U.S. patent application number 11/073347 was filed with the patent office on 2005-11-24 for anti-neovasculature preparations for cancer.
Invention is credited to Diamond, David C., Simard, John J. L..
Application Number | 20050260234 11/073347 |
Document ID | / |
Family ID | 23046599 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260234 |
Kind Code |
A1 |
Simard, John J. L. ; et
al. |
November 24, 2005 |
Anti-neovasculature preparations for cancer
Abstract
Disclosed herein are immunogenic compositions, methods of
designing immunogenic compositions, methods of treatment using
immunogenic compositions, methods of evaluating cell-mediated
immunity resulting from immunogenic compositions, research models,
and methods of making research models, all of which relate to
targeting tumor vasculature.
Inventors: |
Simard, John J. L.;
(Northridge, CA) ; Diamond, David C.; (West Hills,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
23046599 |
Appl. No.: |
11/073347 |
Filed: |
June 30, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11073347 |
Jun 30, 2005 |
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10094699 |
Mar 7, 2002 |
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60274063 |
Mar 7, 2001 |
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Current U.S.
Class: |
424/277.1 |
Current CPC
Class: |
A01K 67/0271 20130101;
A61K 39/3955 20130101; A01K 67/0276 20130101; A61P 35/00 20180101;
A61K 39/39558 20130101; A61P 35/04 20180101; A61K 2039/57 20130101;
A61K 39/001109 20180801; C07K 14/70539 20130101 |
Class at
Publication: |
424/277.1 |
International
Class: |
A61K 039/00 |
Claims
What is claimed is:
1. A method of treating neoplastic disease comprising the step of
immunizing a mammal to induce a cellular immune response directed
against a first antigen differentially expressed by
tumor-associated neovasculature and a second antigen associated
with a tumor, wherein the immunization comprises delivering at
least a first immunogen corresponding to the first antigen and a
second immunogen corresponding to the second antigen.
2. The method of claim 1, wherein the cellular immune response
comprises a CTL response.
3. The method of claim 1 further comprising the step of detecting
the cellular immune response.
4. The method of claim 3, wherein the detecting step comprises
detection of tumor growth inhibition, tumor size reduction,
inhibition of tumor metastasis, or increase in life expectancy of
the mammal.
5. The method of claim 3, wherein the detecting step comprises an
assay selected from the group consisting of a cytokine assay, a
chromium release assay, an immunofluorescence assay, a cytotoxic T
lymphocyte (CTL) assay, an Elispot assay, and observation of the
health of the mammal.
6. A method of treating neoplastic disease comprising the step of
immunizing a mammal to induce a cellular immune response directed
against an antigen differentially expressed by tumor-associated
neovasculature, wherein the immunization comprises delivering a
first immunogen comprising at least one housekeeping epitope and a
second immunogen comprising at least one immune epitope, wherein
the housekeeping and immune epitopes are derived from said antigen
differentially expressed by tumor-associated neovasculature.
7. The method of claim 6, wherein the first immunogen and the
second immunogen are the same.
8. The method of claim 6, further comprising the step of treating
the mammal with an anti-tumor therapy active directly against
cancerous cells.
9. The method of claim 8, wherein the anti-tumor therapy comprises
immunization against a tumor-associated antigen.
10. The method of claim 6, wherein the cellular immune response
comprises a CTL response.
11. The method of claim 6, further comprising the step of detecting
the cellular immune response.
12. The method of claim 11, wherein the detecting step comprises
detection of tumor growth inhibition, tumor size reduction,
inhibition of tumor metastasis, or increase in life expectancy of
the mammal.
13. The method of claim 11, wherein the detecting step comprises an
assay selected from the group consisting of a cytokine assay, a
chromium release assay, an immunofluorescence assay, a cytotoxic T
lymphocyte (CTL) assay, an Elispot assay, and observation of the
health of the mammal.
14. A method of treating neoplastic disease comprising the step of
immunizing a mammal to induce a cellular immune response directed
against an antigen differentially expressed by tumor-associated
neovasculature, wherein the immunization comprises delivering an
immunogen comprising at least one housekeeping epitope derived from
said antigen differentially expressed by tumor-associated
neovasculature.
15. The method of claim 14, wherein the cellular immune response
comprises a CTL response.
16. The method of claim 14, further comprising the step of
detecting the cellular immune response.
17. The method of claim 16, wherein the detecting step comprises
detection of tumor growth inhibition, tumor size reduction,
inhibition of tumor metastasis, or increase in life expectancy of
the mammal.
18. The method of claim 16, wherein the detecting step comprises an
assay selected from the group consisting of a cytokine assay, a
chromium release assay, an immunofluorescence assay, a cytotoxic T
lymphocyte (CTL) assay, an Elispot assay, and observation of the
health of the mammal.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 10/094,699, filed Mar. 7, 2002, entitled "ANTI-NEOVASCULATURE
PREPARATIONS FOR CANCER," which claims priority under 35 U.S.C.
.sctn. 119(e) to U.S. Provisional Patent Application Ser. No.
60/274,063, filed on Mar. 7, 2001, entitled "ANTI-NEOVASCULATURE
PREPARATIONS FOR CANCER," each of which is hereby incorporated by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Description of the Related Art
[0003] The treatment of cancer has remained challenging despite the
advances in biomedicine. In recent years two approaches have been
described showing much promise: therapeutic vaccines and
anti-angiogenesis.
[0004] Therapeutic vaccines rely on the observation that cancerous
tissues generally express certain antigens preferentially,
collectively tumor-associated antigens (TuAA). TuAA include
proteins normally expressed selectively by the tissue from which
the cancer derives (differentiation antigens), proteins that are
associated with a different stage of development (oncofetal and
cancer-testis antigens), proteins that are created by aberrant
chromosomal rearrangement, or proteins that are derived from
oncogenic viruses. These TuAA, or fragments of them, are then used
as immunogens in vaccines intended to stimulate cellular immunity,
particularly cytotoxic T lymphocytes (CTL), capable of killing the
tumor cells.
[0005] The anti-angiogenesis approach takes advantage of the need
of tumors to recruit a blood supply to support their continued
growth. To accomplish this, tumors secrete angiogenic factors that
promote the growth of new blood vessels. The anti-angiogenesis
approach aims to disrupt a tumor's supply of nutrients to cause it
to die, or at least limit its growth. Attempts at this approach
have sought chemotherapeutic drugs used directly against a variety
of anti-angiogenic factors and angiogenesis.
SUMMARY OF THE INVENTION
[0006] The invention disclosed herein is directed to compositions
designed to stimulate cellular immune responses targeting
tumor-associated neovasculature (TuNV). In one embodiment of the
invention the compositions stimulate a CTL response. Such
compositions may include one or more epitopes of the target
antigen. One aspect of this embodiment specifically includes a
housekeeping epitope, another specifically includes an immune
epitope or epitope cluster, and another aspect specifically
combines housekeeping and immune epitopes.
[0007] Embodiments of the invention relate to the use of prostate
specific membrane antigen (PSMA) as the target antigen of the
composition. Aspects of this embodiment include various epitopes
derived from PSMA provided directly as polypeptide, or as a nucleic
acid capable of conferring expression of the epitope. Other
embodiments relate to the use of other TuNV-associated
antigens.
[0008] In other embodiments of the invention, compositions are
directed against both the TuNV and against TuAA expressed by the
cancerous cells, by combining immunogens derived from both sources
into a single formulation or method or treatment.
[0009] Preclinical evaluation of the compositions of this invention
can be accomplished using adoptive transfer of immunized T cells
into SCID mice bearing microvasculature formed from implanted human
dermal microvascular endothelial cells (HDMEC). Preclinical
evaluation can also be accomplished through the use of
HLA-transgenic mice immunized with compositions comprised of
epitopes conserved between mice and humans.
[0010] Embodiments of the invention relate to methods of evaluating
cell-mediated immunity. The methods can include the steps of
implanting vascular cells into an immunodeficient mammal;
establishing an immune response in the mammal; and assaying a
characteristic to determine cell-mediated immunity in the mammal.
The cell-mediated immunity can be directed against a neovasculature
antigen, for example. The neovasculature antigen can be
preferentially expressed by tumor-associated neovasculature, for
example, and in preferred embodiments can be prostate specific
membrane antigen (PSMA), vascular endothelial growth factor
receptor 2 (VEGFR2), and the like. The establishing step can be
achieved, for example, by adoptive transfer of T-cells to the
mammal, by contacting the mammal with an antigen, and the like. The
cell-mediated immunity can be mediated by cytotoxic T lymphocytes.
The vascular cells can be vascular endothelial cells, such as, for
example, human dermal microvascular endothelial cells (HDMEC),
telomerase-transformed endothelial cells, and the like. The
immunodeficient mammal can be a mouse, such as for example a SCID
mouse. The characterizing step can include assessing a parameter,
such as for example, vessel formation, vessel destruction, vessel
density, proportion of vessels carrying blood of the host mammal,
and the like.
[0011] The methods can further include the step of implanting tumor
cells or tumor tissue in the mouse. The characterizing step can
include assessing a parameter, such as, for example, tumor
presence, tumor growth, tumor size, rapidity of tumor appearance,
dose of vaccine required to inhibit or prevent tumor establishment,
tumor vascularization, a proportion of necrotic tissue within the
tumor, and the like.
[0012] The methods can further include the steps of providing a
first population of mammals and a second populations of mammals;
establishing cell-mediated immunity in the first population;
differentially establishing cell-mediated immunity in the second
population; and comparing a result obtained from the first
population of mammals to a result obtained from the second
population of mammals. The cell-mediated immunity of the first
population can include, for example, nave immunity, immunity to an
irrelevant epitope, and the like.
[0013] Other embodiments relate to methods of evaluating
cell-mediated immunity, including immunity directed against a
neovasculature antigen. The methods can include the steps of
implanting or injecting MHC-transgenic tumor cells into an
MHC-transgenic mammal; establishing an immune response in the
mammal; and assaying a characteristic to determine cell-mediated
immunity in the mammal. The MHC-transgenic mammal can be an
HLA-transgenic mammal, such as, for example an HLA-A2 transgenic
mammal. In preferred embodiments the mammal can be a mouse. The
cell-mediated immunity can be established by vaccination, which in
preferred embodiments can take place prior to, concurrent with, or
subsequent to transfer of the tumor cells, for example. In
preferred embodiments the cell-mediated immunity can be mediated by
cytotoxic T lymphocytes. The neovasculature antigen can be
preferentially expressed by tumor-associated neovasculature and can
also be a tumor-associated antigen. Preferably, the antigen can be
the ED-B domain of fibronectin. The characterizing step can
include, for example, assessing a parameter, including tumor
presence, tumor growth, tumor size, rapidity of tumor appearance,
dose of vaccine required to inhibit or prevent tumor establishment,
tumor vascularization, a proportion of necrotic tissue within the
tumor, and the like. The methods can further include the steps of
providing a first population of mammals and a second populations of
mammals; establishing cell-mediated immunity in the first
population; differentially establishing cell-mediated immunity in
the second population; and comparing a result obtained from the
first population of mammals to a result obtained from the second
population of mammals. The cell-mediated immunity of the first
population can include nave immunity, immunity to an irrelevant
epitope, and the like.
[0014] Still further embodiments relate to methods of treating
neoplastic disease, including the step of immunizing a mammal to
induce a cellular immune response directed against an antigen
differentially expressed by tumor-associated neovasculature. The
differentially expressed antigen can be a protein, such as, for
example prostate specific membrane antigen, vascular endothelial
growth factor receptor 2 (VEGFR2), and the like. In other preferred
embodiments, the antigen can be the ED-B domain of fibronectin. The
immunization can be carried out, for example, with at least one
peptide derived from the sequence of the protein, with a nucleic
acid capable of conferring expression of the protein or peptides,
and the like. The at least one peptide can include a housekeeping
epitope, for example, and in preferred embodiments can be
co-C-terminal with the housekeeping epitope. The methods can
further include at least one additional peptide, wherein the at
least one additional peptide includes an immune epitope. The
methods can include an additional step wherein the mammal is
treated with an anti-tumor therapy active directly against
cancerous cells. The anti-tumor therapy can be immunization against
a tumor-associated antigen. Preferably, the cellular immune
response can include a CTL response.
[0015] Other embodiments relate to immunogenic compositions. The
immunogenic compositions can include at least one immunogen
corresponding to an antigen expressed by tumor-associated
neovasculature, wherein the composition can induce a cellular
immune response. The immunogen can be one that is not associated
with a cell conspecific with the recipient. The antigen can be a
protein, such as, for example prostate specific membrane antigen,
vascular endothelial growth factor receptor 2 (VEGFR2), and the
like. In other preferred embodiments the antigen can be the ED-B
domain of fibronectin. The immunogen can include at least one
peptide. The compositions can include a nucleic acid capable of
conferring expression of the antigen, and wherein the antigen is a
protein or a peptide. The compositions can include at least one
peptide that includes a housekeeping epitope, and in preferred
embodiments the at least one peptide can be co-C-terminal with the
housekeeping epitope. Also, the compositions can additionally
include at least one peptide that includes an immune epitope. The
compositions can include at least one immunogen corresponding to a
tumor-associated antigen. In preferred embodiments the cellular
immune response can include a CTL response.
[0016] Embodiments relate to methods of anti-tumor vaccine design.
The methods can include the steps of identifying an antigen
differentially expressed by tumor-associated neovasculature; and
incorporating a component of the antigen into a vaccine. The
component can include, for example, a polypeptide fragment of the
antigen, a nucleic acid encoding the antigen or a fragment of the
antigen, and the like.
[0017] Further embodiments relate to methods of making a research
model. The methods can include implanting a vascular cell and a
tumor cell into an immunodeficient mammal. The tumor cell and the
vascular cell can be implanted adjacent to one another. The
vascular cell can be a vascular endothelial cell, such as for
example HDMEC. In preferred embodiments the vascular endothelial
cell can be telomerase-transformed. The immunodeficient mammal can
be a mouse, such as, for example, a SCID mouse.
