92; 436/501; 530/324; 530/350;
530/387.9; 536/23.5 |
Current CPC
Class: |
A61K 35/13 20130101;
C07K 2317/34 20130101; C07K 16/18 20130101; C07K 14/47 20130101;
G01N 33/57484 20130101; G01N 2333/4703 20130101; G01N 33/574
20130101; G01N 2800/52 20130101; C07K 14/4703 20130101; A61K
38/1709 20130101; A61K 48/00 20130101; A61P 35/00 20180101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61K 38/17 20060101 A61K038/17; C07K 16/18 20060101
C07K016/18; G01N 33/574 20060101 G01N033/574 |
Claims
1. A nucleic acid encoding a DOC1 polypeptide comprising a fragment
of SEQ ID NO: 1, wherein the DOC1 polypeptide is not the
full-length DOC1 polypeptide sequence, or encoding a polypeptide
comprising an amino acid sequence that is at least about 95%
identical to the DOC1 polypeptide.
2. A vector comprising the nucleic acid of claim 1.
3. A host cell comprising the vector of claim 2.
4. A method of inhibiting angiogenesis in a subject comprising
administering to a subject a nucleic acid encoding a DOC1
polypeptide, whereby a cell in the subject produces the DOC1
polypeptide, thus inhibiting angiogenesis.
5. The method of claim 4, wherein the nucleic acid is administered
locally.
6. The method of claim 4, wherein the nucleic acid is administered
systemically.
7. The method of claim 4, wherein the nucleic acid is administered
in a vector.
8. The method of claim 4, wherein the nucleic acid encodes SEQ ID
NO: 1.
9. The method of claim 4, wherein the nucleic acid encodes a
fragment of SEQ ID NO: 1.
10. The method of claim 9, wherein the fragment of SEQ ID NO: 1 is
selected from the group consisting of amino acids amino acids 1-790
of SEQ ID NO: 1, amino acids 1-650 of SEQ ID NO: 1, amino acids
1-512 of SEQ ID NO: 1, amino acids 65-893 of SEQ ID NO: 1, amino
acids 127-893 of SEQ ID NO: 1, and amino acids 127-650 of SEQ ID
NO: 1.
11. A method of inhibiting tumor growth in a subject comprising
administering to a subject a nucleic acid encoding a DOC1
polypeptide, whereby a cell in the subject produces the DOC1
polypeptide, thus inhibiting tumor growth.
12. The method of claim 11, wherein the nucleic acid is
administered locally.
13. The method of claim 11, wherein the nucleic acid is
administered systemically.
14. The method of claim 19, wherein the nucleic acid is
administered in a vector.
15. The method of claim 11, wherein the nucleic acid encodes SEQ ID
NO: 1.
16. The method of claim 11, wherein the nucleic acid encodes a
fragment of SEQ ID NO: 1.
17. The method of claim 16, wherein the fragment of SEQ ID NO: 1 is
selected from the group consisting of amino acids 1-790 of SEQ ID
NO: 1, amino acids 1-650 of SEQ ID NO: 1, amino acids 1-512 of SEQ
ID NO: 1, amino acids 65-893 of SEQ ID NO: 1, amino acids 127-893
of SEQ ID NO: 1, and amino acids 127-650 of SEQ ID NO: 1.
18. An antibody that specifically binds to a DOC1 polypeptide
comprising a fragment of SEQ ID NO: 1, wherein the DOC1 polypeptide
is not the full-length DOC1 polypeptide sequence, with the proviso
that the antibody does not specifically bind a fragment consisting
of amino acids 3-52 or amino acids 510-893 of SEQ ID NO:1.
19. The antibody of claim 18, wherein the antibody specifically
binds to a polypeptide selected from the group consisting of amino
acids amino acids 1-790 of SEQ ID NO: 1, amino acids 1-650 of SEQ
ID NO: 1, amino acids 1-512 of SEQ ID NO: 1, amino acids 65-893 of
SEQ ID NO: 1, amino acids 127-893 of SEQ ID NO: 1, and amino acids
127-650 of SEQ ID NO: 1.
20. A method for classifying a cancer in a subject comprising: a)
contacting a test sample from a subject with an anti-DOC1 antibody;
b) detecting the binding of the anti-DOC1 antibody to a DOC1
polypeptide in the sample; c) comparing the expression level of the
DOC1 polypeptide in the test sample with the expression level of a
DOC1 polypeptide in a reference sample for which a cancer
classification is known; and d) identifying a difference, if
present, in the expression level of the DOC1 polypeptide in the
test sample and reference sample, thereby classifying the cancer in
the subject.
21. The method of claim 20, wherein a difference in the expression
level of the DOC1 polypeptide in the test sample as compared to the
reference sample indicates that the test sample has a different
classification as the reference sample.
22. The method of claim 20, wherein a similar expression level of
the DOC1 polypeptide in the sample as compared to the reference
sample indicates that the test sample has the same classification
as the reference sample.
23. The method of claim 20, wherein the reference sample is a
sample from a subject with a known classification.
24. The method of claim 20, wherein the reference sample is from a
database.
25. The method of claim 20, wherein the reference sample is a
reference sample classified as a normal sample.
26. The method of claim 20, wherein the reference sample is a
reference sample classified as cancerous.
27. A method for identifying a stage of cancer in a subject
comprising: a) contacting a test sample from a subject with an
anti-DOC1 antibody; b) detecting the binding of the anti-DOC1
antibody to a DOC1 polypeptide in the sample; c) comparing the
expression level of the DOC1 polypeptide in the test sample with
the expression level of a DOC1 polypeptide in a reference sample
for which a stage of cancer is known; and d) identifying a
difference, if present, in the expression level of the DOC1
polypeptide in the test sample and reference sample, thereby
identifying the stage of cancer in the subject.
28. The method of claim 27, wherein a difference in the expression
level of the DOC1 polypeptide in the test sample as compared to the
reference sample indicates that the test sample has a different
stage of cancer than the reference sample.
29. The method of claim 27, wherein a similar expression level of
the DOC1 polypeptide in the test sample as compared to the
reference sample indicates that the test sample has the same stage
of cancer as the reference sample.
30. The method of claim 27, wherein the reference sample is a
sample from a patient with a known stage of cancer.
31. The method of claim 27, wherein the reference sample is from a
database.
32. A method of determining the efficacy of an anti-cancer
treatment comprising: a) determining the expression level of a DOC1
polypeptide in a sample obtained from the subject; b) administering
the anti-cancer treatment; c) determining the expression level of a
DOC1 polypeptide in a sample obtained from the subject after
administration of the anti-cancer treatment; and d) comparing the
expression level of the DOC1 polypeptide in the sample obtained
after the anti-cancer treatment with the sample obtained in step a)
such that if there is a change in the expression level of the DOC1
polypeptide in the sample obtained after the anti-cancer treatment
as compared to the sample obtained before administration of the
anti-cancer treatment, wherein the change is associated with an
improvement in the subject, the anti-cancer therapeutic is an
effective anti-cancer therapeutic.
33. The method of claim 32, wherein the change is an increase in
the expression levels of the DOC1 polypeptide.
34. The method of claim 32, wherein the change is a decrease in the
expression levels of the DOC1 polypeptide.
35. A method of identifying a modulator of DOC1 expression
comprising: a) contacting a cell that is capable of expressing DOC1
with a putative modulator; b) measuring DOC1 expression, wherein a
change in DOC1 expression in the cell of step a) as compared to a
cell that was not contacted with the putative modulator indicates
the presence of a modulator of DOC1 expression.
36. The method of claim 35, wherein the change in DOC1 expression
is an increase in DOC1 expression.
37. The method of claim 35, wherein the change in DOC1 expression
is a decrease in DOC1 expression.
38. The method of claim 35, wherein DOC1 expression is measured by
amplifying a DOC1 nucleic acid.
39. The method of claim 35, wherein DOC1 expression is measured by
detecting a DOC1 polypeptide.
40. A purified DOC1 polypeptide comprising a fragment of SEQ ID NO:
2, wherein the DOC1 polypeptide is not the full-length DOC1
polypeptide sequence, and the fragment is at least 51 amino acids
in length.
41. A purified DOC1 polypeptide comprising a fragment of SEQ ID NO:
3, wherein the DOC1 polypeptide is not the full-length DOC1
polypeptide sequence, and the fragment is at least 51 amino acids
in length.
42. A purified DOC1 polypeptide comprising a fragment of SEQ ID NO:
1, wherein the DOC1 polypeptide is not the full-length DOC1
polypeptide sequence, and the fragment comprises amino acids
127-512 of SEQ ID NO:1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/952,192, filed Jul. 26, 2013, which is a divisional of
U.S. patent application Ser. No. 12/745,279 filed Oct. 8, 2010
which is the National Stage of International Application No.:
PCT/IB2008/005015, filed Dec. 8, 2008, which claims the benefit of
U.S. Provisional Application No. 61/005,363, filed Dec. 3, 2007.
The aforementioned applications are incorporated herein by
reference in their entireties for all purposes.
[0002] This application includes a Sequence Listing as a text file
named "1003345-SEQLIST.txt" created Feb. 23, 2016, and containing
48,067 bytes. The material contained in this text file is
incorporated by reference in its entirety for all purposes.
BACKGROUND
[0003] Angiogenesis, the process of the formation of new blood
vessels from pre-existing capillaries, is required for the
sustained growth, invasion and spread of tumors. Thus, the
inhibition of tumor angiogenesis has been considered to be one of
the important targets in anticancer therapy. Utilizing cDNA
microarray analysis, it has been demonstrated that the expression
of genes such as DOC1, KLF4 and TC-1 was rapidly modulated by the
angiogenesis inhibitors, endostatin and fumagillin. In addition,
DOC1 (downregulated in ovarian cancer 1; also known as FILIP1L
(filamin A interacting protein 1-like)) was shown to be an upstream
regulator of KLF4 and TC-1 following endostatin treatment (Mazzanti
C M et al. Genome Res (2004) 14:1585). Expression of DOC1 was
rapidly regulated by another angiogenesis inhibitor EMAP II (Tandle
A T et al. Cytokine (2005) 30:347). DOC1 mRNA expression has been
shown to be consistently absent in ovarian carcinoma cells (Mok S C
et al. Gynecol Oncol (1994) 52:247). In addition, it has been shown
to be induced in senescent human prostate epithelial cells, but
significantly repressed in immortalized prostate epithelial cells
(Schwarze S R et al. J Biol Chem (2002) 277:14877; and Schwarze S R
et al. Neoplasia (2005) 7:816). Furthermore, DOC1 mRNA expression
was shown to be downregulated in microvascular endothelial cells
infected with Kaposi's sarcoma-associated herpesvirus as well as
during B-cell transformation (Poole L I et al. J Virol (2002)
76:3395 7; and Klener P et al. J Virol (2006) 80:1922). The
function of DOC1, however, is completely unknown.
SUMMARY
[0004] In accordance with the purpose of this invention, as
embodied and broadly described herein, this invention relates to
compositions and methods for treating cancer using DOC1 (FILIP1L)
and DOC1 polypeptides.
[0005] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0007] FIG. 1A shows that overexpression of FILIP1L in endothelial
cells leads to inhibition of cell proliferation. Inhibition of cell
proliferation by overexpression of FILIP1L in HUVECs (human
umbilical vein endothelial cells) was analyzed by BrdU ELISA 24 h
after transfection. Error bars indicate SEM (n=4, P<0.0001). The
result is representative of three independent experiments.
[0008] FIG. 1B shows that overexpression of FILIP1L in endothelial
cells leads to an increase in apoptosis. Increased apoptosis by
overexpression of FILIP1L in HUVECs was analyzed by annexin V-FITC
and 7-AAD staining followed by flow cytometry analysis 48 h after
transfection. The numbers 15.2 for control and 44.4 for FILIP1L
indicate the percentage of cells in late apoptosis. The result is a
representative of two independent experiments.
[0009] FIG. 2A shows that FILIP1L truncation mutants have
differential activity in mediating anti-proliferative activity.
Differential inhibition of HUVECs proliferation by FILIP1L
truncation mutants was analyzed by BrdU ELISA 24 h after
transfection. Error bars indicate SEM (n=4). FILIP1L truncation
mutants 1-790 (P=0.0001), 1-650 (P=0.004), 1-512 (P=0.0114) and
127-893 (P=0.0021) significantly inhibited cell proliferation
compared to control. C-terminal mutant 1-790 was more potent than
wild-type FILIP1L in mediating anti-proliferative activity
(P=0.001). The result is a representative of two independent
experiments.
[0010] FIG. 2B shows that FILIP1L N-terminal truncation mutants
have differential activity in mediating anti-proliferative
activity. FILIP1L N-terminal truncation mutants comprising amino
acids 1-893, 65-893, 127-893, 190-893, 250-893, and 310-893 were
tested. Experimental conditions were as in FIG. 2A.
[0011] FIG. 2C shows that FILIP1L C-terminal truncation mutants
have differential activity in mediating anti-proliferative activity
C-terminal (C) mutants were also tested. FILIP1L N-terminal
truncation mutants comprising amino acids 127-893, 127-790,
127-720, 127-650, 127-580, 127-512, 127-442, and 127-369 were
tested. Experimental conditions were as in FIG. 2A.
[0012] FIG. 3 shows that overexpression of FILIP1L.DELTA.C103 in
HUVECs leads to inhibition of cell migration.
FILIP1L.DELTA.C103-transfected HUVECs (dark line) showed a
significantly slower migration rate than control vector-transfected
HUVECs (light line) as measured by Electric Cell-Substrate
Impedance Sensing System in real time (P<0.0001). The square box
within the drawing indicates the linear range in the curve that was
used for analysis. The result is representative of three
independent experiments.
[0013] FIG. 4 shows that FILIP1L protein is present in human
endothelial cells. A 110 kDa FILIP1L protein was detected in the
cytoplasm, membrane and nucleus of HUVECs by Western blot using
FILIP1L antibody. A purified FILIP1L protein was used as a
standard.
[0014] FIG. 5 shows targeted expression of FILIP1L C-terminal
mutant 1-790 in tumor vasculature results in inhibition of tumor
growth in vivo. Tumors from RGD-4C-FILIP1L (1-790) AAVP-treated
mice (empty circles) were significantly smaller than those from
PBS-treated mice (empty triangles) in M21 xenograft model by day 14
(P=0.0062). Tumors from RGD-4C-FILIP1L (1-790) AAVP-treated mice
(empty circles) were also significantly smaller than those from
RGD-4C AAVP-treated mice (filled triangles; P=0.0344) and
RGD-4C-FILIP1L (1-650) AAVP-treated mice (filled circles; P=0.0053)
by day 14. Error bars indicate SEM (n=11). The result is a
representative of two independent experiments.
[0015] FIG. 6A shows the upregulation of FILIP1L protein by
endostatin A, Increased expression of FILIP1L protein was detected
in HUVECs treated with endostatin for 2, 4 and 8 h by western blot
using anti-FILIP1L antibody. GAPDH blot is shown as the loading
control. The numbers underneath the blot are the densitometric
values calculated as FILIP1L-GAPDH ratios using ImageQuant
software. The result is representative of two independent
experiments.
[0016] FIG. 6B shows the upregulation of FILIP1L protein by
endostatin. FILIP1L expression in endostatin-treated HUVECs was
significantly more than that in vehicle-treated control cells
(P=0.0012). Five images from each treatment group were analyzed.
Axiovision 4.6 software (Zeiss) was used to quantify the percent
area with FILIP1L-positive staining. Box & whiskers plot
(GraphPad Prism 3.0) is shown. Experimental conditions are as in
FIG. 6A.
[0017] FIG. 7 shows that FILIP1L truncation mutants have
differential antiproliferative activity. Expression of FILIP1L
mutants was confirmed in HEK293 cells transfected with constructs
expressing wild type 1-893, 1-790, 1-650, 1-512, 1-369, 127-893,
369-893, and 512-893 each construct by western blot analysis using
anti-HA tag antibody. A GAPDH blot is shown as the loading
control.
[0018] FIG. 8A shows that overexpression of FILIP1L.DELTA.C103 in
DU145 prostate cancer cells leads to inhibition of cell migration.
Real time RT-PCR analysis for FILIP1L on cDNA from DU145 clones
transduced with FILIP1L.DELTA.C103-lentivirus. Each clone was
treated with either PBS (w/o Dox) or 1 .mu.g/mL doxycycline (w/Dox)
for 48 h prior to harvest. The y-axis represents a ratio between
each clone and the parental Tet repressor-expressing DU145 cells
where each value was standardized with housekeeping gene GAPDH.
Each bar is an average of three experiments.
[0019] FIG. 8B shows that overexpression of FILIP1L.DELTA.C103 in
DU145 prostate cancer cells leads to inhibition of cell migration.
A 90 kDa FILIP1L.DELTA.C103 protein was detected in Tet
repressor-expressing DU145 cells transduced with
FILIP1L.DELTA.C103-lentivirus, but not control lentivirus, by
western blot using anti-FILIP1L antibody. The result shown is a
representative (clone #12) from several clones. GAPDH blot is shown
as the loading control.
[0020] FIG. 8C shows that overexpression of FILIP1L.DELTA.C103 in
DU145 prostate cancer cells leads to inhibition of cell migration.
All three FILIP1L.DELTA.C103 clones, but not mixed population of
control cells, showed a significantly slower migration in the
presence of doxycycline. Error bars indicate SEM (n=3). P value
comparison between in the presence and in the absence of
doxycycline were: control cells (P=0.141), clone #2 (P=0.0014),
clone #12 (P=0.0005) and clone #13 (P<0.0001). The result is
representative of two independent experiments.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0021] The disclosed method and compositions may be understood more
readily by reference to the following detailed description of
particular embodiments and the Example included therein and to the
Figures and their previous and following description.
[0022] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed method and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutation of these compounds may not be explicitly disclosed,
each is specifically contemplated and described herein. For
example, if a DOC1 polypeptide or nucleic acid (e.g., DOC1
wild-type, variant 2, isoform 1 or isoform 4) is disclosed and
discussed and a number of modifications that can be made to a
number of molecules including the DOC1 polypeptide or nucleic acid
(e.g., DOC1 wild-type, variant 2, isoform 1 or isoform 4) are
discussed, each and every combination and permutation of DOC1
polypeptide or nucleic acid (e.g., DOC1 wild-type, variant 2,
isoform 1 or isoform 4) and the modifications that are possible are
specifically contemplated unless specifically indicated to the
contrary. Thus, if a class of molecules A, B, and C are disclosed
as well as a class of molecules D, E, and F and an example of a
combination molecule, A-D is disclosed, then even if each is not
individually recited, each is individually and collectively
contemplated. Thus, is this example, each of the combinations A-E,
A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated
and should be considered disclosed from disclosure of A, B, and C;
D, E, and F; and the example combination A-D. Likewise, any subset
or combination of these is also specifically contemplated and
disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E
are specifically contemplated and should be considered disclosed
from disclosure of A, B, and C; D, E, and F; and the example
combination A-D. This concept applies to all aspects of this
application including, but not limited to, steps in methods of
making and using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed it is understood
that each of these additional steps can be performed with any
specific embodiment or combination of embodiments of the disclosed
methods, and that each such combination is specifically
contemplated and should be considered disclosed.
[0023] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
[0024] It is understood that the disclosed method and compositions
are not limited to the particular methodology, protocols, and
reagents described as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
DEFINITIONS
[0025] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present method and compositions, the particularly useful
methods, devices, and materials are as described. Publications
cited herein and the material for which they are cited are hereby
specifically incorporated by reference. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such disclosure by virtue of prior invention.
No admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and
applicants reserve the right to challenge the accuracy and
pertinence of the cited documents.
[0026] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells;
reference to "the DOC1 polypeptide" is a reference to one or more
DOC1 polypeptides and equivalents thereof known to those skilled in
the art, and so forth.
[0027] "Optional" or "optionally" means that the subsequently
described event, circumstance, or material may or may not occur or
be present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0028] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0029] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
[0030] The term "preventing" as used herein refers to administering
a compound prior to the onset of clinical symptoms of a disease or
conditions so as to prevent a physical manifestation of aberrations
associated with the disease or condition.
[0031] The term "treating" as used herein refers to administering a
compound after the onset of clinical symptoms. Treating can include
a partial improvement in symptoms (e.g., a reduction in tumor size
or in the number of tumors or a slowing of tumor growth), or may be
a complete cessation of symptoms (e.g., eradication of any
cancer).
[0032] The term "in need of treatment" as used herein refers to a
judgment made by a caregiver (e.g. physician, nurse, nurse
practitioner, or individual in the case of humans; veterinarian in
the case of animals, including non-human mammals) that an
individual or animal requires or will benefit from treatment. This
judgment is made based on a variety of factors that are in the
realm of a care giver's expertise, but that include the knowledge
that the individual or animal is ill, or will be ill, as the result
of a condition that is treatable by the compounds of the invention.
A similar judgment may be mad by a caregiver to determine if a
subject (individual) is "in need of prevention."
[0033] The terms "individual" and "subject" as used herein refer to
a mammal, including animals, preferably mice, rats, other rodents,
rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,
particularly humans.
[0034] The terms "higher," "increases," "elevates," or "elevation"
refer to increases above basal levels, e.g., as compared to a
control. The terms "low," "lower," "reduces," or "reduction" refer
to decreases below basal levels, e.g., as compared to a
control.
[0035] "Anti-angiogenic" as used herein means either the prevention
of or a reduction in the growth of new blood vessels, or a
reduction in existing vessels, e.g., via necrosis, or both.
Compositions
[0036] Functional DOC1 Polypeptides
[0037] Provided is a purified DOC1 (also referred to as FILIP1L)
polypeptide comprising a fragment of SEQ ID NO:1, wherein the DOC1
polypeptide is not the full-length DOC1 protein disclosed in that
sequence. SEQ ID NO:1 is the wild-type DOC1 protein sequence also
known as isoform 2, and found under GenBank Accession No.
NP_055705.2. Provided is a purified DOC1 (also referred to as
FILIP1L) polypeptide comprising a fragment of SEQ ID NO:2 (also
known as variant 2, also formerly found under GenBank Accession No.
NP_055705) wherein the DOC1 polypeptide is not the full-length DOC1
protein disclosed in that sequence. Provided is a purified DOC1
(also referred to as FILIP1L) polypeptide comprising a fragment of
SEQ ID NO:3 (also known as isoform 1, and found under GenBank
Accession No. NP_878913.2) wherein the DOC1 polypeptide is not the
full-length DOC1 protein disclosed in that sequence. Provided is a
purified DOC1 (also referred to as FILIP1L) polypeptide comprising
a fragment of SEQ ID NO:4 (also known as isoform 3, and found under
GenBank Accession No. NP_001035924.1) wherein the DOC1 polypeptide
is not the full-length DOC1 protein disclosed in that sequence.
Thus, the FILIP1L protein referred to herein can be the protein
found under Accession Nos. NP_055705.2, NP_001035924.1,
NP_878913.2, and NP_055705. The FILIP1L polypeptide sequences,
nucleic acid sequences encoding FILIP1L, and the additional
information set forth under GenBank Accession Nos. NP_055705.2,
NP_001035924.1, NP_878913.2, and NP_055705 are hereby incorporated
by reference.
[0038] Provided is a purified DOC1 (also referred to as FILIP1L)
polypeptide comprising a fragment of SEQ ID NO:1, wherein the DOC1
polypeptide is not the full-length DOC1 protein disclosed in that
sequence, and the fragment is at least 400 amino acids in length.
Provided is a purified DOC1 (also referred to as FILIP1L)
polypeptide comprising a fragment of SEQ ID NO:1, wherein the DOC1
polypeptide is not the full-length DOC1 protein disclosed in that
sequence, with the proviso that the fragment does not consist of
amino acids 3-32, 3-52, 43-52, or 510-893 of SEQ ID NO:1. A
purified DOC1 polypeptide comprising a fragment of SEQ ID NO: 2,
wherein the DOC1 polypeptide is not the full-length DOC1
polypeptide sequence, and the fragment is at least 51 amino acids
in length. Also provided is a purified DOC1 polypeptide comprising
a fragment of SEQ ID NO: 3, wherein the DOC1 polypeptide is not the
full-length DOC1 polypeptide sequence, and the fragment is at least
51 amino acids in length. Also provided is a purified DOC1
polypeptide comprising a fragment of SEQ ID NO: 4, wherein the DOC1
polypeptide is not the full-length DOC1 polypeptide sequence, and
the fragment is at least 400 amino acids in length. Also provided
is a purified DOC1 polypeptide comprising a fragment of SEQ ID NO:
1, wherein the DOC1 polypeptide is not the full-length DOC1
polypeptide sequence, and the fragment comprises amino acids
127-512 of SEQ ID NO:1.
[0039] The disclosed DOC1 polypeptide can comprise at least 10
contiguous amino acids of DOC1 (e.g., DOC1 of SEQ ID NO:1, DOC1 of
SEQ ID NO:2, DOC1 of SEQ ID NO:3 or DOC1 of SEQ ID NO:4) and can
range up to 751 contiguous amino acids (for fragments on SEQ ID
NO:2), 892 contiguous amino acids (for fragments on SEQ ID NO:1),
1132 contiguous amino acids (for fragments on SEQ ID NO:4) or 1134
contiguous amino acids (for fragments on SEQ ID NO:3) of DOC1,
including every number of amino acids in between. For example, the
fragment of DOC1 can be up to 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100; 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400; 20, 40, 60, 80,
100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340,
360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600,
620, 640, 660, 680, 700, 710, 720, 730, 740, 750, 760, 770, 780,
790, 800, 810, 820, 839, 840, 850, 860, 870, 880, 890, 900, 910,
920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030,
1040, 1050, 1060, 1080, 1090, 1100, 1110, 1120 or 1130 amino acids
in length. A fragment of a reference protein/polypeptide has a
sequence that is identical to a region of the reference protein. As
used herein, a fragment of DOC1 is a subpart of DOC1. Thus, it does
not include any variation in the amino acid sequence of DOC1 (e.g.,
SEQ ID NO:1), but is only shorter than the native DOC1
polypeptide.
[0040] In one aspect, the DOC1 polypeptide has one or more
functions of full-length DOC1, that is, it is a functional fragment
of DOC1.
[0041] For example, the DOC1 polypeptide can have the
anti-angiogenic function of native DOC1. Angiogenesis is a process
where blood vessels are formed from existing blood vessels. This
involves proliferation, differentiation and migration of
endothelial cells and possibly other cells found in the
vasculature, such as smooth muscle cells and fibroblasts.
Anti-angiogenic function also includes a reduction in existing
vessels, for example, via necrosis of existing vessels. Alteration
of this process, either its potentiation or its inhibition, can
have benefits be beneficial for the therapy or treatment of human
diseases, such as cancer, macular degeneration, rheumatoid
arthritis, Alzheimer's disease, wound healing, atherosclerosis and
ischemia.
[0042] Alternatively or in addition the DOC1 polypeptide can have
the apoptotic activity of native DOC1.
[0043] Alternatively or in addition the DOC1 polypeptide can have
anti-tumor (anti-proliferative) activity of native DOC1.
[0044] Alternatively or in addition the DOC1 polypeptide can have
the anti-migration activity of native DOC1 (see, e.g., FIG. 3 and
FIG. 5).
[0045] A DOC1 polypeptide of the invention that retains at least
50% of the level of at least one function of the native DOC1 is
considered to be functional. The DOC1 polypeptide can exhibit a
level of activity that is 60%, 70%, 80%, 90%, 100% or greater than
the level of the same activity of the full-length DOC1.
[0046] For example, disclosed is a purified DOC1 polypeptide, which
is a fragment of SEQ ID NO: 1 comprising amino acids selected from
the group consisting of amino acids amino acids 1-790 of SEQ ID NO:
1, amino acids 1-650 of SEQ ID NO: 1, amino acids 1-512 of SEQ ID
NO: 1, amino acids 65-893 of SEQ ID NO: 1, amino acids 127-512 of
SEQ ID NO:1, amino acids 127-893 of SEQ ID NO: 1, and amino acids
127-650 of SEQ ID NO: 1, and amino acids 127-790 of SEQ ID NO:1.
More specifically, the DOC1 polypeptide having anti-proliferative
activity can be selected from the group consisting of amino acids
1-790 of SEQ ID NO:1, amino acids 1-650 of SEQ ID NO:1, amino acids
1-512 of SEQ ID NO:1, and amino acids 127-893 of SEQ ID NO:1.
[0047] Also disclosed is a purified DOC1 polypeptide, which is a
fragment of SEQ ID NO: 2 comprising amino acids selected from the
group consisting of amino acids amino acids 1-746 of SEQ ID NO: 2,
amino acids 1-752 of SEQ ID NO: 2, amino acids 1-650 of SEQ ID NO:
2, amino acids 1-512 of SEQ ID NO:2, amino acids 65-752 of SEQ ID
NO:2, amino acids 127-512 of SEQ ID NO:2, amino acids 127-752 of
SEQ ID NO:2, and amino acids 127-650 of SEQ ID NO:2, and amino
acids 127-752 of SEQ ID NO:2. More specifically, the DOC1
polypeptide having anti-proliferative activity can be selected from
the group consisting of amino acids 1-746 of SEQ ID NO: 2, amino
acids 1-752 of SEQ ID NO:2, amino acids 1-650 of SEQ ID NO:2, amino
acids 1-512 of SEQ ID NO:2, and amino acids 127-752 of SEQ ID NO:2.
Also disclosed is a purified DOC1 polypeptide, which is a fragment
of SEQ ID NO: 3 comprising amino acids selected from the group
consisting of amino acids amino acids 241-1128 of SEQ ID NO:3.
[0048] Also disclosed is a purified DOC1 polypeptide, which is a
fragment of SEQ ID NO: 4 comprising amino acids selected from the
group consisting of amino acids amino acids 241-1133 of SEQ ID
NO:4.
[0049] A coiled-coil region (residues 3-542 of SEQ ID NO:1), two
leucine zipper motifs (residues 83-111 and 218-253 of SEQ ID NO:1)
and a prefoldin domain (residues 465-535 of SEQ ID NO:1) could be
recognized in N-terminal half of DOC1 protein. In addition, NCBI
conserved domain search reveals that DOC1 has a SbcC (COG0419;
ATPase involved in DNA repair; residues 19-576) conserved domain in
its N-terminal half and a Herpes_BLLF1 (pfam05109; Herpes virus
major outer envelope glycoprotein; residues 640-829) conserved
domain in its C-terminal half. A DOC1 polypeptide that retains the
coiled-coil region (3-542 of SEQ ID NO:1) is expected to retain
function.
[0050] Functional DOC1 Variants
[0051] Provided is a purified polypeptide comprising an amino acid
sequence that is a functional variant of DOC1 or a functional
variant of a fragment of DOC1. A functional variant of DOC1 or a
functional variant of a DOC1 polypeptide possess at least one
function of full-length DOC1 (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3 or SEQ ID NO:4). Examples of functions of DOC1 or DOC1
polypeptide are recited herein. In general, variants of the DOC1
proteins disclosed herein typically have at least, about 70, 71,
72, 73, 74, 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 percent similarity to
the stated sequence or the native sequence. For example, the
variant polypeptide can be at least about 95% identical to the
amino acid sequence of SEQ ID NO:1 or functional fragment of SEQ ID
NO:1 as described herein. For example, the variant polypeptide can
be at least about 96% identical to the amino acid sequence of SEQ
ID NO:1 or functional fragment of SEQ ID NO:1 as described herein.
For example, the variant polypeptide can be at least about 97%
identical to the amino acid sequence of SEQ ID NO:1 or functional
fragment of SEQ ID NO:1 as described herein. For example, the
variant polypeptide can be at least about 98% identical to the
amino acid sequence of SEQ ID NO:1 or functional fragment of SEQ ID
NO:1 as described herein. For example, the variant polypeptide can
be at least about 99% identical to the amino acid sequence of SEQ
ID NO:1 or functional fragment of SEQ ID NO:1 as described
herein.
[0052] In a further example, the variant polypeptide can be at
least about 95% identical to the amino acid sequence of SEQ ID NO:2
or functional fragment of SEQ ID NO:2 as described herein. For
example, the variant polypeptide can be at least about 96%
identical to the amino acid sequence of SEQ ID NO:2 or functional
fragment of SEQ ID NO:2 as described herein. For example, the
variant polypeptide can be at least about 97% identical to the
amino acid sequence of SEQ ID NO:2 or functional fragment of SEQ ID
NO:2 as described herein. For example, the variant polypeptide can
be at least about 98% identical to the amino acid sequence of SEQ
ID NO:2 or functional fragment of SEQ ID NO:2 as described herein.
For example, the variant polypeptide can be at least about 99%
identical to the amino acid sequence of SEQ ID NO:2 or functional
fragment of SEQ ID NO:2 as described herein.
[0053] In a further example, the variant polypeptide can be at
least about 95% identical to the amino acid sequence of SEQ ID NO:3
or functional fragment of SEQ ID NO:3 as described herein. For
example, the variant polypeptide can be at least about 96%
identical to the amino acid sequence of SEQ ID NO:3 or functional
fragment of SEQ ID NO:3 as described herein. For example, the
variant polypeptide can be at least about 97% identical to the
amino acid sequence of SEQ ID NO:3 or functional fragment of SEQ ID
NO:3 as described herein. For example, the variant polypeptide can
be at least about 98% identical to the amino acid sequence of SEQ
ID NO:3 or functional fragment of SEQ ID NO:3 as described herein.
For example, the variant polypeptide can be at least about 99%
identical to the amino acid sequence of SEQ ID NO:3 or functional
fragment of SEQ ID NO:3 as described herein.
[0054] In a further example, the variant polypeptide can be at
least about 95% identical to the amino acid sequence of SEQ ID NO:4
or functional fragment of SEQ ID NO:4 as described herein. For
example, the variant polypeptide can be at least about 96%
identical to the amino acid sequence of SEQ ID NO:4 or functional
fragment of SEQ ID NO:4 as described herein. For example, the
variant polypeptide can be at least about 97% identical to the
amino acid sequence of SEQ ID NO:4 or functional fragment of SEQ ID
NO:4 as described herein. For example, the variant polypeptide can
be at least about 98% identical to the amino acid sequence of SEQ
ID NO:4 or functional fragment of SEQ ID NO:4 as described herein.
For example, the variant polypeptide can be at least about 99%
identical to the amino acid sequence of SEQ ID NO:4 or functional
fragment of SEQ ID NO:4 as described herein.
[0055] Those of skill in the art readily understand how to
determine the sequence similarity of two proteins or nucleic acids.
For example, the homology can be calculated after aligning the two
sequences so that the homology is at its highest level. Another way
of calculating homology can be performed by published algorithms.
Optimal alignment of sequences for comparison may be conducted by
the local homology algorithm of Smith and Waterman Adv. Appl. Math.
2: 482 (1981), by the homology alignment algorithm of Needleman and
Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity
method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:
2444 (1988), by computerized implementations of these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by inspection. The same types of homology can be obtained for
nucleic acids by for example the algorithms disclosed in Zuker, M.
Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment.
[0056] Provided is a purified polypeptide comprising a fragment of
SEQ ID NO:1, wherein the polypeptide has the formula
R.sub.1-fragment of SEQ ID NO:1-R.sub.2, wherein R.sub.1 or R.sub.2
comprise, H, acyl, or an amino acid sequence that does not flank
the fragment of SEQ ID NO:1 in any naturally occurring DOC1
polypeptide. For example, the following list is exemplary of the
DOC1 polypeptides of the invention:
[0057] R.sub.1-amino acids 1-790 of SEQ ID NO:1-R.sub.2;
[0058] R.sub.1-amino acids 1-650 of SEQ ID NO:1-R.sub.2;
[0059] R.sub.1-amino acids 1-512 of SEQ ID NO:1-R.sub.2;
[0060] R.sub.1-amino acids 127-512 of SEQ ID NO:1-R.sub.2;
[0061] R.sub.1-amino acids 127-893 of SEQ ID NO:1-R.sub.2;
[0062] R.sub.1-amino acids 127-650 of SEQ ID NO:1-R.sub.2; and
[0063] R.sub.1-amino acids 127-790 of SEQ ID NO:1-R.sub.2.
[0064] Fusion Protein
[0065] For example, the polypeptide having the general formula
R.sub.1-fragment of SEQ ID NO:1-R.sub.2 can be a fusion protein.
Fusion proteins can include targeting sequences. The targeting
molecule can be an RGD, NGR, GFP or any targeting sequence. Any
molecule that can target a specific tissue can be used as the
targeting molecule of the present fusion protein. For example, the
targeting molecule can be a molecule (e.g., an antibody or aptamer)
that interacts with human epithelial cell mucin (Muc-1; a 20 amino
acid core repeat for Muc-1 glycoprotein, present on breast cancer
cells and pancreatic cancer cells), the Ha-ras oncogene product,
p53, carcino-embryonic antigen (CEA), the raf oncogene product,
gp100/pmel 17, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, BAGE, GAGE,
tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 &
2, HPV-F4, 6, 7, prostate-specific antigen (PSA), HPV-16, MUM,
alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras
oncogene product, HPV E7, Wilm's tumor antigen-1, telomerase,
melanoma gangliosides, or a simple transmembrane sequence.
[0066] Selective delivery of therapeutic agents to cancer cells in
a living body is another area of research where targeting of cancer
specific biomarkers is intensively studied. (E. Mastrobattista, G.
A. Koning, and G. Storm, "Immonoliposomes for the Targeted Delivery
of Antitumor Drugs," Adv Drug Delivery Reviews 1999, 40: 103-27; J.
Sudimack and R. J. Lee, "Targeted Drug Delivery Via Folate
Receptor," Adv Drug Delivery Reviews 2000, 41: 147-62; S. P. Vyas
and V. Sihorkar, "Endogenous Carriers and Ligands in
Non-Immunogenic Site-Specific Drug Delivery," Adv Drug Delivery
Reviews 2000, 43: 101-64.). Immunoliposome-mediated targeting using
monoclonal antibodies to folate receptor, (E. Mastrobattista, G. A.
Koning, and G. Storm, "Immonoliposomes for the Targeted Delivery of
Antitumor Drugs," Adv Drug Delivery Reviews 1999, 40: 103-27; J.
Sudimack and R. J. Lee, "Targeted Drug Delivery Via Folate
Receptor," Adv Drug Delivery Reviews 2000, 41: 147-62) CA-125, (E.
Mastrobattista, G. A. Koning, and G. Storm, "Immonoliposomes for
the Targeted Delivery of Antitumor Drugs," Adv Drug Delivery
Reviews 1999, 40: 103-27) and HER2/neu antigen (D. B. Kirpotin, J.
W. Park, K. Hong, S. Zalipsky, W. L. Li, P. Carter, C. C. Benz, and
D. Papahadjopoulos, "Sterically Stabilized anti-HER2
immunoliposomes: design and targeting to human breast cancer cells
in vitro," Biochemistry 1997, 36: 66-75) have been described.
[0067] The targeting molecule of the instant invention is an
optional feature that presents an extra measure of selectivity. The
targeting molecule directs the fusion protein to the target cell,
where the DOC1 polypeptide can cause apoptosis or inhibit
proliferation or both. The targeting molecule may be added to the
N- or C-terminus, or both.
[0068] In some embodiments, the targeting molecule is an antibody.
For example, the antibody can be an anti-fibronectin ED-B antibody,
thereby directing the DOC1 fragment to the extracellular matrix
associated with neo-vessel formation.
[0069] Targeting molecules that target the neo-vasculature can also
be linked to the DOC1 fragment. Molecules that target the
neo-vasculature can easily be identified by screening phage display
libraries. Any such peptide would be a suitable targeting molecule
of the present invention.
[0070] Targeting molecules which bind specifically to integrins are
one class of signal sequences that can be found on cells of the
vasculature. These peptides bear the signal sequence based on
Arg-Gly-Asp (RGD). Accordingly, sequences that bind certain
integrins can serve as useful targeting molecules to endothelial
cells and other cells of the neo-vasculature. For example, the
targeting molecule can be an RGD targeting sequence.
[0071] Non-structural spacers may be a feature of the targeting
molecule. Such spacers typically comprise glycine and/or proline
residues. Lengths of these spacers can range from about one to
about 5 amino acids. In addition, it is often preferable to
physically constrain the targeting molecule by cyclization, which
usually results in increased binding. This is usually accomplished
by a pair of cysteine residues, flanking the RGD core at a distance
of about 4 (having only RGD in between) to 10 amino acids from one
another. For example, the pair of cysteine residues, flanking the
RGD core can be at a distance of 7 amino acids from one
another.
[0072] Thus, a typical targeting molecule would have the following
structure: -XRGDYX- wherein X is zero to five amino acids and Y is
a one or two amino acids, selected from cysteine, serine, threonine
and methionine. In a particularly useful embodiment, X is comprised
of glycine residues, but optionally contains at least one, and
typically one or two, free thiol- or amine-containing amino acids
and/or a single hydrophobic amino acid. Thiol-containing residues
include methionine and cysteine; amine-containing residues include
lysine and (at least one additional) arginine; and hydrophobic
residues include leucine, isoleucine, alanine and
phenylalanine.
[0073] Targeting molecules which bind specifically to integrins are
one class of signal sequences that can be found on cells of the
vasculature. These peptides bear the signal sequence based on
Asn-Gly-Arg (NGR). Accordingly, sequences that bind certain
integrins can serve as useful targeting molecules to endothelial
cells and other cells of the neo-vasculature. For example, the
targeting molecule can be an NGR targeting sequence.
[0074] Non-structural spacers may be a feature of the targeting
molecule. Such spacers typically comprise glycine and/or proline
residues. Lengths of these spacers can range from about one to
about 5 amino acids. In addition, it is often preferable to
physically constrain the targeting molecule by cyclization, which
usually results in increased binding. This is usually accomplished
by a pair of cysteine residues, flanking the NGR core at a distance
of about 4 (having only NGR in between) to 10 amino acids from one
another. For example, the pair of cysteine residues, flanking the
NGR core can be at a distance of 7 amino acids from one
another.
[0075] Thus, a typical targeting molecule would have the following
structure: -XNGRYX- wherein X is zero to five amino acids and Y is
a one or two amino acids, selected from cysteine, serine, threonine
and methionine. In a particularly useful embodiment, X is comprised
of glycine residues, but optionally contains at least one, and
typically one or two, free thiol- or amine-containing amino acids
and/or a single hydrophobic amino acid. Thiol-containing residues
include methionine and cysteine; amine-containing residues include
lysine and (at least one additional) arginine; and hydrophobic
residues include leucine, isoleucine, alanine and
phenylalanine.
[0076] The targeting molecule can be an aptamer or an antibody
specific for the target. Traditionally, the identification of
biomarkers and development of antibodies for their specific
targeting has been a difficult and time-consuming process that does
not always provide the best result for a particular application.
For example, although many cancer related biomarkers have been
identified, only few of them have shown promising results for
cancer screening and prognosis, and it has been recognized that it
may be true that only combinations of these biomarkers can provide
the best discrimination between cancerous and normal tissue.
[0077] Numerous reviews have been written about the practice and
products of in vitro selection of aptamers. (Conrad, R. C., L.
Giver, et al. (1996). "In vitro selection of nucleic acid aptamers
that bind proteins." Methods Enzymol 267: 336-67; Osborne, S. E.,
I. Matsumura, et al. (1997). "Aptamers as therapeutic and
diagnostic reagents: problems and prospects." Curr Opin Chem Biol
1(1): 5-9; Famulok, M. and G. Mayer (1999). "Aptamers as tools in
molecular biology and immunology." Curr Top Microbiol Immunol 243:
123-36; Hesselberth, J., M. P. Robertson, et al. (2000). "In vitro
selection of nucleic acids for diagnostic applications [In Process
Citation]." J Biotechnol 74(1): 15-25.). The methods of the present
invention may utilize aptamers with unique or improved binding
characteristics to a target that is unique to or over represented
(as compared to a normal or non-target cell) in, around or on a
cell of interest. An "aptamer" as used herein refers to a nucleic
acid that binds a target molecule through interactions or
conformations other than those of nucleic acid
annealing/hybridization described herein. Methods for making and
modifying aptamers, and assaying the binding of an aptamer to a
target molecule may be assayed or screened for by any mechanism
known to those of skill in the art (see for example, U.S. Pat. Nos.
6,111,095, 5,861,501, 5,840,867, 5,792,613, 5,780,610, 5,780,449,
5,756,291 5,631,146 and 5,582,981; as well as PCT Publication Nos.
WO92/14843, WO91/19813, and WO92/05285, each of which is
incorporated herein by reference).
[0078] Aptamers are single- or double-stranded DNA or
single-stranded RNA molecules that recognize and bind to a desired
target molecule by virtue of their shapes. See, e.g., PCT
Publication Nos. WO92/14843, WO91/19813, and WO92/05285. The SELEX
procedure, described in U.S. Pat. No. 5,270,163 to Gold et al.,
Tuerk et al. (1990) Science 249:505-510, Szostak et al. (1990)
Nature 346:818-822 and Joyce (1989) Gene 82:83-87, can be used to
select for RNA or DNA aptamers that are target-specific. In the
SELEX procedure, an oligonucleotide is constructed wherein an
n-mer, preferably a random sequence tract of nucleotides thereby
forming a "randomer pool" of oligonucleotides, is flanked by two
polymerase chain reaction (PCR) primers. The construct is then
contacted with a target molecule under conditions which favor
binding of the oligonucleotides to the target molecule. Those
oligonucleotides which bind the target molecule are: (a) separated
from those oligonucleotides which do not bind the target molecule
using conventional methods such as filtration, centrifugation,
chromatography, or the like; (b) dissociated from the target
molecule; and (c) amplified using conventional PCR technology to
form a ligand-enriched pool of oligonucleotides. Further rounds of
binding, separation, dissociation and amplification are performed
until an aptamer with the desired binding affinity, specificity or
both is achieved. The final aptamer sequence identified can then be
prepared chemically or by in vitro transcription.
[0079] The length of a random sequence tract can range from 20 to
over 150 residues, and can be even longer if multiple, random
oligonucleotides are combined into a single pool by ligation or
other methods. (Bartel, D. P. and J. W. Szostak (1993). "Isolation
of new ribozymes from a large pool of random sequences [see
comment]." Science 261(5127): 1411-8.). The number of individuals
in a random sequence population is typically at least 10.sup.13 and
can easily be over 10.sup.15. For most pools, this means that
upwards of all possible 25-mers are present, and a proportionately
smaller number of motifs longer than 25. Because of the redundancy
of biological sequences, the sequence diversity of most random
sequence pools likely rivals the sequence diversity of the Earth's
biosphere.
[0080] Aptamers have been selected against a surprising range of
targets, ranging from ions to small organics to peptides to
proteins to supramolecular structures such as viruses and tissues.
(Famulok, M. and G. Mayer (1999). "Aptamers as tools in molecular
biology and immunology." Curr Top Microbiol Immunol 243: 123-36;
Xu, W. and A. D. Ellington (1996). "Anti-peptide aptamers recognize
amino acid sequence and bind a protein epitope." Proc Natl Acad Sci
USA 93(15): 7475-80; Weiss, S., D. Proske, et al. (1997). "RNA
aptamers specifically interact with the prion protein PrP." J Virol
71(11): 8790-7; Convery, M. A., S. Rowsell, et al. (1998). "Crystal
structure of an RNA aptamer-protein complex at 2.8 A resolution."
Nat Struct Biol 5(2): 133-9; Homann, M. and H. U. Goringer (1999).
"Combinatorial selection of high affinity RNA ligands to live
African trypanosomes." Nucleic Acids Res 27(9): 2006-14.). In
particular, aptamers have been selected against a wide variety of
proteins, including many nucleic acid binding proteins, such as T4
DNA polymerase (Tuerk, C. and L. Gold (1990). "Systematic evolution
of ligands by exponential enrichment: RNA ligands to bacteriophage
T4 DNA polymerase." Science 249(4968): 505-10.) and HIV-1 Rev,
(Giver, L., D. Bartel, et al. (1993). "Selective optimization of
the Rev-binding element of HIV-1." Nucleic Acids Res 21(23):
5509-16.) and multiple non-nucleic acid binding proteins. In
general, anti-protein aptamers seem to recognize basic patches on
protein surfaces. For example, the arginine-rich motifs (ARMs) of
many viral proteins are recognized by aptamers (reviewed in
Ellington, A. D., F. Leclerc, et al. (1996). "An RNA groove
[news]." Nat Struct Biol 3(12): 981-4.), the phosphate-binding
pockets of both kinases (Conrad, R., L. M. Keranen, et al. (1994).
"Isozyme-specific inhibition of protein kinase C by RNA aptamers."
J Biol Chem 269(51): 32051-4.) and phosphatases, (Bell, S. D., J.
M. Denu, et al. (1998). "RNA molecules that bind to and inhibit the
active site of a tyrosine phosphatase." J Biol Chem 273(23):
14309-14.) and the heparin-binding sites on many surface proteins
and cytokines, such as basic fibroblast growth factor (Jellinek,
D., C. K. Lynott, et al. (1993). "High-affinity RNA ligands to
basic fibroblast growth factor inhibit receptor binding." Proc Natl
Acad Sci USA 90(23): 11227-31; Jellinek, D., L. S. Green, et al.
(1995). "Potent 2'-amino-2'-deoxypyrimidine RNA inhibitors of basic
fibroblast growth factor." Biochemistry 34(36): 11363-72.) and
vascular endothelial growth factor. (Jellinek, D., L. S. Green, et
al. (1994). "Inhibition of receptor binding by high-affinity RNA
ligands to vascular endothelial growth factor." Biochemistry
33(34): 10450-6; Green, L. S., D. Jellinek, et al. (1995).
"Nuclease-resistant nucleic acid ligands to vascular permeability
factor/vascular endothelial growth factor." Chem Biol 2(10):
683-95.).
[0081] Aptamers also seem to have an affinity for pockets or cusps
on protein surfaces, such as the combining sites of antibodies
(Tsai, D. E., D. J. Kenan, et al. (1992). "In vitro selection of an
RNA epitope immunologically cross-reactive with a peptide." Proc
Natl Acad Sci USA 89(19): 8864-8) or the active sites of enzymes.
(Tuerk, C., S. MacDougal, et al. (1992). "RNA pseudoknots that
inhibit human immunodeficiency virus type 1 reverse transcriptase."
Proc Natl Acad Sci USA 89(15): 6988-92.). Almost all proteins have
either surface pockets or basic patches (indeed, even proteins with
negative pI's, such as T4 DNA polymerase, typically contain sites
that can elicit aptamers). Most aptamer: target complexes have
dissociation constants in the nanomolar range. Moreover, aptamers
recognize their targets with high specificity, and can typically
discriminate between protein targets that are highly homologous or
differ by only a few amino acids. (Conrad, R., L. M. Keranen, et
al. (1994). "Isozyme-specific inhibition of protein kinase C by RNA
aptamers." J Biol Chem 269(51): 32051-4; Eaton, B. E., L. Gold, et
al. (1995). "Let's get specific: the relationship between
specificity and affinity." Chem Biol 2(10): 633-8; Hirao, I., M.
Spingola, et al. (1998). "The limits of specificity: an
experimental analysis with RNA aptamers to MS2 coat protein
variants." Mol Divers 4(2): 75-89.).
[0082] Thus, an example of a fusion protein comprises amino acids
127-512 of SEQ ID NO:1, flanked N-terminally or C-terminally with
RGD, e.g.,
[0083] RGD-amino acids 127-52 of SEQ ID NO:1 or
[0084] amino acids 127-52 of SEQ ID NO:1-RGD.
[0085] Further examples of the fusion protein include:
[0086] [targeting molecule]-amino acids 1-790 of SEQ ID NO: 1,
[0087] amino acids 1-790 of SEQ ID NO: 1-[targeting molecule],
[0088] [targeting molecule]-amino acids 1-650 of SEQ ID NO: 1,
[0089] amino acids 1-650 of SEQ ID NO: 1-[targeting molecule],
[0090] [targeting molecule]-amino acids 1-512 of SEQ ID NO: 1,
[0091] amino acids 1-512 of SEQ ID NO: 1-[targeting molecule],
[0092] [targeting molecule]-amino acids 65-893 of SEQ ID NO: 1,
[0093] amino acids 65-893 of SEQ ID NO: 1-[targeting molecule],
[0094] [targeting molecule]-amino acids 127-893 of SEQ ID NO:
1,
[0095] amino acids 127-790 of SEQ ID NO: 1-[targeting
molecule],
[0096] [targeting molecule]-amino acids 127-790 of SEQ ID NO:
1,
[0097] amino acids 127-893 of SEQ ID NO: 1-[targeting molecule]
[0098] [targeting molecule]-amino acids 127-650 of SEQ ID NO: 1,
and
[0099] amino acids 127-650 of SEQ ID NO: 1-[targeting molecule],
wherein the targeting molecule is as described herein. It will be
understood that linkers (e.g., amino acids) can be used to link the
DOC1 peptide region of the fusion protein to the non-DOC1 region of
the fusion protein. A nucleic acid encoding each of the above
polypeptides is also provided.
[0100] Sequence Similarities
[0101] It is understood that as discussed herein the use of the
terms homology and identity mean the same thing as similarity.
Thus, for example, if the the word homology is used to compare two
non-natural sequences it is understood that this is not necessarily
indicating an evolutionary relationship between these two
sequences, but rather is looking at the similarity or relatedness
between their nucleic acid (or amino acid) sequences. Many of the
methods for determining homology between two evolutionarily related
molecules are routinely applied to any two or more nucleic acids or
proteins for the purpose of measuring sequence similarity
regardless of whether they are evolutionarily related or not.
[0102] In general, it is understood that one way to define any
known variants and derivatives or those that might arise, of the
disclosed genes and proteins herein, is through defining the
variants and derivatives in terms of similarity to specific known
sequences. The similarity of particular sequences disclosed herein
is discussed elsewhere herein. In general, variants of genes and
proteins herein disclosed typically have at least, about 70, 71,
72, 73, 74, 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 percent similarity to
the stated sequence or the native sequence. Those of skill in the
art readily understand how to determine the similarity of two
proteins or nucleic acids, such as genes. For example, the
similarity can be calculated after aligning the two sequences so
that the homology is at its highest level.
[0103] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0104] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used and that in certain
instances the results of these various methods may differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity, and be disclosed herein.
[0105] For example, as used herein, a sequence recited as having a
particular percent homology to another sequence refers to sequences
that have the recited homology as calculated by any one or more of
the calculation methods described above. For example, a first
sequence has 80 percent homology, as defined herein, to a second
sequence if the first sequence is calculated to have 80 percent
homology to the second sequence using the Zuker calculation method
even if the first sequence does not have 80 percent homology to the
second sequence as calculated by any of the other calculation
methods. As another example, a first sequence has 80 percent
homology, as defined herein, to a second sequence if the first
sequence is calculated to have 80 percent homology to the second
sequence using both the Zuker calculation method and the Pearson
and Lipman calculation method even if the first sequence does not
have 80 percent homology to the second sequence as calculated by
the Smith and Waterman calculation method, the Needleman and Wunsch
calculation method, the Jaeger calculation methods, or any of the
other calculation methods. As yet another example, a first sequence
has 80 percent homology, as defined herein, to a second sequence if
the first sequence is calculated to have 80 percent homology to the
second sequence using each of calculation methods (although, in
practice, the different calculation methods will often result in
different calculated homology percentages).
[0106] Similarity can also be determined based on a nucleotide by
nucleotide (amino acid by amino acid) comparison. This comparison
can include a comparison of all of the nucleotides/amino acids in
one sequence against all of the nucleotides/amino acids in the
other sequence (overall homology); thus, the similarity will be
reduced by differences in the length of the sequences being
compared. For example, in such a comparison, a 50 nt nucleic acid
and a 100 nt nucleic acid have a region of overlap defined by the
shorter sequence; if nt in that overlap are identical, the
sequences will have 50% similarity. In contrast, the comparison can
be based on only the overlapping region. For example, in such a
comparison, a 50 nt nucleic acid and a 100 nt nucleic acid that
have an identical overlapping region of 50 nt, will have 100%
similarity. As a further example of comparison in the region of
overlap, a 50 nt nucleic acid and a 100 nt nucleic acid that have
25 nucleotides that differ in the overlapping region, will have 50%
similarity.
[0107] Nucleic Acids
[0108] There are a variety of molecules disclosed herein that are
nucleic acid based, including for example the nucleic acids that
encode, for example DOC1, or fragments thereof, as well as various
functional nucleic acids.
[0109] Provided are nucleic acids encoding the full-length DOC1
protein of SEQ ID NO:1. The nucleic acids encoding DOC1 have
sequences defined by the amino acid sequence of DOC1 in accordance
with the degeneracy of the genetic code. Also, provided are nucleic
acids encoding functional fragments of DOC1, i.e., encoding the
DOC1 polypeptide of the invention. The length of these nucleic
acids is commensurate with the length of the polypeptide they
encode. For example the nucleic acids encode polypeptides having a
length from 10 to 1133 amino acids. Thus, the nucleic acids
encoding these polypeptides range from 30 to 3399 nucleotides in
length, including all numbers of nucleotides in between based on
the number of codons required.
[0110] Provided are nucleic acids encoding a DOC1
fragment-containing fusion protein disclosed herein. The length of
these nucleic acids are commensurate with the length of the fusion
polypeptide they encode. The nucleic acid encodes, for example,
both the DOC1 fragment containing region and the coding region for
a targeting moiety.
[0111] The disclosed nucleic acids can be made up of for example,
nucleotides, nucleotide analogs, or nucleotide substitutes.
Non-limiting examples of these and other molecules are discussed
herein. It is understood that for example, when a vector is
expressed in a cell, the expressed mRNA will typically be made up
of A, C, G, and U. Likewise, it is understood that if, for example,
an antisense molecule is introduced into a cell or cell environment
through for example exogenous delivery, it is advantageous that the
antisense molecule be made up of nucleotide analogs that reduce the
degradation of the antisense molecule in the cellular
environment.
[0112] Nucleotides and Related Molecules
[0113] A nucleotide is a molecule that contains a base moiety, a
sugar moiety and a phosphate moiety. Nucleotides can be linked
together through their phosphate moieties and sugar moieties
creating an internucleoside linkage. The base moiety of a
nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl
(G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a
nucleotide is a ribose or a deoxyribose. The phosphate moiety of a
nucleotide is pentavalent phosphate. An non-limiting example of a
nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP
(5'-guanosine monophosphate). There are many varieties of these
types of molecules available in the art and available herein.
[0114] A nucleotide analog is a nucleotide which contains some type
of modification to either the base, sugar, or phosphate moieties.
Modifications to nucleotides are well known in the art and would
include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as
modifications at the sugar or phosphate moieties. There are many
varieties of these types of molecules available in the art and
available herein.
[0115] Nucleotide substitutes are molecules having similar
functional properties to nucleotides, but which do not contain a
phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide
substitutes are molecules that will recognize nucleic acids in a
Watson-Crick or Hoogsteen manner, but which are linked together
through a moiety other than a phosphate moiety. Nucleotide
substitutes are able to conform to a double helix type structure
when interacting with the appropriate target nucleic acid. There
are many varieties of these types of molecules available in the art
and available herein.
[0116] It is also possible to link other types of molecules
(conjugates) to nucleotides or nucleotide analogs to enhance for
example, cellular uptake. Conjugates can be chemically linked to
the nucleotide or nucleotide analogs. Such conjugates include but
are not limited to lipid moieties such as a cholesterol moiety.
(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86,
6553-6556). There are many varieties of these types of molecules
available in the art and available herein.
[0117] A Watson-Crick interaction is at least one interaction with
the Watson-Crick face of a nucleotide, nucleotide analog, or
nucleotide substitute. The Watson-Crick face of a nucleotide,
nucleotide analog, or nucleotide substitute includes the C2, N1,
and C6 positions of a purine based nucleotide, nucleotide analog,
or nucleotide substitute and the C2, N3, C4 positions of a
pyrimidine based nucleotide, nucleotide analog, or nucleotide
substitute.
[0118] A Hoogsteen interaction is the interaction that takes place
on the Hoogsteen face of a nucleotide or nucleotide analog, which
is exposed in the major groove of duplex DNA. The Hoogsteen face
includes the N7 position and reactive groups (NH2 or O) at the C6
position of purine nucleotides.
[0119] Sequences
[0120] There are a variety of sequences related to the protein
molecules for DOC1, for example SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3 or SEQ ID NO:4, or any of the nucleic acids disclosed herein
for producing DOC1, all of which are encoded by nucleic acids or
are nucleic acids. The sequences for the human analogs of these
genes, as well as other analogs, and alleles of these genes, and
splice variants and other types of variants, are available in a
variety of protein and gene databases, including GenBank. Those
sequences available at the time of filing this application at
GenBank are herein incorporated by reference in their entireties as
well as for individual subsequences contained therein. Those of
skill in the art understand how to resolve sequence discrepancies
and differences and to adjust the compositions and methods relating
to a particular sequence to other related sequences. Primers and/or
probes can be designed for any given sequence given the information
disclosed herein and known in the art.
[0121] Primers and Probes
[0122] Disclosed are compositions including primers and probes,
which are capable of interacting with the disclosed nucleic acids,
such as the DOC1 nucleic acids as disclosed herein. In certain
embodiments the primers are used to support DNA amplification
reactions. Typically the primers will be capable of being extended
in a sequence specific manner. Extension of a primer in a sequence
specific manner includes any methods wherein the sequence and/or
composition of the nucleic acid molecule to which the primer is
hybridized or otherwise associated directs or influences the
composition or sequence of the product produced by the extension of
the primer. Extension of the primer in a sequence specific manner
therefore includes, but is not limited to, PCR, DNA sequencing, DNA
extension, DNA polymerization, RNA transcription, or reverse
transcription. Techniques and conditions that amplify the primer in
a sequence specific manner are preferred. In certain embodiments
the primers are used for the DNA amplification reactions, such as
PCR or direct sequencing. It is understood that in certain
embodiments the primers can also be extended using non-enzymatic
techniques, where for example, the nucleotides or oligonucleotides
used to extend the primer are modified such that they will
chemically react to extend the primer in a sequence specific
manner. Typically the disclosed primers hybridize with the
disclosed nucleic acids or region of the nucleic acids or they
hybridize with the complement of the nucleic acids or complement of
a region of the nucleic acids.
[0123] The size of the primers or probes for interaction with the
nucleic acids in certain embodiments can be any size that supports
the desired enzymatic manipulation of the primer, such as DNA
amplification or the simple hybridization of the probe or primer. A
typical primer or probe would be at least 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, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375,
400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750 or 3000
nucleotides long.
[0124] In other embodiments a primer or probe can be less than or
equal to 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750,
2000, 2250, 2500, 2750 or 3000 nucleotides long.
[0125] The primers for the DOC1 gene typically will be used to
produce an amplified DNA product that contains a region of the DOC1
gene or the complete gene. In general, typically the size of the
product will be such that the size can be accurately determined to
within 3, or 2 or 1 nucleotides.
[0126] In certain embodiments this product is at least 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,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600,
650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000,
2250, 2500, 2750, 3000 or 3405 nucleotides long.
[0127] In other embodiments the product is less than or equal to
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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500,
1750, 2000, 2250, 2500, 2750, 3000 or 3405 nucleotides long.
[0128] Functional Nucleic Acids
[0129] Functional nucleic acids are nucleic acid molecules that
have a specific function, such as binding a target molecule or
catalyzing a specific reaction. Functional nucleic acid molecules
can be divided into the following categories, which are not meant
to be limiting. For example, functional nucleic acids include
antisense molecules, aptamers, ribozymes, triplex forming
molecules, RNAi, and external guide sequences. The functional
nucleic acid molecules can act as affectors, inhibitors,
modulators, and stimulators of a specific activity possessed by a
target molecule, or the functional nucleic acid molecules can
possess a de novo activity independent of any other molecules.
[0130] Functional nucleic acid molecules can interact with any
macromolecule, such as DNA, RNA, polypeptides, or carbohydrate
chains. Often functional nucleic acids are designed to interact
with other nucleic acids based on sequence homology between the
target molecule and the functional nucleic acid molecule. In other
situations, the specific recognition between the functional nucleic
acid molecule and the target molecule is not based on sequence
homology between the functional nucleic acid molecule and the
target molecule, but rather is based on the formation of tertiary
structure that allows specific recognition to take place.
[0131] Antisense molecules are designed to interact with a target
nucleic acid molecule through either canonical or non-canonical
base pairing. The interaction of the antisense molecule and the
target molecule is designed to promote the destruction of the
target molecule through, for example, RNAseH mediated RNA-DNA
hybrid degradation. Alternatively the antisense molecule is
designed to interrupt a processing function that normally would
take place on the target molecule, such as transcription or
replication. Antisense molecules can be designed based on the
sequence of the target molecule. Numerous methods for optimization
of antisense efficiency by finding the most accessible regions of
the target molecule exist. Exemplary methods would be in vitro
selection experiments and DNA modification studies using DMS and
DEPC. It is preferred that antisense molecules bind the target
molecule with a dissociation constant (K.sub.d) less than or equal
to 10.sup.-6, 10.sup.-8, 10.sup.-10, or 10.sup.-12. A
representative sample of methods and techniques which aid in the
design and use of antisense molecules can be found in U.S. Pat.
Nos. 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317,
5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590,
5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522,
6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004,
6,046,319, and 6,057,437.
[0132] Aptamers are molecules that interact with a target molecule,
preferably in a specific way. Typically aptamers are small nucleic
acids ranging from 15-50 bases in length that fold into defined
secondary and tertiary structures, such as stem-loops or
G-quartets. Aptamers can bind small molecules, such as ATP (U.S.
Pat. No. 5,631,146) and theophiline (U.S. Pat. No. 5,580,737), as
well as large molecules, such as reverse transcriptase (U.S. Pat.
No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Aptamers can
bind very tightly with K.sub.d's from the target molecule of less
than 10-12 M. It is preferred that the aptamers bind the target
molecule with a K.sub.d less than 10.sup.-6, 10.sup.-8, 10.sup.-10,
or 10.sup.-12. Aptamers can bind the target molecule with a very
high degree of specificity. For example, aptamers have been
isolated that have greater than a 10,000 fold difference in binding
affinities between the target molecule and another molecule that
differ at only a single position on the molecule (U.S. Pat. No.
5,543,293). It is preferred that the aptamer have a K.sub.d with
the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold
lower than the K.sub.d with a background binding molecule. It is
preferred when doing the comparison for a polypeptide for example,
that the background molecule be a different polypeptide.
Representative examples of how to make and use aptamers to bind a
variety of different target molecules can be found in U.S. Pat.
Nos. 5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228,
5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026,
5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130,
6,028,186, 6,030,776, and 6,051,698.
[0133] Ribozymes are nucleic acid molecules that are capable of
catalyzing a chemical reaction, either intramolecularly or
intermolecularly. Ribozymes are thus catalytic nucleic acid. It is
preferred that the ribozymes catalyze intermolecular reactions.
There are a number of different types of ribozymes that catalyze
nuclease or nucleic acid polymerase type reactions which are based
on ribozymes found in natural systems, such as hammerhead
ribozymes, (U.S. Pat. Nos. 5,334,711, 5,436,330, 5,616,466,
5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463,
5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193,
5,998,203; International Patent Application Nos. WO 9858058 by
Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312
by Ludwig and Sproat) hairpin ribozymes (for example, U.S. Pat.
Nos. 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188,
5,866,701, 5,869,339, and 6,022,962), and tetrahymena ribozymes
(for example, U.S. Pat. Nos. 5,595,873 and 5,652,107). There are
also a number of ribozymes that are not found in natural systems,
but which have been engineered to catalyze specific reactions de
novo (for example, U.S. Pat. Nos. 5,580,967, 5,688,670, 5,807,718,
and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates,
and more preferably cleave RNA substrates. Ribozymes typically
cleave nucleic acid substrates through recognition and binding of
the target substrate with subsequent cleavage. This recognition is
often based mostly on canonical or non-canonical base pair
interactions. This property makes ribozymes particularly good
candidates for target specific cleavage of nucleic acids because
recognition of the target substrate is based on the target
substrates sequence. Representative examples of how to make and use
ribozymes to catalyze a variety of different reactions can be found
in U.S. Pat. Nos. 5,646,042, 5,693,535, 5,731,295, 5,811,300,
5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704,
5,989,906, and 6,017,756.
[0134] Triplex forming functional nucleic acid molecules are
molecules that can interact with either double-stranded or
single-stranded nucleic acid. When triplex molecules interact with
a target region, a structure called a triplex is formed, in which
there are three strands of DNA forming a complex dependant on both
Watson-Crick and Hoogsteen base-pairing. Triplex molecules are
preferred because they can bind target regions with high affinity
and specificity. It is preferred that the triplex forming molecules
bind the target molecule with a K.sub.d less than 10-6, 10-8,
10-10, or 10-12. Representative examples of how to make and use
triplex forming molecules to bind a variety of different target
molecules can be found in U.S. Pat. Nos. 5,176,996, 5,645,985,
5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566,
and 5,962,426.
[0135] External guide sequences (EGSs) are molecules that bind a
target nucleic acid molecule forming a complex, and this complex is
recognized by RNase P, which cleaves the target molecule. EGSs can
be designed to specifically target a RNA molecule of choice. RNAse
P aids in processing transfer RNA (tRNA) within a cell. Bacterial
RNAse P can be recruited to cleave virtually any RNA sequence by
using an EGS that causes the target RNA:EGS complex to mimic the
natural tRNA substrate. (WO 92/03566 by Yale, and Forster and
Altman, Science 238:407-409 (1990)).
[0136] Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA
can be utilized to cleave desired targets within eukarotic cells.
(Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO
93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBO J
14:159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. (USA)
92:2627-2631 (1995)). Representative examples of how to make and
use EGS molecules to facilitate cleavage of a variety of different
target molecules be found in U.S. Pat. Nos. 5,168,053, 5,624,824,
5,683,873, 5,728,521, 5,869,248, and 5,877,162.
[0137] Gene expression can also be effectively silenced in a highly
specific manner through RNA interference (RNAi). This silencing was
originally observed with the addition of double stranded RNA
(dsRNA) (Fire, A., et al. (1998) Nature, 391:806-11; Napoli, C., et
al. (1990) Plant Cell 2:279-89; Hannon, G. J. (2002) Nature,
418:244-51). Once dsRNA enters a cell, it is cleaved by an RNase
III-like enzyme, Dicer, into double stranded small interfering RNAs
(siRNA) 21-23 nucleotides in length that contains 2 nucleotide
overhangs on the 3' ends (Elbashir, S. M., et al. (2001) Genes
Dev., 15:188-200; Bernstein, E., et al. (2001) Nature, 409:363-6;
Hammond, S. M., et al. (2000) Nature, 404:293-6). In an ATP
dependent step, the siRNAs become integrated into a multi-subunit
protein complex, commonly known as the RNAi induced silencing
complex (RISC), which guides the siRNAs to the target RNA sequence
(Nykanen, A., et al. (2001) Cell, 107:309-21). At some point the
siRNA duplex unwinds, and it appears that the antisense strand
remains bound to RISC and directs degradation of the complementary
mRNA sequence by a combination of endo and exonucleases (Martinez,
J., et al. (2002) Cell, 110:563-74). However, the effect of iRNA or
siRNA or their use is not limited to any type of mechanism.
[0138] Short Interfering RNA (siRNA) is a double-stranded RNA that
can induce sequence-specific post-transcriptional gene silencing,
thereby decreasing or even inhibiting gene expression. In one
example, an siRNA triggers the specific degradation of homologous
RNA molecules, such as mRNAs, within the region of sequence
identity between both the siRNA and the target RNA. For example, WO
02/44321 discloses siRNAs capable of sequence-specific degradation
of target mRNAs when base-paired with 3' overhanging ends, herein
incorporated by reference for the method of making these siRNAs.
Sequence specific gene silencing can be achieved in mammalian cells
using synthetic, short double-stranded RNAs that mimic the siRNAs
produced by the enzyme dicer (Elbashir, S. M., et al. (2001)
Nature, 411:494 498) (Ui-Tei, K., et al. (2000) FEBS Lett
479:79-82). siRNA can be chemically or in vitro-synthesized or can
be the result of short double-stranded hairpin-like RNAs (shRNAs)
that are processed into siRNAs inside the cell. Synthetic siRNAs
are generally designed using algorithms and a conventional DNA/RNA
synthesizer. Suppliers include Ambion (Austin, Tex.), ChemGenes
(Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research
(Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo
(Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can
also be synthesized in vitro using kits such as Ambion's
SILENCER.RTM. siRNA Construction Kit.
[0139] The production of siRNA from a vector is more commonly done
through the transcription of a short hairpin RNAs (shRNAs). Kits
for the production of vectors comprising shRNA are available, such
as, for example, Imgenex's GENESUPPRESSOR.TM. Construction Kits and
Invitrogen's BLOCK-IT.TM. inducible RNAi plasmid and lentivirus
vectors. Disclosed herein are any shRNA designed as described above
based on the sequences for the herein disclosed inflammatory
mediators.
[0140] Examples of siRNAs for DOC1 include pSiRNA-Neo-DOC1:
5'AGCGTAACCAAGGAGAGAGAT3' (accession number XM_002964, position
1172-1192; SEQ ID NO:5); and pSiRNA-Neo-Control:
5'ATTCATTCATTCATTCACCAT3' (accession number D00269, position
1192-1212; SEQ ID NO:6)
[0141] Hybridization/Selective Hybridization
[0142] The term hybridization typically means a sequence driven
interaction between at least two nucleic acid molecules, such as a
primer or a probe and a gene. Sequence driven interaction means an
interaction that occurs between two nucleotides or nucleotide
analogs or nucleotide derivatives in a nucleotide specific manner.
For example, G interacting with C or A interacting with T are
sequence driven interactions. Typically sequence driven
interactions occur on the Watson-Crick face or Hoogsteen face of
the nucleotide. The hybridization of two nucleic acids is affected
by a number of conditions and parameters known to those of skill in
the art. For example, the salt concentrations, pH, and temperature
of the reaction all affect whether two nucleic acid molecules will
hybridize.
[0143] Parameters for selective hybridization between two nucleic
acid molecules are well known to those of skill in the art. For
example, in some embodiments selective hybridization conditions can
be defined as stringent hybridization conditions. For example,
stringency of hybridization is controlled by both temperature and
salt concentration of either or both of the hybridization and
washing steps. For example, the conditions of hybridization to
achieve selective hybridization may involve hybridization in high
ionic strength solution (6.times.SSC or 6.times.SSPE) at a
temperature that is about 12-25.degree. C. below the Tm (the
melting temperature at which half of the molecules dissociate from
their hybridization partners) followed by washing at a combination
of temperature and salt concentration chosen so that the washing
temperature is about 5.degree. C. to 20.degree. C. below the Tm.
The temperature and salt conditions are readily determined
empirically in preliminary experiments in which samples of
reference DNA immobilized on filters are hybridized to a labeled
nucleic acid of interest and then washed under conditions of
different stringencies. Hybridization temperatures are typically
higher for DNA-RNA and RNA-RNA hybridizations. The conditions can
be used as described above to achieve stringency, or as is known in
the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.,
1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is
herein incorporated by reference for material at least related to
hybridization of nucleic acids). A preferable stringent
hybridization condition for a DNA:DNA hybridization can be at about
68.degree. C. (in aqueous solution) in 6.times.SSC or 6.times.SSPE
followed by washing at 68.degree. C. Stringency of hybridization
and washing, if desired, can be reduced accordingly as the degree
of complementarity desired is decreased, and further, depending
upon the G-C or A-T richness of any area wherein variability is
searched for. Likewise, stringency of hybridization and washing, if
desired, can be increased accordingly as homology desired is
increased, and further, depending upon the G-C or A-T richness of
any area wherein high homology is desired, all as known in the
art.
[0144] Another way to define selective hybridization is by looking
at the amount (percentage) of one of the nucleic acids bound to the
other nucleic acid. For example, in some embodiments selective
hybridization conditions would be when at least about, 60, 65, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the
limiting nucleic acid is bound to the non-limiting nucleic acid.
Typically, the non-limiting primer is in for example, 10 or 100 or
1000 fold excess. This type of assay can be performed at under
conditions where both the limiting and non-limiting primer are for
example, 10 fold or 100 fold or 1000 fold below their k.sub.d, or
where only one of the nucleic acid molecules is 10 fold or 100 fold
or 1000 fold or where one or both nucleic acid molecules are above
their k.sub.d.
[0145] Another way to define selective hybridization is by looking
at the percentage of primer that gets enzymatically manipulated
under conditions where hybridization is required to promote the
desired enzymatic manipulation. For example, in some embodiments
selective hybridization conditions would be when at least about,
60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100
percent of the primer is enzymatically manipulated under conditions
which promote the enzymatic manipulation, for example if the
enzymatic manipulation is DNA extension, then selective
hybridization conditions would be when at least about 60, 65, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the
primer molecules are extended. Preferred conditions also include
those suggested by the manufacturer or indicated in the art as
being appropriate for the enzyme performing the manipulation.
[0146] Just as with homology, it is understood that there are a
variety of methods herein disclosed for determining the level of
hybridization between two nucleic acid molecules. It is understood
that these methods and conditions may provide different percentages
of hybridization between two nucleic acid molecules, but unless
otherwise indicated meeting the parameters of any of the methods
would be sufficient. For example if 80% hybridization was required
and as long as hybridization occurs within the required parameters
in any one of these methods it is considered disclosed herein.
[0147] It is understood that those of skill in the art understand
that if a composition or method meets any one of these criteria for
determining hybridization either collectively or singly it is a
composition or method that is disclosed herein.
[0148] Cell Delivery Systems
[0149] There are a number of compositions and methods which can be
used to deliver nucleic acids to cells, either in vitro or in vivo.
These methods and compositions can largely be broken down into two
classes: viral based delivery systems and non-viral based delivery
systems. For example, the nucleic acids can be delivered through a
number of direct delivery systems such as, electroporation,
lipofection, calcium phosphate precipitation, plasmids, viral
vectors, viral nucleic acids, phage nucleic acids, phages, cosmids,
or via transfer of genetic material in cells or carriers such as
cationic liposomes. Appropriate means for transfection, including
viral vectors, chemical transfectants, or physico-mechanical
methods such as electroporation and direct diffusion of DNA, are
described by, for example, Wolff, J. A., et al., Science, 247,
1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991).
Such methods are well known in the art and readily adaptable for
use with the compositions and methods described herein. In certain
cases, the methods will be modified to specifically function with
large DNA molecules. Further, these methods can be used to target
certain diseases and cell populations by using the targeting
characteristics of the carrier.
[0150] For example, provided is a vector comprising a nucleic acid
encoding the DOC1 polypeptide of SEQ ID NO:1 or a nucleic acid that
encodes a functional fragment of DOC1 as described herein.
[0151] Based on the provision of such a vector, provided is a host
cell containing a vector comprising a nucleic acid encoding the
DOC1 polypeptide of SEQ ID NO:1 or a nucleic acid that encodes a
functional fragment of DOC1 as described herein.
[0152] A vector comprising a nucleic acid encoding the fusion
protein containing a functional DOC1 fragment is provided.
[0153] Based on the provision of such a vector, provided is a host
cell containing a vector comprising a nucleic acid encoding the
fusion protein containing a functional DOC1 fragment disclosed
herein.
[0154] Nucleic Acid Based Delivery Systems
[0155] Transfer vectors can be any nucleotide construction used to
deliver genes into cells (e.g., a plasmid), or as part of a general
strategy to deliver genes, e.g., as part of recombinant retrovirus
or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
[0156] As used herein, plasmid or viral vectors are agents that
transport the disclosed nucleic acids, such as DOC1 coding sequence
and expression-related sequences into the cell without degradation
and include a promoter yielding expression of the gene in the cells
into which it is delivered. In some embodiments the vectors are
derived from either a virus or a retrovirus. Viral vectors are, for
example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia
virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and
other RNA viruses, including these viruses with the HIV backbone.
Also preferred are any viral families which share the properties of
these viruses which make them suitable for use as vectors.
Retroviruses include Murine Maloney Leukemia virus, MMLV, and
retroviruses that express the desirable properties of MMLV as a
vector. Retroviral vectors are able to carry a larger genetic
payload, i.e., a transgene or marker gene, than other viral
vectors, and for this reason are a commonly used vector. However,
they are not as useful in non-proliferating cells. Adenovirus
vectors are relatively stable and easy to work with, have high
titers, and can be delivered in aerosol formulation, and can
transfect non-dividing cells. Pox viral vectors are large and have
several sites for inserting genes, they are thermostable and can be
stored at room temperature. A preferred embodiment is a viral
vector which has been engineered so as to suppress the immune
response of the host organism, elicited by the viral antigens.
Preferred vectors of this type will carry coding regions for
Interleukin 8 or 10.
[0157] Viral vectors can have higher transaction (ability to
introduce genes) abilities than chemical or physical methods to
introduce genes into cells. Typically, viral vectors contain,
nonstructural early genes, structural late genes, an RNA polymerase
III transcript, inverted terminal repeats necessary for replication
and encapsidation, and promoters to control the transcription and
replication of the viral genome. When engineered as vectors,
viruses typically have one or more of the early genes removed and a
gene or gene/promotor cassette is inserted into the viral genome in
place of the removed viral DNA. Constructs of this type can carry
up to about 8 kb of foreign genetic material. The necessary
functions of the removed early genes are typically supplied by cell
lines which have been engineered to express the gene products of
the early genes in trans.
[0158] Retroviral Vectors
[0159] A retrovirus is an animal virus belonging to the virus
family of Retroviridae, including any types, subfamilies, genus, or
tropisms. Retroviral vectors, in general, are described by Verma,
I. M., Retroviral vectors for gene transfer. In Microbiology-1985,
American Society for Microbiology, pp. 229-232, Washington, (1985),
which is incorporated by reference herein. Examples of methods for
using retroviral vectors for gene therapy are described in U.S.
Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and
WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the
teachings of which are incorporated herein by reference.
[0160] A retrovirus is essentially a package which has packed into
it nucleic acid cargo. The nucleic acid cargo carries with it a
packaging signal, which ensures that the replicated daughter
molecules will be efficiently packaged within the package coat. In
addition to the package signal, there are a number of molecules
which are needed in cis, for the replication, and packaging of the
replicated virus. Typically a retroviral genome, contains the gag,
pol, and env genes which are involved in the making of the protein
coat. It is the gag, pol, and env genes which are typically
replaced by the foreign DNA that it is to be transferred to the
target cell. Retrovirus vectors typically contain a packaging
signal for incorporation into the package coat, a sequence which
signals the start of the gag transcription unit, elements necessary
for reverse transcription, including a primer binding site to bind
the tRNA primer of reverse transcription, terminal repeat sequences
that guide the switch of RNA strands during DNA synthesis, a purine
rich sequence 5' to the 3' LTR that serve as the priming site for
the synthesis of the second strand of DNA synthesis, and specific
sequences near the ends of the LTRs that enable the insertion of
the DNA state of the retrovirus to insert into the host genome. The
removal of the gag, pol, and env genes allows for about 8 kb of
foreign sequence to be inserted into the viral genome, become
reverse transcribed, and upon replication be packaged into a new
retroviral particle. This amount of nucleic acid is sufficient for
the delivery of a one to many genes depending on the size of each
transcript. It is preferable to include either positive or negative
selectable markers along with other genes in the insert.
[0161] Since the replication machinery and packaging proteins in
most retroviral vectors have been removed (gag, pol, and env), the
vectors are typically generated by placing them into a packaging
cell line. A packaging cell line is a cell line which has been
transfected or transformed with a retrovirus that contains the
replication and packaging machinery, but lacks any packaging
signal. When the vector carrying the DNA of choice is transfected
into these cell lines, the vector containing the gene of interest
is replicated and packaged into new retroviral particles, by the
machinery provided in cis by the helper cell. The genomes for the
machinery are not packaged because they lack the necessary
signals.
[0162] Adenoviral Vectors
[0163] The construction of replication-defective adenoviruses has
been described (Berkner et al., J. Virology 61:1213-1220 (1987);
Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et
al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology
61:1226-1239 (1987); Zhang "Generation and identification of
recombinant adenovirus by liposome-mediated transfection and PCR
analysis" BioTechniques 15:868-872 (1993)). The benefit of the use
of these viruses as vectors is that they are limited in the extent
to which they can spread to other cell types, since they can
replicate within an initial infected cell, but are unable to form
new infectious viral particles. Recombinant adenoviruses have been
shown to achieve high efficiency gene transfer after direct, in
vivo delivery to airway epithelium, hepatocytes, vascular
endothelium, CNS parenchyma and a number of other tissue sites
(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.
Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092
(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle,
Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem.
267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993);
Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation
Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10
(1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J.
Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology
74:501-507 (1993)). Recombinant adenoviruses achieve gene
transduction by binding to specific cell surface receptors, after
which the virus is internalized by receptor-mediated endocytosis,
in the same manner as wild type or replication-defective adenovirus
(Chardonnet and Dales, Virology 40:462-477 (1970); Brown and
Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J.
Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655
(1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et
al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell
73:309-319 (1993)).
[0164] A viral vector can be one based on an adenovirus which has
had the E1 gene removed and these virons are generated in a cell
line such as the human 293 cell line. In another preferred
embodiment both the E1 and E3 genes are removed from the adenovirus
genome.
[0165] Adeno-Associated Viral Vectors
[0166] Another type of viral vector is based on an adeno-associated
virus (AAV). This defective parvovirus is a preferred vector
because it can infect many cell types and is nonpathogenic to
humans. AAV type vectors can transport about 4 to 5 kb and wild
type AAV is known to stably insert into chromosome 19. Vectors
which contain this site specific integration property are
preferred. An especially preferred embodiment of this type of
vector is the P4.1 C vector produced by Avigen, San Francisco,
Calif., which can contain the herpes simplex virus thymidine kinase
gene, HSV-tk, and/or a marker gene, such as the gene encoding the
green fluorescent protein, GFP.
[0167] In another type of AAV virus, the AAV contains a pair of
inverted terminal repeats (ITRs) which flank at least one cassette
containing a promoter which directs cell-specific expression
operably linked to a heterologous gene. Heterologous in this
context refers to any nucleotide sequence or gene which is not
native to the AAV or B19 parvovirus.
[0168] Typically the AAV and B19 coding regions have been deleted,
resulting in a safe, noncytotoxic vector. The AAV ITRs, or
modifications thereof, confer infectivity and site-specific
integration, but not cytotoxicity, and the promoter directs
cell-specific expression. U.S. Pat. No. 6,261,834 is herein
incorporated by reference for material related to the AAV
vector.
[0169] The disclosed vectors thus provide DNA molecules which are
capable of integration into a mammalian chromosome without
substantial toxicity.
[0170] The inserted genes in viral and retroviral usually contain
promoters, and/or enhancers to help control the expression of the
desired gene product. A promoter is generally a sequence or
sequences of DNA that function when in a relatively fixed location
in regard to the transcription start site. A promoter contains core
elements required for basic interaction of RNA polymerase and
transcription factors, and may contain upstream elements and
response elements.
[0171] Large Payload Viral Vectors
[0172] Molecular genetic experiments with large human herpesviruses
have provided a means whereby large heterologous DNA fragments can
be cloned, propagated and established in cells permissive for
infection with herpesviruses (Sun et al., Nature genetics 8: 33-41,
1994; Cotter and Robertson, Curr Opin Mol Ther 5: 633-644, 1999).
These large DNA viruses (herpes simplex virus (HSV) and
Epstein-Barr virus (EBV), have the potential to deliver fragments
of human heterologous DNA >150 kb to specific cells. EBV
recombinants can maintain large pieces of DNA in the infected
B-cells as episomal DNA. Individual clones carried human genomic
inserts up to 330 kb appeared genetically stable The maintenance of
these episomes requires a specific EBV nuclear protein, EBNA1,
constitutively expressed during infection with EBV. Additionally,
these vectors can be used for transfection, where large amounts of
protein can be generated transiently in vitro. Herpesvirus amplicon
systems are also being used to package pieces of DNA >220 kb and
to infect cells that can stably maintain DNA as episomes.
[0173] Other useful systems include, for example, replicating and
host-restricted non-replicating vaccinia virus vectors.
[0174] Nucleic acids that are delivered to cells which are to be
integrated into the host cell genome, typically contain integration
sequences. These sequences are often viral related sequences,
particularly when viral based systems are used. These viral
intergration systems can also be incorporated into nucleic acids
which are to be delivered using a non-nucleic acid based system of
deliver, such as a liposome, so that the nucleic acid contained in
the delivery system can become integrated into the host genome.
[0175] Other general techniques for integration into the host
genome include, for example, systems designed to promote homologous
recombination with the host genome. These systems typically rely on
sequence flanking the nucleic acid to be expressed that has enough
homology with a target sequence within the host cell genome that
recombination between the vector nucleic acid and the target
nucleic acid takes place, causing the delivered nucleic acid to be
integrated into the host genome. These systems and the methods
necessary to promote homologous recombination are known to those of
skill in the art.
[0176] Non-Nucleic Acid Based Systems
[0177] The disclosed compositions can be delivered to the target
cells in a variety of ways. For example, the compositions can be
delivered through electroporation, or through lipofection, or
through calcium phosphate precipitation. The delivery mechanism
chosen will depend in part on the type of cell targeted and whether
the delivery is occurring for example in vivo or in vitro.
[0178] Thus, the compositions can comprise, in addition to the
disclosed DOC1 or vectors encoding DOC1 for example, lipids such as
liposomes, such as cationic liposomes (e.g., DOTMA, DOPE,
DC-cholesterol) or anionic liposomes. Liposomes can further
comprise proteins to facilitate targeting a particular cell, if
desired. Administration of a composition comprising a compound and
a cationic liposome can be administered to the blood afferent to a
target organ or inhaled into the respiratory tract to target cells
of the respiratory tract. Regarding liposomes, see, e.g., Brigham
et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner et
al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.
4,897,355. Furthermore, the compound can be administered as a
component of a microcapsule that can be targeted to specific cell
types, such as macrophages, or where the diffusion of the compound
or delivery of the compound from the microcapsule is designed for a
specific rate or dosage.
[0179] In the methods described above which include the
administration and uptake of exogenous DNA into the cells of a
subject (i.e., gene transduction or transfection), delivery of the
compositions to cells can be via a variety of mechanisms. As one
example, delivery can be via a liposome, using commercially
available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE
(GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc.
Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,
Wis.), as well as other liposomes developed according to procedures
standard in the art. In addition, the disclosed nucleic acid or
vector can be delivered in vivo by electroporation, the technology
for which is available from Genetronics, Inc. (San Diego, Calif.)
as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical
Corp., Tucson, Ariz.).
[0180] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These may
be targeted to a particular cell type via antibodies, receptors, or
receptor ligands. The following references are examples of the use
of this technology to target specific proteins to tumor tissue, the
principles of which can be applied to targeting of other cells
(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe,
K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.,
35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews,
129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
42:2062-2065, (1991)). These techniques can be used for a variety
of other specific cell types. Vehicles such as "stealth" and other
antibody conjugated liposomes (including lipid mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA
through cell specific ligands, lymphocyte directed tumor targeting,
and highly specific therapeutic retroviral targeting of murine
glioma cells in vivo. The following references are examples of the
use of this technology to target specific proteins to tumor tissue
(Hughes et al., Cancer Research, 49:6214-6220, (1989); and
Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187,
(1992)). In general, receptors are involved in pathways of
endocytosis, either constitutive or ligand induced. These receptors
cluster in clathrin-coated pits, enter the cell via clathrin-coated
vesicles, pass through an acidified endosome in which the receptors
are sorted, and then either recycle to the cell surface, become
stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as
nutrient uptake, removal of activated proteins, clearance of
macromolecules, opportunistic entry of viruses and toxins,
dissociation and degradation of ligand, and receptor-level
regulation. Many receptors follow more than one intracellular
pathway, depending on the cell type, receptor concentration, type
of ligand, ligand valency, and ligand concentration. Molecular and
cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409
(1991)).
[0181] Nucleic acids that are delivered to cells which are to be
integrated into the host cell genome, typically contain integration
sequences. These sequences are often viral related sequences,
particularly when viral based systems are used. These viral
intergration systems can also be incorporated into nucleic acids
which are to be delivered using a non-nucleic acid based system of
deliver, such as a liposome, so that the nucleic acid contained in
the delivery system can become integrated into the host genome.
[0182] Other general techniques for integration into the host
genome include, for example, systems designed to promote homologous
recombination with the host genome. These systems typically rely on
sequence flanking the nucleic acid to be expressed that has enough
homology with a target sequence within the host cell genome that
recombination between the vector nucleic acid and the target
nucleic acid takes place, causing the delivered nucleic acid to be
integrated into the host genome. These systems and the methods
necessary to promote homologous recombination are known to those of
skill in the art.
[0183] Expression Systems
[0184] The nucleic acids that are delivered to cells typically
contain expression controlling systems. For example, the inserted
genes in viral and retroviral systems usually contain promoters,
and/or enhancers to help control the expression of the desired gene
product. A promoter is generally a sequence or sequences of DNA
that function when in a relatively fixed location in regard to the
transcription start site. A promoter contains core elements
required for basic interaction of RNA polymerase and transcription
factors, and may contain upstream elements and response
elements.
[0185] Viral Promoters and Enhancers
[0186] Preferred promoters controlling transcription from vectors
in mammalian host cells may be obtained from various sources, for
example, the genomes of viruses such as: polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis-B virus and most
preferably cytomegalovirus, or from heterologous mammalian
promoters, e.g. beta actin promoter. The early and late promoters
of the SV40 virus are conveniently obtained as an SV40 restriction
fragment which also contains the SV40 viral origin of replication
(Fiers et al., Nature, 273: 113 (1978)). The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:
355-360 (1982)). Of course, promoters from the host cell or related
species also are useful herein.
[0187] Enhancer generally refers to a sequence of DNA that
functions at no fixed distance from the transcription start site
and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci.
78: 993 (1981)) or 3' (Lusky, M. L., et al., Mol. Cell Bio. 3: 1108
(1983)) to the transcription unit. Furthermore, enhancers can be
within an intron (Banerji, J. L. et al., Cell 33: 729 (1983)) as
well as within the coding sequence itself (Osborne, T. F., et al.,
Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300
by in length, and they function in cis. Enhancers f unction to
increase transcription from nearby promoters. Enhancers also often
contain response elements that mediate the regulation of
transcription. Promoters can also contain response elements that
mediate the regulation of transcription. Enhancers often determine
the regulation of expression of a gene. While many enhancer
sequences are now known from mammalian genes (globin, elastase,
albumin, .alpha.-fetoprotein and insulin), typically one will use
an enhancer from a eukaryotic cell virus for general expression.
Preferred examples are the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0188] The promotor and/or enhancer may be specifically activated
either by light or specific chemical events which trigger their
function. Systems can be regulated by reagents such as tetracycline
and dexamethasone. There are also ways to enhance viral vector gene
expression by exposure to irradiation, such as gamma irradiation,
or alkylating chemotherapy drugs.
[0189] In certain embodiments the promoter and/or enhancer region
can act as a constitutive promoter and/or enhancer to maximize
expression of the region of the transcription unit to be
transcribed. In certain constructs the promoter and/or enhancer
region be active in all eukaryotic cell types, even if it is only
expressed in a particular type of cell at a particular time. A
preferred promoter of this type is the CMV promoter (650 bases).
Other preferred promoters are SV40 promoters, cytomegalovirus (full
length promoter), and retroviral vector LTR.
[0190] It has been shown that all specific regulatory elements can
be cloned and used to construct expression vectors that are
selectively expressed in specific cell types such as melanoma
cells. The glial fibrillary acetic protein (GFAP) promoter has been
used to selectively express genes in cells of glial origin.
[0191] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells) may also
contain sequences necessary for the termination of transcription
which may affect mRNA expression. These regions are transcribed as
polyadenylated segments in the untranslated portion of the mRNA
encoding tissue factor protein. The 3' untranslated regions also
include transcription termination sites. It is preferred that the
transcription unit also contain a polyadenylation region. One
benefit of this region is that it increases the likelihood that the
transcribed unit will be processed and transported like mRNA. The
identification and use of polyadenylation signals in expression
constructs is well established. It is preferred that homologous
polyadenylation signals be used in the transgene constructs. In
certain transcription units, the polyadenylation region is derived
from the SV40 early polyadenylation signal and consists of about
400 bases. It is also preferred that the transcribed units contain
other standard sequences alone or in combination with the above
sequences improve expression from, or stability of, the
construct.
[0192] Markers
[0193] The viral vectors can include nucleic acid sequence encoding
a marker product. This marker product is used to determine if the
gene has been delivered to the cell and once delivered is being
expressed. Preferred marker genes are the E. Coli lacZ gene, which
encodes .beta.-galactosidase, and green fluorescent protein.
[0194] In some embodiments the marker may be a selectable marker.
Examples of suitable selectable markers for mammalian cells are
dihydrofolate reductase (DHFR), thymidine kinase, neomycin,
neomycin analog G418, hydromycin, and puromycin. When such
selectable markers are successfully transferred into a mammalian
host cell, the transformed mammalian host cell can survive if
placed under selective pressure. There are two widely used distinct
categories of selective regimes. The first category is based on a
cell's metabolism and the use of a mutant cell line which lacks the
ability to grow independent of a supplemented media. Two examples
are: CHO DHFR-cells and mouse LTK-cells. These cells lack the
ability to grow without the addition of such nutrients as thymidine
or hypoxanthine. Because these cells lack certain genes necessary
for a complete nucleotide synthesis pathway, they cannot survive
unless the missing nucleotides are provided in a supplemented
media. An alternative to supplementing the media is to introduce an
intact DHFR or TK gene into cells lacking the respective genes,
thus altering their growth requirements. Individual cells which
were not transformed with the DHFR or TK gene will not be capable
of survival in non-supplemented media.
[0195] The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use
of a mutant cell line. These schemes typically use a drug to arrest
growth of a host cell. Those cells which have a novel gene would
express a protein conveying drug resistance and would survive the
selection. Examples of such dominant selection use the drugs
neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327
(1982)), mycophenolic acid, (Mulligan, R. C. and Berg, P. Science
209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell.
Biol. 5: 410-413 (1985)). The three examples employ bacterial genes
under eukaryotic control to convey resistance to the appropriate
drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or
hygromycin, respectively. Others include the neomycin analog G418
and puramycin.
[0196] Peptides
[0197] Protein Variants
[0198] As discussed herein there are numerous variants of the DOC1
protein that are known and herein contemplated. In addition, to the
known functional DOC1 variants there are derivatives of the DOC1
proteins which also function in the disclosed methods and
compositions. Protein variants and derivatives are well understood
to those of skill in the art and in can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Immunogenic fusion protein derivatives, such as those described in
the examples, are made by fusing a polypeptide sufficiently large
to confer immunogenicity to the target sequence by cross-linking in
vitro or by recombinant cell culture transformed with DNA encoding
the fusion. Deletions are characterized by the removal of one or
more amino acid residues from the protein sequence. Typically, no
more than about from 2 to 6 residues are deleted at any one site
within the protein molecule. These variants ordinarily are prepared
by site specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example M13 primer mutagenesis
and PCR mutagenesis. Amino acid substitutions are typically of
single residues, but can occur at a number of different locations
at once; insertions usually will be on the order of about from 1 to
10 amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Such substitutions generally are made in accordance with the
following Tables 1 and 2 and are referred to as conservative
substitutions.
TABLE-US-00001 TABLE 1 Amino Acid Abbreviations Amino Acid
Abbreviations Alanine Ala A Allosoleucine AIle Arginine Arg R
Asparagine Asn N aspartic acid Asp D Cysteine Cys C glutamic acid
Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isolelucine Ile
I Leucine Leu L Lysine Lys K phenylalanine Phe F Proline Pro P
Pyroglutamic acid pGlu Serine Ser S Threonine Thr T Tyrosine Tyr Y
Tryptophan Trp W Valine Val V
TABLE-US-00002 TABLE 2 Amino Acid Substitutions Exemplary
Conservative Substitutions, Original Residue others are known in
the art. Ala Ser Arg Lys; Gln Asn Gln; His Asp Glu Cys Ser Gln Asn,
Lys Glu Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg;
Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp;
Phe Val Ile; Leu
[0199] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those in Table 2, i.e., selecting residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in the protein properties will be
those in which (a) a hydrophilic residue, e.g. seryl or threonyl,
is substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine, in this case, (e) by increasing the
number of sites for sulfation and/or glycosylation.
[0200] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the mosaic
polypeptides provided herein.
[0201] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
may be desirable. Deletions or substitutions of potential
proteolysis sites, e.g. Arg, is accomplished for example by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0202] Certain post-translational derivatizations are the result of
the action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
[0203] It is understood that one way to define the variants and
derivatives of the disclosed proteins herein is through defining
the variants and derivatives in terms of homology/identity to
specific known sequences. For example, SEQ ID NO:1 sets forth a
particular sequence of DOC1 and SEQ ID NO:2 sets forth a particular
sequence of a variant DOC1 protein, SEQ ID NO:3 sets forth a
particular sequence of another variant DOC1 protein, and SEQ ID
NO:4 sets forth a particular sequence of another variant DOC1
protein. Specifically disclosed are variants of these and other
proteins herein disclosed which have at least, 70% or 75% or 80% or
85% or 90% or 95% sequence similarity (also referred to as
homology) to the stated sequence. Those of skill in the art readily
understand how to determine the homology of two proteins. For
example, the homology can be calculated after aligning the two
sequences so that the homology is at its highest level.
[0204] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0205] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment.
[0206] It is understood that the description of conservative
mutations and homology can be combined together in any combination,
such as embodiments that have at least 70% homology to a particular
sequence wherein the variants are conservative mutations.
[0207] As this specification discusses various proteins and protein
sequences it is understood that the nucleic acids that can encode
those protein sequences are also disclosed. This would include all
degenerate sequences related to a specific protein sequence, i.e.
all nucleic acids having a sequence that encodes one particular
protein sequence as well as all nucleic acids, including degenerate
nucleic acids, encoding the disclosed variants and derivatives of
the protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence. For example, one of the
many nucleic acid sequences that can encode the protein sequence
set forth in SEQ ID NO:1 is set forth in SEQ ID NO:7. Other nucleic
acids that encode the same protein sequence set forth in SEQ ID
NO:1 are known to the skilled person based on degeneracy of the
genetic code.
[0208] In addition, for example, a disclosed conservative
derivative of SEQ ID NO:1 is shown in SEQ ID NO: 8, where the
isoleucine (I) at position 20 is changed to a valine (V). It is
understood that for this mutation all of the nucleic acid sequences
that encode this particular derivative of DOC1 are also disclosed.
It is also understood that while no amino acid sequence indicates
what particular DNA sequence encodes that protein within an
organism, where particular variants of a disclosed protein are
disclosed herein, the known nucleic acid sequence that encodes that
protein is also known and herein disclosed and described.
[0209] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
compositions. For example, there are numerous D amino acids or
amino acids which have a different functional substituent then the
amino acids shown in Table 1 and Table 2. The opposite stereo
isomers of naturally occurring peptides are disclosed, as well as
the stereo isomers of peptide analogs. These amino acids can
readily be incorporated into polypeptide chains by charging tRNA
molecules with the amino acid of choice and engineering genetic
constructs that utilize, for example, amber codons, to insert the
analog amino acid into a peptide chain in a site specific way
(Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller,
Current Opinion in Biotechnology, 3:348-354 (1992); Ibba,
Biotechnology & Genetic Engineering Reviews 13:197-216 (1995),
Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech,
12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682
(1994) all of which are herein incorporated by reference at least
for material related to amino acid analogs).
[0210] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2, --CH.dbd.CH--
(cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and
--CHH.sub.2SO--(These and others can be found in Spatola, A. F. in
Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins,
B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);
Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide
Backbone Modifications (general review); Morley, Trends Pharm Sci
(1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res
14:177-185 (1979) (--CH.sub.2NH--, CH.sub.2CH.sub.2--); Spatola et
al. Life Sci 38:1243-1249 (1986) (--CH H.sub.2--S); Hann J. Chem.
Soc Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans);
Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH.sub.2--);
Jennings-White et al. Tetrahedron Lett 23:2533 (1982)
(--COCH.sub.2--); Szelke et al. European Appln, EP 45665 CA (1982):
97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay et al. Tetrahedron.