[0018] Other embodiments relate to research models. The research
models can include an immunodeficient mammal. The mammal can
include an implanted vascular cell and an implanted tumor cell. The
vascular cell and the tumor cell can be implanted adjacent to one
another.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A, B, and C show results of N-terminal pool sequencing
of a T=60 min. time point aliquot of the PSMA.sub.163-192
proteasomal digest.
[0020] FIG. 2 shows binding curves for HLA-A2:PSMA.sub.168-177 and
HLA-A2:PSMA.sub.288-297 with controls.
[0021] FIG. 3 shows results of N-terminal pool sequencing of a T=60
min. time point aliquot of the PSMA.sub.281-310 proteasomal
digest.
[0022] FIG. 4 shows binding curves for HLA-A2:PSMA.sub.461-469,
HLA-A2:PSMA.sub.460-469, and HLA-A2:PSMA.sub.663-671, with
controls.
[0023] FIG. 5 shows the results of a .gamma.(gamma)-IFN-based
ELISPOT assay detecting PSMA.sub.463-471-reactive HLA-A1.sup.+
CD8.sup.+ T cells.
[0024] FIG. 6 shows blocking of reactivity of the T cells used in
FIG. 10 by anti-HLA-A1 mAb, demonstrating HLA-A1-restricted
recognition.
[0025] FIG. 7 shows a binding curve for HLA-A2:PSMA.sub.663-671,
with controls.
[0026] FIG. 8 shows a binding curve for HLA-A2:PSMA.sub.662-671,
with controls.
[0027] FIG. 9 shows epitope specific lysis by CTL from HHD-A2 mice
immunized with ED-B 29-38 peptide.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0028] Embodiments of the invention disclosed herein provide
compositions, methods of composition or vaccine design, and methods
of treatment related to the generation of a cellular immune
response, preferably, a T cell response and, more preferably, a CTL
response, directed against the neovasculature of tumors. Such
methods and compositions are particularly useful in the treatment
and prevention of cancer. Other embodiments relate to composition
evaluation models.
[0029] Compositions, Composition Design, and Treatment Using the
Compositions
[0030] Embodiments of the invention relate to immunogenic
compositions, including vaccines, for the generation of a cellular
immune response, particularly a T cell response and specifically a
CTL response, directed against tumor neovasculature (TuNV). "Tumor
neovasculature" is broadly meant to include any vasculature found
in or around tumor masses, vasculature which supports or is
necessary for tumor growth, and the like. It should be noted, and
one of skill in the art will appreciate, that although the
discussion herein refers generally to the tumors and tumor
neovasculature, the embodiments of the present invention also can
be applied to other conditions or disease states associated with
inappropriate angiogenesis.
[0031] Until now the design of anti-tumor vaccines has concentrated
on antigens expressed by the malignant cells themselves. However,
larger tumors are complex structures and not simply a homogeneous
mass of cells. All cells, particularly rapidly growing cells, need
a supply of nutrients (oxygen, glucose, amino acids, etc.), as well
as a means of removal of metabolic wastes, in order to remain
metabolically active and intact. This is normally accomplished by
the flow of blood and lymph through the various organs of the body.
At a cellular level, the tissues of the body are permeated by a
fine network of capillaries--tiny vessels through which nutrients
and waste products can be exchanged with the surrounding cells by
diffusion. Diffusion is effective over relatively short distances.
The capillary beds are so extensive that generally cells are at
most located only a few cells away from a capillary. If a tumor
merely grew by propagation of its malignant cells, soon those cells
in the interior of the mass would be unable to sustain themselves.
In fact, the interiors of unvascularized tumors often contain
necrotic tissue. Thus, in order to grow unchecked, tumors secrete
factors that promote the in-growth of new blood vessels, namely
TuNV. Since the TuNV expresses antigens differentiating it from
other tissues, cancer can be treated with therapeutic compositions
directed against the TuNV, instead of directly targeting the
cancerous cells themselves. Suitable TuNV antigens can include
those that are expressed generally in neovasculature or
preferentially by TuNV, for example.
[0032] In some embodiments of the invention the compositions can
include, for example, an epitopic peptide or peptides. Immune
epitopes may be provided embedded in epitope clusters and protein
fragments. Housekeeping epitopes can be provided with the proper
C-terminus. In other embodiments of the invention the compositions
can include nucleic acids capable of conferring expression of these
epitopes on pAPC, for example.
[0033] In preferred embodiments, the compositions can be
administered directly to the lymphatic system of a mammal being
treated. This can be applied to both polypeptide and nucleic acid
based compositions. Administration methods of this type, and
related technologies, are disclosed in U.S. patent application Ser.
No. 09/380,534, filed on Sep. 1, 1999, and a Continuation-in-Part
thereof, filed on Feb. 2, 2001; U.S. patent application Ser. No.
09/776,232, both entitled "A METHOD OF INDUCING A CTL RESPONSE,"
which are incorporated by reference in their entirety.
[0034] In a preferred embodiment, destruction of the blood vessels
in a tumor by action of a composition of the invention can
eliminate all of the cells in a tumor. However, small tumors,
including micrometastases, are typically unvasculaturized.
Additionally, unvascularized tumors that instead apparently rely on
blood flow through channels penetrating the tumor mass have been
reported (Maniotis, A. J., et al. Am. J. Pathol. 155: 739-752,
1999). Thus in other embodiments, the compositions are generally
effective as tumor control agents that may not eradicate all cancer
cells. Accordingly, the invention provides tools for eliminating
tumors, controlling tumor growth, reducing tumor burden, improving
overall clinical status, and the like. In some embodiments, it can
be desirable to combine these compositions with other treatments
that target the cancerous cells directly. Additionally there is
evidence that the vasculature in tumors can be mosaic in nature
consisting of both endothelial and cancer cells (Chang, Y. S., et
al. Proc. Natl. Acad. Sci. USA 97:14608-14613, 2000). Thus, in some
embodiments of the invention a course of composition treatment can
be followed by administration of a bio- or chemotherapeutic agent.
In a particularly preferred embodiment, treatment can include
administration of a TuAA directed composition concurrent or
subsequent to administration of the anti-TuNV composition.
[0035] As mentioned above, suitable TuNV antigens for the
compositions can include those that are expressed generally in
neovasculature or preferentially by TuNV, for example. A variety of
techniques for discovery of TuAA are known in the art. Examples of
these techniques include, without limitation, differential
hybridization and subtractive hybridization, including use of
microarrays; expression cloning; SAGE (serial analysis of gene
expression); SEREX (serological identification of antigens by
recombinant expression cloning); in situ RT-PCT;
immunohistochemistry (as was the case for PSMA); EST analysis;
variously using bulk, sectioned, and/or microdissected tissue; and
the like. Utilization of these and other methods provides one of
skill in the art the techniques necessary to identify genes and
gene products contained within a target cell that may be used as
antigens of immunogenic compositions. The techniques are applicable
to TuAA discovery regardless of whether the target cell is a cancer
cell or an endothelial cell. Any identified antigen can be
scrutinized for epitopes, which can be used in embodiments of the
invention.
[0036] The endothelial cells making up the lining of the
vasculature can express housekeeping proteasomes. Thus,
compositions targeting endothelial cells can be comprised of
peptides, or nucleic acids conferring expression of the peptides,
corresponding to the digestion products of the housekeeping
proteasome (i.e. housekeeping epitopes). IFN-.gamma. (gamma),
secreted by activated cells of the immune system, can induce
expression of the immunoproteasome in the target cells. Generally,
the immunoproteasome is constitutive in professional antigen
presenting cells (pAPC). Thus, it can be helpful to include immune
epitopes or epitope clusters in CTL-inducing compositions to ensure
that there are CTL able to recognize the target cell regardless of
the state that the target cell is in. This can be particularly true
with endothelial cells, which readily assume antigen presentation
functions. These concepts are more fully explained in U.S. patent
application Ser. No. 09/560,465, filed on Apr., 28, 2000; Ser. No.
10/005,905, filed on Nov. 7, 2001; and a continuation thereof, U.S.
application Ser. No. 10/026,066, filed on Dec. 7, 2001, each of
which is entitled "EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING
CELLS," and each of which is hereby incorporated by reference in
its entirety.
[0037] As discussed above, the immunogenic compositions, including
in preferred embodiments, vaccines, can include TuNV antigens and
epitopes, for example. The epitopes can include one or more
housekeeping epitopes and/or one or more immune epitopes. Specific
epitopes useful in compositions can be identified using the methods
disclosed in U.S. patent application Ser. No. 09/561,074 entitled
"METHOD OF EPITOPE DISCOVERY," filed on Apr. 28, 2000. For example,
peptide sequences that are known or predicted to bind to some MHC
restriction element can be compared to fragments produced by
proteasomal digestion in order to identify those that are
co-C-terminal.
[0038] Examples of useful epitopes for the embodiments of the
invention, including epitopes of ED-B and PSMA, are disclosed in a
U.S. Provisional Patent Application No. 60/363,210, entitled
"EPITOPE SEQUENCES," filed on Mar. 7, 2002, and two U.S.
Provisional Patent Applications, each entitled "EPITOPE SEQUENCES;"
Application No. 60/282,211, filed on Apr. 6, 2001 and 60/337,017,
filed on Nov. 7, 2001. Each of these applications is incorporated
herein by reference in its entirety.
[0039] PSMA is one example of a TuAA that can be targeted in some
embodiments. PSMA is expressed in the neovasculature of most tumor
types, but not by the vascular endothelium of normal tissues
(Chang, S. M. et al., Cancer Res. 59(13):3192-8,1999; Clin Cancer
Res. 10:2674-81, 1999). PSMA is a membrane antigen, and as such, it
may be possible to attack PSMA-expressing TuNV with monoclonal
antibody (mAb). However, the effectiveness of mAb in the treatment
of cancer has proved to be more difficult than initially
anticipated. Moreover, as other antigens are discovered to be
associated with the TuNV, it is likely that many of them will prove
not to be expressed at the vasculature surface, making them
inaccessible to mAb attack.
[0040] T cells, particularly CTL, on the other hand, survey the
expression of internal components of the cell through the process
of major histocompatability complex (MHC)-restricted antigen
presentation. The parameters for determining the effectiveness of T
cell-activating vaccines and compositions against self-antigens are
subtle. Some of the critical features and parameters relating to
appropriate epitope selection are disclosed in U.S. patent
application Ser. No. 09/560,465 entitled "EPITOPE SYNCHRONIZATION
IN ANTIGEN PRESENTING CELLS," filed on Apr. 28, 2001; U.S. patent
application Ser. No. 09/561,074 entitled "METHOD OF EPITOPE
DISCOVERY," filed on Apr. 28, 2001; and U.S. patent application
Ser. No. 09/561,571 entitled "EPITOPE CLUSTERS," filed on Apr. 28,
2001. Features of DNA vaccine design promoting epitope
synchronization are disclosed in U.S. patent application Ser. No.
09/561,572 entitled "EXPRESSION VECTORS ENCODING EPITOPES OF
TARGET-ASSOCIATED ANTIGENS," filed on Apr. 28, 2001 and U.S.
Provisional Application No. 60/336,968 entitled "EXPRESSION VECTORS
ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR
THEIR DESIGN," filed on Nov. 7, 2001. Particularly effective means
of vaccine delivery are described in U.S. patent application Ser.
No. 09/380,534, filed on Sep. 1, 1999, and a Continuation-in-Part
thereof, U.S. patent application Ser. No. 09/776,232, filed on Feb.
2, 2001, both entitled "A METHOD OF INDUCING A CTL RESPONSE." Each
of the above-mentioned references is incorporated herein by
reference in its entirety.
[0041] Another example of a TuNV antigen that can be used in
embodiments is fibronectin, preferably the ED-B domain. Fibronectin
is subject to developmentally regulated alternative splicing, with
the ED-B domain being encoded by a single exon that is used
primarily in oncofetal tissues. Matsuura, H. and S. Hakomori Proc.
Natl. Acad. Sci. USA 82:6517-6521, 1985; Carnemolla, B. et al. J.
Cell Biol. 108:1139-1148, 1989; Loridon-Rosa, B. et al. Cancer Res.
50:1608-1612, 1990; Nicolo, G. et al. Cell Differ. Dev. 32:401-408,
1990; Borsi, L. et al. Exp. Cell Res. 199:98-105, 1992; Oyama, F.
et al. Cancer Res. 53:2005-2011, 1993; Mandel, U. et al. APMIS
102:695-702, 1994; Famoud, M. R. et al. Int. J. Cancer 61:27-34,
1995; Pujuguet, P. et al. Am. J. Pathol. 148:579-592, 1996; Gabler,
U. et al. Heart 75:358-362, 1996; Chevalier, X. Br. J. Rheumatol.
35:407-415, 1996; Midulla, M. Cancer Res. 60:164-169, 2000.
[0042] The ED-B domain is also expressed in fibronectin of the
neovasculature Kaczmarek, J. et al. Int. J. Cancer 59:11-16, 1994;
Castellani, P. et al. Int. J. Cancer 59:612-618, 1994; Neri, D. et
al. Nat. Biotech. 15:1271-1275, 1997; Karelina, T. V. and A. Z.
Eisen Cancer Detect. Prev. 22:438-444, 1998; Tarli, L. et al. Blood
94:192-198, 1999; Castellani, P. et al. Acta Neurochir. (Wien)
142:277-282, 2000. As an oncofetal domain, the ED-B domain is
commonly found in the fibronectin expressed by neoplastic cells, in
addition to being expressed by the TuNV. Therefore, CTL-inducing
compositions targeting the ED-B domain can exhibit two mechanisms
of action: direct lysis of tumor cells, and disruption of tumor
blood supply through destruction of the TuNV.
[0043] It should be noted that expression of the fibronectin ED-B
domain has been reported in both tumor-associated and normal
neovasculature (Castellani, P. et al. Int. J. Cancer 59:612-618,
1994). Thus, compositions based on it, or similarly expressed
antigens, can be effective against other conditions associated with
inappropriate angiogenesis. Further, as CTL activity can decay
rapidly after withdrawal of the composition, interference with
normal angiogenesis can be minimal.
[0044] Other targets for the immunogenic compositions include
growth factor receptors, including those associated with vascular
cells. One such example is the vascular endothelial growth factor
receptor 2 (VEGFR2). U.S. Pat. No. 6,342,221 includes discussion of
VEGF and VEGFR2, and is hereby incorporated by reference in its
entirety. One of skill in the art will appreciate that any other
antigen or protein associated with vascular cells can be a target
for the immunogenic compositions, including those that are
presently known and those yet to be identified.
[0045] Animal Models, Methods of Making the Models, and Composition
Evaluation
[0046] Compositions designed based upon the preceding
considerations are effective against the various targets. However,
additional evaluation can be easily performed at any time, but
preferably in a pre-clinical setting. For example, such evaluation
can be used in order to further aid in composition design. Other
embodiments of the invention relate to methods of evaluating the
immunogenic compositions. The compositions of the present invention
can be easily evaluated by one of skill in the art using animal
models for composition evaluation. For example, following the
routine procedures below, one of skill in the art can evaluate TuNV
compositions quickly and efficiently. Thus, using the models or
guidance described herein, one of skill in the art can evaluate any
TuNV composition for any TuNV antigen with little or no
experimentation. Further embodiments relate to methods of making
the animal research models. Other embodiments relate to the
research model animals. These embodiments are set forth more fully
below.
[0047] Xenotransplanted Human Vasculature-Based Model
[0048] Some embodiments relate to a model system for studying the
mechanisms of human microvessel formation. For example, in some
embodiments, the model system can be used for preclinical
evaluation of compositions. The model involves the subcutaneous
implantation of telomerase-transformed human dermal microvascular
endothelial cells (HDMEC) mixed with MATRIGEL (Becton Dickinson)
into SCID mice. Subcutaneous implantation of telomerase-transformed
HCMEC is described in Yang, J. et al. Nature Biotech 19:219-224,
2001, which is hereby incorporated by reference in its entirety. T
cells activated by the compositions of this invention can be
adoptively transferred, for example, into such implanted mice, and
the ability of the T cells to destroy, or prevent the formation of,
such human microvessels can be assessed. In other embodiments, the
mouse can be directly vaccinated and evaluated. Also, in still
further embodiments, the model system can be further adapted for
testing compositions effective in non-human species by substituting
DMEC from other species and species-matched telomerase, and by
using analogous reagents to those described below for the human
system.
[0049] The MHC-restriction elements presenting the epitopes of the
composition being tested, preferably, are shared by the HDMEC line
implanted into the mice. The T cells can be derived from in vitro
immunization of human T cells, or by immunization of HLA-transgenic
mice (procedures for which are well known in the art and examples
of which are provided in the above incorporated patent
applications). Use of T cells generated in HLA-transgeneic mice
allows matching of genetic backgrounds between the adoptively
transferred T cells and the host, thereby reducing the possibility
of allogeneic or xenogeneic reactions that might complicate
interpretation of the results. However, depending on the mouse
strains available, this might require cross-breeding to get the
HLA-transgene and SCID phenotype on the same genetic background. In
the alternative, the donor T cells (human or murine) can be
subjected to one or more rounds of in vitro stimulation to enrich
for the desired population or establish a clone, and thereby
similarly avoid undesired reactivities.
[0050] Techniques for in vitro immunization are know in the art,
for example, Stauss et al., Proc. Natl. Acad. Sci. USA
89:7871-7875, 1992; Salgaller et al. Cancer Res. 55:4972-4979,
1995; Tsai et al., J. Immunol. 158:1796-1802, 1997; and Chung et
al., J Immunother. 22:279-287, 1999. Once generated, whether in
vivo or in vitro, sufficient numbers of such T cells can be
obtained by expansion in vitro through stimulation with the
compositions of this invention and/or cytokines (see for example
Kurokawa, T. et al., Int. J. Cancer 91:749-746, 2001) or other
mitogens. These T cells can constitute a clone or a polyclonal
population recognizing one or more epitopes. In preferred
embodiments, on the order of 10.sup.5 to 10.sup.8 cells are
transferred for adoptive transfer experiments in mice. (See for
example Drobyski, W. R. et al. Blood 97:2506-2513, 2001; Seeley B.
M. et al. Otolaryngol. Head Neck Surg. 124:436-441, 2001; Kanwar,
J. R. et al. Cancer Res. 61:1948-1956, 2001). Clones and otherwise
more enriched populations generally require the transfer of fewer
cells.
[0051] Transfer of the T cells can take place shortly before,
concurrent with, or subsequent to implantation or establishment of
the HDMEC. Parameters that can be assessed to evaluate
effectiveness of the compositions include vessel formation, changes
in vessel density, and ability to carry mouse blood (as described
in Yang et al.), and the like. Assessment can be carried out as
early as one week, and at least as long as 6 weeks, after
implantation of telomerase-transformed HDMEC, preferably after 2
weeks; and from a day to more than 6 weeks after T cell transfer,
preferably after 1 to 3 weeks. Generally, assessment can include
comparison of mice receiving T cells reactive with the target
antigen with mice receiving nave (including sham-immunized), or
irrelevant epitope-reactive T cells.
[0052] Relevant antigens can be expressed generally in
neovasculature or preferentially by TuNV. Expression can be
confirmed by a variety of techniques known in the art, including
immunohistochemistry and RT-PCR. For example, tumor cells can be
implanted along with the HDMEC. This can result in inducing
expression of antigens preferentially expressed by TuNV. In one
example, this can be accomplished by implanting a block of tumor
tissue adjacent to the HDMEC-containing MATRIGEL implant, injecting
tumor cells at the site of the implant, implanting tumor
cell-containing MATRIGEL adjacent to the HDMEC-containing MATRIGEL
implant, incorporating both tumor cells and HDMEC into the same
MATRIGEL implant or by any other suitable method. As discussed
above, in some embodiments, tumor cells can be implanted along with
vascular cells. The animals so made, can be used as research
models. Additional variations will be apparent to one of skill in
the art.
[0053] HLA-transgenic Animal Model
[0054] For antigens that are conserved, in sequence and/or
expression profile, between human and the model species,
HLA-transgenic strains allow another approach, namely vaccination
of the model animal to combat a syngeneic tumor. The ED-B domain of
fibronectin provides such an opportunity, as it is a marker of
angiogenesis and has identical amino acid sequence in both humans
and mice (Nilsson, F. et al. Cancer Res. 61:711-716, 2001).
Moreover, spontaneous tumor tissue from a strain of HLA-A2
transgenic mice has been isolated and propagated. Epitope discovery
and selection, and composition design and delivery for CTL inducing
compositions are discussed above.
[0055] The tumor cell line, M1, is derived from a spontaneous
salivary glandular cystadenocarcinoma. The M1 tumor cell line and
methods of using the same is disclosed in U.S. Provisional
Application No. 60/363,131, Mar. 7, 2002, entitled "AN
HLA-TRANSGENIC MURINE TUMOR CELL LINE," which is hereby expressly
incorporated by reference in its entirety. The tumor cell line, can
arise in individuals of the HHD-A2 transgenic mouse strain of S.
Pascolo et al. (J. Exp. Med. 185:2043-2051, 1997). These mice
express a single monochain class I molecule comprising human .beta.
(beta).sub.2-microglobulin, and .alpha.1 (alpha-1), and .alpha.2
(alpha-2) domains of HLA-A2.1 with the balance of the molecule
derived from the murine class I molecule H2 D.sup.b. Blocks of
tumor can be transplanted into new individuals where the tumor will
re-grow, commonly within 1-3 weeks, with 3 mm blocks growing to 3
cm. Alternatively, tumor tissue can be disaggregated and the tumor
cells grown in vitro. Upon harvest, the tumor cells can be injected
subcutaneously into the neck or abdomen (2.5.times.10.sup.6 cells
for 1-3 successive days), to result in a visible tumor in
approximately 5-12 weeks for early passage cells. After the cells
have become better adapted to growth in vitro, single injections of
1.times.10.sup.6 to 1.times.10.sup.7 cells lead to visible tumor in
ten days. Generally, the initial tumor consistently occurs in the
vicinity of the salivary glands, but secondary tumors can also
occur in a variety of locations, including kidney, lung, liver, and
abdominal muscle.
[0056] To evaluate the efficacy of a composition, it can be
administered before, concurrent with, or subsequent to
establishment of the tumor, depending on the expected mechanism of
the composition. For therapeutic compositions intended to be used
with some sort of debulking technique (e.g. surgery), concurrent
administration can be appropriate. The better established the tumor
is before treatment begins, the more stringent the test.
[0057] Both animal evaluation models have been described for the
testing of human compositions. However, application to veterinary
compositions is analogous, requiring only the substitution of
species-matched endothelial cells, MHC, TuAA, etc.
[0058] All patents, patent applications, and publications referred
to herein are hereby incorporated by reference in their
entirety.
[0059] The following examples are intended for illustration
purposes only, and should not be construed as limiting the scope of
the invention in any way.
EXAMPLES
Example 1
[0060] A preclinical study was carried out using the already
identified antigens PSMA and ED-B disclosed herein. The results of
the study revealed excellent candidate epitopes. See table 9
below.
Example 1.1
[0061] Cluster Analysis (PSMA.sub.163-192)
[0062] A peptide, AFSPQGMPEGDLVYVNYARTEDFFKLERDM, PSMA.sub.163-192,
(SEQ ID NO. 3), containing an A1 epitope cluster from prostate
specific membrane antigen, PSMA.sub.168-190 (SEQ ID NO. 4) was
synthesized using standard solid-phase F-moc chemistry on a 433A
ABI Peptide synthesizer. After side chain deprotection and cleavage
from the resin, peptide first dissolved in formic acid and then
diluted into 30% Acetic acid, was run on a reverse-phase
preparative HPLC C4 column at following conditions: linear AB
gradient (5% B/min) at a flow rate of 4 ml/min, where eluent A is
0.1% aqueous TFA and eluent B is 0.1% TFA in acetonitrile. A
fraction at time 16.642 min containing the expected peptide, as
judged by mass spectrometry, was pooled and lyophilized. The
peptide was then subjected to proteasome digestion and mass
spectrum analysis essentially as described above. Prominent peaks
from the mass spectra are summarized in Table 1.
1TABLE 1 PSMA.sub.163-192 Mass Peak Identification. CALCU- SEQ
LATED ID MASS NO. PEPTIDE SEQUENCE (MH.sup.+) 110 163-177
AFSPQGMPEGDLVYV 1610.0 111 178-189 NYARTEDFFKLE 1533.68 112 170-189
PEGDLVYVNYARTEDFFKLE 2406.66 113 178-191 NYARTEDFFKLERD 1804.95 114
170-191 PEGDLVYVNYARTEDFFKLERD 2677.93 115 178-192 NYARTEDFFKLERDM
1936.17 116 163-176 AFSPQGMPEGDLVY 1511.70 117 177-192
VNYARTEDFFKLERDM 2035.30 118 163-179 AFSPQGMPEGDLVYVNY 1888.12 119
180-192 ARTEDFFKLERDM 1658.89 120 163-183 AFSPQGMPEGDLVYVNYARTE
2345.61 121 184-192 DFFKLERDM 1201.40 122 176-192 YVNYARTEDFFKLERDM
2198.48 123 167-185 QGMPEGDLVYVNYARTEDF 2205.41 124 178-186
NYARTEDFF 1163.22 Boldface sequences correspond to peptides
predicted to bind to MHC, see Table 2.
[0063] N-Terminal Pool Sequence Analysis
[0064] One aliquot at one hour of the proteasomal digestion was
subjected to N-nal terminal amino acid sequence analysis by an ABI
473A Protein Sequencer (Applied Biosystems, Foster City, Calif.).
Determination of the sites and efficiencies of cleavage was based
on consideration of the sequence cycle, the repetitive yield of the
protein sequencer, and the relative yields of amino acids unique in
the analyzed sequence. That is if the unique (in the analyzed
sequence) residue X appears only in the nth cycle a cleavage site
exists n-1 residues before it in the N-terminal direction. In
addition to helping resolve any ambiguity in the assignment of mass
to sequences, these data also provide a more reliable indication of
the relative yield of the various fragments than does mass
spectrometry.
[0065] For PSMA.sub.163-192 (SEQ ID NO. 3) this pool sequencing
supports a single major cleavage site after V.sub.177 and several
minor cleavage sites, particularly one after Y.sub.179. Reviewing
the results presented in FIGS. 1A-C reveals the following:
[0066] S at the 3.sup.rd cycle indicating presence of the
N-terminus of the substrate.
[0067] Q at the 5.sup.th cycle indicating presence of the
N-terminus of the substrate.
[0068] N at the 1.sup.st cycle indicating cleavage after
V.sub.177.
[0069] N at the 3.sup.rd cycle indicating cleavage after V.sub.175.
Note the fragment 176-192 in Table 1.
[0070] T at the 5.sup.th cycle indicating cleavage after
V.sub.177.
[0071] T at the 1.sup.st-3.sup.rd cycles, indicating increasingly
common cleavages after R.sub.181, A.sub.180 and Y.sub.179. Only the
last of these correspond to peaks detected by mass spectrometry;
163-179 and 180-192, see Table 1. The absence of the others can
indicate that they are on fragments smaller than were examined in
the mass spectrum.
[0072] K at the 4.sup.th, 8.sup.th, and 10.sup.th cycles indicating
cleavages after E.sub.183, Y.sub.179, and V.sub.177, respectively,
all of which correspond to fragments observed by mass spectroscopy.
See Table 1.
[0073] A at the 1.sup.st and 3.sup.rd cycles indicating presence of
the N-terminus of the substrate and cleavage after V.sub.177,
respectively.
[0074] P at the 4.sup.th and 8.sup.th cycles indicating presence of
the N-terminus of the substrate.
[0075] G at the 6.sup.th and 10.sup.th cycles indicating presence
of the N-terminus of the substrate.
[0076] M at the 7.sup.th cycle indicating presence of the
N-terminus of the substrate and/or cleavage after F.sub.185.
[0077] M at the 15.sup.th cycle indicating cleavage after
V.sub.177.
[0078] The 1.sup.st cycle can indicate cleavage after D.sub.191,
see Table 1.
[0079] R at the 4.sup.th and 13.sup.th cycle indicating cleavage
after V.sub.177.