Lett 24:4401-4404 (1983) (--C(OH)CH.sub.2--); and Hruby Life Sci
31:189-199 (1982) (--CH.sub.2--S--); each of which is incorporated
herein by reference. A particularly preferred non-peptide linkage
is --CH.sub.2NH--. It is understood that peptide analogs can have
more than one atom between the bond atoms, such as b-alanine,
g-aminobutyric acid, and the like.
[0211] Amino acid analogs and analogs and peptide analogs often
have enhanced or desirable properties, such as, more economical
production, greater chemical stability, enhanced pharmacological
properties (half-life, absorption, potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity, and others.
[0212] D-amino acids can be used to generate more stable peptides,
because D amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides.
Cysteine residues can be used to cyclize or attach two or more
peptides together. This can be beneficial to constrain peptides
into particular conformations. (Rizo and Gierasch Ann. Rev.
Biochem. 61:387 (1992), incorporated herein by reference).
[0213] Antibodies
[0214] Provided is an antibody that specifically binds a
full-length wild-type DOC1, or a full-length variant of DOC1 as
defined herein. "Specifically binds" is used herein to mean an
antibody that binds the amino acid sequence of the peptide
specified, and not any other peptide with a substantially different
polypeptide sequence. Also provided is an antibody that
specifically binds to a DOC1 polypeptide disclosed herein. Provided
is an antibody that can specifically bind to a fragment of DOC1,
wherein the fragment comprises a polypeptide selected from the
group consisting of amino acids amino acids 1-790 of SEQ ID NO: 1,
amino acids 1-650 of SEQ ID NO: 1, amino acids 1-512 of SEQ ID NO:
1, amino acids 65-893 of SEQ ID NO: 1, amino acids 127-893 of SEQ
ID NO: 1, and amino acids 127-650 of SEQ ID NO: 1. Provided is an
antibody that specifically binds the DOC1 polypeptide consisting of
amino acids 1-790 or SEQ ID NO:1 and the DOC1 polypeptide
consisting of amino acids 1-650 or SEQ ID NO:1. Provided is an
antibody that binds to a fragment of DOC1, with the proviso that
the antibody does specifically not bind to a fragment consisting of
amino acids 3-32, 3-52, 43-52, or 510-893 of SEQ ID NO:1.
[0215] Provided is an antibody that has the binding characteristics
of the antibody that binds the DOC1 polypeptide comprising amino
acids 1-790 of SEQ ID NO:1, e.g., the antibody produced by the
hybridoma deposited with the ATCC under deposit number
SD-5990/5991. Further provided is an antibody that has the binding
characteristics of the antibody that binds wild-type DOC1 (SEQ ID
NO:1), e.g., the antibody antibody produced by the hybridoma
deposited with the American Type Culture Collection (ATCC), P.O.
Box 1549, Manassas, Va. 20108 under deposit number SD-5990/5991.
Binding characteristics of an antibody include its binding
specificity. The binding specificity can be specificity for the
antigen or it can be specificity based on the epitope recognized by
the antibody. Since both the former and the latter are inherent
characteristics of an antibody, the disclosure of the present
antibodies provides definition of both epitope and antigen
specificity. Thus, provided are an antibody that has the binding
specificity of the antibody produced by the hybridoma deposited
with the ATCC under deposit number SD-5990/5991 and an antibody
that has the binding specificity of the antibody produced by the
hybridoma deposited with the ATCC under deposit number
SD-5990/5991. Reference to the binding specificity of a deposited
monoclonal antibody is the equivalent of reference to the specific
epitope on DOC1 to which that antibody binds. The binding
specificity of any individual monoclonal antibody is an inherent
property of any other monoclonal antibody of the sub-genus defined
by the disclosed, deposited antibody. Methods of identifying the
binding specificity of a given antibody are well known in the art.
Further methods of measuring avidity and other characteristics of
antibody binding are well known.
[0216] Antibodies Generally
[0217] The term "antibodies" is used herein in a broad sense and
includes both polyclonal and monoclonal antibodies. In addition to
intact immunoglobulin molecules, also included in the term
"antibodies" are fragments or polymers of those immunoglobulin
molecules, and human or humanized versions of immunoglobulin
molecules or fragments thereof, as long as they are chosen for
their ability to interact with DOC1 or DOC1 fragment. Antibodies
that bind the disclosed regions of DOC1 are disclosed. The
antibodies can be tested for their desired activity using the in
vitro assays described herein, or by analogous methods, after which
their in vivo therapeutic and/or prophylactic activities are tested
according to known clinical testing methods.
[0218] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a substantially homogeneous population of
antibodies, i.e., the individual antibodies within the population
are identical except for possible naturally occurring mutations
that may be present in a small subset of the antibody molecules.
The monoclonal antibodies herein specifically include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, as long as they exhibit the desired antagonistic
activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc.
Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
[0219] The disclosed monoclonal antibodies can be made using any
procedure which produces mono clonal antibodies. For example,
disclosed monoclonal antibodies can be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse or other appropriate
host animal is typically immunized with an immunizing agent to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the immunizing agent.
Alternatively, the lymphocytes may be immunized in vitro, e.g.,
using the HIV Env-CD4-co-receptor complexes described herein.
[0220] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567
(Cabilly et al.). DNA encoding the disclosed monoclonal antibodies
can be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). Libraries of antibodies or active antibody fragments
can also be generated and screened using phage display techniques,
e.g., as described in U.S. Pat. No. 5,804,440 to Burton et al. and
U.S. Pat. No. 6,096,441 to Barbas et al.
[0221] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art. For instance, digestion can be
performed using papain. Examples of papain digestion are described
in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
Papain digestion of antibodies typically produces two identical
antigen binding fragments, called Fab fragments, each with a single
antigen binding site, and a residual Fc fragment. Pepsin treatment
yields a fragment that has two antigen combining sites and is still
capable of cross-linking antigen.
[0222] The fragments, whether attached to other sequences or not,
can also include insertions, deletions, substitutions, or other
selected modifications of particular regions or specific amino
acids residues, provided the activity of the antibody or antibody
fragment is not significantly altered or impaired compared to the
non-modified antibody or antibody fragment. These modifications can
provide for some additional property, such as to remove/add amino
acids capable of disulfide bonding, to increase its bio-longevity,
to alter its secretory characteristics, etc. In any case, the
antibody or antibody fragment must possess a bioactive property,
such as specific binding to its cognate antigen. Functional or
active regions of the antibody or antibody fragment may be
identified by mutagenesis of a specific region of the protein,
followed by expression and testing of the expressed polypeptide.
Such methods are readily apparent to a skilled practitioner in the
art and can include site-specific mutagenesis of the nucleic acid
encoding the antibody or antibody fragment. (Zoller, M. J. Curr.
Opin. Biotechnol. 3:348-354, 1992).
[0223] As used herein, the term "antibody" or "antibodies" can also
refer to a human antibody and/or a humanized antibody. Many
non-human antibodies (e.g., those derived from mice, rats, or
rabbits) are naturally antigenic in humans, and thus can give rise
to undesirable immune responses when administered to humans.
Therefore, the use of human or humanized antibodies in the methods
serves to lessen the chance that an antibody administered to a
human will evoke an undesirable immune response.
[0224] Human Antibodies
[0225] The disclosed human antibodies can be prepared using any
technique. Examples of techniques for human monoclonal antibody
production include those described by Cole et al. (Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by
Boerner et al. (J. Immunol., 147(1):86-95, 1991). Human antibodies
(and fragments thereof) can also be produced using phage display
libraries (Hoogenboom et al., J. Mol. Biol., 227:381, 1991; Marks
et al., J. Mol. Biol., 222:581, 1991).
[0226] The disclosed human antibodies can also be obtained from
transgenic animals. For example, transgenic, mutant mice that are
capable of producing a full repertoire of human antibodies, in
response to immunization, have been described (see, e.g.,
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993);
Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al.,
Year in Immunol., 7:33 (1993)). Specifically, the homozygous
deletion of the antibody heavy chain joining region (J(H)) gene in
these chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production, and the successful
transfer of the human germ-line antibody gene array into such
germ-line mutant mice results in the production of human antibodies
upon antigen challenge. Antibodies having the desired activity are
selected using Env-CD4-co-receptor complexes as described
herein.
[0227] Humanized Antibodies
[0228] Antibody humanization techniques generally involve the use
of recombinant DNA technology to manipulate the DNA sequence
encoding one or more polypeptide chains of an antibody molecule.
Accordingly, a humanized form of the mouse or other non-human
antibody (or a fragment thereof) is a chimeric antibody or antibody
chain (or a fragment thereof, such as an Fv, Fab, Fab', or other
antigen-binding portion of an antibody) which contains a portion of
an antigen binding site from a non-human (donor) antibody
integrated into the framework of a human (recipient) antibody.
[0229] To generate a humanized antibody, residues from one or more
complementarity determining regions (CDRs) of a recipient (human)
antibody molecule are replaced by residues from one or more CDRs of
a donor (non-human) antibody molecule that is known to have desired
antigen binding characteristics (e.g., a certain level of
specificity and affinity for the target antigen). In some
instances, Fv framework (FR) residues of the human antibody are
replaced by corresponding non-human residues. Humanized antibodies
may also contain residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. Generally,
a humanized antibody has one or more amino acid residues introduced
into it from a source which is non-human. In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies. Humanized antibodies
generally contain at least a portion of an antibody constant region
(Fc), typically that of a human antibody (Jones et al., Nature,
321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988),
and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).
[0230] Methods for humanizing non-human antibodies are well known
in the art. For example, humanized antibodies can be generated
according to the methods of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327
(1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Methods that can be used to produce
humanized antibodies are also described in U.S. Pat. No. 4,816,567
(Cabilly et al.), U.S. Pat. No. 5,565,332 (Hoogenboom et al.), U.S.
Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No. 5,837,243 (Deo et
al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.), U.S. Pat. No.
6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377 (Morgan
et al.).
[0231] Administration of Antibodies
[0232] Administration of the antibodies can be done as disclosed
herein. Nucleic acid approaches for antibody delivery also exist.
The anti-DOC1 antibodies and antibody fragments can also be
administered to patients or subjects as a nucleic acid preparation
(e.g., DNA or RNA) that encodes the antibody or antibody fragment,
such that the patient's or subject's own cells take up the nucleic
acid and produce and secrete the encoded antibody or antibody
fragment. The delivery of the nucleic acid can be by any means, as
disclosed herein, for example.
[0233] Effectors
[0234] The herein provided compositions can further comprise an
effector molecule. By "effector molecule" is meant a substance that
acts upon the target cell(s) or tissue to bring about a desired
effect. The effect can, for example, be the labeling, activating,
repressing, or killing of the target cell(s) or tissue. Thus, the
effector molecule can, for example, be a small molecule,
pharmaceutical drug, toxin, fatty acid, detectable marker,
conjugating tag, nanoparticle, or enzyme. The effector can be a
known anti-cancer therapeutic that can be administered with the
disclosed full-length DOC1 polypeptide or DOC1 polypeptide
comprising a DOC1 fragment.
[0235] Examples of small molecules and pharmaceutical drugs that
can be conjugated to a targeting peptide are known in the art. The
effector can be a cytotoxic small molecule or drug that kills the
target cell. The small molecule or drug can be designed to act on
any critical cellular function or pathway. For example, the small
molecule or drug can inhibit the cell cycle, activate protein
degredation, induce apoptosis, modulate kinase activity, or modify
cytoskeletal proteins. Any known or newly discovered cytotoxic
small molecule or drugs is contemplated for use with the targeting
peptides.
[0236] The effector can be a toxin that kills the targeted cell.
Non-limiting examples of toxins include abrin, modeccin, ricin and
diphtheria toxin. Other known or newly discovered toxins are
contemplated for use with the provided compositions.
[0237] Fatty acids (i.e., lipids) that can be conjugated to the
provided compositions include those that allow the efficient
incorporation of the peptide into liposomes. Generally, the fatty
acid is a polar lipid. Thus, the fatty acid can be a phospholipid
The provided compositions can comprise either natural or synthetic
phospholipid. The phospholipids can be selected from phospholipids
containing saturated or unsaturated mono or disubstituted fatty
acids and combinations thereof. These phospholipids can be
dioleoylphosphatidylcholine, dioleoylphosphatidylserine,
dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol,
dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine,
palmitoyloleoylphosphatidylserine,
palmitoyloleoylphosphatidylethanolamine,
palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic
acid, palmitelaidoyloleoylphosphatidylcholine,
palmitelaidoyloleoylphosphatidylserine,
palmitelaidoyloleoylphosphatidylethanolamine,
palmitelaidoyloleoylphosphatidylglycerol,
palmitelaidoyloleoylphosphatidic acid,
myristoleoyloleoylphosphatidylcholine,
myristoleoyloleoylphosphatidylserine,
myristoleoyloleoylphosphatidylethanoamine,
myristoleoyloleoylphosphatidylglycerol,
myristoleoyloleoylphosphatidic acid,
dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine,
dilinoleoylphosphatidylethanolamine,
dilinoleoylphosphatidylglycerol, dilinoleoylphosphatidic acid,
palmiticlinoleoylphosphatidylcholine,
palmiticlinoleoylphosphatidylserine,
palmiticlinoleoylphosphatidylethanolamine,
palmiticlinoleoylphosphatidylglycerol,
palmiticlinoleoylphosphatidic acid. These phospholipids may also be
the monoacylated derivatives of phosphatidylcholine
(lysophophatidylidylcholine), phosphatidylserine
(lysophosphatidylserine), phosphatidylethanolamine
(lysophosphatidylethanolamine), phophatidylglycerol
(lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidic
acid). The monoacyl chain in these lysophosphatidyl derivatives may
be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or
myristoleoyl. The phospholipids can also be synthetic. Synthetic
phospholipids are readily available commercially from various
sources, such as AVANTI Polar Lipids (Alabaster, Ala.); Sigma
Chemical Company (St. Louis, Mo.). These synthetic compounds may be
varied and may have variations in their fatty acid side chains not
found in naturally occurring phospholipids. The fatty acid can have
unsaturated fatty acid side chains with C14, C16, C18 or C20 chains
length in either or both the PS or PC. Synthetic phospholipids can
have dioleoyl (18:1)-PS; palmitoyl (16:0)-oleoyl (18:1)-PS,
dimyristoyl (14:0)-PS; dipalmitoleoyl (16:1)-PC, dipalmitoyl
(16:0)-PC, dioleoyl (18:1)-PC, palmitoyl (16:0)-oleoyl (18:1)-PC,
and myristoyl (14:0)-oleoyl (18:1)-PC as constituents. Thus, as an
example, the provided compositions can comprise palmitoyl 16:0.
[0238] Detectable markers include any substance that can be used to
label or stain a target tissue or cell(s). Non-limiting examples of
detectable markers include radioactive isotopes, enzymes,
fluorochromes, and quantum dots (Qdot.RTM.). Other known or newly
discovered detectable markers are contemplated for use with the
provided compositions.
[0239] The effector molecule can be a nanoparticle, such as a heat
generating nanoshell. As used herein, "nanoshell" is a nanoparticle
having a discrete dielectric or semi-conducting core section
surrounded by one or more conducting shell layers. U.S. Pat. No.
6,530,944 is hereby incorporated by reference herein in its
entirety for its teaching of the methods of making and using metal
nanoshells. Nanoshells can be formed with a core of a dielectric or
inert material such as silicon, coated with a material such as a
highly conductive metal which can be excited using radiation such
as near infrared light (approximately 800 to 1300 nm). Upon
excitation, the nanoshells emit heat. The resulting hyperthermia
can kill the surrounding cell(s) or tissue. The combined diameter
of the shell and core of the nanoshells ranges from the tens to the
hundreds of nanometers. Near infrared light is advantageous for its
ability to penetrate tissue. Other types of radiation can also be
used, depending on the selection of the nanoparticle coating and
targeted cells. Examples include x-rays, magnetic fields, electric
fields, and ultrasound. The problems with the existing methods for
hyperthermia, especially for use in cancer therapy, such as the use
of heated probes, microwaves, ultrasound, lasers, perfusion,
radiofrequency energy, and radiant heating is avoided since the
levels of radiation used as described herein is insufficient to
induce hyperthermia except at the surface of the nanoparticles,
where the energy is more effectively concentrated by the metal
surface on the dielectric. The particles can also be used to
enhance imaging, especially using infrared diffuse photon imaging
methods. Targeting molecules can be antibodies or fragments
thereof, ligands for specific receptors, or other proteins
specifically binding to the surface of the cells to be
targeted.
[0240] The effector molecule can be covalently linked to the
disclosed peptide. The effector molecule can be linked to the amino
terminal end of the disclosed peptide. The effector molecule can be
linked to the carboxy terminal end of the disclosed peptide. The
effector molecule can be linked to an amino acid within the
disclosed peptide. The herein provided compositions can further
comprise a linker connecting the effector molecule and disclosed
peptide. The disclosed peptide can also be conjugated to a coating
molecule such as bovine serum albumin (BSA) (see Tkachenko et al.,
(2003) J Am Chem Soc, 125, 4700-4701) that can be used to coat the
Nanoshells with the peptide.
[0241] Protein crosslinkers that can be used to crosslink the
effector molecule to the disclosed peptide are known in the art and
are defined based on utility and structure and include DSS
(Disuccinimidylsuberate), DSP (Dithiobis(succinimidylpropionate)),
DTSSP (3,3'-Dithiobis (sulfosuccinimidylpropionate)), SULFO BSOCOES
(Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BSOCOES
(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST
(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO
EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene
glycolbis(sulfosuccinimidylsuccinate)), DPDPB
(1,2-Di[3'-(2'-pyridyldithio) propionamido]butane), BSSS
(Bis(sulfosuccinimdyl) suberate), SMPB
(Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB
(Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS
(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS
(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB
(N-Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SIAB
(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC
(Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate),
SULFO SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate), NHS LC SPDP
(Succinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate),
SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio)
propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio)
propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate),
NHS IODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH
(4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH
(4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide
hydrochloride), MBH (m-Maleimidobenzoic acid
hydrazidehydrochloride), SULFO EMCS
(N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS
(N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI
(N-(p-Maleimidophenyl) isocyanate), KMUH
(N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC
(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate-
)), SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide
ester), SMPH
(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS
(N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS
(N-(gamma-Maleimidobutyrloxy) succinimide), DMP
(Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate
hydrochloride), MHBH (Wood's Reagent) (Methyl-p-hydroxybenzimidate
hydrochloride, 98%), DMA (Dimethyladipimidate hydrochloride).
[0242] Carriers
[0243] The disclosed DOC polypeptide or DOC1 polypeptide fragments
can be combined, conjugated or coupled with or to carriers and
other compositions to aid administration, delivery or other aspects
of the inhibitors and their use. For convenience, such composition
will be referred to herein as carriers. Carriers can, for example,
be a small molecule, pharmaceutical drug, fatty acid, detectable
marker, conjugating tag, nanoparticle, or enzyme.
[0244] The disclosed compositions can be used therapeutically in
combination with a pharmaceutically acceptable carrier. By
"pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material can be
administered to a subject, along with the composition, without
causing any undesirable biological effects or interacting in a
deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained. The carrier
would naturally be selected to minimize any degradation of the
active ingredient and to minimize any adverse side effects in the
subject, as would be well known to one of skill in the art.
[0245] Suitable carriers and their formulations are described in
Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.
R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically,
an appropriate amount of a pharmaceutically-acceptable salt is used
in the formulation to render the formulation isotonic. Examples of
the pharmaceutically-acceptable carrier include, but are not
limited to, saline, Ringer's solution and dextrose solution. The pH
of the solution is preferably from about 5 to about 8, and more
preferably from about 7 to about 7.5. Further carriers include
sustained release preparations such as semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers may be more preferable depending upon,
for instance, the route of administration and concentration of
composition being administered.
[0246] Pharmaceutical carriers are known to those skilled in the
art. These most typically would be standard carriers for
administration of drugs to humans, including solutions such as
sterile water, saline, and buffered solutions at physiological pH.
The compositions can be administered intramuscularly or
subcutaneously. Other compounds can be administered according to
standard procedures used by those skilled in the art.
[0247] Pharmaceutical compositions can include carriers,
thickeners, diluents, buffers, preservatives, surface active agents
and the like in addition to the molecule of choice. Pharmaceutical
compositions can also include one or more active ingredients such
as antimicrobial agents, antiinflammatory agents, anesthetics, and
the like.
[0248] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives can also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0249] Formulations for topical administration can include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0250] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0251] Some of the compositions can potentially be administered as
a pharmaceutically acceptable acid- or base-addition salt, formed
by reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0252] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These can
be targeted to a particular cell type via antibodies, receptors, or
receptor ligands. The following references are examples of the use
of this technology to target specific proteins to tumor tissue
(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe,
K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.,
35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews,
129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
42:2062-2065, (1991)). Vehicles such as "stealth" and other
antibody conjugated liposomes (including lipid mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA
through cell specific ligands, lymphocyte directed tumor targeting,
and highly specific therapeutic retroviral targeting of murine
glioma cells in vivo. The following references are examples of the
use of this technology to target specific proteins to tumor tissue
(Hughes et al., Cancer Research, 49:6214-6220, (1989); and
Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187,
(1992)). In general, receptors are involved in pathways of
endocytosis, either constitutive or ligand induced. These receptors
cluster in clathrin-coated pits, enter the cell via clathrin-coated
vesicles, pass through an acidified endosome in which the receptors
are sorted, and then either recycle to the cell surface, become
stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as
nutrient uptake, removal of activated proteins, clearance of
macromolecules, opportunistic entry of viruses and toxins,
dissociation and degradation of ligand, and receptor-level
regulation. Many receptors follow more than one intracellular
pathway, depending on the cell type, receptor concentration, type
of ligand, ligand valency, and ligand concentration. Molecular and
cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409
(1991)).
[0253] The carrier molecule can be covalently linked to the
disclosed inhibitors. The carrier molecule can be linked to the
amino terminal end of the disclosed peptides. The carrier molecule
can be linked to the carboxy terminal end of the disclosed
peptides. The carrier molecule can be linked to an amino acid
within the disclosed peptides. The herein provided compositions can
further comprise a linker connecting the carrier molecule and
disclosed inhibitors. The disclosed inhibitors can also be
conjugated to a coating molecule such as bovine serum albumin (BSA)
(see Tkachenko et al., (2003) J Am Chem Soc, 125, 4700-4701) that
can be used to coat microparticles, nanoparticles of nanoshells
with the inhibitors.
[0254] Protein crosslinkers that can be used to crosslink the
carrier molecule to the inhibitors, such as the disclosed peptides,
are known in the art and are defined based on utility and structure
and include DSS (Disuccinimidylsuberate), DSP
(Dithiobis(succinimidylpropionate)), DTSSP (3,3'-Dithiobis
(sulfosuccinimidylpropionate)), SULFO BSOCOES
(Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BSOCOES
(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST
(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO
EGS (Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethylene
glycolbis(sulfosuccinimidylsuccinate)), DPDPB
(1,2-Di[3'-(2'-pyridyldithio) propionamido]butane), BSSS
(Bis(sulfosuccinimdyl) suberate), SMPB
(Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB
(Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS
(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS
(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), STAB
(N-Succinimidyl(4-iodoacetyl) aminobenzoate), SULFO SIAB
(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC
(Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate),
SULFO SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate), NHS LC SPDP
(Succinimidyl-6-[3-(2-pyridyldithio) propionamido) hexanoate),
SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio)
propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio)
propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate),
NHS IODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH
(4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH
(4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid hydrazide
hydrochloride), MBH (m-Maleimidobenzoic acid
hydrazidehydrochloride), SULFO EMCS
(N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide), EMCS
(N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI
(N-(p-Maleimidophenyl) isocyanate), KMUH
(N-(kappa-Maleimidoundecanoic acid) hydrazide), LC SMCC
(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate-
)), SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide
ester), SMPH
(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS
(N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS
(N-(gamma-Maleimidobutyrloxy) succinimide), DMP
(Dimethylpimelimidate hydrochloride), DMS (Dimethylsuberimidate
hydrochloride), MHBH (Wood's Reagent) (Methyl-p-hydroxybenzimidate
hydrochloride, 98%), DMA (Dimethyladipimidate hydrochloride).
[0255] Nanoparticles, Microparticles, and Microbubbles
[0256] The term "nanoparticle" refers to a nanoscale particle with
a size that is measured in nanometers, for example, a nanoscopic
particle that has at least one dimension of less than about 100 nm.
Examples of nanoparticles include paramagnetic nanoparticles,
superparamagnetic nanoparticles, metal nanoparticles,
fullerene-like materials, inorganic nanotubes, dendrimers (such as
with covalently attached metal chelates), nanofibers, nanohoms,
nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle
can produce a detectable signal, for example, through absorption
and/or emission of photons (including radio frequency and visible
photons) and plasmon resonance.
[0257] Microspheres (or microbubbles) can also be used with the
methods disclosed herein. Microspheres containing chromophores have
been utilized in an extensive variety of applications, including
photonic crystals, biological labeling, and flow visualization in
microfluidic channels. See, for example, Y. Lin, et al., Appl. Phys
Lett. 2002, 81, 3134; D. Wang, et al., Chem. Mater. 2003, 15, 2724;
X. Gao, et al., J. Biomed. Opt. 2002, 7, 532; M. Han, et al.,
Nature Biotechnology. 2001, 19, 631; V. M. Pai, et al., Mag. &
Magnetic Mater. 1999, 194, 262, each of which is incorporated by
reference in its entirety. Both the photostability of the
chromophores and the monodispersity of the microspheres can be
important.
[0258] Nanoparticles, such as, for example, silica nanoparticles,
metal nanoparticles, metal oxide nanoparticles, or semiconductor
nanocrystals can be incorporated into microspheres. The optical,
magnetic, and electronic properties of the nanoparticles can allow
them to be observed while associated with the microspheres and can
allow the microspheres to be identified and spatially monitored.
For example, the high photostability, good fluorescence efficiency
and wide emission tunability of colloidally synthesized
semiconductor nanocrystals can make them an excellent choice of
chromophore. Unlike organic dyes, nanocrystals that emit different
colors (i.e. different wavelengths) can be excited simultaneously
with a single light source. Colloidally synthesized semiconductor
nanocrystals (such as, for example, core-shell CdSe/ZnS and CdS/ZnS
nanocrystals) can be incorporated into microspheres. The
microspheres can be monodisperse silica microspheres.
[0259] The nanoparticle can be a metal nanoparticle, a metal oxide
nanoparticle, or a semiconductor nanocrystal. The metal of the
metal nanoparticle or the metal oxide nanoparticle can include
titanium, zirconium, hafnium, vanadium, niobium, tantalum,
chromium, molybdenum, tungsten, manganese, technetium, rhenium,
iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel,
palladium, platinum, copper, silver, gold, zinc, cadmium, scandium,
yttrium, lanthanum, a lanthanide series or actinide series element
(e.g., cerium, praseodymium, neodymium, promethium, samarium,
europium, gadolinium, terbium, dysprosium, holmium, erbium,
thulium, ytterbium, lutetium, thorium, protactinium, and uranium),
boron, aluminum, gallium, indium, thallium, silicon, germanium,
tin, lead, antimony, bismuth, polonium, magnesium, calcium,
strontium, and barium. In certain embodiments, the metal can be
iron, ruthenium, cobalt, rhodium, nickel, palladium, platinum,
silver, gold, cerium or samarium. The metal oxide can be an oxide
of any of these materials or combination of materials. For example,
the metal can be gold, or the metal oxide can be an iron oxide, a
cobalt oxide, a zinc oxide, a cerium oxide, or a titanium oxide.
Preparation of metal and metal oxide nanoparticles is described,
for example, in U.S. Pat. Nos. 5,897,945 and 6,759,199, each of
which is incorporated by reference in its entirety.
[0260] For example, DOC1 can be immobilized on silica nanoparticles
(SNPs). SNPs have been widely used for biosensing and catalytic
applications owing to their favorable surface area-to-volume ratio,
straightforward manufacture and the possibility of attaching
fluorescent labels, magnetic nanoparticles (Yang, H. H. et al.
2005) and semiconducting nanocrystals (Lin, Y. W., et al.
2006).
[0261] The nanoparticle can also be, for example, a heat generating
nanoshell. As used herein, "nanoshell" is a nanoparticle having a
discrete dielectric or semi-conducting core section surrounded by
one or more conducting shell layers. U.S. Pat. No. 6,530,944 is
hereby incorporated by reference herein in its entirety for its
teaching of the methods of making and using metal nanoshells.
[0262] Targeting molecules can be attached to the disclosed
compositions and/or carriers. For example, the targeting molecules
can be antibodies or fragments thereof, ligands for specific
receptors, or other proteins specifically binding to the surface of
the cells to be targeted.
[0263] Liposomes
[0264] "Liposome" as the term is used herein refers to a structure
comprising an outer lipid bi- or multi-layer membrane surrounding
an internal aqueous space. Liposomes can be used to package any
biologically active agent for delivery to cells.
[0265] Materials and procedures for forming liposomes are
well-known to those skilled in the art. Upon dispersion in an
appropriate medium, a wide variety of phospholipids swell, hydrate
and form multilamellar concentric bilayer vesicles with layers of
aqueous media separating the lipid bilayers. These systems are
referred to as multilamellar liposomes or multilamellar lipid
vesicles ("MLVs") and have diameters within the range of 10 nm to
100 p.m. These MLVs were first described by Bangham, et al., J Mol.
Biol. 13:238-252 (1965). In general, lipids or lipophilic
substances are dissolved in an organic solvent. When the solvent is
removed, such as under vacuum by rotary evaporation, the lipid
residue forms a film on the wall of the container. An aqueous
solution that typically contains electrolytes or hydrophilic
biologically active materials is then added to the film. Large MLVs
are produced upon agitation. When smaller MLVs are desired, the
larger vesicles are subjected to sonication, sequential filtration
through filters with decreasing pore size or reduced by other forms
of mechanical shearing. There are also techniques by which MLVs can
be reduced both in size and in number of lamellae, for example, by
pressurized extrusion (Barenholz, et al., FEBS Lett. 99:210-214
(1979)).
[0266] Liposomes can also take the form of unilamnellar vesicles,
which are prepared by more extensive sonication of MLVs, and
consist of a single spherical lipid bilayer surrounding an aqueous
solution. Unilamellar vesicles ("ULVs") can be small, having
diameters within the range of 20 to 200 nm, while larger ULVs can
have diameters within the range of 200 nm to 2 p.m. There are
several well-known techniques for making unilamellar vesicles. In
Papahadjopoulos, et al., Biochim et Biophys Acta 135:624-238
(1968), sonication of an aqueous dispersion of phospholipids
produces small ULVs having a lipid bilayer surrounding an aqueous
solution. Schneider, U.S. Pat. No. 4,089,801 describes the
formation of liposome precursors by ultrasonication, followed by
the addition of an aqueous medium containing amphiphilic compounds
and centrifugation to form a biomolecular lipid layer system.
[0267] Small ULVs can also be prepared by the ethanol injection
technique described by Batzri, et al., Biochim et Biophys Acta
298:1015-1019 (1973) and the ether injection technique of Deamer,
et al., Biochim et Biophys Acta 443:629-634 (1976). These methods
involve the rapid injection of an organic solution of lipids into a
buffer solution, which results in the rapid formation of
unilamellar liposomes. Another technique for making ULVs is taught
by Weder, et al. in "Liposome Technology", ed. G. Gregoriadis, CRC
Press Inc., Boca Raton, Fla., Vol. I, Chapter 7, pg. 79-107 (1984).
This detergent removal method involves solubilizing the lipids and
additives with detergents by agitation or sonication to produce the
desired vesicles.
[0268] Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes
the preparation of large ULVs by a reverse phase evaporation
technique that involves the formation of a water-in-oil emulsion of
lipids in an organic solvent and the drug to be encapsulated in an
aqueous buffer solution. The organic solvent is removed under
pressure to yield a mixture which, upon agitation or dispersion in
an aqueous media, is converted to large ULVs. Suzuki et al., U.S.
Pat. No. 4,016,100, describes another method of encapsulating
agents in unilamellar vesicles by freezing/thawing an aqueous
phospholipid dispersion of the agent and lipids.
[0269] In addition to the MLVs and ULVs, liposomes can also be
multivesicular. Described in Kim, et al., Biochim et Biophys Acta
728:339-348 (1983), these multivesicular liposomes are spherical
and contain internal granular structures. The outer membrane is a
lipid bilayer and the internal region contains small compartments
separated by bilayer septum. Still yet another type of liposomes
are oligolamellar vesicles ("OLVs"), which have a large center
compartment surrounded by several peripheral lipid layers. These
vesicles, having a diameter of 2-15 .mu.m, are described in Callo,
et al., Cryobiology 22(3):251-267 (1985).
[0270] Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also
describe methods of preparing lipid vesicles. More recently, Hsu,
U.S. Pat. No. 5,653,996 describes a method of preparing liposomes
utilizing aerosolization and Yiournas, et al., U.S. Pat. No.
5,013,497 describes a method for preparing liposomes utilizing a
high velocity-shear mixing chamber. Methods are also described that
use specific starting materials to produce ULVs (Wallach, et al.,
U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848
and 5,628,936).
[0271] A comprehensive review of all the aforementioned lipid
vesicles and methods for their preparation are described in
"Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca
Raton, Fla., Vol. I, II & III (1984). This and the
aforementioned references describing various lipid vesicles
suitable for use in the invention are incorporated herein by
reference.
[0272] Fatty acids (i.e., lipids) that can be conjugated to the
provided compositions include those that allow the efficient
incorporation of the proprotein convertase inhibitors into
liposomes. Generally, the fatty acid is a polar lipid. Thus, the
fatty acid can be a phospholipid The provided compositions can
comprise either natural or synthetic phospholipid. The
phospholipids can be selected from phospholipids containing
saturated or unsaturated mono or disubstituted fatty acids and
combinations thereof. These phospholipids can be
dioleoylphosphatidylcholine, dioleoylphosphatidylserine,
dioleoylphosphatidylethanolamine, dioleoylphosphatidylglycerol,
dioleoylphosphatidic acid, palmitoyloleoylphosphatidylcholine,
palmitoyloleoylphosphatidylserine,
palmitoyloleoylphosphatidylethanolamine,
palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic
acid, palmitelaidoyloleoylphosphatidylcholine,
palmitelaidoyloleoylphosphatidylserine,
palmitelaidoyloleoylphosphatidylethanolamine,
palmitelaidoyloleoylphosphatidylglycerol,
palmitelaidoyloleoylphosphatidic acid,
myristoleoyloleoylphosphatidylcholine,
myristoleoyloleoylphosphatidylserine,
myristoleoyloleoylphosphatidylethanoamine,
myristoleoyloleoylphosphatidylglycerol,
myristoleoyloleoylphosphatidic acid,
dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine,
dilinoleoylphosphatidylethanolamine,
dilinoleoylphosphatidylglycerol, dilinoleoylphosphatidic acid,
palmiticlinoleoylphosphatidylcholine,
palmiticlinoleoylphosphatidylserine,
palmiticlinoleoylphosphatidylethanolamine,
palmiticlinoleoylphosphatidylglycerol,
palmiticlinoleoylphosphatidic acid. These phospholipids may also be
the monoacylated derivatives of phosphatidylcholine
(lysophophatidylidylcholine), phosphatidylserine
(lysophosphatidylserine), phosphatidylethanolamine
(lysophosphatidylethanolamine), phophatidylglycerol
(lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidic
acid). The monoacyl chain in these lysophosphatidyl derivatives may
be palimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or
myristoleoyl. The phospholipids can also be synthetic. Synthetic
phospholipids are readily available commercially from various
sources, such as AVANTI Polar Lipids (Alabaster, Ala.); Sigma
Chemical Company (St. Louis, Mo.). These synthetic compounds may be
varied and may have variations in their fatty acid side chains not
found in naturally occurring phospholipids. The fatty acid can have
unsaturated fatty acid side chains with C14, C16, C18 or C20 chains
length in either or both the PS or PC. Synthetic phospholipids can
have dioleoyl (18:1)-PS; palmitoyl (16:0)-oleoyl (18:1)-PS,
dimyristoyl (14:0)-PS; dipalmitoleoyl (16:1)-PC, dipalmitoyl
(16:0)-PC, dioleoyl (18:1)-PC, palmitoyl (16:0)-oleoyl (18:1)-PC,
and myristoyl (14:0)-oleoyl (18:1)-PC as constituents. Thus, as an
example, the provided compositions can comprise palmitoyl 16:0.