[0080] R at the 2.sup.nd and 11.sup.th cycle indicating cleavage
after Y.sub.179.
[0081] V at the 2.sup.nd, 6.sup.th, and 13.sup.th cycle indicating
cleavage after V.sub.175, M.sub.169 and presence of the N-terminus
of the substrate, respectively. Note fragments beginning at 176 and
170 in Table 1.
[0082] Y at the 1.sup.st, 2.sup.nd, and 14.sup.th cycles indicating
cleavage after V.sub.175, V.sub.177, and presence of the N-terminus
of the substrate, respectively.
[0083] L at the 11.sup.th and 12.sup.th cycles indicating cleavage
after V.sub.177, and presence of the N-terminus of the substrate,
respectively, is the interpretation most consistent with the other
data. Comparing to the mass spectrometry results we see that
[0084] L at the 2.sup.nd, 5.sup.th, and 9.sup.th cycles is
consistent with cleavage after F.sub.186, E.sub.183 or M.sub.169,
and Y.sub.179, respectively. See Table 1.
[0085] Epitope Identification
[0086] Fragments co-C-terminal with 8-10 amino acid long sequences
predicted to bind HLA by the SYFPEITHI or NIH algorithms were
chosen for further analysis. The digestion and prediction steps of
the procedure can be usefully practiced in any order. Although the
substrate peptide used in proteasomal digest described here was
specifically designed to include a predicted HLA-A1 binding
sequence, the actual products of digestion can be checked after the
fact for actual or predicted binding to other MHC molecules.
Selected results are shown in Table 2.
2TABLE 2 Predicted HLA binding by proteasomally generated fragments
I. SEQ ID NO II. PEPTIDE HLA SYFPEITHI NIH 5 & (6) (G)MPEGDLVYV
A*0201 17(27) (2605) B*0702 20 <5 B*5101 22 314 7 & (8)
(Q)GMPEGDLVY A1 24(26) <5 A3 16(18) 36 B*2705 17 25 9 MPEGDLVY
B*5101 15 NP.dagger. 10 & (11) (P)EGDLVYVNY A1 27(15) 12 A26
23(17) NP 12 LVYVNYARTE A3 21 <5 13 & (14) (Y)VNYARTEDF A26
(20) NP B*08 15 <5 B*2705 12 50 15 NYARTEDFF A24 NP.dagger. 100
Cw*0401 NP 120 16 YARTEDFF B*08 16 <5 17 RTEDFFKLE A1 21 <5
A26 15 NP .dagger.No prediction
[0087] HLA-A*0201 Binding Assay:
[0088] Binding of the candidate epitope PSMA.sub.168-177,
GMPEGDLVYV, (SEQ ID NO. 6) to HLA-A2.1 was assayed using a
modification of the method of Stauss et al., (Proc Natl Acad Sci
USA 89(17):7871-5 (1992)). Specifically, T2 cells, which express
empty or unstable MHC molecules on their surface, were washed twice
with Iscove's modified Dulbecco's medium (IMDM) and cultured
overnight in serum-free AIM-V medium (Life Technologies, Inc.,
Rockville, Md.) supplemented with human .beta.2-microglobulin at 3
.mu.g/ml (Sigma, St. Louis, Mo.) and added peptide, at 800, 400,
200, 100, 50, 25, 12.5, and 6.25 .mu.g/ml.in a 96-well flat-bottom
plate at 3.times.10.sup.5 cells/200 .mu.l/well. Peptide was mixed
with the cells by repipeting before distributing to the plate
(alternatively peptide can be added to individual wells), and the
plate was rocked gently for 2 minutes. Incubation was in a 5%
CO.sub.2 incubator at 37.degree. C. The next day the unbound
peptide was removed by washing twice with serum free RPMI medium
and a saturating amount of anti-class I HLA monoclonal antibody,
fluorescein isothiocyanate (FITC)-conjugated anti-HLA A2, A28 (One
Lambda, Canoga Park, Calif.) was added. After incubation for 30
minutes at 4.degree. C., cells were washed 3 times with PBS
supplemented with 0.5% BSA, 0.05% (w/v) sodium azide, pH 7.4-7.6
(staining buffer). (Alternatively W6/32 (Sigma) can be used as the
anti-class I HLA monoclonal antibody the cells washed with staining
buffer and then incubated with fluorescein isothiocyanate
(FITC)-conjugated goat F(ab') antimouse-IgG (Sigma) for 30 min at
4.degree. C. and washed 3 times as before.) The cells were
resuspended in 0.5 ml staining buffer. The analysis of surface
HLA-A2.1 molecules stabilized by peptide binding was performed by
flow cytometry using a FACScan (Becton Dickinson, San Jose,
Calif.). If flow cytometry is not to be performed immediately the
cells can be fixed by adding a quarter volume of 2%
paraformaldehyde and storing in the dark at 4.degree. C.
[0089] As seen in FIG. 2, this epitope exhibits significant binding
at even lower concentrations than the positive control peptides.
The Melan-A peptide used as a control in this assay (and throughout
this disclosure), ELAGIGILTV (SEQ ID NO: 106), is actually a
variant of the natural sequence (EAAGIGILTV; SEQ ID NO: 107)) and
exhibits a high affinity in this assay. The known A2.1 binder
FLPSDYFPSV (HBV.sub.18-27; SEQ ID NO: 107) was also used as a
positive control. An HLA-B44 binding peptide, AEMGKYSFY (SEQ ID NO:
109), was used as a negative control. The fluorescence obtained
from the negative control was similar to the signal obtained when
no peptide was used in the assay. Positive and negative control
peptides were chosen from Table 18.3.1 in Current Protocols in
Immunology p. 18.3.2, John Wiley and Sons, New York, 1998.
Example 1.2
[0090] Cluster Analysis (PSMA.sub.281-310).
[0091] Another peptide, RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG,
PSMA.sub.281-310, (SEQ ID NO. 18), containing an A1 epitope cluster
from prostate specific membrane antigen, PSMA.sub.283-307 (SEQ ID
NO. 19), was synthesized using standard solid-phase F-moc chemistry
on a 433A ABI Peptide synthesizer. After side chain deprotection
and cleavage from the resin, peptide in ddH2O was run on a
reverse-phase preparative HPLC C18 column at following conditions:
linear AB gradient (5% B/min) at a flow rate of 4 ml/min, where
eluent A is 0.1% aqueous TFA and eluent B is 0.1% TFA in
acetonitrile. A fraction at time 17.061 min containing the expected
peptide as judged by mass spectrometry, was pooled and lyophilized.
The peptide was then subjected to proteasome digestion and mass
spectrum analysis essentially as described above. Prominent peaks
from the mass spectra are summarized in Table 3.
3TABLE 3 PSMA.sub.281-310 Mass Peak Identification. CALCU- SEQ
LATED ID MASS NO. PEPTIDE SEQUENCE (MH.sup.+) 125 281-297
RGIAEAVGLPSIPVHPI* 1727.07 126 286-297 AVGLPSIPVHPI** 1200.46 127
287-297 VGLPSIPVHPI 1129.38 128 288-297 GLPSIPVHPI.sup..dagger.
1030.25 129 298-310 GYYDAQKLLEKMG.noteq. 1516.5 130 298-305
GYYDAQKL.sctn. 958.05 131 281-305 RGIAEAVGLPSIPVHPIGYYDAQ- KL
2666.12 132 281-307 RGIAEAVGLPSIPVHPIGYYDAQKLLE 2908.39 133 286-307
AVGLPSIPVHPIGYYDAQKLLE.paragraph. 2381.78 134 287-307
VGLPSIPVHPIGYYDAQKLLE 2310.70 135 288-307 GLPSIPVHPIGYYDAQKLLE#
2211.57 136 281-299 RGIAEAVGLPSIPVHPIGY 1947 137 286-299
AVGLPSIPVHPIGY 1420.69 138 287-299 VGLPSIPVHPIGY 1349.61 139
288-299 GLPSIPVHPIGY 1250.48 140 287-310 VGLPSIPVHPIGYYDAQKLLEKMG
2627.14 141 288-310 GLPSIPVHPIGYYDAQKLLEKMG 2528.01 Boldface
sequences correspond to peptides predicted to bind to MHC, see
Table 4. *By mass alone this peak could also have been 296-310 or
288-303. **By mass alone this peak could also have been 298-307.
Combination of HPLC and mass spectrometry show that at some later
time points this peak is a mixture of both species. .sup..dagger.By
mass alone this peak could also have been 289-298. .noteq.By mass
alone this peak could also have been 281-295 or 294-306. .sctn.By
mass alone this peak could also have been 297-303. .paragraph.By
mass alone this peak could also have been 285-306. #By mass alone
this peak could also have been 288-303. None of these alternate
assignments are supported N-terminal pool sequence analysis.
[0092] N-Terminal Pool Sequence Analysis
[0093] One aliquot at one hour of the proteasomal digestion (see
Example 3 part 3 above) was subjected to N-terminal amino acid
sequence analysis by an ABI 473A Protein Sequencer (Applied
Biosystems, Foster City, Calif.). Determination of the sites and
efficiencies of cleavage was based on consideration of the sequence
cycle, the repetitive yield of the protein sequencer, and the
relative yields of amino acids unique in the analyzed sequence.
That is if the unique (in the analyzed sequence) residue X appears
only in the nth cycle a cleavage site exists n-1 residues before it
in the N-terminal direction. In addition to helping resolve any
ambiguity in the assignment of mass to sequences, these data also
provide a more reliable indication of the relative yield of the
various fragments than does mass spectrometry.
[0094] For PSMA.sub.281-310 (SEQ ID NO. 18) this pool sequencing
supports two major cleavage sites after V.sub.287 and 1297 among
other minor cleavage sites. Reviewing the results presented in FIG.
3 reveals the following:
[0095] S at the 4.sup.th and 11.sup.th cycles indicating cleavage
after V.sub.287 and presence of the N-terminus of the substrate,
respectively.
[0096] H at the 8.sup.th cycle indicating cleavage after V.sub.287.
The lack of decay in peak height at positions 9 and 10 versus the
drop in height present going from 10 to 11 can suggest cleavage
after A.sub.286 and E.sub.285 as well, rather than the peaks
representing latency in the sequencing reaction.
[0097] D at the 2.sup.nd, 4.sup.th, and 7.sup.th cycles indicating
cleavages after Y.sub.299, 1297, and V.sub.294, respectively. This
last cleavage is not observed in any of the fragments in Table 4 or
in the alternate assignments in the notes below.
[0098] Q at the 6.sup.th cycle indicating cleavage after 1297.
[0099] M at the 10.sup.th and 12.sup.th cycle indicating cleavages
after Y.sub.299 and I.sub.297, respectively.
[0100] Epitope Identification
[0101] Fragments co-C-terminal with 8-10 amino acid long sequences
predicted to bind HLA by the SYFPEITHI or NIH algorithms were
chosen for further study. The digestion and prediction steps of the
procedure can be usefully practiced in any order. Although the
substrate peptide used in proteasomal digest described here was
specifically designed to include a predicted HLA-A1 binding
sequence, the actual products of digestion can be checked after the
fact for actual or predicted binding to other MHC molecules.
Selected results are shown in Table 4.
4TABLE 4 Predicted HLA binding by proteasomally generated
fragments: PSMA.sub.281-310 III. SEQ ID NO. IV. PEPTIDE HLA
SYFPEITHI NIH 20 & (21) (G)LPSIPVHPI A*0201 16(24) (24)
B*0702/B7 23 12 B*5101 24 572 Cw*0401 NP.dagger. 20 22 & (23)
(P)IGYYDAQKL A*0201 (16) <5 A26 (20) NP B*2705 16 25 B*2709 15
NP B*5101 21 57 Cw*0301 NP 24 24 & (25) (P)SIPVHPIGY A1 21(27)
<5 A26 22 NP A3 16 <5 26 IPVHPIGY B*5101 16 NP 27 YYDAQKLLE
A1 22 <5 .dagger.No prediction
[0102] As seen in Table 4, N-terminal addition of authentic
sequence to epitopes can often generate still useful, even better
epitopes, for the same or different MHC restriction elements. Note
for example the pairing of (G)LPSIPVHPI with HLA-A*0201, where the
10-mer can be used as a vaccine useful with several MHC types by
relying on N-terminal trimming to create the epitopes for HLA-B7,
-B*5101, and Cw*0401.
[0103] HLA-A*0201 Binding Assay:
[0104] HLA-A*0201 binding studies were preformed with
PSMA.sub.288-297, GLPSIPVHPI, (SEQ ID NO. 21) essentially as
described in Example 1.1 above. As seen in FIG. 2, this epitope
exhibits significant binding at even lower concentrations than the
positive control peptides.
Example 1.3
[0105] Cluster Analysis (PSMA.sub.454-481).
[0106] Another peptide, SSIEGNYTLRVDCTPLMYSLVHLTKEL,
PSMA.sub.454-481, (SEQ ID NO. 28) containing an epitope cluster
from prostate specific membrane antigen, was synthesized by MPS
(purity >95%) and subjected to proteasome digestion and mass
spectrum analysis as described above. Prominent peaks from the mass
spectra are summarized in Table 5.
5TABLE 5 PSMA.sub.454-481 Mass Peak Identification. SEQ ID MS PEAK
CALCULATED NO. (measured) PEPTIDE SEQUENCE MASS (MH.sup.+) 142
1238.5 454-464 SSIEGNYTLRV 1239.78 143 1768.38 .+-. 0.60 454-469
SSIEGNYTLRVDCTPL 1768.99 144 1899.8 454-470 SSIEGNYTLRVDCTPLM
1900.19 145 1097.63 .+-. 0.91 463-471 RVDCTPLMY 1098.32 146 2062.87
.+-. 0.68 454-471* SSIEGNYTLRVDCTPLMY 2063.36 147 1153 472-481**
SLVHNLTKEL 1154.36 148 1449.93 .+-. 1.79 470-481 MYSLVHNLTKEL
1448.73 Boldface sequence correspond to peptides predicted to bind
to MHC, see Table 6. *On the basis of mass alone this peak could
equally well be assigned to the peptide 455-472 however proteasomal
removal of just the N-terminal amino acid is considered unlikely.
If the issue were important it could be resolved by N-terminal
sequencing. **On the basis of mass this fragment might also
represent 455-464.
[0107] Epitope Identification
[0108] Fragments co-C-terminal with 8-10 amino acid long sequences
predicted to bind HLA by the SYFPEITHI or NIH algorithms were
chosen for further study. The digestion and prediction steps of the
procedure can be usefully practiced in any order. Although the
substrate peptide used in proteasomal digest described here was
specifically designed to include predicted HLA-A2.1 binding
sequences, the actual products of digestion can be checked after
the fact for actual or predicted binding to other MHC molecules.
Selected results are shown in Table 6.
6TABLE 6 Predicted HLA binding by proteasomally generated fragments
V. SEQ ID NO VI. PEPTIDE HLA SYFPEITHI NIH 29 & (30)
(S)IEGNYTLRV A1 (19) <5 A*0201 16(22) <5 31 EGNYTLRV B*5101
15 NP.dagger. 32 & (33) (Y)TLRVDCTPL A*0201 20(18) (5) A26
16(18) NP B7 14 40 B8 23 <5 B*2705 12 30 Cw*0301 NP (30) 34
LRVDCTPLM B*2705 20 600 B*2709 20 NP 35 & (36) (L)RVDCTPLMY A1
32(22) 125(13.5) A3 25 <5 A26 22 NP B*2702 NP (200) B*2705
13(NP) (1000) .dagger.No prediction
[0109] As seen in Table 6, N-terminal addition of authentic
sequence to epitopes can often generate still useful, even better
epitopes, for the same or different MHC restriction elements. Note
for example the pairing of (L)RVDCTPLMY (SEQ ID NOS 35 and (36))
with HLA-B*2702/5, where the 10-mer has substantial predicted
halftimes of dissociation and the co-C-terminal 9-mer does not.
Also note the case of SIEGNYTLRV (SEQ ID NO 30) a predicted
HLA-A*0201 epitope which can be used as a vaccine useful with
HLA-B*5101 by relying on N-terminal trimming to create the
epitope.
[0110] HLA-A*0201 Binding Assay
[0111] HLA-A*0201 binding studies were preformed, essentially as
described in Example 1.1 above, with PSMA.sub.460-469, YTLRVDCTPL,
(SEQ ID NO.33). As seen in FIG. 4, this epitope was found to bind
HLA-A2.1 to a similar extent as the known A2.1 binder FLPSDYFPSV
(HBV.sub.18-27; SEQ ID NO: 108) used as a positive control.
Additionally, PSMA.sub.461-469, (SEQ ID NO. 32) binds nearly as
well.
[0112] ELISPOT analysis: PSMA.sub.463-471 (SEQ ID NO. 35)
[0113] The wells of a nitrocellulose-backed microtiter plate were
coated with capture antibody by incubating overnight at 4.degree.
C. using 50 .mu.l/well of 4 .mu.g/ml murine anti-human
.quadrature.-IFN monoclonal antibody in coating buffer (35 mM
sodium bicarbonate, 15 mM sodium carbonate, pH 9.5). Unbound
antibody was removed by washing 4 times 5 min. with PBS. Unbound
sites on the membrane then were blocked by adding 200 .mu.l/well of
RPMI medium with 10% serum and incubating 1 hr. at room
temperature. Antigen stimulated CD8.sup.+ T cells, in 1:3 serial
dilutions, were seeded into the wells of the microtiter plate using
100 .mu.l/well, starting at 2.times.10.sup.5 cells/well. (Prior
antigen stimulation was essentially as described in Scheibenbogen,
C. et al. Int. J. Cancer 71:932-936, 1997; which is incorporated
herein by reference in its entirety.) PSMA.sub.462-471 (SEQ ID NO.
36) was added to a final concentration of 10 .mu.g/ml and IL-2 to
100 U/ml and the cells cultured at 37.degree. C. in a 5% CO.sub.2,
water-saturated atmosphere for 40 hrs. Following this incubation
the plates were washed with 6 times 200 .mu.l/well of PBS
containing 0.05% Tween-20 (PBS-Tween). Detection antibody, 50
.mu.l/well of 2 g/ml biotinylated murine anti-human
.quadrature.-IFN monoclonal antibody in PBS+10% fetal calf serum,
was added and the plate incubated at room temperature for 2 hrs.
Unbound detection antibody was removed by washing with 4 times 200
.mu.l of PBS-Tween. 100 .mu.l of avidin-conjugated horseradish
peroxidase (Pharmingen, San Diego, Calif.) was added to each well
and incubated at room temperature for 1 hr. Unbound enzyme was
removed by washing with 6 times 200 .mu.l of PBS-Tween. Substrate
was prepared by dissolving a 20 mg tablet of 3-amino
9-ethylcoarbasole in 2.5 ml of N,N-dimethylformamide and adding
that solution to 47.5 ml of 0.05 M phosphate-citrate buffer (pH
5.0). 25 .mu.l of 30% H.sub.2O.sub.2 was added to the substrate
solution immediately before distributing substrate at 100
.mu.l/well and incubating the plate at room temperature. After
color development (generally 15-30 min.), the reaction was stopped
by washing the plate with water. The plate was air dried and the
spots counted using a stereomicroscope.
[0114] FIG. 5 shows the detection of PSMA.sub.463-471 (SEQ ID NO.
35)-reactive HLA-A1.sup.+ CD8.sup.+ T cells previously generated in
cultures of HLA-A1.sup.+ CD8.sup.+ T cells with autologous
dendritic cells plus the peptide. No reactivity is detected from
cultures without peptide (data not shown). In this case it can be
seen that the peptide reactive T cells are present in the culture
at a frequency between 1 in 2.2.times.10.sup.4 and 1 in
6.7.times.10.sup.4. That this is truly an HLA-A1-restricted
response is demonstrated by the ability of anti-HLA-A1 monoclonal
antibody to block .quadrature.-IFN production; see FIG. 6.
Example 1.4
[0115] Cluster Analysis (PSMA.sub.653-687).
[0116] Another peptide, FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY
PSMA.sub.653-687, (SEQ ID NO. 37) containing an A2 epitope cluster
from prostate specific membrane antigen, PSMA.sub.660-681 (SEQ ID
NO. 38), was synthesized by MPS (purity>95%) and subjected to
proteasome digestion and mass spectrum analysis as described above.
Prominent peaks from the mass spectra are summarized in Table
7.
7TABLE 7 PSMA.sub.653-687 Mass Peak Identification. SEQ ID MS PEAK
CALCULATED NO. (measured) PEPTIDE SEQUENCE MASS (MH.sup.+) 149
906.17 .+-. 0.65 681-687** LPDRPFY 908.05 150 1287.73 .+-. 0.76
677-687** DPLGLPDRPFY 1290.47 151 1400.3 .+-. 1.79 676-687
IDPLGLPDRPFY 1403.63 152 1548.0 .+-. 1.37 675-687 FIDPLGLPDRPFY
1550.80 153 1619.5 .+-. 1.51 674-687** AFIDPLGLPDRPFY 1621.88 154
1775.48 .+-. 1.32 673-687* RAFIDPLGLPDRPFY 1778.07 155 2440.2 .+-.
1.3 653-672 FDKSNPIVLRMMNDQLMFLE 2442.932313.82 156 1904.63 .+-.
1.56 672-687* ERAFIDPLGLPDRPFY 1907.19 157 2310.6 .+-. 2.5 653-671
FDKSNPIVLRMMNQLMFL 2313.82 158 2017.4 .+-. 1.94 671-687
LERAFIDPLGLPDRPFY 2020.35 159 2197.43 .+-. 1.78 653-670
FDKSNPIVLRMMNDQLMF 2200.66 Boldface sequence correspond to peptides
predicted to bind to MHC, see Table 7. *On the basis of mass alone
this peak could equally well be assigned to a peptide beginning at
654, however proteasomal removal of just the N-terminal amino acid
is considered unlikely. If the issue were important it could be
resolved by N-terminal sequencing. **On the basis of mass alone
these peaks could have been assigned to internal fragments, but
given the overall pattern of digestion it was considered
unlikely.
[0117] Epitope Identification
[0118] Fragments co-C-terminal with 8-10 amino acid long sequences
predicted to bind HLA by the SYFPEITHI or NIH algorithms were
chosen for further study. The digestion and prediction steps of the
procedure can be usefully practiced in any order. Although the
substrate peptide used in proteasomal digest described here was
specifically designed to include predicted HLA-A2.1 binding
sequences, the actual products of digestion can be checked after
the fact for actual or predicted binding to other MHC molecules.
Selected results are shown in Table 8.
8TABLE 8 Predicted HLA binding by proteasomally generated fragments
VII. SEQ ID NO VIII. PEPTIDE HLA SYFPEITHI NIH 39 & (40)
(R)MMNDQLMFL A*0201 24(23) 1360(722) A*0205 NP.dagger. 71(42) A26
15 NP B*2705 12 50 41 RMMNDQLMF B*2705 17 75 .dagger.No
prediction
[0119] As seen in Table 8, N-terminal addition of authentic
sequence to epitopes can generate still useful, even better
epitopes, for the same or different MHC restriction elements. Note
for example the pairing of (R)MMNDQLMFL (SEQ ID NOS. 39 and (40))
with HLA-A*02, where the 10-mer retains substantial predicted
binding potential.
[0120] HLA-A*0201 Binding Assay
[0121] HLA-A*0201 binding studies were preformed, essentially as
described in Example 1.1 above, with PSMA.sub.663-671, (SEQ ID NO.
39) and PSMA.sub.662-671, RMMNDQLMFL (SEQ NO. 67). As seen in FIGS.
4, 7 and 8, this epitope exhibits significant binding at even lower
concentrations than the positive control peptide (FLPSDYFPSV
(HBV.sub.18-27); SEQ ID NO. 108). Though not run in parallel,
comparison to the controls suggests that PSMA.sub.662-671 (which
approaches the Melan A peptide in affinity) has the superior
binding activity of these two PSMA peptides.
Example 2
[0122] A multi-center clinical study is carried out using
compositions as disclosed herein. The results of the study show the
compositions to be useful and effective for debulking solid tumors
and for generally inducing anti-angiogenic activity.
Example 3
Evaluation of a PSMA composition in the Xenotransplanted Human
Vasculature Model
[0123] Generation of Target Antigen-Reactive CTL
[0124] A. In Vivo Immunization of Mice.
[0125] HHD1 transgenic A*0201 mice (Pascolo, S., et al. J. Exp.
Med. 185:2043-2051, 1997) were anesthetized and injected
subcutaneously at the base of the tail, avoiding lateral tail
veins, using 100 .mu.l containing 100 nmol of PSMA.sub.288-297 (SEQ
ID NO. 21) and 20 .mu.g of a HTL epitope peptide in PBS emulsified
with 50 .mu.l of IFA (incomplete Freund's adjuvant).
[0126] B. Preparation of Stimulating Cells (LPS Blasts).
[0127] Using spleens from 2 nave mice for each group of immunized
mice, un-immunized mice were sacrificed and their carcasses placed
in alcohol. Using sterile instruments, the top dermal layer of skin
on the mouse's left side (lower mid-section) was cut through,
exposing the peritoneum. The peritoneum was saturated with alcohol,
and the spleen aseptically extracted. The spleens were placed in a
petri dish with serum-free media. Splenocytes were isolated by
using sterile plungers from 3 ml syringes to mash the spleens.
Cells were collected in a 50 ml conical tubes in serum-free media,
rinsing dish well. Cells were centrifuged (12000 rpm, 7 min) and
washed one time with RPMI. Fresh spleen cells were resuspended to a
concentration of 1.times.10.sup.6 cells per ml in RPMI-10% FCS
(fetal calf serum). 25 g/ml lipopolysaccharide and 7 .mu.g/ml
Dextran Sulfate were added. Cell were incubated for 3 days in T-75
flasks at 37.degree. C., with 5% CO.sub.2. Splenic blasts were
collected in 50 ml tubes pelleted (12,000 rpm, 7 min) and
resuspended to 3.times.10.sup.7/ml in RPMI. The blasts were pulsed
with the priming peptide at 50 .mu.g/ml, RT 4 hr. mitomycin
C-treated at 25 .mu.g/ml, 37.degree. C., 20 min and washed three
times with DMEM.
[0128] C. In Vitro Stimulation.
[0129] Three days after LPS stimulation of the blast cells and the
same day as peptide loading, the primed mice were sacrificed (at 14
days post immunization) to remove spleens as above.
3.times.10.sup.6 splenocytes were co-cultured with 1.times.10.sup.6
LPS blasts/well in 24-well plates at 37.degree. C., with 5%
CO.sub.2 in DMEM media supplemented with 10% FCS, 5.times.10.sup.-5
M .beta.(beta)-mercaptoethanol, 100 .mu.g/ml streptomycin and 100
.mu.g/ml penicillin. Cultures were fed 5% (vol/vol) ConA
supernatant on day 3 and can be transferred on day 7. An aliquot of
the CTL are also tested in a standard chromium release assay to
ensure activity.
[0130] Implantation and Adoptive Transfer
[0131] 1.times.10.sup.6 telomerase-transformed HDMEC in 10 .mu.l of
EGM-2-VM medium (Clonetics, San Diego, Calif.) are mixed with 0.5
ml of MATRIGEL (Becton Dickinson) on ice. The mixture is injected
subcutaneously, through a 25 gauge needle, along the ventral
midline of the thorax of SCID mice. One week later 1.times.10.sup.7
T cells (target epitope-reactive or sham-immunized) in 0.2 ml are
injected intravenously (alternatively they can be injected
intraperitoneally).
[0132] Assessment (Micromorphometry)
[0133] At one and two weeks after transfer remove implants, fix in
10% buffered overnight, embed in paraffin, and section. For
immunofluorescence detection of human microvessels using anti-human
type IV collagen IgG and fluorescently-labeled secondary antibody,
deparifinize and retrieve antigen by microwaving thin sections
2.times.7 minutes in 10 mM citric acid, pH 6.0. Vessel density is
assessed as a function of the average number of positively stained
annular structures observed in five separate, randomly selected
20.times. fields-of-view, from at least three sections per
implant.