[0273] In Vivo/Ex Vivo
[0274] As described above, the compositions can be administered in
a pharmaceutically acceptable carrier and can be delivered to the
subject's cells in vivo and/or ex vivo by a variety of mechanisms
well known in the art (e.g., uptake of naked DNA, liposome fusion,
intramuscular injection of DNA via a gene gun, endocytosis and the
like).
[0275] If ex vivo methods are employed, cells or tissues can be
removed and maintained outside the body according to standard
protocols well known in the art. The compositions can be introduced
into the cells via any gene transfer mechanism, such as, for
example, calcium phosphate mediated gene delivery, electroporation,
microinjection or proteoliposomes. The transduced cells can then be
infused (e.g., in a pharmaceutically acceptable carrier) or
homotopically transplanted back into the subject per standard
methods for the cell or tissue type. Standard methods are known for
transplantation or infusion of various cells into a subject.
Methods
[0276] Treatment and Prevention Methods
[0277] A method of inhibiting angiogenesis in a subject is provided
comprising increasing the activity of DOC1 in the subject by an
amount sufficient to inhibit angiogenesis. The activity of DOC1 can
be increased by delivering exogenous DOC1 polypeptide to the
subject or by delivering a molecule that enhances expression of
DOC1 in the subject or in a relevant tissue of the subject.
[0278] A method of inhibiting angiogenesis in a subject is provided
comprising administering to a subject a nucleic acid encoding a
DOC1 polypeptide as disclosed herein, whereby a cell in the subject
produces the DOC1 polypeptide, thus inhibiting angiogenesis. Any of
the DOC1 polypeptides disclosed herein can by used in the present
methods. For example, the wild-type DOC1 isoform 2 (NP_055705.2
(893 aa), is disclosed herein as SEQ ID NO:1.
[0279] In the disclosed methods, the administration of DOC1
polypeptide can be direct (e.g., delivery of the DOC1 polypeptide
to a subject), or it can be indirect (e.g., delivery of a
DOC1-expressing nucleic acid construct to the subject or delivery
of a nucleic acid encoding a molecule that increases expression of
DOC1 to the subject). The administration of DOC-1 polypeptide can
be by any therapeutically effective mode of administration,
including but not limited to oral, intravascular, intrathecal,
subcutaneous, and intratumor.
[0280] In the method that calls for delivering a nucleic acid, the
nucleic acid can be administered locally (e.g., to the site of a
tumor). In another aspect of the method, the nucleic acid can be
administered systemically. Whether delivered locally or
systemically, the nucleic can be administered in a vector. The
vector can be any suitable vector, including the bacteriophage AAV
hybrid or other vectors disclosed herein. In one example, the
nucleic acid encodes SEQ ID NO: 1. In further examples, the nucleic
acid encodes a fragment of SEQ ID NO: 1. In further examples the
nucleic acid encodes SEQ ID NO:2 or a fragment thereof. In further
examples the nucleic acid encodes SEQ ID NO:3 or a fragment
thereof. In further examples the nucleic acid encodes SEQ ID NO:4
or a fragment thereof.
[0281] In the method where a nucleic acid encoding a fragment of
SEQ ID NO:1 is administered, the fragment of SEQ ID NO: 1 can be
selected from the group consisting of amino acids amino acids 1-790
of SEQ ID NO: 1, amino acids 1-650 of SEQ ID NO: 1, amino acids
1-512 of SEQ ID NO: 1, amino acids 65-893 of SEQ ID NO: 1, amino
acids 127-893 of SEQ ID NO: 1, and amino acids 127-650 of SEQ ID
NO: 1.
[0282] Provided is a method of inhibiting tumor growth in a subject
comprising increasing the activity of DOC1 in the subject by an
amount sufficient to inhibit tumor growth. It is recognized that
the subject being treated is in need of tumor growth inhibition,
for example, after the patient is diagnosed with a tumor. The
activity of DOC1 can be increased directly or indirectly in the
subject by several means, including those described herein, each of
which is contemplated as a manner of carrying out the
invention.
[0283] Thus, provided is a method of inhibiting tumor growth in a
subject is provided, comprising administering to a subject a
nucleic acid encoding a DOC1 polypeptide, whereby a cell in the
subject produces the DOC1 polypeptide, thus inhibiting tumor
growth.
[0284] In the method that calls for delivering a nucleic acid, the
nucleic acid can be administered locally (e.g., to the site of a
tumor), orally, intravascularly, intrathecally, and subcutaneously.
In another aspect of the method, the nucleic acid can be
administered systemically. Whether delivered locally or
systemically, the nucleic can be administered in a vector. The
vector can be any suitable vector, including the bacteriophage AAV
hybrid or other vectors disclosed herein. In one example, the
nucleic acid encodes SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. In
a further example, the nucleic acid encodes SEQ ID NO: 2 or a
fragment of SEQ ID NO: 2. In a further example, the nucleic acid
encodes SEQ ID NO: 3 or a fragment of SEQ ID NO: 3. In a further
example, the nucleic acid encodes SEQ ID NO: 4 or a fragment of SEQ
ID NO: 4.
[0285] In the method where a nucleic acid encoding a fragment of
SEQ ID NO:1 is administered, the fragment of SEQ ID NO: 1 can be
selected from the group consisting of amino acids amino acids 1-790
of SEQ ID NO: 1, amino acids 1-650 of SEQ ID NO: 1, amino acids
1-512 of SEQ ID NO: 1, amino acids 65-893 of SEQ ID NO: 1, amino
acids 127-893 of SEQ ID NO: 1, and amino acids 127-650 of SEQ ID
NO: 1.
[0286] The inhibition of tumor growth by administration of DOC1
polypeptide can be via inhibition of cell motility and via
inhibition of cell migration. The tumor growth inhibition by
administration of DOC1 polypeptide can also be via increased cell
apoptosis and inhibition of cell proliferation. These effects are
seen on both endothelial cells and tumor cells. For example,
inhibition of cell migration, apoptosis and proliferation in both
endothelial cells and tumor cells are demonstrated in vitro. In
vivo administration of a DOC1 fragment that targets tumor
vasculature resulted in necrosis in the tumor and inhibition of
tumor growth.
[0287] Provided is a method of inhibiting cell migration comprising
administering a DOC1 polypeptide, or fragment thereof, or nucleic
acid encoding a DOC-1 polypeptide or fragment thereof. In one
embodiment, a method of inhibiting the cell migration of
endothelial cells is provided. A further embodiment is a method of
inhibiting the cell migration of DU145 prostate cancer cells. A
further embodiment is a method of inhibiting the cell migration of
cancer cells. A further embodiment is a method of inhibiting the
metastasis of cancer cells. A further embodiment is a method of
inhibiting the cell migration of immune system cells. A further
embodiment is a method of inhibiting inflammatory disease by
inhibiting the cell migration of immune system cells. In a further
embodiment is a method of inhibiting cell migration of
disease-causing cells
[0288] Provided is a method of increasing apoptosis. In one
embodiment a method of increasing apoptosis of endothelial cells is
provided, comprising administering a DOC1 polypeptide, or fragment
thereof, or nucleic acid encoding a DOC-1 polypeptide or fragment
thereof. In a further embodiment is a method of increasing
apoptosis in cancer cells. In a further embodiment is a method of
increasing apoptosis in disease-causing cells.
[0289] Provided is a method of treating cancer in a subject
comprising increasing the activity of DOC1 in the subject by an
amount sufficient to treat cancer. It is recognized that the
subject being treated is in need of treatment for cancer, for
example, after the patient is diagnosed with cancer. The activity
of DOC1 can be increased in the subject by several means, including
those described herein, each of which is contemplated as a manner
of carrying out the invention.
[0290] A representative but non-limiting list of cancers that the
disclosed methods compositions can be used to treat is the
following: lymphoma (Hodgkins and non-Hodgkins), B cell lymphoma, T
cell lymphoma, AIDS-related lymphomas, hematopoietic cancers,
mycosis fungoides, Hodgkin's Disease, leukemias, myeloid leukemia,
myelomas, carcinomas of solid tissues bladder cancer, brain cancer,
nervous system cancer, head and neck cancer, squamous cell
carcinoma of head and neck, kidney cancer, lung cancers such as
small cell lung cancer and non-small cell lung cancer,
neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,
prostate cancer, skin cancer, liver cancer, melanoma, colon cancer,
cervical cancer, cervical carcinoma, breast cancer, and epithelial
cancer, renal cancer, genitourinary cancer, pulmonary cancer,
esophageal carcinoma, head and neck carcinoma, large bowel cancer,
hematopoietic cancers, testicular cancer, colon and rectal cancers,
prostatic cancer, or pancreatic cancer, squamous cell carcinomas,
squamous cell carcinomas of the mouth, throat, larynx, and lung,
adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas,
neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,
hypoxic tumors, AIDS-related sarcomas, metastatic cancers, or
cancers in general.
[0291] In the cancer treatment method that calls for delivering a
nucleic acid, the nucleic acid can be administered locally (e.g.,
to the site of a tumor). In another aspect of the method, the
nucleic acid can be administered systemically. Whether delivered
locally or systemically, the nucleic can be administered in a
vector. The vector can be any suitable vector, including the
bacteriophage AAV hybrid or other vectors disclosed herein. In one
example, the nucleic acid encodes SEQ ID NO: 1. For example the
nucleic acid can be any nucleic acid shown in SEQ ID NO:7 or any
other coding sequence for SEQ ID NO:1.
[0292] In further examples, the nucleic acid encodes a fragment of
SEQ ID NO: 1. In the method where a nucleic acid encoding a
fragment of SEQ ID NO:1 is administered, the nucleic acid encoding
a fragment of SEQ ID NO: 1 can be selected from the group
consisting of nucleic acids that encode amino acids 1-790 of SEQ ID
NO: 1, amino acids 1-650 of SEQ ID NO: 1, amino acids 1-512 of SEQ
ID NO: 1, amino acids 127-512 of SEQ ID NO:1, amino acids 65-893 of
SEQ ID NO: 1, amino acids 127-893 of SEQ ID NO: 1, amino acids
127-650 of SEQ ID NO: 1 and amino acids 127-790 of SEQ ID NO:1.
More specifically, the nucleic acid encoding a DOC1 polypeptide
having anti-proliferative activity can be selected from the group
of nucleic acids consisting nucleic acids encoding amino acids
1-790 of SEQ ID NO:1, amino acids 1-650 of SEQ ID NO:1, amino acids
1-512 of SEQ ID NO:1, and amino acids 127-893 of SEQ ID NO:1.
[0293] Numerous anti-cancer drugs are available for combination
with the present method and compositions. The following are lists
of anti-cancer (anti-neoplastic) drugs that can be used in
conjunction with the presently disclosed DOC1 activity-enhancing or
expression-enhancing methods.
[0294] Antineoplastic: Acivicin; Aclarubicin; Acodazole
Hydrochloride; AcrQnine; Adozelesin; Aldesleukin; Altretamine;
Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine;
Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine;
Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;
Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;
Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;
Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;
Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;
Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol
Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin;
Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine;
Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin;
Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate;
Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine
Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;
Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;
Estramustine; Estramustine Phosphate Sodium; Etanidazole;
Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine;
Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine;
Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone;
Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au
198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine;
Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;
Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;
Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate;
Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol
Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol;
Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;
Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine;
Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa;
Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;
Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;
Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;
Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin
Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone
Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium;
Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin;
Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide;
Safmgol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate
Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine;
Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89;
Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur;
Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone;
Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;
Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate;
Trestolone Acetate; Triciribine Phosphate; Trimetrexate;
Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride;
Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine
Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate;
Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate;
Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate;
Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.
[0295] Other anti-neoplastic compounds include: 20-epi-1,25
dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;
acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK
antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; atrsacrine; anagrelide;
anastrozole; andrographolide; angiogenesis inhibitors; antagonist
D; antagonist G; antarelix; anti-dorsalizing morphogenetic
protein-1; antiandrogen, prostatic carcinoma; antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate;
apoptosis gene modulators; apoptosis regulators; apurinic acid;
ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;
atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;
azasetron; azatoxin; azatyrosine; baccatin III derivatives;
balanol; batimastat; BCR/ABL antagonists; benzochlorins;
benzoylstaurosporine; beta lactam derivatives; beta-alethine;
betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide;
bisantrene; bisaziridinylspermine; bisnafide; bistratene A;
bizelesin; breflate; bropirimine; budotitane; buthionine
sulfoximine; calcipotriol; calphostin C; camptothecin derivatives;
canarypox IL-2; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorins;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B;
didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-;
dioxamycin; diphenyl spiromustine; docosanol; dolasetron;
doxifluridine; droloxifene; dronabinol; duocannycin SA; ebselen;
ecomustine; edelfosine; edrecolomab; eflornithine; elemene;
emitefur; epirubicin; epristeride; estramustine analogue; estrogen
agonists; estrogen antagonists; etanidazole; etoposide phosphate;
exemestane; fadrozole; fazarabine; fenretinide; filgrastim;
fmasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact;
irsogladine; isobengazole; isohomohalicondrin B; itasetron;
jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;
leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;
leukemia inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance genie inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenyl
acetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome
inhibitors; protein A-based immune modulator; protein kinase C
inhibitor; protein kinase C inhibitors, microalgal; protein
tyrosine phosphatase inhibitors; purine nucleoside phosphorylase
inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin
polyoxyethylene conjugate; raf antagonists; raltitrexed;
ramosetron; ras farnesyl protein transferase inhibitors; ras
inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium
Re 186 etidronate; rhizoxin; ribozymes; RII retinamide;
rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1;
ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim;
Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense
oligonucleotides; signal transduction inhibitors; signal
transduction modulators; single chain antigen binding protein;
sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate;
solverol; somatomedin binding protein; sonermin; sparfosic acid;
spicamycin D; spiromustine; splenopentin; spongistatin 1;
squalamine; stem cell inhibitor; stem-cell division inhibitors;
stipiamide; stromelysin inhibitors; sulfmosine; superactive
vasoactive intestinal peptide antagonist; suradista; suramin;
swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen
methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;
tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide;
teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine;
thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic;
thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid
stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene dichloride; topotecan; topsentin; toremifene; totipotent
stem cell factor; translation inhibitors; tretinoin;
triacetyluridine; triciribine; trimetrexate; triptorelin;
tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins;
UBC inhibitors; ubenimex; urogenital sinus-derived growth
inhibitory factor; urokinase receptor antagonists; vapreotide;
variolin B; vector system, erythrocyte gene therapy; velaresol;
veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin;
vorozole; zanoterone; zeniplatin; zilascorb; zinostatin
stimalamer.
[0296] Administration
[0297] The disclosed compounds and compositions can be administered
in any suitable manner. The manner of administration can be chosen
based on, for example, whether local or systemic treatment is
desired, and on the area to be treated. For example, the
compositions can be administered orally, parenterally (e.g.,
intravenous, subcutaneous, intraperitoneal, or intramuscular
injection), by inhalation, extracorporeally, topically (including
transdermally, ophthalmically, vaginally, rectally, intranasally)
or the like.
[0298] As used herein, "topical intranasal administration" means
delivery of the compositions into the nose and nasal passages
through one or both of the nares and can comprise delivery by a
spraying mechanism or droplet mechanism, or through aerosolization
of the nucleic acid or vector. Administration of the compositions
by inhalant can be through the nose or mouth via delivery by a
spraying or droplet mechanism. Delivery can also be directly to any
area of the respiratory system (e.g., lungs) via intubation.
[0299] Parenteral administration of the composition, if used, is
generally characterized by injection. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution of suspension in liquid prior to
injection, or as emulsions. A more recently revised approach for
parenteral administration involves use of a slow release or
sustained release system such that a constant dosage is maintained.
See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by
reference herein.
[0300] The exact amount of the compositions required can vary from
subject to subject, depending on the species, age, weight and
general condition of the subject, the severity of the allergic
disorder being treated, the particular nucleic acid or vector used,
its mode of administration and the like. Thus, it is not possible
to specify an exact amount for every composition. However, an
appropriate amount can be determined by one of ordinary skill in
the art using only routine experimentation given the teachings
herein. Thus, effective dosages and schedules for administering the
compositions may be determined empirically, and making such
determinations is within the skill in the art. The dosage ranges
for the administration of the compositions are those large enough
to produce the desired effect in which the symptoms disorder are
effected. The dosage should not be so large as to cause adverse
side effects, such as unwanted cross-reactions, anaphylactic
reactions, and the like. Generally, the dosage can vary with the
age, condition, sex and extent of the disease in the patient, route
of administration, or whether other drugs are included in the
regimen, 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 counter indications. Dosage can vary, and can be administered
in one or more dose administrations daily, for one or several days.
Guidance can be found in the literature for appropriate dosages for
given classes of pharmaceutical products.
[0301] For example, a typical daily dosage of the DOC1 or DOC1
polypeptide comprising a DOC1 fragment used alone might range from
about 1 .mu.g/kg to up to 100 mg/kg of body weight or more per day,
depending on the factors mentioned above. More specifically, from
about 0.1 mg to about 10 mg/kg of body weight or more per day can
be administered. In another embodiment a DOC1 polypeptide (e.g.,
aa1-790 of SEQ ID NO:1)-expressing phage is administered directly
to a tumor to increase DOC1 polypeptide presence in the tumor. An
effective amount of DOC1 polypeptide expression in a tumor being
treated can range from 2-300 fold more mRNA expression than in a
corresponding control. For example, a treatment regimen can include
DOC1-expressing phage (1.times.10.sup.11 TU per dose) injected
intravenously twice at day 0 and day 7 and continuing as long as
required.
[0302] Following administration of a disclosed composition for
treating, inhibiting, or preventing cancer, the efficacy of the
therapeutic DOC1 or DOC1 polypeptide comprising a DOC1 fragment can
be assessed in various ways well known to the skilled practitioner.
For instance, one of ordinary skill in the art will understand that
a composition disclosed herein is efficacious in treating cancer or
inhibiting tumor growth in a subject by observing that the
composition reduces tumor size or prevents a further increase in
tumor load. Tumor size, tumor load, etc., can be measured by
methods that are known in the art.
[0303] The disclosed DOC1 polypeptide compositions that inhibit
angiogenesis and reduce tumor size disclosed herein may be
administered prophylactically to patients or subjects who are at
risk for cancer or who have been newly diagnosed with cancer.
[0304] Other molecules that interact with DOC1 which do not have a
specific pharmaceutical function, but which may be used for the
delivery of diagnostic tools for example can be delivered in ways
similar to those described for the pharmaceutical products.
[0305] The disclosed compositions and methods can also be used for
example as tools to isolate and test new drug candidates for a
variety of neoplastic diseases.
[0306] Screening/Diagnostic Methods
[0307] A method of diagnosing cancer in a subject is provided. The
method comprises measuring the amount of DOC1 expression in a
subject, the lower the level of DOC1, the more likely a cancer
exists in the subject. The present diagnostic and classifying
methods are expected to be useful for the cancers described herein,
particularly, epithelial cancers, e.g., colon cancer and prostate
cancer.
[0308] A method for classifying a cancer in a subject is provided.
The method can comprise the steps of contacting a test sample from
a subject with an anti-DOC1 antibody; detecting the binding of the
anti-DOC1 antibody to a DOC1 polypeptide in the test sample;
comparing the expression level of the DOC1 polypeptide in the test
sample with the expression level of a DOC1 polypeptide in a
reference sample for which a cancer classification is known; and
identifying a difference, if present, in the expression level of
the DOC1 polypeptide in the test sample and reference sample,
thereby classifying the cancer in the subject. The difference in
the expression level of the DOC1 polypeptide in the test sample as
compared to the reference sample indicates that the test sample has
a different classification as the reference sample. A similar
expression level of the DOC1 polypeptide in the test sample as
compared to the reference sample indicates that the test sample has
the same classification as the reference sample. In the method of
classifying a cancer, the reference sample can be a sample from a
subject with a known classification. Alternatively, in the method
of classifying a cancer, the reference sample can be from a
database. Further, the reference sample can be classified as a
normal sample or the reference sample can be classified as
cancerous.
[0309] A method for identifying a stage of cancer in a subject is
provided. The method can comprise contacting a test sample from a
subject with an anti-DOC1 antibody; detecting the binding of the
anti-DOC1 antibody to a DOC1 polypeptide in the sample; comparing
the expression level of the DOC1 polypeptide in the test sample
with the expression level of a DOC1 polypeptide in a reference
sample for which a stage of cancer is known; and identifying a
difference, if present, in the expression level of the DOC1
polypeptide in the test sample and reference sample, thereby
identifying the stage of cancer in the subject. A difference in the
expression level of the DOC1 polypeptide in the test sample as
compared to the reference sample indicates that the test sample has
a different stage of cancer than the reference sample. A similar
expression level of the DOC1 polypeptide in the test sample as
compared to the reference sample indicates that the test sample has
the same stage of cancer as the reference sample. The reference
sample can be from a patient with a known stage of cancer.
Alternatively, the reference sample is from a database.
[0310] A method of determining the efficacy of an anti-cancer
treatment is provided, The method can comprise determining the
expression level of a DOC1 polypeptide in a sample obtained from
the subject; administering the anti-cancer treatment; determining
the expression level of a DOC1 polypeptide in a sample obtained
from the subject after administration of the anti-cancer treatment;
and comparing the expression level of the DOC1 polypeptide in the
sample obtained after the anti-cancer treatment with the sample
obtained in step a) such that if there is a change in the
expression level of the DOC1 polypeptide in the sample obtained
after the anti-cancer treatment as compared to the sample obtained
before administration of the anti-cancer treatment, wherein the
change is associated with an improvement in the subject, the
anti-cancer therapeutic is an effective anti-cancer therapeutic.
The change can be an increase in the expression levels of the DOC1
polypeptide.
[0311] There are contexts in which a decrease in the expression
levels of the DOC1 polypeptide is desired. For example, conditions
caused by excessive DOC1-induced apoptosis, can be treated by
reducing/decreasing levels of DOC1.
[0312] Thus, provided is a method of identifying a modulator of
DOC1 expression. The method can comprise a) contacting a cell that
is capable of expressing DOC1 with a putative modulator; measuring
DOC1 expression, wherein a change in DOC1 expression in the cell of
step a) as compared to a cell that was not contacted with the
putative modulator indicates the presence of a modulator of DOC1
expression. The modulator of DOC1 expression can increase DOC1
expression. Angiogenesis inhibitors such as endostatin, fumagillin
and EMAP II upregulate DOC1 expression. For example, the human
prolactin antagonist, G129R, can inhibit human breast cancer cell
proliferation in vitro and to slow the growth rate of tumors in
mice (Beck et al., Cancer Res. 2003 Jul. 1; 63(13):3598-604). The
modulator of DOC1 expression can decrease DOC1 expression. For
example, DOC1 expression can be silenced using small interfering
RNA (Tandle et al. Cytokine. 2005 Jun. 21; 30(6):347-58). The siRNA
used in this experiment were pSiRNA-Neo-DOC1:
5'AGCGTAACCAAGGAGAGAGAT3' (accession number XM 002964, position
1172-1192; SEQ ID NO:5); and pSiRNA-Neo-Control:
5'ATTCATTCATTCATTCACCAT3' (accession number D00269, position
1192-1212; SEQ ID NO:6)
[0313] DOC1 expression can be measured by amplifying a DOC1 nucleic
acid. As the sequence of the nucleic acid is provided, the
generation and use of DOC1 amplifying primers is routine. DOC1
expression can be measured by detecting a DOC1 polypeptide. In the
method that measures DOC1 expression by detecting DOC1 polypeptide,
the DOC1 polypeptide can be detected using anti-DOC1 antibody, for
example the antibody described herein.
[0314] Thus, method of detecting DOC1 in a fluid or tissue of a
subject is provided, comprising: contacting a sample obtained from
the subject with an anti-DOC1 antibody; and determining the
presence of a DOC1 polypeptide in the sample.
Kits
[0315] The materials described above as well as other materials can
be packaged together in any suitable combination as a kit useful
for performing, or aiding in the performance of, the disclosed
method. It is useful if the kit components in a given kit are
designed and adapted for use together in the disclosed method. For
example disclosed are kits for detecting DOC1 in a tissue or blood,
the kit comprising anti-DOC1 antibody. The kits also can contain a
detectable labels for detecting an antibody-DOC1 complex.
Uses
[0316] The disclosed compositions can be used in a variety of ways
as research tools. For example, the disclosed compositions, such
the anti-DOC1 antibody can be used to study the tissue distribution
of DOC1 to confirm the reliability of using its expression level as
an indicator of cancer or non-cancer status. The antibody can also
be used in a method to stage cancer. The antibody can also be used
in a method a method of determining the efficacy of an anti-cancer
treatment. The antibody can also be used in a method a method of
identifying a modulator of DOC1 expression. Other uses are
disclosed, apparent from the disclosure, and/or will be understood
by those in the art.
Methods of Making the Compositions
[0317] The compositions disclosed herein and the compositions
necessary to perform the disclosed methods can be made using any
method known to those of skill in the art for that particular
reagent or compound unless otherwise specifically noted.
Nucleic Acid Synthesis
[0318] For example, the nucleic acids, such as, the
oligonucleotides to be used as primers can be made using standard
chemical synthesis methods or can be produced using enzymatic
methods or any other known method. Such methods can range from
standard enzymatic digestion followed by nucleotide fragment
isolation (see for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N. Y., 1989) Chapters 5, 6) to purely
synthetic methods, for example, by the cyanoethyl phosphoramidite
method using a Milligen or Beckman System 1Plus DNA synthesizer
(for example, Model 8700 automated synthesizer of
Milligen-Biosearch, Burlington, Mass. or ABI Model 380B). Synthetic
methods useful for making oligonucleotides are also described by
Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984),
(phosphotriester and phosphite-triester methods), and Narang et
al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method).
Protein nucleic acid molecules can be made using known methods such
as those described by Nielsen et al., Bioconjug. Chem. 5:3-7
(1994).
Peptide Synthesis
[0319] One method of producing the disclosed proteins, such as SEQ
ID NO:23, is to link two or more peptides or polypeptides together
by protein chemistry techniques. For example, peptides or
polypeptides can be chemically synthesized using currently
available laboratory equipment using either Fmoc
(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)
chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One
skilled in the art can readily appreciate that a peptide or
polypeptide corresponding to the disclosed proteins, for example,
can be synthesized by standard chemical reactions. For example, a
peptide or polypeptide can be synthesized and not cleaved from its
synthesis resin whereas the other fragment of a peptide or protein
can be synthesized and subsequently cleaved from the resin, thereby
exposing a terminal group which is functionally blocked on the
other fragment. By peptide condensation reactions, these two
fragments can be covalently joined via a peptide bond at their
carboxyl and amino termini, respectively, to form an antibody, or
fragment thereof (Grant G A (1992) Synthetic Peptides: A User
Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B.,
Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc.,
NY (which is herein incorporated by reference at least for material
related to peptide synthesis). Alternatively, the peptide or
polypeptide is independently synthesized in vivo as described
herein. Once isolated, these independent peptides or polypeptides
may be linked to form a peptide or fragment thereof via similar
peptide condensation reactions.
[0320] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide-thioester with another unprotected peptide
segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site (Baggiolini M et al.
(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J.Biol.Chem.,
269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
[0321] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
Process for Making the Compositions
[0322] Disclosed are processes for making the compositions as well
as making the intermediates leading to the compositions. For
example, disclosed are the nucleic acid shown in SEQ ID NO:7, and
every degenerate nucleic encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3 or SEQ ID NO:4. There are a variety of methods that can be
used for making these compositions, such as synthetic chemical
methods and standard molecular biology methods. It is understood
that the methods of making these and the other disclosed
compositions are specifically disclosed.
[0323] Disclosed are nucleic acid molecules produced by the process
comprising linking in an operative way a nucleic acid comprising
the sequence set forth in SEQ ID NO:7 (or functional DOC1
fragment-encoding nucleic acid fragment), or any degenerate
sequence encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID
NO:4, to a sequence controlling the expression of the nucleic
acid.
[0324] Also disclosed are nucleic acid molecules produced by the
process comprising linking in an operative way a nucleic acid
molecule comprising a sequence having 80% identity to a sequence
set forth in SEQ ID NO:7, or 80% identity to any degenerate
sequence encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID
NO:4, and a sequence controlling the expression of the nucleic
acid.
[0325] Disclosed are nucleic acid molecules produced by the process
comprising linking in an operative way a nucleic acid molecule
comprising a sequence that hybridizes under stringent hybridization
conditions to a sequence set forth SEQ ID NO:7, or to any
degenerate sequence encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3
or SEQ ID NO:4, and a sequence controlling the expression of the
nucleic acid.
[0326] Disclosed are nucleic acid molecules produced by the process
comprising linking in an operative way a nucleic acid molecule
comprising a sequence encoding a peptide set forth in SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and a sequence controlling
an expression of the nucleic acid molecule.
[0327] Disclosed are nucleic acid molecules produced by the process
comprising linking in an operative way a nucleic acid molecule
comprising a sequence encoding a peptide having 80%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any peptide set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 and a
sequence controlling an expression of the nucleic acid
molecule.
[0328] Disclosed are nucleic acids produced by the process
comprising linking in an operative way a nucleic acid molecule
comprising a sequence encoding a peptide having 80%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any peptide set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4,
wherein any change from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or
SEQ ID NO:4 are conservative changes and a sequence controlling an
expression of the nucleic acid molecule.
[0329] Disclosed are cells produced by the process of transforming
the cell with any of the disclosed nucleic acids. Disclosed are
cells produced by the process of transforming the cell with any of
the non-naturally occurring disclosed nucleic acids.
[0330] Disclosed are any of the disclosed peptides produced by the
process of expressing any of the disclosed nucleic acids. Disclosed
are any of the non-naturally occurring disclosed peptides produced
by the process of expressing any of the disclosed nucleic acids.
Disclosed are any of the disclosed peptides produced by the process
of expressing any of the non-naturally disclosed nucleic acids.
[0331] Disclosed are animals produced by the process of
transfecting a cell within the animal with any of the nucleic acid
molecules disclosed herein. Disclosed are animals produced by the
process of transfecting a cell within the animal any of the nucleic
acid molecules disclosed herein, wherein the animal is a mammal.
Also disclosed are animals produced by the process of transfecting
a cell within the animal any of the nucleic acid molecules
disclosed herein, wherein the mammal is mouse, rat, rabbit, cow,
sheep, pig, or primate.
[0332] Also disclose are animals produced by the process of adding
to the animal any of the cells disclosed herein.
Examples
[0333] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
disclosure. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
Example 1
Functional Studies of DOC1 and Selected Truncation Fragments; Novel
Proteins that Inhibit Angiogenesis and Inhibit Tumor Growth
[0334] Over-expression of DOC1 in endothelial cells leads to
inhibition of cell proliferation and an increase in apoptosis. In
particular, DOC1 over-expression inhibited cell proliferation by 25
percent compared to controls as determined by BrdU labeling (FIG.
1A), and increased apoptosis 3-fold compared to controls as
determined by Annexin V and 7-AAD staining (FIG. 1B). In addition,
over-expression of DOC1 fragments in a series of DOC1 truncation
mutants showed differential activity in terms of the inhibition of
cell proliferation as determined by BrdU labeling (FIG. 2A-C).
Based on these truncation studies, an active region that mediates
the inhibition of cell proliferation can be placed between aa127
and aa512, and C-terminal mutant 1-790 is more potent than
wild-type DOC1 in mediating anti-proliferative activity. Stable
clones expressing DOC1 C-terminal mutant 1-790 showed slower cell
migration than those expressing control vector (FIG. 3), indicating
that overexpression of DOC1 results in inhibition of cell
migration. Administration of DOC1 truncation mutant (aa1-790) by
vasculature-targeting bacteriophage into tumor-bearing nude mice
resulted in the inhibition of M21 melanoma growth (FIG. 5; Example
3).
Example 2
Mouse Monoclonal DOC1 Antibody
[0335] A mouse monoclonal DOC1 antibody was developed, which
allowed detection of endogenous DOC1 protein in human tissues for
the first time. The antibody, was produced by standard methods.
His6 (SEQ ID NO: 13) and MBP fusion constructs encoding a full
length FILIP1L cDNA were generated. The fusion construct was
expressed in Baculovirus, the fusion protein was purified and was
cleaved by TEV protease. We then used the intact FILIP1L protein as
an antigen to immunize mice. Mouse monoclonal antibodiewere
screened by ELISA and Western blot using the purified FILIP1L
protein, and monoclonal antibodies from clone 3C5 and 1C2 were
selected.
[0336] DOC1 protein is expressed in the cytoplasm, on the membrane
and in the nucleus of endothelial cells (FIG. 4).
Immunohistochemical staining showed that DOC1 protein is expressed
on the vasculature, stroma and muscularis in human cancer patient
specimens. DOC1 appears to be more expressed in foci of
inflammation in cancer stroma (e.g. chronic prostatitis) in these
cancer patient specimens. In normal human colon, FILIP1L was
expressed in the vasculature and muscularis mucosa (smooth muscle
type stroma) determined by immunohistochemical staining of FILIP1L.
FILIP1L appears to be more expressed in foci of inflammation in
human prostate cancer stroma (e.g. chronic prostatitis).
[0337] The antibodies from clones 3C5 and 1C2 tested for binding
against DOC1 polypeptides. 293 cells were transfected with the
plasmid encoding each construct, harvested cells after 24 h, and
the cell lysates were subjected to Western blot analysis using the
antibody. In addition to binding the DOC1 of SEQ ID NO:1, this
antibody specifically detects wild-type, 1-790 (SEQ ID NO:1) and
1-650 (SEQ ID NO:2).