Example 4
A Fibronectin ED-B Vaccine in the HLA-transgenic Mouse Model
[0134] A. Establishment of Tumor
[0135] M1 tumor cells grown in complete RPMI plus 10% serum were
harvested and washed with PBS by centrifugation. The cells were
suspended in PBS at 5.times.10.sup.6 cells/ml and 0.5 ml of the
suspension (early passage) was injected subcutaneously into the
abdomen.
[0136] B. Vaccination
[0137] A nucleotide sequence encoding an HLA-A2-restricted
fibronectin ED-B domain-derived housekeeping epitope, for example
ED-B.sub.29-38 (SEQ ID NO. 103), is inserted into an appropriate
vaccine vector (e.g. pVAX1 (Invitrogen Inc, Carlsbad, Calif.) or
one of the vectors described in U.S. patent application Ser. No.
09/561,572 entitled "EXPRESSION VECTORS ENCODING EPITOPES OF
TARGET-ASSOCIATED ANTIGENS," filed on Apr. 28, 2001, and
incorporated by reference above. HHD-A2 mice are injected
intranodally in the inguinal lymph node with 0, 2, 10, 50, 100, and
200 .mu.g of vector in PBS every other day over 8 days (4
injections) alternating sides for each injection (single dosage per
mouse or group of mice). Injection series are started the day of
tumor cell injection, at 2 weeks before, and at 4 and 10 weeks
after.
[0138] C. Evaluation
[0139] At approximately 12 weeks after injection of tumor cells
visible tumors are observed in the mice receiving the vehicle
instead of the vaccine. Effectiveness of the vaccine is expressed
as the proportion of vaccinated animals that fail to develop a
tumor in the same time frame, the relative size of tumors at the
same time point, the delay in time before tumors appear in the
vaccinated animals, and the dose and number of composition cycles
needed to inhibit or prevent the establishment of tumor.
[0140] D. Alternative Schedule
[0141] The availability of more aggressive later passage M1 cells
enables a more compressed experimental schedule. Instead mice are
vaccinated on the day of tumor cell inoculation, 1 and 2 weeks
before, and 3 or 4 days after injections of 1.times.10.sup.6 cells.
Effectiveness of vaccination is assessed at approximately 10 days
after tumor cell inoculation.
[0142] E. Immunization with peptide
[0143] HHD-A2 mice were immunized with ED-B29-38 (SEQ ID NO. 103)
in complete Freund's adjuvants and spleen cells were harvested and
re-stimulated in vitro using standard methodology. The resulting
CTL were able to specifically lyse peptide cells, which are
HLA-A2+(FIG. 9).
Example 5
Epitopes and Epitope Clusters
[0144] Table 9 discloses epitopes and epitope clusters from PSMA
and ED-B that can be useful in construction of compositions
according to the present invention.
9TABLE 9 SEQ ID NOS.* SEQ ID NO ENTITY SEQUENCE 1 PSMA protein
Accession number**: NP_004467 2 PSMA cDNA Accession number:
NM_004476 3 PSMA 163-192 AFSPQGMPEGDLVYVNYARTEDFFKLERDM 4 PSMA
168-190 GMPEGDLVYVNYARTEDFFKLER 5 PSMA 169-177 MPEGDLVYV 6 PSMA
168-177 GMPEGDLVYV 7 PSMA 168-176 GMPEGDLVY 8 PSMA 167-176
QGMPEGDLVY 9 PSMA 169-176 MPEGDLVY 10 PSMA 171-179 EGDLVYVNY 11
PSMA 170-179 PEGDLVYVNY 12 PSMA 174-183 LVYVNYARTE 13 PSMA 177-185
VNYARTEDF 14 PSMA 176-185 YVNYARTEDF 15 PSMA 178-186 NYARTEDFF 16
PSMA 179-186 YARTEDFF 17 PSMA 181-189 RTEDFFKLE 18 PSMA 281-310
RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG 19 PSMA 283-307
IAEAVGLPSIPVHPIGYYDAQKLLE 20 PSMA 289-297 LPSIPVHPI 21 PSMA 288-297
GLPSIPVHPI 22 PSMA 297-305 IGYYDAQKL 23 PSMA 296-305 PIGYYDAQKL 24
PSMA 291-299 SIPVHPIGY 25 PSMA 290-299 PSIPVHPIGY 26 PSMA 292-299
IPVHPIGY 27 PSMA 299-307 YYDAQKLLE 28 PSMA 454-481
SSIEGNYTLRVDCTPLMYSLVHLTKEL 29 PSMA 456-464 IEGNYTLRV 30 PSMA
455-464 SIEGNYTLRV 31 PSMA 457-464 EGNYTLRV 32 PSMA 461-469
TLRVDCTPL 33 PSMA 460-469 YTLRVDCTPL 34 PSMA 462-470 LRVDCTPLM 35
PSMA 463-471 RVDCTPLMY 36 PSMA 462-471 LRVDCTPLMY 37 PSMA 653-687
FDKSNPIVLRMMNDQLMFLERAFIDPLGLP- DRPFY 38 PSMA 660-681
VLRMMNDQLMFLERAFIDPLGL 39 PSMA 663-671 MMNDQLMFL 40 PSMA 662-671
RMMNDQLMFL 41 PSMA 662-670 RMMNDQLMF 42 PSMA 4-12 LLHETDSAV 43 PSMA
13-21 ATARRPRWL 44 PSMA 53-61 TPKHNMKAF 45 PSMA 64-73 ELKAENIKKF 46
PSMA 69-77 NIKKFLH.sup.1NF 47 PSMA 68-77 ENIKKFLH.sup.1NF 48 PSMA
220-228 AGAKGVILY 49 PSMA 468-477 PLMYSLVHNL 50 PSMA 469-477
LMYSLVHNL 51 PSMA 463-471 RVDCTPLMY 52 PSMA 465-473 DCTPLMYSL 53
PSMA 507-515 SGMPRISKL 54 PSMA 506-515 FSGMPRISKL 55 PSMA 211-218
GNKVKNAQ 56 PSMA 202-209 IARYGKVF 57 PSMA 217-225 AQLAGAKGV 58 PSMA
207-215 KVFRGNKVK 59 PSMA 211-219 GNKVKNAQL 60 PSMA 269-277
TPGYPANEY 61 PSMA 268-277 LTPGYPANEY 62 PSMA 271-279 GYPANEYAY 63
PSMA 270-279 PGYPANEYAY 64 PSMA 266-274 DPLTPGYPA 65 PSMA 492-500
SLYESWTKK 66 PSMA 491-500 KSLYESWTKK 67 PSMA 486-494 EGFEGKSLY 68
PSMA 485-494 DEGFEGKSLY 69 PSMA 498-506 TKKSPSPEF 70 PSMA 497-506
WTKKSPSPEF 71 PSMA 492-501 SLYESWTKKS 72 PSMA 725-732 WGEVKRQI 73
PSMA 724-732 AWGEVKRQI 74 PSMA 723-732 KAWGEVKRQI 75 PSMA 723-730
KAWGEVKR 76 PSMA 722-730 SKAWGEVKR 77 PSMA 731-739 QIYVAAFTV 78
PSMA 733-741 YVAAFTVQA 79 PSMA 725-733 WGEVKRQIY 80 PSMA 727-735
EVKRQIYVA 81 PSMA 738-746 TVQAAAETL 82 PSMA 737-746 FTVQAAAETL 83
PSMA 729-737 KRQIYVAAF 84 PSMA 721-729 PSKAWGEVK 85 PSMA 723-731
KAWGEVKRQ 86 PSMA 100-108 WKEFGLDSV 87 PSMA 99-108 QWKEFGLDSV 88
PSMA 102-111 EFGLDSVELA 89 ED-B domain of
EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRI Fibronectin
TVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID YDISVITLINGGESAPTTLTQQT 90 ED-B
domain of CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIP Fibronectin with
EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRI flanking sequence
TVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGID from Fribronectin
YDISVITLINGGESAPTTLTQQT AVPPPTDLRFTNIGPDTMRVTW 91 ED-B domain of
Accession number: X07717 Fibronectin cds 92 ED-B 4'-5 TIIPEVPQL 93
ED-B 5'-5 DTTIIPEVPQL 94 ED-B 1-10 EVPQLTDLSF 95 ED-B 23-30
TPLNSSTI 96 ED-B 18-25 IGLRWTPL 97 ED-B 17-25 SIGLRWTPL 98 ED-B
25-33 LNSSTHGY 99 ED-B 24-33 PLNSSTIIGY 100 ED-B 23-31 TPLNSSTII
101 ED-B 31-38 IGYRITVV 102 ED-B 30-38 IIGYRITVV 103 ED-B 29-38
TIIGYRITVV 104 ED-B 31-39 IGYRITVVA 105 ED-B 30-39 IIGYRITVVA 106
Melan-A 26-35.sub.A > L ELAGIGILTV 107 Melan-A 26-35 EAAGIGILTV
108 HBV 18-27 FLPSDYFPSV 109 HLA-B44 binder AEMGKYSFY .sup.1This H
was reported as Y in the SWISSPROT database. *Any of SEQ ID NOS.
5-17, 20-27, 29-36, 39-88, and 92-105 can be useful as epitopes in
the various embodiments of the invention. Any of SEQ ID NOS. 3, 4,
18, 19, 28, 37, 38, 89 and 90 can be useful as sequences containing
epitopes or epitope clusters, as described in various embodiments
of the invention. **All accession numbers used here and throughout
can be accessed through the NCBI databases, for example, through
the Entrez seek and retrieval system on the world wide web.
[0145]
10 PSMA LOCUS NM_004476 2653 bp mRNA PRI 01-NOV-2000 DEFINITION
Homo sapiens folate hydrolase (prostate-specific membrane antigen)
1 (FOLH1), mRNA. ACCESSION NM_004476 VERSION NM_004476.1 GI:4758397
KEYWORDS . SOURCE human. ORGANISM Homo sapiens Eukaryota; Metazoa;
Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria;
Primates; Catarrhini; Hominidae; Homo. REFERENCE 1 (bases 1 to
2653) AUTHORS Israeli, R. S., Powell, C. T., Fair, W. R. and
Heston, W. D. TITLE Molecular cloning of a complementary DNA
encoding a prostate-specific membrane antigen JOURNAL Cancer Res.
53 (2), 227-230 (1993) MEDLINE 93113576 REFERENCE 2 (bases 1 to
2653) AUTHORS Rinker-Schaeffer C. W., Hawkins A. L., Su S. L.,
Israeli R. S., Griffin C. A., Isaacs J. T. and Heston W. D. TITLE
Localization and physical mapping of the prostate-specific membrane
antigen (PSM) gene to human chromosome 11 JOURNAL Genomics 30 (1),
105-108 (1995) MEDLINE 96129312 PUBMED 8595888 REFERENCE 3 (bases 1
to 2653) AUTHORS O'Keefe D. S., Su S. L., Bacich D. J., Horiguchi
Y., Luo Y., Powell C. T., Zandvliet D., Russell P. J., Molloy P.
L., Nowak N. J., Shows T. B., Mullins C., Vonder Haar R. A., Fair
W. R. and Heston W. D. TITLE Mapping, genomic organization and
promoter analysis of the human prostate-specific membrane antigen
gene JOURNAL Biochim. Biophys. Acta 1443 (1-2), 113-127 (1998)
MEDLINE 99057588 PUBMED 9838072 REFERENCE 4 (bases 1 to 2653)
AUTHORS Maraj B. H., Leek J. P., Karayi M., Ali M., Lench N. J. and
Markham A. F. TITLE Detailed genetic mapping around a putative
prostate-specific membrane antigen locus on human chromosome
11p11.2 JOURNAL Cytogenet. Cell Genet. 81 (1), 3-9 (1998) MEDLINE
98358137 PUBMED 9691167 COMMENT PROVISIONAL REFSEQ: This record has
not yet been subject to final NCBI review. The reference sequence
was derived from M99487.1. FEATURES Location/Qualifiers source 1 .
. . 2653 /organism="Homo sapiens" /db_xref="taxon:9606"
/chromosome="11" /map="11p11.2" /sex="male" /cell_line="LNCaP-ATCC"
/cell_type="prostate" /tissue_type="prostatic carcinoma metastatic
lymph node" /tissue_lib="LNCaP cDNA of Ron Israeli" gene 1 . . .
2653 /gene="FOLH1" /note="FOLH; PSM; PSMA" /db_xref="LocusID:2346"
/db_xref="MIM:600934" CDS 262 . . . 2514 /gene="FOLH1"
/note="folate hydrolase 1 (prostate-specific membrane antigen)"
/codon_start=1 /db_xref="LocusID:2346" /db_xref="MIM:600934"
/evidence=experimental /product="folate hydrolase
(prostate-specific membrane antigen) 1" /protein_id="NP_004467.1"
/db_xref="GI:4758398" (SEQ ID NO. 1)
/translation="MWNLLHETDSAVATARRPRWLCAG-
ALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIK
KFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLF-
EPPPPGYENVSD IVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIV-
IARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKS
YPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAP-
PDSSWRGSLKVP YNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRY-
VILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWR
PRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSP-
DEGFEGKSLYES WTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNW-
ETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRG
GMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFD-
KSNPIVLRMMND QLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDA-
LFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA" misc feature 778 . . .