Example 3
In Vivo Activity of DOC1 Functional Fragments
[0338] An in vivo experiment using M1 phage to treat mice provided
the results shown in FIG. 5. A bacteriophage AAV hybrid vector
containing a DOC1 fragment that targets tumor vasculature was
administered. This resulted in necrosis in the tumor and subsequent
inhibition of tumor growth.
[0339] It has been recently shown that a new hybrid
adeno-associated virus/phage (AAVP) specifically targets tumor
vasculature in a mouse model (Hajitou A et al. 2006 Cell
125:385-398)). Specifically, a eukaryotic gene cassette from
adeno-associated virus was inserted into RGD-4C phage which
displays the double-cyclic peptide CDCRGDCFC (RGD-4C) (SEQ ID NO:
14). Since the RGD-4C peptide binds to .quadrature.v integrins
which vasculature, the hybrid AAVP could specifically target tumor
vasculature. AAVP specifically targets tumor vasculature in several
different tumor models, and in both immunosuppressed and
immunocompetent mice. To test if targeted expression of DOC1 in
tumor vasculature resulted in inhibition of tumor growth, the AAVP
system was utilized. M21 melanoma cells were injected
subcutaneously onto the female athymic nude mice, and grown to the
average size of 100 mm3. Mice were randomly sorted to four groups
(n=11 for each group), phages (1.times.1011 TU per dose) were
injected intravenously twice at day 0 and day 7, and the tumors
were measured in a blinded manner. Four groups were tested: PBS,
RGD-4C AAVP, RGD-4C-DOC1 (1-650) and RGD-4CDOC1 (1-790) AAVP. The
PBS control tumors were grown fast and some of the tumors started
to show necrosis in the center of tumor by day 14, so the tumors
were measured until day 14. Only tumors from RGD-4C-DOC1 (1-790)
AAVP-treated mice were significantly smaller than those from
PBS-treated mice by day 14 (P=0.0062). In addition, tumors from
RGD-4C-DOC1 (1-790) AAVP-treated mice were significantly smaller
than those from RGD-4C AAVP-treated mice (P=0.0344) and RGD-4C-DOC1
(1-650) AAVP-treated mice (P=0.0053) by day 14.
[0340] In this experiment, it was shown that 1-790 had a potent
anti-tumor activity. Therefore, DOC1 and its truncation fragments
are shown herein to be mediators of the anti-proliferative and
apoptotic activity in endothelial cells and tumor cells. The DOC1
fragments can be utilized as cancer therapeutics.
[0341] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this pertains. The references disclosed are also individually
and specifically incorporated by reference herein for the material
contained in them that is discussed in the sentence in which the
reference is relied upon.
Example 4
Functional Characterization of Filamin A Interacting Protein 1-Like
Production of Mouse Monoclonal Anti-FILIP1L Antibody
[0342] The construct encoding a full length FILIP1L cDNA was
generated and expressed in Baculovirus. The purified full-length
FILIP1L protein (893 amino acids) was used as an antigen to
immunize mice. Immunization of mice, production of hybridoma cells,
screening by enzyme-linked immunosorbent assay (ELISA) and
purification of monoclonal antibody were performed by Green
Mountain Antibodies Inc. Antibodies that recognize FILIP1L were
further tested by western blot and a monoclonal antibody was
selected.
[0343] Cell Culture
[0344] HUVECs were cultured in complete EGM-2 medium as recommended
by the manufacturer (Lonza). HEK293 cells were grown in Dulbecco
modified Eagle medium containing 10% FBS. DU145 human prostate
carcinoma cells and M21 human melanoma cells were grown in RPMI
1640 medium containing 10% FBS.
[0345] Western Blot
[0346] HUVECs were cultured, harvested and fractionated with
ProteoExtract Subcellular Proteome Extraction Kit according to the
manufacturer's protocol (Calbiochem). For endostatin experiment,
HUVECs were starved in EGM-2 basal medium containing 1% FBS for 16
h, treated with 1 .mu.g/ml endostatin for 2, 4 and 8 h, and lysed
with radioimmuno precipitation assay (RIPA) buffer. HEK293 cells
were transfected, using Lipofectamine 2000 (Invitrogen), with a
series of N-terminal and C-terminal truncation mutants of FILIP1L
containing a C-terminal hemagglutinin (HA) tag, harvested at 24 h
and lysed with RIPA buffer. Empty lentivirus- or lentivirus
expressing FILIP1L mutant 1-790 (here-after referred to as
FILIP1L.DELTA.C103)-transduced DU145 clones were cultured in the
presence or absence of 1 .mu.g/ml doxycycline and lysed with RIPA
buffer. Tumors from PBS-, null-adeno-associated virus-phage
(here-after referred to as AAVP-null)- and AAVP expressing
FILIP1L.DELTA.C103 (here-after referred to as
AAVP-.DELTA.C103)-treated mice were removed 4 days after tail vein
injection and snap frozen. Whole tumor lysates were prepared from
RIPA buffer lysis of 60 .mu.m tumor section. 25-50 .mu.g cellular
fractionation, whole cell lysates or whole tumor lysates prepared
by above methods were separated on SDS-PAGE and transferred to
nitrocellulose membrane. The membranes were blotted with antibodies
against FILIP1L, HA tag (Covance) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) (Chemicon) followed by incubation with
anti-mouse antibody conjugated to horseradish peroxidase. The
signal was detected using chemiluminescence (Millipore).
[0347] Immunohistochemistry
[0348] Frozen human colon tumors and their adjacent normal colon
samples were obtained under an IRB approved protocol. 10 .mu.m
tissue sections were fixed with 4% paraformaldehyde for 20 min and
stained with mouse monoclonal antibodies against FILIP1L (7.5
.mu.g/ml) and CD31 (10 DAKO). After visualization of staining by
DAB (3,3'-diaminobenzidine tetrahydrochloride), the slides were
counterstained with hematoxylin. Images were acquired by Axioplan 2
microscope using a 20.times./0.75 objective with Axiovision 4.1
software (Zeiss).
[0349] Immunofluorescence and Vessel Density Determination
[0350] HUVECs were starved in EGM-2 basal medium containing 1% FBS
for 16 h and treated with 1 .mu.g/ml endostatin for 4 h. The cells
were fixed with 4% paraformaldehyde for 10 min followed by
permeabilization with 0.1% Triton X-100 for 5 min. The cells were
washed with PBS, blocked with 5% bovine serum albumin in PBS and
treated with mouse anti-FILIP1L antibody (4 .mu.g/ml) preincubated
with 500 fold molar excess of either bovine serum albumin (BSA) or
FILIP1L. The cells were then incubated with 2 .mu.g/ml Alexa Fluor
488 anti-mouse IgG (Invitrogen) and treated with
4,6-diamidino-2-phenylindole (DAPI) mounting media (Vector
Laboratories). Images were acquired on a LSM-510 confocal
microscope using a 25.times./0.8, 40.times./1.3 or 63.times./1.4
objective and analyzed by AxioVision LE software (Zeiss). For
AAVP-targeting experiment, tumors from PBS-, AAVP-.DELTA.C103- and
AAVP-.DELTA.C243-treated mice were removed at 30 min and 6 days
after tail vein injection (30 min only for PBS-treated tumor), snap
frozen and cut into 10 .mu.m tumor sections. These tumor sections
were subjected to immunofluorescent staining as described above
except rabbit anti-AAVP antibody (Sigma) followed by Alexa Fluor
594 anti-rabbit IgG (Invitrogen) and rat anti-CD31 antibody (BD
Pharmingen) followed by Alexa Fluor 488 anti-rat IgG (Invitrogen)
were used.
[0351] CD31-stained tumor sections from PBS-, AAVP-null- and
AAVP-.DELTA.C103-treated mice were analyzed for vessel density as
described in Blansfield J A et al. Clin Cancer Res 2008; 14:270-80,
which is herein incorporated by reference for at least material
related to the analysis of vessel density. Three tumors from each
treatment group were analyzed. Five random fields per tumor were
imaged by Axiovert 200M microscope using a 10.times./0.3 objective
(Zeiss). Axiovision 4.6 software (Zeiss) was used to quantify
CD31-positive vessels. In addition, TUNEL staining was also
performed on these AAVP-treated tumors as recommended by the
manufacturer (Promega Corporation). Images were taken by Axiovert
200M microscope using a 5.times./0.15 objective (Zeiss).
[0352] Transfection of HUVECs with FILIP1L Plasmids
[0353] Plasmids encoding wild-type as well as truncation mutants of
FILIP1L were purified using Endo-free maxiprep kit (Qiagen). HUVECs
were transfected with equimolar amount of each DNA using HUVEC
nucleofector solution and Nucleofector II machine as provided by
the manufacturer (Amaxa). Transfection efficiency was verified
using a plasmid with an enhanced green fluorescent protein (eGFP)
marker (2 .mu.g) as was calculated by the GFP expression. The
percentage of transfection reached by this method was 50.+-.10%.
After transfection, the cells were subjected to proliferation,
apoptosis or migration assays.
[0354] Bromodeoxyuridine (BrdU) ELISA Cell Proliferation Assay
[0355] The transfected HUVECs were plated with 2.times.10.sup.4
cells/well in 96 well culture plates and incubated for 24 h. Cell
proliferation was measured by Cell Proliferation Biotrak ELISA (GE
Healthcare) as recommended by the manufacturer.
[0356] Apoptosis Assay
[0357] The transfected HUVECs were plated with 2.5.times.10.sup.4
cells/well in white-walled 96 well culture plates and incubated for
24 h. Early apoptosis was determined by the measurement of caspase
3/7 activity using the Caspase-Glo 3/7 Assay (Promega Corporation)
following manufacturer's instructions. The transfected HUVECs were
plated with 2.times.10.sup.6 cells/100 mm culture dishes and
incubated for 48 h. Late stage apoptosis was determined by the
staining of annexin V-fluorescein isothiocyanate (FITC) and
7-amino-actinomycin D (7-AAD) staining using the Annexin V-FITC
Apoptosis Detection Kit (BD Pharmingen) following manufacturer's
instructions. The stained cells were subjected to flow cytometric
analysis using a FACSCalibur (BD) and analyzed by the CELLQuest
program.
[0358] Migration Assay
[0359] The migratory potential of the transfected HUVECs was
assessed by Electric Cell-Substrate Impedance Sensing (ECIS Model
9600, Applied Biophysics Inc. (Giaever I and Keese C R. Nature
1993; 366:591-2; Keese C R et al. Proc Natl Acad Sci USA 2004;
101:1554-9). 1.1.times.10.sup.5 cells were inoculated in 8W1E+
plates in complete EGM-2 medium. The cells were allowed to
completely adhere to the electrodes which produced maximum and non
variable readings of impedance. The monolayers were then wounded
(30 seconds, 4.0 V, 60 kHz) where impedance became a minimum. As
cells migrated to heal the wound, the impedance was recorded at 15
kHz every 5 seconds for 10 hours in real time. The differences in
migration rate were evaluated by comparison of the slopes of the
curves in linear range for early time points.
[0360] DU145 clones transduced with either empty lentivirus or
lentivirus expressing FILIP1L.DELTA.C103 (7.5.times.10.sup.4
cells/chamber) were plated in the presence or absence of 1 .mu.g/ml
doxycycline in upper chamber. Migration toward 10% FBS was measured
at 15 h by QCM 24-well colorimetric cell migration assay kit
(Chemicon) as recommended by the manufacturer.
[0361] Cloning of FILIP1L and its Truncation Mutants
[0362] Genes for FILIP1L and its truncation mutants were cloned
into Gateway entry clones using multi-step PCR. The subsequent
entry clones were sequence verified throughout the entire cloned
region. Entry clones were then subcloned by Gateway LR
recombination using the manufacturer's protocols (Invitrogen) into
different expression vectors.
[0363] Lentivirus Generation and Development of Inducible Clones
Overexpressing FILIP1L.DELTA.c103
[0364] A lentiviral construct encoding FILIP1L.DELTA.C103 was used
to generate lentivirus expressing FILIP1L.DELTA.C103 by the
ViraPower.TM. T-REx.TM. Lentiviral Expression System (Invitrogen)
using the manufacturer's protocols. DU145 cells were transduced
with the Tet repressor-lentivirus, and screened for clones that
expressed the Tet repressor by western blot analysis. Tet
repressor-expressing DU145 cells were then transduced with either
empty lentivirus or lentivirus expressing FILIP1L.DELTA.C103, and
stable clones were screened by real time RT-PCR analysis.
[0365] Quantitative Real-Time RT-PCR
[0366] DU145 clones transduced with lentivirus expressing
FILIP1L.DELTA.C103 were cultured in the presence or absence of 1
.mu.g/ml doxycycline for 48 h and harvested. Total RNA was prepared
by RNeasy kit (Qiagen) and cDNA was prepared by Superscript II
reverse transcriptase (Invitrogen). qPCR was performed using ABI
7500 SDS real-time PCR instrument following manufacturer's
instructions (Applied Biosystems). The expression of the FILIP1L
gene was normalized to GAPDH expression. The primers used were:
5'-AACGCTGGTATCATGGCTGAA-3' (SEQ ID NO:9) and
5'-ATCTCTGCACTGCTCCTCCATT-3' (SEQ ID NO:10) for FILIP1L;
5'-TCACCAGGGCTGCTTTTAACTC-3' (SEQ ID NO:11) and
5'-GGAATCATATTGGAACATGTAAACCA-3' (SEQ ID NO:12) for GAPDH.
[0367] Construction and Generation of Targeted AAVP Particles
[0368] Cloning of both FILIP1L.DELTA.C103 (amino acid 1-790) and
FILIP1L.DELTA.C243 (amino acid 1-650) mutant cDNA into the AAVP
vector and the production of AAVP was performed as described
previously (Hajitou A et al. Nat Protoc 2007; 2:523-31; Hajitou A
et al. Cell 2006; 125:385-98).
[0369] Xenograft Assay
[0370] M21 human melanoma cells were injected subcutaneously into
female athymic nude mice, and grown to an average size of 100
mm.sup.3. Mice were randomly sorted into four groups (n=11 for each
group), AAVP (1.times.10.sup.11 transducing units per dose) was
injected intravenously at day 0 and day 7, and tumors were measured
in a blinded manner. Tumor volume was calculated as the product of
(length.times.width.times.height).times.0.52. All animal
experiments were conducted according to protocols approved by the
NIH Animal Care and Use Committee.
[0371] Statistical Analysis
[0372] Statistical analyses were performed using a two-tailed
Student's t test (GraphPad Prism 3.0) and differences were
considered to be statistically significant at a value of P less
than 0.05. Xenograft and vessel density data were analyzed using
One-way analysis of variance with Newman-Keuls Multiple Comparison
Test. A P value less than 0.05 was considered significant.
Results
[0373] Expression of FILIP1L Protein in HUVECs and Human Tissue
[0374] Although FILIP1L mRNA expression has been shown to be
upregulated in human endothelial cells in response to different
angiogenesis inhibitors (Mazzanti C M et al. Genome Res 2004;
14:1585-93; Tandle A T et al. Cytokine 2005; 30:347-58), the
expression of FILIP1L protein has not been previously investigated.
To determine whether FILIP1L protein is endogenously expressed in
human tissue, monoclonal antibodies that specifically recognize
FILIP1L were produced. To detect FILIP1L protein in endothelial
cells and to determine its subcellular localization, HUVECs were
fractionized and a western blot was performed using anti-FILIP1L
antibody. A specific 110 kDa band, identical size to the purified
FILIP1L protein, was detected by anti-FILIP1L antibody, suggesting
that HUVECs express a full length FILIP1L protein (FIG. 4). In
addition, FILIP1L was expressed predominantly in the cytoplasm with
less expression in the membrane and nucleus. Having demonstrated
the expression of FILIP1L protein in cultured endothelial cells,
the expression of FILIP1L in human tissue was then examined.
Immunohistochemical analysis was performed on 15 frozen human colon
cancers and matched normal colon tissues using anti-FILIP1L
antibody. In normal colon, FILIP1L was expressed in the vasculature
and muscularis mucosa. In colon cancer, FILIP1L was strongly
expressed in tumor stroma and the vasculature. Thus, these data
demonstrate that FILIP1L is expressed in vasculature and smooth
muscle, and in desmoplastic stroma in response to tumor invasion.
Endogenous FILIP1L protein in HUVECs was immunofluorescently
stained with FILIP1L antibody, which showed a punctate distribution
in the cytoplasm. Preincubation of FILIP1L antibody with FILIP1L
protein, but not with BSA, abrogated the staining.
[0375] Upregulation of FILIP1L Protein by Endostatin
[0376] Previous studies have shown that FILIP1L mRNA expression is
upregulated in HUVECs within 1 h following the treatment of
endothelial cells with the angiogenesis inhibitors endostatin,
fumagillin and EMAP II (Mazzanti). To further confirm that FILIP1L
protein expression is upregulated in endothelial cells in response
to angiogenesis inhibitors, HUVECs were treated with endostatin,
harvested cells at 2, 4 and 8 h, and performed western blot
analysis using anti-FILIP1L antibody on whole cell lysates.
Compared to vehicle-treated controls, endostatin-treated HUVECs
expressed more FILIP1L protein at all the time points tested
(densitometric quantitation values are also shown in FIG. 6A). To
examine if endostatin treatment affects cellular distribution of
FILIP1L protein in endothelial cells, HUVECs were serum starved to
synchronize them. HUVECs were treated with endostatin and
immunofluorescently stained them with anti-FILIP1L antibody at 4 h.
Serum-starved, vehicle-treated control cells demonstrated weak
cytoplasmic staining, whereas endostatin-treated cells showed a
stronger punctate distribution of staining in the cytoplasm.
FILIP1L expression measured by immunofluorescent staining in
endostatin-treated HUVECs was significantly more than that in
vehicle-treated control cells (P=0.0012) (FIG. 6B). In addition,
this staining was FILIP1L-specific as anti-FILIP1L antibody
preincubated with FILIP1L protein, but not with BSA control, failed
to demonstrate the staining. These results suggest that FILIP1L
protein expression is increased following endostatin treatment and
support the mRNA results. The punctate distribution in the
cytoplasm was detected in HUVECs treated with endostatin for 4 h by
immunofluorescent staining using anti-FILIP1L antibody.
Vehicle-treated control cells showed diffused cytoplasmic staining.
Preincubation of anti-FILIP1L antibody with FILIP1L protein, but
not with BSA, abrogated the punctuate staining seen in HUVECs
treated with endostatin.
Overexpression of FILIP1L in Endothelial Cells Leads to Inhibition
of Cell Proliferation and an Increase in Apoptosis
[0377] HUVECs were transfected with a plasmid encoding FILIP1L cDNA
and measured cell proliferation by BrdU ELISA 24 h after
transfection. Transfection efficiency was 50.+-.10%, as verified by
GFP expression following the transfection of HUVECs with a control
plasmid encoding an eGFP. Compared to control empty
vector-transfected cells, FILIP1L-transfected cells showed a
decrease in cell proliferation (P<0.0001). To determine if
overexpression of FILIP1L in endothelial cells results in an
increase in apoptosis, caspase 3/7 activity was measured at 24 h
following transfection of HUVECs with FILIP1L cDNA. Caspase 3/7
activity was measured by luminescence. Although caspase 3/7
activity in control vector-transfected cells was present due to the
cytotoxicity caused by the transfection procedure,
FILIP1L-transfected cells showed significantly more activity
(P<0.001). To further detect apoptosis in these cells, the
transfected cells were stained with annexin V-FITC and 7-AAD at 48
h following transfection, and measured staining using flow
cytometry. FILIP1L overexpression resulted in increased staining of
both annexin V-FITC and 7-AAD (44.4% vs. 15.2%), indicating that
late stage apoptosis is increased in FILIP1L-transfected cells
compared to control vector-transfected cells.
[0378] FILIP1L Truncation Mutants have Differential
Antiproliferative Activity
[0379] A coiled-coil region (residues 3-542), two leucine zipper
motifs (residues 83-111 and 218-253) and a prefoldin domain
(residues 465-535) can be recognized in the N-terminal half of the
FILIP1L protein. In addition, an NCBI conserved domain search
reveals that FILIP1L has a SbcC (COG0419; ATPase involved in DNA
repair; residues 19-576) conserved domain in its N-terminal half
and a Herpes_BLLF1 (pfam05109; Herpes virus major outer envelope
glycoprotein; residues 640-829) conserved domain in its C-terminal
half.
[0380] To examine which part of the FILIP1L protein mediates the
antiproliferative activity in endothelial cells, a series of
N-terminal and C-terminal truncation mutants of FILIP1L as a fusion
protein containing a C-terminal HA tag were generated. To determine
if these mutant constructs produce proteins in cells, HEK293 cells
were transfected with each construct and western blot analysis was
performed using anti-HA tag antibody. All the constructs produced
proteins of the predicted size, although N-terminal truncation
mutants 127-893 and 512-893 showed low levels of expression (FIG.
7). To determine if these proteins are functional, HUVECs were
transfected with the plasmid encoding each FILIP1L mutant, and
measured cell proliferation by BrdU ELISA 24 h after transfection.
FILIP1L truncation mutants 1-790, 1-650, 1-512 and 127-893
significantly inhibited cell proliferation compared to control.
C-terminal truncation mutant 1-790 was more potent in its ability
to inhibit cell proliferation than wild-type (P=0.001) (FIG. 2A).
To examine if overexpression of FILIP1L.DELTA.C103 (amino acids
1-790) in endothelial cells results in an increase in apoptosis, we
measured caspase 3/7 activity at 24 h following transfection of
HUVECs with FILIP1L.DELTA.C103 cDNA. FILIP1L.DELTA.C103-transfected
cells showed significantly more apoptotic activity than control
cells (P<0.0001).
[0381] Overexpression of FILIP1L.DELTA.C103 in HUVECs as Well as
DU145 Prostate Cancer Cells Leads to Inhibition of Cell
Migration
[0382] Since inhibition of cell migration is one of the important
characteristics of angiogenesis inhibitors, it was tested whether
overexpression of FILIP1L.DELTA.C103 results in inhibition of cell
migration. To do this, HUVECs were transfected with a plasmid
encoding FILIP1L.DELTA.C103 cDNA and cell migration was measured by
Electric Cell-Substrate Impedance Sensing system (Applied
Biophysics Inc. (Giaever I and Keese C R. Nature 1993; 366:591-2;
Keese C R et al. Proc Natl Acad Sci USA 2004; 101:1554-9)).
Compared to control empty vector-transfected cells,
FILIP1L.DELTA.C103-transfected cells showed a significantly slower
migration rate (P<0.0001) (FIG. 3), indicating that
overexpression of FILIP1L.DELTA.C103 in HUVECs results in
inhibition of cell migration.
[0383] The effects of overexpression of FILIP1L.DELTA.C103 on
migration of neoplastic cell lines was also tested. DU145 prostate
cancer cells were selected as a model system because FILIP1L mRNA
expression was shown to be repressed in immortalized prostate
epithelial cells (Schwarze S R et al. J Biol Chem 2002;
277:14877-83; Schwarze S R et al. Neoplasia 2005; 7:816-23), and
FILIP1L mRNA expression is relatively low in this cell line
compared to other cancer cell lines. Overexpression of
FILIP1L.DELTA.C103 in DU145 cells also resulted in inhibition of
cell proliferation. Thus, inducible
FILIP1L.DELTA.C103-overexpressing clones were developed. The system
used was the ViraPower.TM. T-REx.TM. Lentiviral Expression System
(Invitrogen). In this system the expression of a gene of interest
is repressed by a Tet repressor in the absence of tetracycline (or
doxycycline), whereas it is derepressed in the presence of
tetracycline (or doxycycline). Clones were screened by real time
RT-PCR analysis. Several clones showed a 2.5-7 fold increase in
FILIP1L.DELTA.C103 mRNA expression following doxycycline induction
compared to the uninduced condition (FIG. 8A).
[0384] Unexpectedly, however, it was observed that, at the
uninduced basal level, most of these clones expressed 20-60 fold
more FILIP1L.DELTA.C103 mRNA than parental Tet repressor-expressing
DU145 cells (FIG. 8A). FILIP1L.DELTA.C103 protein levels were shown
to be increased considerably by doxycycline, but the basal level
expression was also detectable in these clones (FIG. 8B). Control
cells that were a mixed population from empty lentivirus-transduced
Tet repressor-expressing DU145, however, did not produce any
FILIP1L.DELTA.C103 protein (FIG. 8B). To measure cell migration for
these cells, the Boyden chamber assay was utilized. Using this
system, cell migration was measured for the FILIP1L.DELTA.C103
clones #2, 12 and 13 as well as control cells. All the
FILIP1L.DELTA.C103 clones, but not control cells, showed a
significantly slower migration in the presence of doxycycline
(P<0.005) (FIG. 8C). Therefore, these data indicate that
overexpression of FILIP1L.DELTA.C103 in DU145 cells also results in
inhibition of cell migration.
[0385] Targeted Expression of FILIP1L.DELTA.C103 in Tumor
Vasculature Results in Inhibition of Tumor Growth In Vivo
[0386] It has been demonstrated that overexpression of FILIP1L
results in inhibition of cell proliferation and migration, and
increased apoptosis in vitro. The effects of targeted FILIP1L
expression in vivo were then evaluated. In particular, it was
chosen to selectively deliver FILIP1L to tumor vasculature in order
to determine if overexpression of FILIP1L in tumor vasculature
leads to an antitumor effect. To achieve this, a hybrid
adeno-associated virus/phage (AAVP) vector was utilized, which has
been shown to specifically target tumor vasculature in an RGD
peptide-restricted manner (Hajitou A et al. Nat Protoc 2007;
2:523-31; Hajitou A et al. Cell 2006; 125:385-98; Soghomonyan S et
al. Nat Protoc 2007; 2:416-23). These hybrid vectors rely on the
specific binding relationship between an RGD peptide and a,
integrin expressed on the surface of tumor vasculature. These
vectors have been shown to specifically traffic to, and
specifically transfect, tumor associated endothelial cells without
evidence of transfection of normal endothelial cells. Two FILIP1L
mutants were tested for this purpose: FILIP1L.DELTA.C103 (amino
acid 1-790) and FILIP1L.DELTA.C243 (amino acid 1-650). Each FILIP1L
mutant cDNA was cloned into the AAVP vector, produced RGD-targeted
AAVP and screened for the AAVP which showed the highest expression
of each mutant by real time RT-PCR analysis.
[0387] In order to examine whether the AAVP specifically targets
tumor vasculature in our M21 human melanoma model, the RGD-targeted
AAVP was injected into the tail vein of female athymic nude mice
harboring a 100-150 mm.sup.3 subcutaneous M21 tumor. After
injection, tumors were harvested by time-course, and analyzed by
immunofluorescent staining with anti-CD31 and anti-AAVP antibodies.
Both RGD-4C-FILIP1L.DELTA.C103 AAVP (AAVP expressing FILIP1L mutant
1-790; referred to as AAVP-.DELTA.C103) and
RGD-4C-FILIP1L.DELTA.C243 AAVP (AAVP expressing FILIP1L mutant
1-650; here-after referred to as AAVP-.DELTA.C243) were shown to
specifically target tumor vasculature, but not tumor cells or the
vasculature of normal organs. FILIP1L protein expression was also
measured in these tumors by western blot analysis using
anti-FILIP1L antibody on whole tumor lysates. Tumors from
AAVP-.DELTA.C103-treated mice, but not PBS- and RGD-4C AAVP
(control null AAVP; referred to as AAVP-null)-treated mice, showed
FILIP1L protein expression. Therefore, these data are consistent
with the observation that the FILIP1L mutant is expressed in the
tumor vasculature of mice treated with AAVP-.DELTA.C103.
AAVP-.DELTA.C103 injected intravenously specifically targeted tumor
vasculature. Tumors from PBS- and AAVP-.DELTA.C103-treated mice
were immunofluorescently stained with anti-AAVP antibody followed
by Alexa Fluor 594 anti-rabbit IgG antibody followed by Alexa Fluor
488 anti-rat IgG (green for the blood vessel staining). A 90 kDa
FILIP1L.DELTA.C103 protein was detected in whole tumor lysates from
AAVP-.DELTA.C103-treated mice, but not PBS- and AAVP-null-treated
mice, by western blot using anti-FILIP1L antibody. Three different
tumors per group were analyzed. Vessel density was significantly
decreased in tumors from AAVP-.DELTA.C103-treated mice compared to
those from AAVP-null-treated mice (P<0.01) and PBS-treated mice
(P<0.001) at day 4. (FIG. 5)
[0388] To evaluate the efficacy of the FILIP1L mutant-AAVP
treatment on tumor growth inhibition in vivo, M21 melanoma cells
were injected subcutaneously into female athymic nude mice, and
grown to an average size of 100 mm.sup.3. Mice were randomly sorted
into four groups (n=11 for each group), AAVP (1.times.10.sup.11
transducing units per dose) was injected intravenously at day 0 and
day 7, and tumors were measured in a blinded manner. The following
groups were tested: PBS, AAVP-null, AAVP-.DELTA.C243 and
AAVP-.DELTA.C103. PBS control tumors grew aggressively and started
to show central necrosis by day 14. Thus, the experiments were
terminated at day 14. Only tumors from AAVP-.DELTA.C103-treated
mice were significantly smaller than those from PBS-treated mice by
day 14 (P<0.01) (FIG. 9). Although tumors from AAVP-null-treated
mice and AAVP-.DELTA.C243-treated mice were smaller than those from
PBS-treated mice, the differences were not statistically
significant. In addition, tumors from AAVP-.DELTA.C103-treated mice
were significantly smaller than those from AAVP-null-treated mice
(P<0.05) and AAVP-.DELTA.C243-treated mice (P<0.05) by day 14
(FIG. 9). These results indicate that targeted expression of
FILIP1L.DELTA.C103 in tumor vasculature results in inhibition of
M21 melanoma growth in vivo.
[0389] In order to confirm that the inhibition of tumor growth is
dependent on the antivascular effects of FILIP1L.DELTA.C103, vessel
density was analyzed for these AAVP-treated tumors. The percentage
area of CD31 positive cells was used as a measure of vessel density
(Blansfield J A et al. Clin Cancer Res 2008; 14:270-80). Vessel
density from AAVP-.DELTA.C103-treated tumors was significantly less
than those from PBS-treated tumors (P<0.001) and
AAVP-null-treated tumors (P<0.01) at day 4, indicating that the
inhibition of tumor vasculature by AAVP-.DELTA.C103 leads to the
inhibition of M21 tumor growth. In addition,
AAVP-.DELTA.C103-treated tumors showed extensive apoptosis as
measured by TUNEL staining compared to PBS- or AAVP-null-treated
tumors, further indicating that the inhibition of tumor vasculature
by AAVP-.DELTA.C103 results in induction of apoptosis and necrosis
in these M21 tumors.
[0390] a. Sequences
TABLE-US-00003 DOC1 Isoform 2 (SEQ ID NO: 1; GenBank Accession No.
NP_055705.2 (893 aa)): mvvdeqqrlt aqltlqrqki qelttnaket htklalaear
vqeeeqkatr lekelqtqtt kfhqdqdtim akltnedsqn rqlqqklaal srqideleet
nrslrkaeee lqdikekisk geygnagima eveelrkrvl dmegkdeeli kmeeqcrdln
krleretlqs kdfklevekl skrimalekl edafnkskqe cyslkcnlek ermttkqlsq
eleslkvrik eleaiesrle kteftlkedl tklktltvmf vderktmsek lkktedklqa
assqlqveqn kvttvtekli eetkralksk tdveekmysv tkerddlknk lkaeeekgnd
llsrvnmlkn rlqsleaiek dflknklnqd sgksttalhq ennkikelsq everlklklk
dmkaieddlm ktedeyetle rryanerdka qflskelehv kmelakykla ektetsheqw
lfkrlgeeea ksghlsrevd alkekiheym atedlichlq gdhsvlqkkl nqqenrnrdl
greienitke leryrhfsks lrpslngrri sdpqvfskev qteavdnepp dykslipler
avingqlyee senqdedpnd egsvlsfkcs qstpcpvnrk lwipwmkske ghlqngkmqt
kpnanfvqpg dlvlshtpgq plhikvtpdh vqntatleit spttesphsy tstavipncg
tpkqritilq nasitpvksk tstedlmnle qgmspitmat faraqtpesc gsltpertms
piqvlavtgs asspeqgrsp epteisakha ifrvspdrqs swqfqrsnsn sssvittedn
kihihlgspy mqavaspvrp aspsaplqdn rtqglingal nkttnkvtss ititptatpl
prqsqitvsn iyn DOC1 variant 2 (GenBank Accession No. NP_055705; SEQ
ID NO: 2 (752 aa)) mvvdeqqrlt aqltlqrqki qelttnaket htklalaear
vqeeeqkatr lekelqtqtt kfhqdqdtim akltnedsqn rqlqqklaal srqideleet
nrslrkaeee lqdikekisk geygnagima eveelikmee gcrdlnkrle retlqskdfk
leveklskri malekledaf nkskqecysl kcnlekermt tkqlsqeles lkvrikelea
iesrlektef tlkedltklk tltvmfvder ktmseklkkt edklqaassq lqveqnkvtt
vteklieetk ralksktdve ekmysvtker ddlknklkae eekgndllsr vnmlknrlqs
leaiekdflk nklnqdsgks ttalhqennk ikelsqever lklklkdmka ieddlmkted
eyetlerrya nerdkaqfls kelehvkmel akyklaekte tsheqwlfkr lqeeeaksgh
lsrevdalke kiheymated lichlqgdhs vckkklnqqe nrnrdlgrei enltkelery
rhfskslrps lngrrisdpq vfskevqtea vdneppdyks lipleravin gqlyeesenq
dedpndegsv lsfkcsgstp cpvnrklwip wmkskeghlq ngkmqtkpna nfvqpgdlvl
shtpgqplhi kvtpdhvqnt atleitsptt esphsytsta vipncgtpkq ritilqnasi
tpvksktste dlmnleqgms pitmatfara qtpescgslt pertmslfrf wl DOC1
Isoform 1 (GenBank Accession No. NP_878913.2; SEQ ID NO: 3 (1135
aa)) mrsrgsdteg saqkkfprht kghsfqgpkn mkhrqqdkds psesdvilpc
pkaekphsgn ghqaedlsrd dllfllsile gelqardevi gilkaekmdl alleaqygfv
tpkkvlealq rdafqakstp wqediyekpm neldkvvekh kesyrrilgq llvaeksrrq
tileleeekr khkeymeksd eficlleqec erlkklidge iksqeekeqe kekrvttlke
eltklksfal mvvdeqqrlt aqltlqrqki qelttnaket htklalaear vqeeeqkatr
lekelqtqtt kfhqdqdtim akltnedsqn rqlqqklaal srqideleet nrslrkaeee
lqdikekisk geygnagima eveelrkrvl dmegkdeeli kmeeqcrdln krleretlqs
kdfklevekl skrimalekl edafnkskqe cyslkcnlek ermttkqlsq eleslkvrik
eleaiesrle kteftlkedl tklktltvmf vderktmsek lkktedklqa assqlqveqn
kvttvtekli eetkralksk tdveekmysv tkerddlknk lkaeeekgnd llsrvnmlkn
rlqsleaiek dflknklnqd sgksttalhq ennkikelsq everlklklk dmkaieddlm
ktedeyetle rryanerdka qflskelehv kmelakykla ektetsheqw lfkrlgeeea
ksghlsrevd alkekiheym atedlichlq gdhsvlqkkl nggenrnrdl greienitke
leryrhfsks lrpslngrri sdpqvfskev qteavdnepp dykslipler avingglyee
sengdedpnd egsvlsfkcs qstpcpvnrk lwipwmkske ghlqngkmqt kpnanfvqpg
dlvlshtpgq plhikvtpdh vqntatleit spttesphsy tstavipncg tpkgritilq
nasitpvksk tstedlmnle qgmspitmat faraqtpesc gsltpertms piqvlavtgs
asspeqgrsp epteisakha ifrvspdrqs swqfqrsnsn sssvittedn kihihlgspy
mqavaspvrp aspsaplqdn rtqglingal nkttnkvtss ititptatpl prqsgitvep
lllph DOC1 Isoform 3 (GenBank Accession No. NP_001035924.1; SEQ ID
NO: 4 (1133 aa)) mrsrgsdteg saqkkfprht kghsfqgpkn mkhrqqdkds
psesdvilpc pkaekphsgn ghqaedlsrd dllfllsile gelqardevi gilkaekmdl
alleaqygfv tpkkvlealq rdafqakstp wqediyekpm neldkvvekh kesyrrilgq
llvaeksrrq tileleeekr khkeymeksd eficllegec erlkklidge iksgeekege
kekrvttlke eltklksfal mvvdeqqrlt aqltlqrqki qelttnaket htklalaear
vgeeegkatr lekelqtqtt kfhqdqdtim akltnedsqn rqlqqklaal srqideleet
nrslrkaeee lqdikekisk geygnagima eveelrkrvl dmegkdeeli kmeeqcrdln
krleretlqs kdfklevekl skrimalekl edafnkskqe cyslkcnlek ermttkqlsq
eleslkvrik eleaiesrle kteftlkedl tklktltvmf vderktmsek lkktedklqa
assqlgvegn kvttvtekli eetkralksk tdveekmysv tkerddlknk lkaeeekgnd
llsrvnmlkn rlqsleaiek dflknklnqd sgksttalhq ennkikelsq everlklklk
dmkaieddlm ktedeyetle rryanerdka qflskelehv kmelakykla ektetsheqw
lfkrlgeeea ksghlsrevd alkekiheym atedlichlq gdhsvlqkkl nggenrnrdl
greienitke leryrhfsks lrpslngrri sdpqvfskev qteavdnepp dykslipler
avingglyee sengdedpnd egsvlsfkcs qstpcpvnrk lwipwmkske ghlqngkmqt
kpnanfvqpg dlvlshtpgq plhikvtpdh vqntatleit spttesphsy tstavipncg
tpkgritilq nasitpvksk tstedlmnle qgmspitmat faraqtpesc gsltpertms
piqvlavtgs asspeqgrsp epteisakha ifrvspdrqs swqfqrsnsn sssvittedn
kihihlgspy mqavaspvrp aspsaplqdn rtqglingal nkttnkvtss ititptatpl
prqsgitvsn iyn SEQ ID NO: 5: AGCGTAACCAAGGAGAGAGAT SEQ ID NO: 6
ATTCATTCATTCATTCACCAT SEQ ID NO: 7 1 ataggccggg cgcgctcagc
gccccgctcg cattgttcgg gcgactctcg gagcgcgcac 61 agtcggctcg
cagcgcggca ctacagcggc cccggcccgg cccccgcccg gccccggcgc 121
aggcagttca gattaaagaa gctaattgat caagaaatca agtctcagga ggagaaggag
181 caagaaaagg agaaaagggt caccaccctg aaagaggagc tgaccaagct
gaagtctttt 241 gctttgatgg tggtggatga acagcaaagg ctgacggcac
agctcaccct tcaaagacag 301 aaaatccaag agctgaccac aaatgcaaag
gaaacacata ccaaactagc ccttgctgaa 361 gccagagttc aggaggaaga
gcagaaggca accagactag agaaggaact gcaaacgcag 421 accacaaagt
ttcaccaaga ccaagacaca attatggcga agctcaccaa tgaggacagt 481
caaaatcgcc agcttcaaca aaagctggca gcactcagcc ggcagattga tgagttagaa
541 gagacaaaca ggtctttacg aaaagcagaa gaggagctgc aagatataaa
agaaaaaatc 601 agtaagggag aatatggaaa cgctggtatc atggctgaag
tggaagagct caggaaacgt 661 gtgctagata tggaagggaa agatgaagag
ctcataaaaa tggaggagca gtgcagagat 721 ctcaataaga ggcttgaaag
ggagacgtta cagagtaaag actttaaact agaggttgaa 781 aaactcagta
aaagaattat ggctctggaa aagttagaag acgctttcaa caaaagcaaa 841
caagaatgct actctctgaa atgcaattta gaaaaagaaa ggatgaccac aaagcagttg
901 tctcaagaac tggagagttt aaaagtaagg atcaaagagc tagaagccat
tgaaagtcgg 961 ctagaaaaga cagaattcac tctaaaagag gatttaacta
aactgaaaac attaactgtg 1021 atgtttgtag atgaacggaa aacaatgagt
gaaaaattaa agaaaactga agataaatta 1081 caagctgctt cttctcagct
tcaagtggag caaaataaag taacaacagt tactgagaag 1141 ttaattgagg
aaactaaaag ggcgctcaag tccaaaaccg atgtagaaga aaagatgtac 1201
agcgtaacca aggagagaga tgatttaaaa aacaaattga aagcggaaga agagaaagga
1261 aatgatctcc tgtcaagagt taatatgttg aaaaataggc ttcaatcatt
ggaagcaatt 1321 gagaaagatt tcctaaaaaa caaattaaat caagactctg
ggaaatccac aacagcatta 1381 caccaagaaa acaataagat taaggagctc
tctcaagaag tggaaagact gaaactgaag 1441 ctaaaggaca tgaaagccat
tgaggatgac ctcatgaaaa cagaagatga atatgagact 1501 ctagaacgaa
ggtatgctaa tgaacgagac aaagctcaat ttttatctaa agagctagaa 1561
catgttaaaa tggaacttgc taagtacaag ttagcagaaa agacagagac cagccatgaa
1621 caatggcttt tcaaaaggct tcaagaagaa gaagctaagt cagggcacct
ctcaagagaa 1681 gtggatgcat taaaagagaa aattcatgaa tacatggcaa
ctgaagacct aatatgtcac 1741 ctccagggag atcactcagt cctgcaaaaa
aaactaaatc aacaagaaaa caggaacaga 1801 gatttaggaa gagagattga
aaacctcact aaggagttag agaggtaccg gcatttcagt 1861 aagagcctca
ggcctagtct caatggaaga agaatttccg atcctcaagt attttctaaa
1921 gaagttcaga cagaagcagt agacaatgaa ccacctgatt acaagagcct
cattcctctg 1981 gaacgtgcag tcatcaatgg tcagttatat gaggagagtg
agaatcaaga cgaggaccct 2041 aatgatgagg gatctgtgct gtccttcaaa
tgcagccagt ctactccatg tcctgttaac 2101 agaaagctat ggattccctg
gatgaaatcc aaggagggcc atcttcagaa tggaaaaatg 2161 caaactaaac
ccaatgccaa ctttgtgcaa cctggagatc tagtcctaag ccacacacct 2221
gggcagccac ttcatataaa ggttactcca gaccatgtac aaaacacagc cactcttgaa
2281 atcacaagtc caaccacaga gagtcctcac tcttacacga gtactgcagt
gataccgaac 2341 tgtggcacgc caaagcaaag gataaccatc ctccaaaacg
cctccataac accagtaaag 2401 tccaaaacct ctaccgaaga cctcatgaat
ttagaacaag gcatgtcccc aattaccatg 2461 gcaacctttg ccagagcaca
gaccccagag tcttgtggtt ctctaactcc agaaaggaca 2521 atgtccccta
ttcaggtttt ggctgtgact ggttcagcta gctctcctga gcagggacgc 2581
tccccagaac caacagaaat cagtgccaag catgcgatat tcagagtctc cccagaccgg
2641 cagtcatcat ggcagtttca gcgttcaaac agcaatagct caagtgtgat
aactactgag 2701 gataataaaa tccacattca cttaggaagt ccttacatgc
aagctgtagc cagccctgtg 2761 agacctgcca gcccttcagc accactgcag
gataaccgaa ctcaaggctt aattaacggg 2821 gcactaaaca aaacaaccaa
taaagtcacc agcagtatta ctatcacacc aacagccaca 2881 cctcttcctc
gacaatcaca aattacagta agtaatatat ataactgacc acgctcaccc 2941
tcatccagtc catactgata tttttgcaag gaactcaatc cttttttaat catccctcca
3001 tatcccccaa gactgactga actcgtactt tgggaaggtt tgtgcatgaa
ctatacaaga 3061 gtatctgaaa ctaactgttg cctgcatagt catatcgagt
gtgcacttac tgtatatctt 3121 ttcatttaca tacttgtatg gaaaatattt
agtctgcact tgtataaata catctttatg 3181 tatttcattt tccataactc
actttaattt gactgcaact tgtcttggtg aaatacttta 3241 acattataaa
acagtaaata atttgttatt ttta SEQ ID NO: 8 mvvdeqqrlt aqltlqrqkv
qelttnaket htklalaear vgeeegkatr lekelqtqtt kfhqdqdtim akltnedsqn
rqlqqklaal srqideleet nrslrkaeee lqdikekisk geygnagima eveelrkrvl
dmegkdeeli kmeeqcrdln krleretlqs kdfklevekl skrimalekl edafnkskqe
cyslkcnlek ermttkqlsq eleslkvrik eleaiesrle kteftlkedl tklktltvmf
vderktmsek lkktedklqa assqlgvegn kvttvtekli eetkralksk tdveekmysv
tkerddlknk lkaeeekgnd llsrvnmlkn rlqsleaiek dflknklnqd sgksttalhq
ennkikelsq everlklklk dmkaieddlm ktedeyetle rryanerdka qflskelehv
kmelakykla ektetsheqw lfkrlgeeea ksghlsrevd alkekiheym atedlichlq
gdhsvlqkkl nggenrnrdl greienitke leryrhfsks lrpslngrri sdpqvfskev
qteavdnepp dykslipler avingglyee sengdedpnd egsvlsfkcs qstpcpvnrk
lwipwmkske ghlqngkmqt kpnanfvqpg dlvlshtpgq plhikvtpdh vqntatleit
spttesphsy tstavipncg tpkgritilq nasitpvksk tstedlmnle qgmspitmat
faraqtpesc gsltpertms piqvlavtgs asspeqgrsp epteisakha ifrvspdrqs
swqfqrsnsn sssvittedn kihihlgspy mqavaspvrp aspsaplqdn rtqglingal
nkttnkvtss ititptatpl prqsgitvsn iyn SEQ ID NO: 9
AACGCTGGTATCATGGCTGAA SEQ ID NO: 10 ATCTCTGCACTGCTCCTCCATT SEQ ID
NO: 11 TCACCAGGGCTGCTTTTAACTC SEQ ID NO: 12
GGAATCATATTGGAACATGTAAACCA
[0391] Information for DOC1 (FILIP1L) Amino Acid and Gene:
TABLE-US-00004 LOCUS NM_014890 3274 bp mRNA linear PRI 03-SEP-2007
DEFINITION Homo sapiens filamin A interacting protein 1-like
(FILIP1L), transcript variant 2, mRNA. ACCESSION NM_014890 VERSION
NM_014890.2 GI:109659848 KEYWORDS . SOURCE Homo sapiens (human)
ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;
Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;
Primates; Haplorrhini; Catarrhini; Hominidae; Homo. REFERENCE 1
(bases 1 to 3274) AUTHORS Tandle,A.T., Mazzanti,C., Alexander,H.R.,
Roberts,D.D. and Libutti,S.K. TITLE Endothelial monocyte activating
polypeptide-II induced gene expression changes in endothelial cells
JOURNAL Cytokine 30 (6), 347-358 (2005) PUBMED 15935955 REMARK
GeneRIF: DOC1 might play a role in mediating some of the effects of
EMAP-II on endothelial cells REFERENCE 2 (bases 1 to 3274) AUTHORS
Suzuki,Y., Yamashita,R., Shirota,M., Sakakibara,Y., Chiba,J.,
Mizushima-Sugano,J., Nakai,K. and Sugano,S. TITLE Sequence
comparison of human and mouse genes reveals a homologous structure
in the promoter regions JOURNAL Genome Res. 14 (9), 1711-1718
(2004) PUBMED 15342556 REFERENCE 3 (bases 1 to 3274) AUTHORS
Mok,S.C., Wong,K.K., Chan,R.K., Lau,C.C., Tsao,S.W., Knapp,R.C. and
Berkowitz,R.S. TITLE Molecular cloning of differentially expressed
genes in human epithelial ovarian cancer JOURNAL Gynecol. Oncol. 52
(2), 247-252 (1994) PUBMED 8314147 COMMENT VALIDATED REFSEQ: This
record has undergone preliminary review of the sequence, but has
not yet been subject to final review. The reference sequence was
derived from BP233250.1 and BC027860.1. On Jun. 27, 2006 this
sequence version replaced gi:7657036. COMPLETENESS: complete on the
3' end. PRIMARY REFSEQ_SPAN PRIMARY_IDENTIFIER PRIMARY_SPAN COMP
1-583 BP233250.1 1-583 584-3274 BC027860.1 516-3206 FEATURES
Location/Qualifiers source 1..3274 /organism=''Homo sapiens''
/mol_type=''mRNA'' /db_xref=''taxon:9606'' /chromosome=''3''
/map=''3q12.1'' gene 1..3274 gene=''FILIP1L'' /note=''filamin A
interacting protein 1-like; synonyms: DOC1, DOC-1, GIP90''
/db_xref=''GeneID:11259'' /db_xref=''HGNC:24589''
/db_xref=''HPRD:16832'' exon 1..131 /gene=''FILIP1L''
/note=''alignment:Splign'' /number=5 exon 132..3274
/gene=''FILIP1L'' /note=''alignment:Splign'' /number=6b CDS
247..2928 /gene=''FILIP1L'' /GO_component=''myosin''
/note=''isoform 2 is encoded by transcript variant 2''
/codon_start=1 /product=''downregulated in ovarian cancer 1 isoform
2'' /protein_id=''NP_055705.7'' /db_xref=''GI:109659849''
/db_xref=''GeneID:11259'' /db xref=''HGNC:24589''
/db_xref=''HPRD:16832'' /translation= (SEQ ID NO: 1)
MVVDEQQRLTAQLTLQRQKIQELTTNAKETHTKLALAEARVQEE
EQKATRLEKELQTQTTKEHQDQDTIMAKLTNEDSQNRQLQQKLAALSRQIDELEETNR
SLRKAEEELQDIKEKISKGEYGNAGIMAEVEELRKRVLDMEGKDEELIKMEEQCRDLN
KRLERETLQSKDFKLEVEKLSKRIMALEKLEDAFNKSKQECYSLKCNLEKERMTTKQL
SQELESLKVRIKELEAIESRLEKTEFTLKEDLTKLKTLTVMFVDERKTMSEKLKKTED
KLQAASSQLQVEQNKVTTVTEKLIEETKRALKSKTDVEEKMYSVTKERDDLKNKLKAE
EEKGNDLLSRVNMLKNRLQSLEATEKDFLKNKLNQDSGKSTTALHQENNKIKELSQEV
ERLKLKLKDMKAIEDDLMKTEDEYETLERRYANERDKAQFLSKELEHVKMELAKYKLA
EKTETSHEQWLFKRLQEEEAKSGHLSREVDALKEKIHEYMATEDLICHLQGDHSVLQK
KLNQQENRNRDLGREIENLTKELERYRHFSKSLRPSLNGRRISDPQVFSKEVQTEAVD
NEPPDYKSLIPLERAVINGQLYEESENQDEDPNDEGSVLSEKCSQSTPCPVNRKLWIP
WMKSKEGHLQNGKMQTKPNANFVQPGDLVLSHTPGQPLHIKVTPDHVQNTATLEITSP
TTESPHSYTSTAVIPNCGTPKQRITILQNASITPVKSKTSTEDLMNLEQGMSPITMAT
FARAQTPESCGSLTPERTMSPIQVLAVTGSASSPEQGRSPEPTEISAKHAIERVSPDR
QSSWQFQRSNSNSSSVITTEDNKIHIHLGSPYMQAVASPVRPASPSAPLQDNRTQGLI
NGALNKTTNKVTSSITITPTATPLPRQSQITVSNIYN STS 2535..2674
/gene=''FILIP1L'' /standard_name=''RH16583''
/db_xref=''UniSTS:72547'' STS 2669..2798 /gene=''FILIP1L''
/standard_name=''SHGC-33580'' /db_xref=''UniSTS:171033'' STS
2822..3237 /gene=''FILIP1L'' /standard_name=''DOC1_9246''
/db_xref=''UniSTS:468384'' 1 ataggccggg cgcgctcagc gccccgctcg
cattgttcgg gcgactctcg gagcgcgcac 61 agtcggctcg cagcgcggca
ctacagcggc cccggcccgg cccccgcccg gccccggcgc 121 aggcagttca
gattaaagaa gctaattgat caagaaatca agtctcagga ggagaaggag 181
caagaaaagg agaaaagggt caccaccctg aaagaggagc tgaccaagct gaagtctttt
241 gctttgatgg tggtggatga acagcaaagg ctgacggcac agctcaccct
tcaaagacag 301 aaaatccaag agctgaccac aaatgcaaag gaaacacata
ccaaactagc ccttgctgaa 361 gccagagttc aggaggaaga gcagaaggca
accagactag agaaggaact gcaaacgcag 421 accacaaagt ttcaccaaga
ccaagacaca attatggcga agctcaccaa tgaggacagt 481 caaaatcgcc
agcttcaaca aaagctggca gcactcagcc ggcagattga tgagttagaa 541
gagacaaaca ggtctttacg aaaagcagaa gaggagctgc aagatataaa agaaaaaatc
601 agtaagggag aatatggaaa cgctggtatc atggctgaag tggaagagct
caggaaacgt 661 gtgctagata tggaagggaa agatgaagag ctcataaaaa
tggaggagca gtgcagagat 721 ctcaataaga ggcttgaaag ggagacgtta
cagagtaaag actttaaact agaggttgaa 781 aaactcagta aaagaattat
ggctctggaa aagttagaag acgctttcaa caaaagcaaa 841 caagaatgct
actctctgaa atgcaattta gaaaaagaaa ggatgaccac aaagcagttg 901
tctcaagaac tggagagttt aaaagtaagg atcaaagagc tagaagccat tgaaagtcgg
961 ctagaaaaga cagaattcac tctaaaagag gatttaacta aactgaaaac
attaactgtg 1021 atgtttgtag atgaacggaa aacaatgagt gaaaaattaa
agaaaactga agataaatta 1081 caagctgctt cttctcagct tcaagtggag
caaaataaag taacaacagt tactgagaag 1141 ttaattgagg aaactaaaag
ggcgctcaag tccaaaaccg atgtagaaga aaagatgtac 1201 agcgtaacca
aggagagaga tgatttaaaa aacaaattga aagcggaaga agagaaagga 1261
aatgatctcc tgtcaagagt taatatgttg aaaaataggc ttcaatcatt ggaagcaatt
1321 gagaaagatt tcctaaaaaa caaattaaat caagactctg ggaaatccac
aacagcatta 1381 caccaagaaa acaataagat taaggagctc tctcaagaag
tggaaagact gaaactgaag 1441 ctaaaggaca tgaaagccat tgaggatgac
ctcatgaaaa cagaagatga atatgagact 1501 ctagaacgaa ggtatgctaa
tgaacgagac aaagctcaat ttttatctaa agagctagaa 1561 catgttaaaa
tggaacttgc taagtacaag ttagcagaaa agacagagac cagccatgaa 1621
caatggcttt tcaaaaggct tcaagaagaa gaagctaagt cagggcacct ctcaagagaa
1681 gtggatgcat taaaagagaa aattcatgaa tacatggcaa ctgaagacct
aatatgtcac 1741 ctccagggag atcactcagt cctgcaaaaa aaactaaatc
aacaagaaaa caggaacaga 1801 gatttaggaa gagagattga aaacctcact
aaggagttag agaggtaccg gcatttcagt 1861 aagagcctca ggcctagtct
caatggaaga agaatttccg atcctcaagt attttctaaa 1921 gaagttcaga
cagaagcagt agacaatgaa ccacctgatt acaagagcct cattcctctg 1981
gaacgtgcag tcatcaatgg tcagttatat gaggagagtg agaatcaaga cgaggaccct
2041 aatgatgagg gatctgtgct gtccttcaaa tgcagccagt ctactccatg
tcctgttaac 2101 agaaagctat ggattccctg gatgaaatcc aaggagggcc
atcttcagaa tggaaaaatg 2161 caaactaaac ccaatgccaa ctttgtgcaa
cctggagatc tagtcctaag ccacacacct 2221 gggcagccac ttcatataaa
ggttactcca gaccatgtac aaaacacagc cactcttgaa 2281 atcacaagtc
caaccacaga gagtcctcac tcttacacga gtactgcagt gataccgaac 2341
tgtggcacgc caaagcaaag gataaccatc ctccaaaacg cctccataac accagtaaag
2401 tccaaaacct ctaccgaaga cctcatgaat ttagaacaag gcatgtcccc
aattaccatg 2461 gcaacctttg ccagagcaca gaccccagag tcttgtggtt
ctctaactcc agaaaggaca 2521 atgtccccta ttcaggtttt ggctgtgact
ggttcagcta gctctcctga gcagggacgc 2581 tccccagaac caacagaaat
cagtgccaag catgcgatat tcagagtctc cccagaccgg 2641 cagtcatcat
ggcagtttca gcgttcaaac agcaatagct caagtgtgat aactactgag 2701
gataataaaa tccacattca cttaggaagt ccttacatgc aagctgtagc cagccctgtg
2761 agacctgcca gcccttcagc accactgcag gataaccgaa ctcaaggctt
aattaacggg
2821 gcactaaaca aaacaaccaa taaagtcacc agcagtatta ctatcacacc
aacagccaca 2881 cctcttcctc gacaatcaca aattacagta agtaatatat
ataactgacc acgctcaccc 2941 tcatccagtc catactgata tttttgcaag
gaactcaatc cttttttaat catccctcca 3001 tatcccccaa gactgactga
actcgtactt tgggaaggtt tgtgcatgaa ctatacaaga 3061 gtatctgaaa
ctaactgttg cctgcatagt catatcgagt gtgcacttac tgtatatctt 3121
ttcatttaca tacttgtatg gaaaatattta gtctgcact tgtataaata catctttatg
3181 tatttcattt tccataactc actttaatttg actgcaact tgtcttggtg
aaatacttta 3241 acattataaa acagtaaata atttgttattt tta (SEQ ID NO:7)
Sequence CWU 1
1
141893PRTHomo sapiens 1Met Val Val Asp Glu Gln Gln Arg Leu Thr Ala
Gln Leu Thr Leu Gln 1 5 10 15 Arg Gln Lys Ile Gln Glu Leu Thr Thr
Asn Ala Lys Glu Thr His Thr 20 25 30 Lys Leu Ala Leu Ala Glu Ala
Arg Val Gln Glu Glu Glu Gln Lys Ala 35 40 45 Thr Arg Leu Glu Lys
Glu Leu Gln Thr Gln Thr Thr Lys Phe His Gln 50 55 60 Asp Gln Asp
Thr Ile Met Ala Lys Leu Thr Asn Glu Asp Ser Gln Asn 65 70 75 80 Arg
Gln Leu Gln Gln Lys Leu Ala Ala Leu Ser Arg Gln Ile Asp Glu 85 90
95 Leu Glu Glu Thr Asn Arg Ser Leu Arg Lys Ala Glu Glu Glu Leu Gln
100 105 110 Asp Ile Lys Glu Lys Ile Ser Lys Gly Glu Tyr Gly Asn Ala
Gly Ile 115 120 125 Met Ala Glu Val Glu Glu Leu Arg Lys Arg Val Leu
Asp Met Glu Gly 130 135 140 Lys Asp Glu Glu Leu Ile Lys Met Glu Glu
Gln Cys Arg Asp Leu Asn 145 150 155 160 Lys Arg Leu Glu Arg Glu Thr
Leu Gln Ser Lys Asp Phe Lys Leu Glu 165 170 175 Val Glu Lys Leu Ser
Lys Arg Ile Met Ala Leu Glu Lys Leu Glu Asp 180 185 190 Ala Phe Asn
Lys Ser Lys Gln Glu Cys Tyr Ser Leu Lys Cys Asn Leu 195 200 205 Glu
Lys Glu Arg Met Thr Thr Lys Gln Leu Ser Gln Glu Leu Glu Ser 210 215
220 Leu Lys Val Arg Ile Lys Glu Leu Glu Ala Ile Glu Ser Arg Leu Glu
225 230 235 240 Lys Thr Glu Phe Thr Leu Lys Glu Asp Leu Thr Lys Leu
Lys Thr Leu 245 250 255 Thr Val Met Phe Val Asp Glu Arg Lys Thr Met
Ser Glu Lys Leu Lys 260 265 270 Lys Thr Glu Asp Lys Leu Gln Ala Ala
Ser Ser Gln Leu Gln Val Glu 275 280 285 Gln Asn Lys Val Thr Thr Val
Thr Glu Lys Leu Ile Glu Glu Thr Lys 290 295 300 Arg Ala Leu Lys Ser
Lys Thr Asp Val Glu Glu Lys Met Tyr Ser Val 305 310 315 320 Thr Lys
Glu Arg Asp Asp Leu Lys Asn Lys Leu Lys Ala Glu Glu Glu 325 330 335
Lys Gly Asn Asp Leu Leu Ser Arg Val Asn Met Leu Lys Asn Arg Leu 340
345 350 Gln Ser Leu Glu Ala Ile Glu Lys Asp Phe Leu Lys Asn Lys Leu
Asn 355 360 365 Gln Asp Ser Gly Lys Ser Thr Thr Ala Leu His Gln Glu
Asn Asn Lys 370 375 380 Ile Lys Glu Leu Ser Gln Glu Val Glu Arg Leu
Lys Leu Lys Leu Lys 385 390 395 400 Asp Met Lys Ala Ile Glu Asp Asp
Leu Met Lys Thr Glu Asp Glu Tyr 405 410 415 Glu Thr Leu Glu Arg Arg
Tyr Ala Asn Glu Arg Asp Lys Ala Gln Phe 420 425 430 Leu Ser Lys Glu
Leu Glu His Val Lys Met Glu Leu Ala Lys Tyr Lys 435 440 445 Leu Ala
Glu Lys Thr Glu Thr Ser His Glu Gln Trp Leu Phe Lys Arg 450 455 460
Leu Gln Glu Glu Glu Ala Lys Ser Gly His Leu Ser Arg Glu Val Asp 465
470 475 480 Ala Leu Lys Glu Lys Ile His Glu Tyr Met Ala Thr Glu Asp
Leu Ile 485 490 495 Cys His Leu Gln Gly Asp His Ser Val Leu Gln Lys
Lys Leu Asn Gln 500 505 510 Gln Glu Asn Arg Asn Arg Asp Leu Gly Arg
Glu Ile Glu Asn Leu Thr 515 520 525 Lys Glu Leu Glu Arg Tyr Arg His
Phe Ser Lys Ser Leu Arg Pro Ser 530 535 540 Leu Asn Gly Arg Arg Ile
Ser Asp Pro Gln Val Phe Ser Lys Glu Val 545 550 555 560 Gln Thr Glu
Ala Val Asp Asn Glu Pro Pro Asp Tyr Lys Ser Leu Ile 565 570 575 Pro
Leu Glu Arg Ala Val Ile Asn Gly Gln Leu Tyr Glu Glu Ser Glu 580 585
590 Asn Gln Asp Glu Asp Pro Asn Asp Glu Gly Ser Val Leu Ser Phe Lys
595 600 605 Cys Ser Gln Ser Thr Pro Cys Pro Val Asn Arg Lys Leu Trp
Ile Pro 610 615 620 Trp Met Lys Ser Lys Glu Gly His Leu Gln Asn Gly
Lys Met Gln Thr 625 630 635 640 Lys Pro Asn Ala Asn Phe Val Gln Pro
Gly Asp Leu Val Leu Ser His 645 650 655 Thr Pro Gly Gln Pro Leu His
Ile Lys Val Thr Pro Asp His Val Gln 660 665 670 Asn Thr Ala Thr Leu
Glu Ile Thr Ser Pro Thr Thr Glu Ser Pro His 675 680 685 Ser Tyr Thr
Ser Thr Ala Val Ile Pro Asn Cys Gly Thr Pro Lys Gln 690 695 700 Arg
Ile Thr Ile Leu Gln Asn Ala Ser Ile Thr Pro Val Lys Ser Lys 705 710
715 720 Thr Ser Thr Glu Asp Leu Met Asn Leu Glu Gln Gly Met Ser Pro
Ile 725 730 735 Thr Met Ala Thr Phe Ala Arg Ala Gln Thr Pro Glu Ser
Cys Gly Ser 740 745 750 Leu Thr Pro Glu Arg Thr Met Ser Pro Ile Gln
Val Leu Ala Val Thr 755 760 765 Gly Ser Ala Ser Ser Pro Glu Gln Gly
Arg Ser Pro Glu Pro Thr Glu 770 775 780 Ile Ser Ala Lys His Ala Ile
Phe Arg Val Ser Pro Asp Arg Gln Ser 785 790 795 800 Ser Trp Gln Phe
Gln Arg Ser Asn Ser Asn Ser Ser Ser Val Ile Thr 805 810 815 Thr Glu
Asp Asn Lys Ile His Ile His Leu Gly Ser Pro Tyr Met Gln 820 825 830
Ala Val Ala Ser Pro Val Arg Pro Ala Ser Pro Ser Ala Pro Leu Gln 835
840 845 Asp Asn Arg Thr Gln Gly Leu Ile Asn Gly Ala Leu Asn Lys Thr
Thr 850 855 860 Asn Lys Val Thr Ser Ser Ile Thr Ile Thr Pro Thr Ala
Thr Pro Leu 865 870 875 880 Pro Arg Gln Ser Gln Ile Thr Val Ser Asn
Ile Tyr Asn 885 890 2752PRTHomo sapiens 2Met Val Val Asp Glu Gln
Gln Arg Leu Thr Ala Gln Leu Thr Leu Gln 1 5 10 15 Arg Gln Lys Ile
Gln Glu Leu Thr Thr Asn Ala Lys Glu Thr His Thr 20 25 30 Lys Leu
Ala Leu Ala Glu Ala Arg Val Gln Glu Glu Glu Gln Lys Ala 35 40 45
Thr Arg Leu Glu Lys Glu Leu Gln Thr Gln Thr Thr Lys Phe His Gln 50
55 60 Asp Gln Asp Thr Ile Met Ala Lys Leu Thr Asn Glu Asp Ser Gln
Asn 65 70 75 80 Arg Gln Leu Gln Gln Lys Leu Ala Ala Leu Ser Arg Gln
Ile Asp Glu 85 90 95 Leu Glu Glu Thr Asn Arg Ser Leu Arg Lys Ala
Glu Glu Glu Leu Gln 100 105 110 Asp Ile Lys Glu Lys Ile Ser Lys Gly
Glu Tyr Gly Asn Ala Gly Ile 115 120 125 Met Ala Glu Val Glu Glu Leu
Ile Lys Met Glu Glu Gln Cys Arg Asp 130 135 140 Leu Asn Lys Arg Leu
Glu Arg Glu Thr Leu Gln Ser Lys Asp Phe Lys 145 150 155 160 Leu Glu
Val Glu Lys Leu Ser Lys Arg Ile Met Ala Leu Glu Lys Leu 165 170 175
Glu Asp Ala Phe Asn Lys Ser Lys Gln Glu Cys Tyr Ser Leu Lys Cys 180
185 190 Asn Leu Glu Lys Glu Arg Met Thr Thr Lys Gln Leu Ser Gln Glu
Leu 195 200 205 Glu Ser Leu Lys Val Arg Ile Lys Glu Leu Glu Ala Ile
Glu Ser Arg 210 215 220 Leu Glu Lys Thr Glu Phe Thr Leu Lys Glu Asp
Leu Thr Lys Leu Lys 225 230 235 240 Thr Leu Thr Val Met Phe Val Asp
Glu Arg Lys Thr Met Ser Glu Lys 245 250 255 Leu Lys Lys Thr Glu Asp
Lys Leu Gln Ala Ala Ser Ser Gln Leu Gln 260 265 270 Val Glu Gln Asn
Lys Val Thr Thr Val Thr Glu Lys Leu Ile Glu Glu 275 280 285 Thr Lys
Arg Ala Leu Lys Ser Lys Thr Asp Val Glu Glu Lys Met Tyr 290 295 300
Ser Val Thr Lys Glu Arg Asp Asp Leu Lys Asn Lys Leu Lys Ala Glu 305
310 315 320 Glu Glu Lys Gly Asn Asp Leu Leu Ser Arg Val Asn Met Leu
Lys Asn 325 330 335 Arg Leu Gln Ser Leu Glu Ala Ile Glu Lys Asp Phe
Leu Lys Asn Lys 340 345 350 Leu Asn Gln Asp Ser Gly Lys Ser Thr Thr
Ala Leu His Gln Glu Asn 355 360 365 Asn Lys Ile Lys Glu Leu Ser Gln
Glu Val Glu Arg Leu Lys Leu Lys 370 375 380 Leu Lys Asp Met Lys Ala
Ile Glu Asp Asp Leu Met Lys Thr Glu Asp 385 390 395 400 Glu Tyr Glu
Thr Leu Glu Arg Arg Tyr Ala Asn Glu Arg Asp Lys Ala 405 410 415 Gln
Phe Leu Ser Lys Glu Leu Glu His Val Lys Met Glu Leu Ala Lys 420 425
430 Tyr Lys Leu Ala Glu Lys Thr Glu Thr Ser His Glu Gln Trp Leu Phe
435 440 445 Lys Arg Leu Gln Glu Glu Glu Ala Lys Ser Gly His Leu Ser
Arg Glu 450 455 460 Val Asp Ala Leu Lys Glu Lys Ile His Glu Tyr Met
Ala Thr Glu Asp 465 470 475 480 Leu Ile Cys His Leu Gln Gly Asp His
Ser Val Cys Lys Lys Lys Leu 485 490 495 Asn Gln Gln Glu Asn Arg Asn
Arg Asp Leu Gly Arg Glu Ile Glu Asn 500 505 510 Leu Thr Lys Glu Leu
Glu Arg Tyr Arg His Phe Ser Lys Ser Leu Arg 515 520 525 Pro Ser Leu
Asn Gly Arg Arg Ile Ser Asp Pro Gln Val Phe Ser Lys 530 535 540 Glu
Val Gln Thr Glu Ala Val Asp Asn Glu Pro Pro Asp Tyr Lys Ser 545 550
555 560 Leu Ile Pro Leu Glu Arg Ala Val Ile Asn Gly Gln Leu Tyr Glu
Glu 565 570 575 Ser Glu Asn Gln Asp Glu Asp Pro Asn Asp Glu Gly Ser
Val Leu Ser 580 585 590 Phe Lys Cys Ser Gln Ser Thr Pro Cys Pro Val
Asn Arg Lys Leu Trp 595 600 605 Ile Pro Trp Met Lys Ser Lys Glu Gly
His Leu Gln Asn Gly Lys Met 610 615 620 Gln Thr Lys Pro Asn Ala Asn
Phe Val Gln Pro Gly Asp Leu Val Leu 625 630 635 640 Ser His Thr Pro
Gly Gln Pro Leu His Ile Lys Val Thr Pro Asp His 645 650 655 Val Gln
Asn Thr Ala Thr Leu Glu Ile Thr Ser Pro Thr Thr Glu Ser 660 665 670
Pro His Ser Tyr Thr Ser Thr Ala Val Ile Pro Asn Cys Gly Thr Pro 675
680 685 Lys Gln Arg Ile Thr Ile Leu Gln Asn Ala Ser Ile Thr Pro Val
Lys 690 695 700 Ser Lys Thr Ser Thr Glu Asp Leu Met Asn Leu Glu Gln
Gly Met Ser 705 710 715 720 Pro Ile Thr Met Ala Thr Phe Ala Arg Ala
Gln Thr Pro Glu Ser Cys 725 730 735 Gly Ser Leu Thr Pro Glu Arg Thr
Met Ser Leu Phe Arg Phe Trp Leu 740 745 750 31135PRTHomo sapiens
3Met Arg Ser Arg Gly Ser Asp Thr Glu Gly Ser Ala Gln Lys Lys Phe 1
5 10 15 Pro Arg His Thr Lys Gly His Ser Phe Gln Gly Pro Lys Asn Met
Lys 20 25 30 His Arg Gln Gln Asp Lys Asp Ser Pro Ser Glu Ser Asp
Val Ile Leu 35 40 45 Pro Cys Pro Lys Ala Glu Lys Pro His Ser Gly
Asn Gly His Gln Ala 50 55 60 Glu Asp Leu Ser Arg Asp Asp Leu Leu
Phe Leu Leu Ser Ile Leu Glu 65 70 75 80 Gly Glu Leu Gln Ala Arg Asp
Glu Val Ile Gly Ile Leu Lys Ala Glu 85 90 95 Lys Met Asp Leu Ala
Leu Leu Glu Ala Gln Tyr Gly Phe Val Thr Pro 100 105 110 Lys Lys Val
Leu Glu Ala Leu Gln Arg Asp Ala Phe Gln Ala Lys Ser 115 120 125 Thr
Pro Trp Gln Glu Asp Ile Tyr Glu Lys Pro Met Asn Glu Leu Asp 130 135
140 Lys Val Val Glu Lys His Lys Glu Ser Tyr Arg Arg Ile Leu Gly Gln
145 150 155 160 Leu Leu Val Ala Glu Lys Ser Arg Arg Gln Thr Ile Leu
Glu Leu Glu 165 170 175 Glu Glu Lys Arg Lys His Lys Glu Tyr Met Glu
Lys Ser Asp Glu Phe 180 185 190 Ile Cys Leu Leu Glu Gln Glu Cys Glu
Arg Leu Lys Lys Leu Ile Asp 195 200 205 Gln Glu Ile Lys Ser Gln Glu
Glu Lys Glu Gln Glu Lys Glu Lys Arg 210 215 220 Val Thr Thr Leu Lys
Glu Glu Leu Thr Lys Leu Lys Ser Phe Ala Leu 225 230 235 240 Met Val
Val Asp Glu Gln Gln Arg Leu Thr Ala Gln Leu Thr Leu Gln 245 250 255
Arg Gln Lys Ile Gln Glu Leu Thr Thr Asn Ala Lys Glu Thr His Thr 260
265 270 Lys Leu Ala Leu Ala Glu Ala Arg Val Gln Glu Glu Glu Gln Lys
Ala 275 280 285 Thr Arg Leu Glu Lys Glu Leu Gln Thr Gln Thr Thr Lys
Phe His Gln 290 295 300 Asp Gln Asp Thr Ile Met Ala Lys Leu Thr Asn
Glu Asp Ser Gln Asn 305 310 315 320 Arg Gln Leu Gln Gln Lys Leu Ala
Ala Leu Ser Arg Gln Ile Asp Glu 325 330 335 Leu Glu Glu Thr Asn Arg
Ser Leu Arg Lys Ala Glu Glu Glu Leu Gln 340 345 350 Asp Ile Lys Glu
Lys Ile Ser Lys Gly Glu Tyr Gly Asn Ala Gly Ile 355 360 365 Met Ala
Glu Val Glu Glu Leu Arg Lys Arg Val Leu Asp Met Glu Gly 370 375 380
Lys Asp Glu Glu Leu Ile Lys Met Glu Glu Gln Cys Arg Asp Leu Asn 385
390 395 400 Lys Arg Leu Glu Arg Glu Thr Leu Gln Ser Lys Asp Phe Lys
Leu Glu 405 410 415 Val Glu Lys Leu Ser Lys Arg Ile Met Ala Leu Glu
Lys Leu Glu Asp 420 425 430 Ala Phe Asn Lys Ser Lys Gln Glu Cys Tyr
Ser Leu Lys Cys Asn Leu 435 440 445 Glu Lys Glu Arg Met Thr Thr Lys
Gln Leu Ser Gln Glu Leu Glu Ser 450 455 460 Leu Lys Val Arg Ile Lys
Glu Leu Glu Ala Ile Glu Ser Arg Leu Glu 465 470 475 480 Lys Thr Glu
Phe Thr Leu Lys Glu Asp Leu Thr Lys Leu Lys Thr Leu 485 490 495 Thr
Val Met Phe Val Asp Glu Arg Lys Thr Met Ser Glu Lys Leu Lys 500 505
510 Lys Thr Glu Asp Lys Leu Gln Ala Ala Ser Ser Gln Leu Gln Val Glu
515 520 525 Gln Asn Lys Val Thr Thr Val Thr Glu Lys Leu Ile Glu Glu
Thr Lys 530 535 540 Arg Ala Leu Lys Ser Lys Thr Asp Val Glu Glu Lys
Met Tyr Ser Val 545 550 555 560 Thr Lys Glu Arg Asp Asp Leu Lys Asn
Lys Leu Lys Ala Glu Glu Glu 565 570 575 Lys Gly Asn Asp Leu Leu Ser
Arg Val Asn Met Leu Lys Asn Arg Leu 580 585 590 Gln Ser Leu Glu Ala
Ile Glu Lys Asp Phe Leu Lys Asn Lys Leu Asn 595 600 605 Gln Asp Ser
Gly Lys Ser Thr Thr Ala Leu His Gln Glu Asn Asn Lys 610 615 620 Ile
Lys Glu Leu Ser Gln Glu Val Glu Arg Leu Lys Leu Lys Leu Lys 625 630
635 640 Asp Met Lys Ala Ile Glu Asp Asp Leu Met Lys Thr Glu Asp Glu
Tyr 645 650
655 Glu Thr Leu Glu Arg Arg Tyr Ala Asn Glu Arg Asp Lys Ala Gln Phe
660 665 670 Leu Ser Lys Glu Leu Glu His Val Lys Met Glu Leu Ala Lys
Tyr Lys 675 680 685 Leu Ala Glu Lys Thr Glu Thr Ser His Glu Gln Trp
Leu Phe Lys Arg 690 695 700 Leu Gln Glu Glu Glu Ala Lys Ser Gly His
Leu Ser Arg Glu Val Asp 705 710 715 720 Ala Leu Lys Glu Lys Ile His
Glu Tyr Met Ala Thr Glu Asp Leu Ile 725 730 735 Cys His Leu Gln Gly
Asp His Ser Val Leu Gln Lys Lys Leu Asn Gln 740 745 750 Gln Glu Asn
Arg Asn Arg Asp Leu Gly Arg Glu Ile Glu Asn Leu Thr 755 760 765 Lys
Glu Leu Glu Arg Tyr Arg His Phe Ser Lys Ser Leu Arg Pro Ser 770 775
780 Leu Asn Gly Arg Arg Ile Ser Asp Pro Gln Val Phe Ser Lys Glu Val
785 790 795 800 Gln Thr Glu Ala Val Asp Asn Glu Pro Pro Asp Tyr Lys
Ser Leu Ile 805 810 815 Pro Leu Glu Arg Ala Val Ile Asn Gly Gln Leu
Tyr Glu Glu Ser Glu 820 825 830 Asn Gln Asp Glu Asp Pro Asn Asp Glu
Gly Ser Val Leu Ser Phe Lys 835 840 845 Cys Ser Gln Ser Thr Pro Cys
Pro Val Asn Arg Lys Leu Trp Ile Pro 850 855 860 Trp Met Lys Ser Lys
Glu Gly His Leu Gln Asn Gly Lys Met Gln Thr 865 870 875 880 Lys Pro
Asn Ala Asn Phe Val Gln Pro Gly Asp Leu Val Leu Ser His 885 890 895
Thr Pro Gly Gln Pro Leu His Ile Lys Val Thr Pro Asp His Val Gln 900
905 910 Asn Thr Ala Thr Leu Glu Ile Thr Ser Pro Thr Thr Glu Ser Pro
His 915 920 925 Ser Tyr Thr Ser Thr Ala Val Ile Pro Asn Cys Gly Thr
Pro Lys Gln 930 935 940 Arg Ile Thr Ile Leu Gln Asn Ala Ser Ile Thr
Pro Val Lys Ser Lys 945 950 955 960 Thr Ser Thr Glu Asp Leu Met Asn
Leu Glu Gln Gly Met Ser Pro Ile 965 970 975 Thr Met Ala Thr Phe Ala
Arg Ala Gln Thr Pro Glu Ser Cys Gly Ser 980 985 990 Leu Thr Pro Glu
Arg Thr Met Ser Pro Ile Gln Val Leu Ala Val Thr 995 1000 1005 Gly
Ser Ala Ser Ser Pro Glu Gln Gly Arg Ser Pro Glu Pro Thr 1010 1015
1020 Glu Ile Ser Ala Lys His Ala Ile Phe Arg Val Ser Pro Asp Arg
1025 1030 1035 Gln Ser Ser Trp Gln Phe Gln Arg Ser Asn Ser Asn Ser
Ser Ser 1040 1045 1050 Val Ile Thr Thr Glu Asp Asn Lys Ile His Ile
His Leu Gly Ser 1055 1060 1065 Pro Tyr Met Gln Ala Val Ala Ser Pro
Val Arg Pro Ala Ser Pro 1070 1075 1080 Ser Ala Pro Leu Gln Asp Asn
Arg Thr Gln Gly Leu Ile Asn Gly 1085 1090 1095 Ala Leu Asn Lys Thr
Thr Asn Lys Val Thr Ser Ser Ile Thr Ile 1100 1105 1110 Thr Pro Thr
Ala Thr Pro Leu Pro Arg Gln Ser Gln Ile Thr Val 1115 1120 1125 Glu
Pro Leu Leu Leu Pro His 1130 1135 41133PRTHomo sapiens 4Met Arg Ser
Arg Gly Ser Asp Thr Glu Gly Ser Ala Gln Lys Lys Phe 1 5 10 15 Pro
Arg His Thr Lys Gly His Ser Phe Gln Gly Pro Lys Asn Met Lys 20 25
30 His Arg Gln Gln Asp Lys Asp Ser Pro Ser Glu Ser Asp Val Ile Leu
35 40 45 Pro Cys Pro Lys Ala Glu Lys Pro His Ser Gly Asn Gly His
Gln Ala 50 55 60 Glu Asp Leu Ser Arg Asp Asp Leu Leu Phe Leu Leu
Ser Ile Leu Glu 65 70 75 80 Gly Glu Leu Gln Ala Arg Asp Glu Val Ile
Gly Ile Leu Lys Ala Glu 85 90 95 Lys Met Asp Leu Ala Leu Leu Glu
Ala Gln Tyr Gly Phe Val Thr Pro 100 105 110 Lys Lys Val Leu Glu Ala
Leu Gln Arg Asp Ala Phe Gln Ala Lys Ser 115 120 125 Thr Pro Trp Gln
Glu Asp Ile Tyr Glu Lys Pro Met Asn Glu Leu Asp 130 135 140 Lys Val
Val Glu Lys His Lys Glu Ser Tyr Arg Arg Ile Leu Gly Gln 145 150 155
160 Leu Leu Val Ala Glu Lys Ser Arg Arg Gln Thr Ile Leu Glu Leu Glu
165 170 175 Glu Glu Lys Arg Lys His Lys Glu Tyr Met Glu Lys Ser Asp
Glu Phe 180 185 190 Ile Cys Leu Leu Glu Gln Glu Cys Glu Arg Leu Lys
Lys Leu Ile Asp 195 200 205 Gln Glu Ile Lys Ser Gln Glu Glu Lys Glu
Gln Glu Lys Glu Lys Arg 210 215 220 Val Thr Thr Leu Lys Glu Glu Leu
Thr Lys Leu Lys Ser Phe Ala Leu 225 230 235 240 Met Val Val Asp Glu
Gln Gln Arg Leu Thr Ala Gln Leu Thr Leu Gln 245 250 255 Arg Gln Lys
Ile Gln Glu Leu Thr Thr Asn Ala Lys Glu Thr His Thr 260 265 270 Lys
Leu Ala Leu Ala Glu Ala Arg Val Gln Glu Glu Glu Gln Lys Ala 275 280
285 Thr Arg Leu Glu Lys Glu Leu Gln Thr Gln Thr Thr Lys Phe His Gln
290 295 300 Asp Gln Asp Thr Ile Met Ala Lys Leu Thr Asn Glu Asp Ser
Gln Asn 305 310 315 320 Arg Gln Leu Gln Gln Lys Leu Ala Ala Leu Ser
Arg Gln Ile Asp Glu 325 330 335 Leu Glu Glu Thr Asn Arg Ser Leu Arg
Lys Ala Glu Glu Glu Leu Gln 340 345 350 Asp Ile Lys Glu Lys Ile Ser
Lys Gly Glu Tyr Gly Asn Ala Gly Ile 355 360 365 Met Ala Glu Val Glu
Glu Leu Arg Lys Arg Val Leu Asp Met Glu Gly 370 375 380 Lys Asp Glu
Glu Leu Ile Lys Met Glu Glu Gln Cys Arg Asp Leu Asn 385 390 395 400
Lys Arg Leu Glu Arg Glu Thr Leu Gln Ser Lys Asp Phe Lys Leu Glu 405
410 415 Val Glu Lys Leu Ser Lys Arg Ile Met Ala Leu Glu Lys Leu Glu
Asp 420 425 430 Ala Phe Asn Lys Ser Lys Gln Glu Cys Tyr Ser Leu Lys
Cys Asn Leu 435 440 445 Glu Lys Glu Arg Met Thr Thr Lys Gln Leu Ser
Gln Glu Leu Glu Ser 450 455 460 Leu Lys Val Arg Ile Lys Glu Leu Glu
Ala Ile Glu Ser Arg Leu Glu 465 470 475 480 Lys Thr Glu Phe Thr Leu
Lys Glu Asp Leu Thr Lys Leu Lys Thr Leu 485 490 495 Thr Val Met Phe
Val Asp Glu Arg Lys Thr Met Ser Glu Lys Leu Lys 500 505 510 Lys Thr
Glu Asp Lys Leu Gln Ala Ala Ser Ser Gln Leu Gln Val Glu 515 520 525
Gln Asn Lys Val Thr Thr Val Thr Glu Lys Leu Ile Glu Glu Thr Lys 530
535 540 Arg Ala Leu Lys Ser Lys Thr Asp Val Glu Glu Lys Met Tyr Ser
Val 545 550 555 560 Thr Lys Glu Arg Asp Asp Leu Lys Asn Lys Leu Lys
Ala Glu Glu Glu 565 570 575 Lys Gly Asn Asp Leu Leu Ser Arg Val Asn
Met Leu Lys Asn Arg Leu 580 585 590 Gln Ser Leu Glu Ala Ile Glu Lys
Asp Phe Leu Lys Asn Lys Leu Asn 595 600 605 Gln Asp Ser Gly Lys Ser
Thr Thr Ala Leu His Gln Glu Asn Asn Lys 610 615 620 Ile Lys Glu Leu
Ser Gln Glu Val Glu Arg Leu Lys Leu Lys Leu Lys 625 630 635 640 Asp
Met Lys Ala Ile Glu Asp Asp Leu Met Lys Thr Glu Asp Glu Tyr 645 650
655 Glu Thr Leu Glu Arg Arg Tyr Ala Asn Glu Arg Asp Lys Ala Gln Phe
660 665 670 Leu Ser Lys Glu Leu Glu His Val Lys Met Glu Leu Ala Lys
Tyr Lys 675 680 685 Leu Ala Glu Lys Thr Glu Thr Ser His Glu Gln Trp
Leu Phe Lys Arg 690 695 700 Leu Gln Glu Glu Glu Ala Lys Ser Gly His
Leu Ser Arg Glu Val Asp 705 710 715 720 Ala Leu Lys Glu Lys Ile His
Glu Tyr Met Ala Thr Glu Asp Leu Ile 725 730 735 Cys His Leu Gln Gly
Asp His Ser Val Leu Gln Lys Lys Leu Asn Gln 740 745 750 Gln Glu Asn
Arg Asn Arg Asp Leu Gly Arg Glu Ile Glu Asn Leu Thr 755 760 765 Lys
Glu Leu Glu Arg Tyr Arg His Phe Ser Lys Ser Leu Arg Pro Ser 770 775
780 Leu Asn Gly Arg Arg Ile Ser Asp Pro Gln Val Phe Ser Lys Glu Val
785 790 795 800 Gln Thr Glu Ala Val Asp Asn Glu Pro Pro Asp Tyr Lys
Ser Leu Ile 805 810 815 Pro Leu Glu Arg Ala Val Ile Asn Gly Gln Leu
Tyr Glu Glu Ser Glu 820 825 830 Asn Gln Asp Glu Asp Pro Asn Asp Glu
Gly Ser Val Leu Ser Phe Lys 835 840 845 Cys Ser Gln Ser Thr Pro Cys
Pro Val Asn Arg Lys Leu Trp Ile Pro 850 855 860 Trp Met Lys Ser Lys
Glu Gly His Leu Gln Asn Gly Lys Met Gln Thr 865 870 875 880 Lys Pro
Asn Ala Asn Phe Val Gln Pro Gly Asp Leu Val Leu Ser His 885 890 895
Thr Pro Gly Gln Pro Leu His Ile Lys Val Thr Pro Asp His Val Gln 900
905 910 Asn Thr Ala Thr Leu Glu Ile Thr Ser Pro Thr Thr Glu Ser Pro
His 915 920 925 Ser Tyr Thr Ser Thr Ala Val Ile Pro Asn Cys Gly Thr
Pro Lys Gln 930 935 940 Arg Ile Thr Ile Leu Gln Asn Ala Ser Ile Thr
Pro Val Lys Ser Lys 945 950 955 960 Thr Ser Thr Glu Asp Leu Met Asn
Leu Glu Gln Gly Met Ser Pro Ile 965 970 975 Thr Met Ala Thr Phe Ala
Arg Ala Gln Thr Pro Glu Ser Cys Gly Ser 980 985 990 Leu Thr Pro Glu
Arg Thr Met Ser Pro Ile Gln Val Leu Ala Val Thr 995 1000 1005 Gly
Ser Ala Ser Ser Pro Glu Gln Gly Arg Ser Pro Glu Pro Thr 1010 1015
1020 Glu Ile Ser Ala Lys His Ala Ile Phe Arg Val Ser Pro Asp Arg
1025 1030 1035 Gln Ser Ser Trp Gln Phe Gln Arg Ser Asn Ser Asn Ser
Ser Ser 1040 1045 1050 Val Ile Thr Thr Glu Asp Asn Lys Ile His Ile
His Leu Gly Ser 1055 1060 1065 Pro Tyr Met Gln Ala Val Ala Ser Pro
Val Arg Pro Ala Ser Pro 1070 1075 1080 Ser Ala Pro Leu Gln Asp Asn
Arg Thr Gln Gly Leu Ile Asn Gly 1085 1090 1095 Ala Leu Asn Lys Thr
Thr Asn Lys Val Thr Ser Ser Ile Thr Ile 1100 1105 1110 Thr Pro Thr
Ala Thr Pro Leu Pro Arg Gln Ser Gln Ile Thr Val 1115 1120 1125 Ser
Asn Ile Tyr Asn 1130 521DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 5agcgtaacca
aggagagaga t 21621DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 6attcattcat tcattcacca t
2173274DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 7ataggccggg cgcgctcagc gccccgctcg
cattgttcgg gcgactctcg gagcgcgcac 60agtcggctcg cagcgcggca ctacagcggc
cccggcccgg cccccgcccg gccccggcgc 120aggcagttca gattaaagaa
gctaattgat caagaaatca agtctcagga ggagaaggag 180caagaaaagg
agaaaagggt caccaccctg aaagaggagc tgaccaagct gaagtctttt
240gctttgatgg tggtggatga acagcaaagg ctgacggcac agctcaccct
tcaaagacag 300aaaatccaag agctgaccac aaatgcaaag gaaacacata
ccaaactagc ccttgctgaa 360gccagagttc aggaggaaga gcagaaggca
accagactag agaaggaact gcaaacgcag 420accacaaagt ttcaccaaga
ccaagacaca attatggcga agctcaccaa tgaggacagt 480caaaatcgcc
agcttcaaca aaagctggca gcactcagcc ggcagattga tgagttagaa
540gagacaaaca ggtctttacg aaaagcagaa gaggagctgc aagatataaa
agaaaaaatc 600agtaagggag aatatggaaa cgctggtatc atggctgaag
tggaagagct caggaaacgt 660gtgctagata tggaagggaa agatgaagag
ctcataaaaa tggaggagca gtgcagagat 720ctcaataaga ggcttgaaag
ggagacgtta cagagtaaag actttaaact agaggttgaa 780aaactcagta
aaagaattat ggctctggaa aagttagaag acgctttcaa caaaagcaaa
840caagaatgct actctctgaa atgcaattta gaaaaagaaa ggatgaccac
aaagcagttg 900tctcaagaac tggagagttt aaaagtaagg atcaaagagc
tagaagccat tgaaagtcgg 960ctagaaaaga cagaattcac tctaaaagag
gatttaacta aactgaaaac attaactgtg 1020atgtttgtag atgaacggaa
aacaatgagt gaaaaattaa agaaaactga agataaatta 1080caagctgctt
cttctcagct tcaagtggag caaaataaag taacaacagt tactgagaag
1140ttaattgagg aaactaaaag ggcgctcaag tccaaaaccg atgtagaaga
aaagatgtac 1200agcgtaacca aggagagaga tgatttaaaa aacaaattga
aagcggaaga agagaaagga 1260aatgatctcc tgtcaagagt taatatgttg
aaaaataggc ttcaatcatt ggaagcaatt 1320gagaaagatt tcctaaaaaa
caaattaaat caagactctg ggaaatccac aacagcatta 1380caccaagaaa
acaataagat taaggagctc tctcaagaag tggaaagact gaaactgaag
1440ctaaaggaca tgaaagccat tgaggatgac ctcatgaaaa cagaagatga
atatgagact 1500ctagaacgaa ggtatgctaa tgaacgagac aaagctcaat
ttttatctaa agagctagaa 1560catgttaaaa tggaacttgc taagtacaag
ttagcagaaa agacagagac cagccatgaa 1620caatggcttt tcaaaaggct
tcaagaagaa gaagctaagt cagggcacct ctcaagagaa 1680gtggatgcat
taaaagagaa aattcatgaa tacatggcaa ctgaagacct aatatgtcac
1740ctccagggag atcactcagt cctgcaaaaa aaactaaatc aacaagaaaa
caggaacaga 1800gatttaggaa gagagattga aaacctcact aaggagttag
agaggtaccg gcatttcagt 1860aagagcctca ggcctagtct caatggaaga
agaatttccg atcctcaagt attttctaaa 1920gaagttcaga cagaagcagt
agacaatgaa ccacctgatt acaagagcct cattcctctg 1980gaacgtgcag
tcatcaatgg tcagttatat gaggagagtg agaatcaaga cgaggaccct
2040aatgatgagg gatctgtgct gtccttcaaa tgcagccagt ctactccatg
tcctgttaac 2100agaaagctat ggattccctg gatgaaatcc aaggagggcc
atcttcagaa tggaaaaatg 2160caaactaaac ccaatgccaa ctttgtgcaa
cctggagatc tagtcctaag ccacacacct 2220gggcagccac ttcatataaa
ggttactcca gaccatgtac aaaacacagc cactcttgaa 2280atcacaagtc
caaccacaga gagtcctcac tcttacacga gtactgcagt gataccgaac
2340tgtggcacgc caaagcaaag gataaccatc ctccaaaacg cctccataac
accagtaaag 2400tccaaaacct ctaccgaaga cctcatgaat ttagaacaag
gcatgtcccc aattaccatg 2460gcaacctttg ccagagcaca gaccccagag
tcttgtggtt ctctaactcc agaaaggaca 2520atgtccccta ttcaggtttt
ggctgtgact ggttcagcta gctctcctga gcagggacgc 2580tccccagaac
caacagaaat cagtgccaag catgcgatat tcagagtctc cccagaccgg
2640cagtcatcat ggcagtttca gcgttcaaac agcaatagct caagtgtgat
aactactgag 2700gataataaaa tccacattca cttaggaagt ccttacatgc
aagctgtagc cagccctgtg 2760agacctgcca gcccttcagc accactgcag
gataaccgaa ctcaaggctt aattaacggg 2820gcactaaaca aaacaaccaa
taaagtcacc agcagtatta ctatcacacc aacagccaca 2880cctcttcctc
gacaatcaca aattacagta agtaatatat ataactgacc acgctcaccc
2940tcatccagtc catactgata tttttgcaag gaactcaatc cttttttaat
catccctcca 3000tatcccccaa gactgactga actcgtactt tgggaaggtt
tgtgcatgaa ctatacaaga 3060gtatctgaaa ctaactgttg cctgcatagt
catatcgagt gtgcacttac tgtatatctt 3120ttcatttaca tacttgtatg
gaaaatattt agtctgcact tgtataaata catctttatg 3180tatttcattt
tccataactc actttaattt gactgcaact tgtcttggtg aaatacttta
3240acattataaa acagtaaata atttgttatt ttta 32748893PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Met Val Val Asp Glu Gln Gln Arg Leu Thr Ala Gln Leu Thr Leu Gln 1
5 10 15 Arg Gln Lys Val Gln Glu Leu Thr Thr Asn Ala Lys Glu Thr His
Thr 20 25 30 Lys Leu Ala Leu Ala Glu Ala Arg Val Gln Glu Glu Glu
Gln Lys Ala 35 40 45 Thr Arg Leu Glu Lys Glu Leu Gln Thr Gln Thr
Thr Lys Phe His Gln 50 55 60 Asp Gln Asp Thr Ile Met Ala Lys Leu
Thr Asn Glu Asp Ser Gln Asn 65 70 75 80 Arg Gln Leu Gln Gln Lys Leu
Ala Ala Leu Ser Arg Gln Ile Asp Glu 85 90 95 Leu Glu Glu Thr Asn
Arg Ser Leu Arg Lys
Ala Glu Glu Glu Leu Gln 100 105 110 Asp Ile Lys Glu Lys Ile Ser Lys
Gly Glu Tyr Gly Asn Ala Gly Ile 115 120 125 Met Ala Glu Val Glu Glu
Leu Arg Lys Arg Val Leu Asp Met Glu Gly 130 135 140 Lys Asp Glu Glu
Leu Ile Lys Met Glu Glu Gln Cys Arg Asp Leu Asn 145 150 155 160 Lys
Arg Leu Glu Arg Glu Thr Leu Gln Ser Lys Asp Phe Lys Leu Glu 165 170
175 Val Glu Lys Leu Ser Lys Arg Ile Met Ala Leu Glu Lys Leu Glu Asp
180 185 190 Ala Phe Asn Lys Ser Lys Gln Glu Cys Tyr Ser Leu Lys Cys
Asn Leu 195 200 205 Glu Lys Glu Arg Met Thr Thr Lys Gln Leu Ser Gln
Glu Leu Glu Ser 210 215 220 Leu Lys Val Arg Ile Lys Glu Leu Glu Ala
Ile Glu Ser Arg Leu Glu 225 230 235 240 Lys Thr Glu Phe Thr Leu Lys
Glu Asp Leu Thr Lys Leu Lys Thr Leu 245 250 255 Thr Val Met Phe Val
Asp Glu Arg Lys Thr Met Ser Glu Lys Leu Lys 260 265 270 Lys Thr Glu
Asp Lys Leu Gln Ala Ala Ser Ser Gln Leu Gln Val Glu 275 280 285 Gln
Asn Lys Val Thr Thr Val Thr Glu Lys Leu Ile Glu Glu Thr Lys 290 295
300 Arg Ala Leu Lys Ser Lys Thr Asp Val Glu Glu Lys Met Tyr Ser Val
305 310 315 320 Thr Lys Glu Arg Asp Asp Leu Lys Asn Lys Leu Lys Ala
Glu Glu Glu 325 330 335 Lys Gly Asn Asp Leu Leu Ser Arg Val Asn Met
Leu Lys Asn Arg Leu 340 345 350 Gln Ser Leu Glu Ala Ile Glu Lys Asp
Phe Leu Lys Asn Lys Leu Asn 355 360 365 Gln Asp Ser Gly Lys Ser Thr
Thr Ala Leu His Gln Glu Asn Asn Lys 370 375 380 Ile Lys Glu Leu Ser
Gln Glu Val Glu Arg Leu Lys Leu Lys Leu Lys 385 390 395 400 Asp Met
Lys Ala Ile Glu Asp Asp Leu Met Lys Thr Glu Asp Glu Tyr 405 410 415
Glu Thr Leu Glu Arg Arg Tyr Ala Asn Glu Arg Asp Lys Ala Gln Phe 420
425 430 Leu Ser Lys Glu Leu Glu His Val Lys Met Glu Leu Ala Lys Tyr
Lys 435 440 445 Leu Ala Glu Lys Thr Glu Thr Ser His Glu Gln Trp Leu
Phe Lys Arg 450 455 460 Leu Gln Glu Glu Glu Ala Lys Ser Gly His Leu
Ser Arg Glu Val Asp 465 470 475 480 Ala Leu Lys Glu Lys Ile His Glu
Tyr Met Ala Thr Glu Asp Leu Ile 485 490 495 Cys His Leu Gln Gly Asp
His Ser Val Leu Gln Lys Lys Leu Asn Gln 500 505 510 Gln Glu Asn Arg
Asn Arg Asp Leu Gly Arg Glu Ile Glu Asn Leu Thr 515 520 525 Lys Glu
Leu Glu Arg Tyr Arg His Phe Ser Lys Ser Leu Arg Pro Ser 530 535 540
Leu Asn Gly Arg Arg Ile Ser Asp Pro Gln Val Phe Ser Lys Glu Val 545
550 555 560 Gln Thr Glu Ala Val Asp Asn Glu Pro Pro Asp Tyr Lys Ser
Leu Ile 565 570 575 Pro Leu Glu Arg Ala Val Ile Asn Gly Gln Leu Tyr
Glu Glu Ser Glu 580 585 590 Asn Gln Asp Glu Asp Pro Asn Asp Glu Gly
Ser Val Leu Ser Phe Lys 595 600 605 Cys Ser Gln Ser Thr Pro Cys Pro
Val Asn Arg Lys Leu Trp Ile Pro 610 615 620 Trp Met Lys Ser Lys Glu
Gly His Leu Gln Asn Gly Lys Met Gln Thr 625 630 635 640 Lys Pro Asn
Ala Asn Phe Val Gln Pro Gly Asp Leu Val Leu Ser His 645 650 655 Thr
Pro Gly Gln Pro Leu His Ile Lys Val Thr Pro Asp His Val Gln 660 665
670 Asn Thr Ala Thr Leu Glu Ile Thr Ser Pro Thr Thr Glu Ser Pro His
675 680 685 Ser Tyr Thr Ser Thr Ala Val Ile Pro Asn Cys Gly Thr Pro
Lys Gln 690 695 700 Arg Ile Thr Ile Leu Gln Asn Ala Ser Ile Thr Pro
Val Lys Ser Lys 705 710 715 720 Thr Ser Thr Glu Asp Leu Met Asn Leu
Glu Gln Gly Met Ser Pro Ile 725 730 735 Thr Met Ala Thr Phe Ala Arg
Ala Gln Thr Pro Glu Ser Cys Gly Ser 740 745 750 Leu Thr Pro Glu Arg
Thr Met Ser Pro Ile Gln Val Leu Ala Val Thr 755 760 765 Gly Ser Ala
Ser Ser Pro Glu Gln Gly Arg Ser Pro Glu Pro Thr Glu 770 775 780 Ile
Ser Ala Lys His Ala Ile Phe Arg Val Ser Pro Asp Arg Gln Ser 785 790
795 800 Ser Trp Gln Phe Gln Arg Ser Asn Ser Asn Ser Ser Ser Val Ile
Thr 805 810 815 Thr Glu Asp Asn Lys Ile His Ile His Leu Gly Ser Pro
Tyr Met Gln 820 825 830 Ala Val Ala Ser Pro Val Arg Pro Ala Ser Pro
Ser Ala Pro Leu Gln 835 840 845 Asp Asn Arg Thr Gln Gly Leu Ile Asn
Gly Ala Leu Asn Lys Thr Thr 850 855 860 Asn Lys Val Thr Ser Ser Ile
Thr Ile Thr Pro Thr Ala Thr Pro Leu 865 870 875 880 Pro Arg Gln Ser
Gln Ile Thr Val Ser Asn Ile Tyr Asn 885 890 921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9aacgctggta tcatggctga a 211022DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 10atctctgcac tgctcctcca tt
221122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11tcaccagggc tgcttttaac tc 221226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12ggaatcatat tggaacatgt aaacca 26136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
13His His His His His His 1 5 149PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 14Cys Asp Cys Arg Gly Asp
Cys Phe Cys 1 5
* * * * *