1029 /note="PA; Region: PA domain" BASE COUNT 782 a 524 c 640 g 707
t ORIGIN (SEQ ID NO. 2) 1 ctcaaaaggg gccggatttc cttctcctgg
aggcagatgt tgcctctctc tctcgctcgg 61 attggttcag tgcactctag
aaacactgct gtggtggaga aactggaccc caggtctgga 121 gcgaattcca
gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac 181
cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag
241 gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc
ggctgtggcc 301 accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg
tgctggcggg tggcttcttt 361 ctcctcggct tcctcttcgg gtggtttata
aaatcctcca atgaagctac taacattact 421 ccaaagcata atatgaaagc
atttttggat gaattgaaag ctgagaacat caagaagttc 481 ttatataatt
ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca 541
aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat
601 gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat
aattaatgaa 661 gatggaaatg agattttcaa cacatcatta tttgaaccac
ctcctccagg atatgaaaat 721 gtttcggata ttgtaccacc tttcagtgct
ttctctcctc aaggaatgcc agagggcgat 781 ctagtgtatg ttaactatgc
acgaactgaa gacttcttta aattggaacg ggacatgaaa 841 atcaattgct
ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag 901
gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga ccctgctgac
961 tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctgg
aggtggtgtc 1021 cagcgtggaa atatcctaaa tctgaatggt gcaggagacc
ctctcacacc aggttaccca 1081 gcaaatgaat atgcttatag gcgtggaatt
gcagaggctg ttggtcttcc aagtattcct 1141 gttcatccaa ttggatacta
tgatgcacag aagctcctag aaaaaatggg tggctcagca 1201 ccaccagata
gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt 1261
actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa tgaagtgaca
1321 agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacag
atatgtcatt 1381 ctgggaggtc accgggactc atgggtgttt ggtggtattg
accctcagag tggagcagct 1441 gttgttcatg aaattgtgag gagctttgga
acactgaaaa aggaagggtg gagacctaga 1501 agaacaattt tgtttgcaag
ctgggatgca gaagaatttg gtcttcttgg ttctactgag 1561 tgggcagagg
agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac 1621
tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgat gtacagcttg
1681 gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaagg
caaatctctt 1741 tatgaaagtt ggactaaaaa aagtccttcc ccagagttca
gtggcatgcc caggataagc 1801 aaattgggat ctggaaatga ttttgaggtg
ttcttccaac gacttggaat tgcttcaggc 1861 agagcacggt atactaaaaa
ttgggaaaca aacaaattca gcggctatcc actgtatcac 1921 agtgtctatg
aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac 1981
ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc
2041 ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa
aatctacagt 2101 atttctatga aacatccaca ggaaatgaag acatacagtg
tatcatttga ttcacttttt 2161 tctgcagtaa agaattttac agaaattgct
tccaagttca gtgagagact ccaggacttt 2221 gacaaaagca acccaatagt
attaagaatg atgaatgatc aactcatgtt tctggaaaga 2281 gcatttattg
atccattagg gttaccagac aggccttttt ataggcatgt catctatgct 2341
ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga tgctctgttt
2401 gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag
acagatttat 2461 gttgcagcct tcacagtgca ggcagctgca gagactttga
gtgaagtagc ctaagaggat 2521 tctttagaga atccgtattg aatttgtgtg
gtatgtcact cagaaagaat cgtaatgggt 2581 atattgataa attttaaaat
tggtatattt gaaataaagt tgaatattat atataaaaaa 2641 aaaaaaaaaa aaa
[0146]
11 ED-B domain of Fibronectin LOCUS HSFIBEDB 2823 bp DNA linear PRI
09-AUG-1999 DEFINITION Human fibronectin gene ED-B region.
ACCESSION X07717 VERSION X07717.1 GI:31406 KEYWORDS alternate
splicing; fibronectin. SOURCE human. ORGANISM Homo sapiens.
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.
REFERENCE 1 (bases 1 to 2823) AUTHORS Paolella, G., Henchcliffe,
C., Sebastio, G. and Baralle, F. E. TITLE Sequence analysis and in
vivo expression show that alternative splicing of ED-B and ED-A
regions of the human fibronectin gene are independent events
JOURNAL Nucleic Acids Res. 16 (8), 3545-3557 (1988) MEDLINE
88233940 FEATURES Location/Qualifiers source 1 . . . 2823
/organism="Homo sapiens" /db_xref="taxon:9606" /clone="MA10" exon 1
. . . 104 /number=1 /product="fibronectin" CDS join(<2 . . .
104, 1375 . . . 1647, 2758 . . . >2823) /codon_start=1
/product="fibronectin" /protein_id="CAB52437.1"
/db_xref=.intg.GI:5725425" (SEQ ID NO. 90)
/translation="CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGL-
RWTPLNSSTI IGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITL-
INGGESAPTTLTQQTAVPPPTDLRFTNIGPDT MRVTW" intron 105 . . . 1374
/number=1 exon 1375 . . . 1647 /note="ED-B exon" /number=2
/product="fibronectin" intron 1648 . . . 2757 /number=2 exon 2758 .
. . 2823 /number=3 /product="fibronectin" BASE COUNT 824 a 556 c
528 g 91 t ORIGIN (SEQ ID NO. 91) 1 ctgcactttt gataacctga
gtcccggcct ggagtacaat gtcagtgttt acactgtcaa 61 ggatgacaag
gaaagtgtcc ctatctctga taccatcatc ccaggtaata gaaaataagc 121
tgctatcctg agagtgacat tccaataaga gtggggatta gcatcttaat ccccagatgc
181 ttaagggtgt caactatatt tgggatttaa ttccgatctc ccagctgcac
tttccaaaac 241 caagaagtca aagcagcgat ttggacaaaa tgcttgctgt
taacactgct ttactgtctg 301 tgcttcactg ggatgctgtg tgttgcagcg
agtatgtaat ggagtggcag ccatggcttt 361 aactctgtat tgtctgctca
catggaagta tgactaaaac actgtcacgt gtctgtactc 421 agtactgata
ggctcaaagt aatatggtaa atgcatccca tcagtacatt tctgcccgat 481
tttacaatcc atatcaattt ccaacagctg cctatttcat cttgcagttt caaatccttc
541 tttttgaaaa ttggatttta aaaaaaagtt aagtaaaagt cacaccttca
gggttgttct 601 ttcttgtggc cttgaaagac aacattgcaa aggcctgtcc
taaggatagg cttgtttgtc 661 cattgggtta taacataatg aaagcattgg
acagatcgtg tccccctttg gactcttcag 721 tagaatgctt ttactaacgc
taattacatg ttttgattat gaatgaacct aaaatagtgg 781 caatggcctt
aacctaggcc tgtctttcct cagcctgaat gtgcttttga atggcacatt 841
tcacaccata cattcataat gcattagcgt tatggccatg atgttgtcat gagttttgta
901 tgggagaaaa aaaatcaatt tatcacccat ttattatttt ttccggttgt
tcatgcaagc 961 ttattttcta ctaaaacagt tttggaatta ttaaaagcat
tgctgatact tacttcagat 1021 attatgtcta ggctctaaga atggtttcga
catcctaaac agccatatga tttttaggaa 1081 tctgaacagt tcaaattgta
ccctttaagg atgttttcaa aatgtaaaaa atatatatat 1141 atatatatat
tccctaaaag aatattcctg tttattcttc tagggaagca aactgttcat 1201
gatgcttagg aagtcttttc agagaattta aaacagattg catattacca tcattgcttt
1261 aacattccac caattttact actagtaacc tgatatacac tgctttattt
tttcctcttt 1321 ttttccctct attttccttt tgcctccccc tccctttgct
ttgtaactca atagaggtgc 1381 cccaactcac tgacctaagc tttgttgata
taaccgattc aagcatcggc ctgaggtgga 1441 ccccgctaaa ctcttccacc
attattgggt accgcatcac agtagttgcg gcaggagaag 1501 gtatccctat
ttttgaagat tttgtggact cctcagtagg atactacaca gtcacagggc 1561
tggagccggg cattgactat gatatcagcg ttatcactct cattaatggc ggcgagagtg
1621 cccctactac actgacacaa caaacgggtg aattttgaaa acttctgcgt
ttgagacata 1681 gatggtgttg catgctgcca ccagttactc cggttaaata
tggatgtttc atgggggaag 1741 tcagcaattg gccaaagatt cagataggtg
gaattggggg gataaggaat caaatgcatc 1801 tgctaaactg attggagaaa
aacacatgca atatcttcag tacactctca tttaaaccac 1861 aagtagatat
aaagcctaga gaaatacaga tgtctgctct gttaaatata aaatagcaaa 1921
tgttcattca atttgaagac ctagaatttt tcttcttaaa taccaaacac gaataccaaa
1981 ttgcgtaagt accaattgat aagaatatat caccaaaatg taccatcatg
ctcttccttc. 2041 taccctttga taaactctac catgctcctt ctttgtagct
aaaaacccat caaaatttag 2101 ggtagagtgg atgggcattg ttttgaggta
ggagaaaagt aaacttggga ccattctagg 2161 ttttgttgct gtcactaggt
aaagaaacac ctctttaacc acagtctggg gacaagcatg 2221 caacattfta
aaggttctct gctgtgcatg ggaaaagaaa catgctgaga accaatttgc 2281
atgaacatgt tcacttgtaa gtagaattca ctgaatggaa ctgtagctct agatatctca
2341 catgggggga agtttaggac cctcttgtct ttttgtctgt gtgcatgtat
ttctttgtaa 2401 agtactgcta tgtttctctt tgctgtgtgg caacttaagc
ctcttcggcc tgggataaaa 2461 taatctgcag tggtattaat aatgtacata
aagtcaacat atttgaaagt agattaaaat 2521 cttttttaaa tatatcaatg
atggcaaaaa ggttaaaggg ggcctaacag tactgtgtgt 2581 agtgttttat
ttttaacagt agtacactat aacttaaaat agacttagat tagactgttt 2641
gcatgattat gattctgttt cctttatgca tgaaatattg attttacctt tccagctact
2701 tcgttagctt taattttaaa atacattaac tgagtcttcc ttcttgttcg
aaaccagctg 2761 ttcctcctcc cactgacctg cgattcacca acattggtcc
agacaccatg cgtgtcacct 2821 ggg //
[0147]
Sequence CWU 1
1
159 1 750 PRT Homo sapien 1 Met Trp Asn Leu Leu His Glu Thr Asp Ser
Ala Val Ala Thr Ala Arg 1 5 10 15 Arg Pro Arg Trp Leu Cys Ala Gly
Ala Leu Val Leu Ala Gly Gly Phe 20 25 30 Phe Leu Leu Gly Phe Leu
Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu 35 40 45 Ala Thr Asn Ile
Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu 50 55 60 Leu Lys
Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile 65 70 75 80
Pro His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile 85
90 95 Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala
His 100 105 110 Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro
Asn Tyr Ile 115 120 125 Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe
Asn Thr Ser Leu Phe 130 135 140 Glu Pro Pro Pro Pro Gly Tyr Glu Asn
Val Ser Asp Ile Val Pro Pro 145 150 155 160 Phe Ser Ala Phe Ser Pro
Gln Gly Met Pro Glu Gly Asp Leu Val Tyr 165 170 175 Val Asn Tyr Ala
Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 180 185 190 Lys Ile
Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val 195 200 205
Phe Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly 210
215 220 Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val
Lys 225 230 235 240 Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly
Val Gln Arg Gly 245 250 255 Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp
Pro Leu Thr Pro Gly Tyr 260 265 270 Pro Ala Asn Glu Tyr Ala Tyr Arg
Arg Gly Ile Ala Glu Ala Val Gly 275 280 285 Leu Pro Ser Ile Pro Val
His Pro Ile Gly Tyr Tyr Asp Ala Gln Lys 290 295 300 Leu Leu Glu Lys
Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg 305 310 315 320 Gly
Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn 325 330
335 Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu Val
340 345 350 Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val
Glu Pro 355 360 365 Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser
Trp Val Phe Gly 370 375 380 Gly Ile Asp Pro Gln Ser Gly Ala Ala Val
Val His Glu Ile Val Arg 385 390 395 400 Ser Phe Gly Thr Leu Lys Lys
Glu Gly Trp Arg Pro Arg Arg Thr Ile 405 410 415 Leu Phe Ala Ser Trp
Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr 420 425 430 Glu Trp Ala
Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala 435 440 445 Tyr
Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 450 455
460 Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu
465 470 475 480 Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu
Tyr Glu Ser 485 490 495 Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser
Gly Met Pro Arg Ile 500 505 510 Ser Lys Leu Gly Ser Gly Asn Asp Phe
Glu Val Phe Phe Gln Arg Leu 515 520 525 Gly Ile Ala Ser Gly Arg Ala
Arg Tyr Thr Lys Asn Trp Glu Thr Asn 530 535 540 Lys Phe Ser Gly Tyr
Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu 545 550 555 560 Leu Val
Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val 565 570 575
Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val 580
585 590 Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr
Ala 595 600 605 Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu
Met Lys Thr 610 615 620 Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala
Val Lys Asn Phe Thr 625 630 635 640 Glu Ile Ala Ser Lys Phe Ser Glu
Arg Leu Gln Asp Phe Asp Lys Ser 645 650 655 Asn Pro Ile Val Leu Arg
Met Met Asn Asp Gln Leu Met Phe Leu Glu 660 665 670 Arg Ala Phe Ile
Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg 675 680 685 His Val
Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser 690 695 700
Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp 705
710 715 720 Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val
Ala Ala 725 730 735 Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu
Val Ala 740 745 750 2 2653 DNA Homo sapien 2 ctcaaaaggg gccggatttc
cttctcctgg aggcagatgt tgcctctctc tctcgctcgg 60 attggttcag
tgcactctag aaacactgct gtggtggaga aactggaccc caggtctgga 120
gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac
180 cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg
gtcccgggag 240 gccggctctg ctcgcgccga gatgtggaat ctccttcacg
aaaccgactc ggctgtggcc 300 accgcgcgcc gcccgcgctg gctgtgcgct
ggggcgctgg tgctggcggg tggcttcttt 360 ctcctcggct tcctcttcgg
gtggtttata aaatcctcca atgaagctac taacattact 420 ccaaagcata
atatgaaagc atttttggat gaattgaaag ctgagaacat caagaagttc 480
ttatataatt ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca
540 aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct
agcacattat 600 gatgtcctgt tgtcctaccc aaataagact catcccaact
acatctcaat aattaatgaa 660 gatggaaatg agattttcaa cacatcatta
tttgaaccac ctcctccagg atatgaaaat 720 gtttcggata ttgtaccacc
tttcagtgct ttctctcctc aaggaatgcc agagggcgat 780 ctagtgtatg
ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa 840
atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag
900 gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga
ccctgctgac 960 tactttgctc ctggggtgaa gtcctatcca gatggttgga
atcttcctgg aggtggtgtc 1020 cagcgtggaa atatcctaaa tctgaatggt
gcaggagacc ctctcacacc aggttaccca 1080 gcaaatgaat atgcttatag
gcgtggaatt gcagaggctg ttggtcttcc aagtattcct 1140 gttcatccaa
ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca 1200
ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt
1260 actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa
tgaagtgaca 1320 agaatttaca atgtgatagg tactctcaga ggagcagtgg
aaccagacag atatgtcatt 1380 ctgggaggtc accgggactc atgggtgttt
ggtggtattg accctcagag tggagcagct 1440 gttgttcatg aaattgtgag
gagctttgga acactgaaaa aggaagggtg gagacctaga 1500 agaacaattt
tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg ttctactgag 1560
tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac
1620 tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgat
gtacagcttg 1680 gtacacaacc taacaaaaga gctgaaaagc cctgatgaag
gctttgaagg caaatctctt 1740 tatgaaagtt ggactaaaaa aagtccttcc
ccagagttca gtggcatgcc caggataagc 1800 aaattgggat ctggaaatga
ttttgaggtg ttcttccaac gacttggaat tgcttcaggc 1860 agagcacggt
atactaaaaa ttgggaaaca aacaaattca gcggctatcc actgtatcac 1920
agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac
1980 ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc
catagtgctc 2040 ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt
atgctgacaa aatctacagt 2100 atttctatga aacatccaca ggaaatgaag
acatacagtg tatcatttga ttcacttttt 2160 tctgcagtaa agaattttac
agaaattgct tccaagttca gtgagagact ccaggacttt 2220 gacaaaagca
acccaatagt attaagaatg atgaatgatc aactcatgtt tctggaaaga 2280
gcatttattg atccattagg gttaccagac aggccttttt ataggcatgt catctatgct
2340 ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga
tgctctgttt 2400 gatattgaaa gcaaagtgga cccttccaag gcctggggag
aagtgaagag acagatttat 2460 gttgcagcct tcacagtgca ggcagctgca
gagactttga gtgaagtagc ctaagaggat 2520 tctttagaga atccgtattg
aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt 2580 atattgataa
attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa 2640
aaaaaaaaaa aaa 2653 3 30 PRT Homo sapien 3 Ala Phe Ser Pro Gln Gly
Met Pro Glu Gly Asp Leu Val Tyr Val Asn 1 5 10 15 Tyr Ala Arg Thr
Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 20 25 30 4 23 PRT Homo
sapien 4 Gly Met Pro Glu Gly Asp Leu Val Tyr Val Asn Tyr Ala Arg
Thr Glu 1 5 10 15 Asp Phe Phe Lys Leu Glu Arg 20 5 9 PRT Homo
sapien 5 Met Pro Glu Gly Asp Leu Val Tyr Val 1 5 6 10 PRT Homo
sapien 6 Gly Met Pro Glu Gly Asp Leu Val Tyr Val 1 5 10 7 9 PRT
Homo sapien 7 Gly Met Pro Glu Gly Asp Leu Val Tyr 1 5 8 10 PRT Homo
sapien 8 Gln Gly Met Pro Glu Gly Asp Leu Val Tyr 1 5 10 9 8 PRT
Homo sapien 9 Met Pro Glu Gly Asp Leu Val Tyr 1 5 10 9 PRT Homo
sapien 10 Glu Gly Asp Leu Val Tyr Val Asn Tyr 1 5 11 10 PRT Homo
sapien 11 Pro Glu Gly Asp Leu Val Tyr Val Asn Tyr 1 5 10 12 10 PRT
Homo sapien 12 Leu Val Tyr Val Asn Tyr Ala Arg Thr Glu 1 5 10 13 9
PRT Homo sapien 13 Val Asn Tyr Ala Arg Thr Glu Asp Phe 1 5 14 10
PRT Homo sapien 14 Tyr Val Asn Tyr Ala Arg Thr Glu Asp Phe 1 5 10
15 9 PRT Homo sapien 15 Asn Tyr Ala Arg Thr Glu Asp Phe Phe 1 5 16
8 PRT Homo sapien 16 Tyr Ala Arg Thr Glu Asp Phe Phe 1 5 17 9 PRT
Homo sapien 17 Arg Thr Glu Asp Phe Phe Lys Leu Glu 1 5 18 30 PRT
Homo sapien 18 Arg Gly Ile Ala Glu Ala Val Gly Leu Pro Ser Ile Pro
Val His Pro 1 5 10 15 Ile Gly Tyr Tyr Asp Ala Gln Lys Leu Leu Glu
Lys Met Gly 20 25 30 19 25 PRT Homo sapien 19 Ile Ala Glu Ala Val
Gly Leu Pro Ser Ile Pro Val His Pro Ile Gly 1 5 10 15 Tyr Tyr Asp
Ala Gln Lys Leu Leu Glu 20 25 20 9 PRT Homo sapien 20 Leu Pro Ser
Ile Pro Val His Pro Ile 1 5 21 10 PRT Homo sapien 21 Gly Leu Pro
Ser Ile Pro Val His Pro Ile 1 5 10 22 9 PRT Homo sapien 22 Ile Gly
Tyr Tyr Asp Ala Gln Lys Leu 1 5 23 10 PRT Homo sapien 23 Pro Ile
Gly Tyr Tyr Asp Ala Gln Lys Leu 1 5 10 24 9 PRT Homo sapien 24 Ser
Ile Pro Val His Pro Ile Gly Tyr 1 5 25 10 PRT Homo sapien 25 Pro
Ser Ile Pro Val His Pro Ile Gly Tyr 1 5 10 26 8 PRT Homo sapien 26
Ile Pro Val His Pro Ile Gly Tyr 1 5 27 9 PRT Homo sapien 27 Tyr Tyr
Asp Ala Gln Lys Leu Leu Glu 1 5 28 27 PRT Homo sapien 28 Ser Ser
Ile Glu Gly Asn Tyr Thr Leu Arg Val Asp Cys Thr Pro Leu 1 5 10 15
Met Tyr Ser Leu Val His Leu Thr Lys Glu Leu 20 25 29 9 PRT Homo
sapien 29 Ile Glu Gly Asn Tyr Thr Leu Arg Val 1 5 30 10 PRT Homo
sapien 30 Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 1 5 10 31 8 PRT
Homo sapien 31 Glu Gly Asn Tyr Thr Leu Arg Val 1 5 32 9 PRT Homo
sapien 32 Thr Leu Arg Val Asp Cys Thr Pro Leu 1 5 33 10 PRT Homo
sapien 33 Tyr Thr Leu Arg Val Asp Cys Thr Pro Leu 1 5 10 34 9 PRT
Homo sapien 34 Leu Arg Val Asp Cys Thr Pro Leu Met 1 5 35 9 PRT
Homo sapien 35 Arg Val Asp Cys Thr Pro Leu Met Tyr 1 5 36 10 PRT
Homo sapien 36 Leu Arg Val Asp Cys Thr Pro Leu Met Tyr 1 5 10 37 35
PRT Homo sapien 37 Phe Asp Lys Ser Asn Pro Ile Val Leu Arg Met Met
Asn Asp Gln Leu 1 5 10 15 Met Phe Leu Glu Arg Ala Phe Ile Asp Pro
Leu Gly Leu Pro Asp Arg 20 25 30 Pro Phe Tyr 35 38 22 PRT Homo
sapien 38 Val Leu Arg Met Met Asn Asp Gln Leu Met Phe Leu Glu Arg
Ala Phe 1 5 10 15 Ile Asp Pro Leu Gly Leu 20 39 9 PRT Homo sapien
39 Met Met Asn Asp Gln Leu Met Phe Leu 1 5 40 10 PRT Homo sapien 40
Arg Met Met Asn Asp Gln Leu Met Phe Leu 1 5 10 41 9 PRT Homo sapien
41 Arg Met Met Asn Asp Gln Leu Met Phe 1 5 42 9 PRT Homo sapien 42
Leu Leu His Glu Thr Asp Ser Ala Val 1 5 43 9 PRT Homo sapien 43 Ala
Thr Ala Arg Arg Pro Arg Trp Leu 1 5 44 9 PRT Homo sapien 44 Thr Pro
Lys His Asn Met Lys Ala Phe 1 5 45 10 PRT Homo sapien 45 Glu Leu
Lys Ala Glu Asn Ile Lys Lys Phe 1 5 10 46 9 PRT Homo sapien VARIANT
(7)...(7) Xaa = His or Tyr 46 Asn Ile Lys Lys Phe Leu Xaa Asn Phe 1
5 47 10 PRT Homo sapien VARIANT (8)...(8) Xaa = His or Tyr 47 Glu
Asn Ile Lys Lys Phe Leu Xaa Asn Phe 1 5 10 48 9 PRT Homo sapien 48
Ala Gly Ala Lys Gly Val Ile Leu Tyr 1 5 49 10 PRT Homo sapien 49
Pro Leu Met Tyr Ser Leu Val His Asn Leu 1 5 10 50 9 PRT Homo sapien
50 Leu Met Tyr Ser Leu Val His Asn Leu 1 5 51 9 PRT Homo sapien 51
Arg Val Asp Cys Thr Pro Leu Met Tyr 1 5 52 9 PRT Homo sapien 52 Asp
Cys Thr Pro Leu Met Tyr Ser Leu 1 5 53 9 PRT Homo sapien 53 Ser Gly
Met Pro Arg Ile Ser Lys Leu 1 5 54 10 PRT Homo sapien 54 Phe Ser
Gly Met Pro Arg Ile Ser Lys Leu 1 5 10 55 8 PRT Homo sapien 55 Gly
Asn Lys Val Lys Asn Ala Gln 1 5 56 8 PRT Homo sapien 56 Ile Ala Arg
Tyr Gly Lys Val Phe 1 5 57 9 PRT Homo sapien 57 Ala Gln Leu Ala Gly
Ala Lys Gly Val 1 5 58 9 PRT Homo sapien 58 Lys Val Phe Arg Gly Asn
Lys Val Lys 1 5 59 9 PRT Homo sapien 59 Gly Asn Lys Val Lys Asn Ala
Gln Leu 1 5 60 9 PRT Homo sapien 60 Thr Pro Gly Tyr Pro Ala Asn Glu
Tyr 1 5 61 10 PRT Homo sapien 61 Leu Thr Pro Gly Tyr Pro Ala Asn
Glu Tyr 1 5 10 62 9 PRT Homo sapien 62 Gly Tyr Pro Ala Asn Glu Tyr
Ala Tyr 1 5 63 10 PRT Homo sapien 63 Pro Gly Tyr Pro Ala Asn Glu
Tyr Ala Tyr 1 5 10 64 9 PRT Homo sapien 64 Asp Pro Leu Thr Pro Gly
Tyr Pro Ala 1 5 65 9 PRT Homo sapien 65 Ser Leu Tyr Glu Ser Trp Thr
Lys Lys 1 5 66 10 PRT Homo sapien 66 Lys Ser Leu Tyr Glu Ser Trp
Thr Lys Lys 1 5 10 67 9 PRT Homo sapien 67 Glu Gly Phe Glu Gly Lys
Ser Leu Tyr 1 5 68 10 PRT Homo sapien 68 Asp Glu Gly Phe Glu Gly
Lys Ser Leu Tyr 1 5 10 69 9 PRT Homo sapien 69 Thr Lys Lys Ser Pro
Ser Pro Glu Phe 1 5 70 10 PRT Homo sapien 70 Trp Thr Lys Lys Ser
Pro Ser Pro Glu Phe 1 5 10 71 10 PRT Homo sapien 71 Ser Leu Tyr Glu
Ser Trp Thr Lys Lys Ser 1 5 10 72 8 PRT Homo sapien 72 Trp Gly Glu
Val Lys Arg Gln Ile 1 5 73 9 PRT Homo sapien 73 Ala Trp Gly Glu Val
Lys Arg Gln Ile 1 5 74 10 PRT Homo sapien 74 Lys Ala Trp Gly Glu
Val Lys Arg Gln Ile 1 5 10 75 8 PRT Homo sapien 75 Lys Ala Trp Gly
Glu Val Lys Arg 1 5 76 9 PRT Homo sapien 76 Ser Lys Ala Trp Gly Glu
Val Lys Arg 1 5 77 9 PRT Homo sapien 77 Gln Ile Tyr Val Ala Ala Phe
Thr Val 1 5 78 9 PRT Homo sapien 78 Tyr Val Ala Ala Phe Thr Val Gln
Ala 1 5 79 9 PRT Homo sapien 79 Trp Gly Glu Val Lys Arg Gln Ile Tyr
1 5 80 9 PRT Homo sapien 80 Glu Val Lys Arg Gln Ile Tyr Val Ala 1 5
81 9 PRT Homo sapien 81 Thr Val Gln Ala Ala Ala Glu Thr Leu 1 5 82
10 PRT Homo sapien 82 Phe Thr Val Gln Ala Ala Ala Glu Thr Leu 1 5
10 83 9 PRT Homo sapien 83 Lys Arg Gln Ile Tyr Val Ala Ala Phe 1 5
84 9 PRT Homo sapien 84 Pro Ser Lys Ala Trp Gly Glu Val Lys 1 5 85
9 PRT Homo sapien 85 Lys Ala Trp Gly Glu Val Lys Arg Gln 1 5 86 9
PRT Homo sapien 86 Trp Lys Glu Phe Gly Leu Asp Ser Val 1 5 87 10
PRT Homo sapien 87 Gln Trp Lys Glu Phe Gly Leu Asp Ser Val 1 5 10
88 10 PRT Homo sapien 88 Glu Phe Gly Leu Asp Ser Val Glu Leu Ala 1
5 10 89 91 PRT Homo sapien 89 Glu Val Pro Gln Leu Thr Asp Leu Ser
Phe Val Asp Ile Thr Asp Ser 1 5 10 15 Ser Ile Gly Leu Arg Trp Thr
Pro Leu Asn Ser Ser Thr Ile Ile Gly 20 25 30 Tyr Arg Ile Thr Val
Val Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu 35 40 45 Asp Phe Val
Asp Ser Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu 50 55 60 Pro
Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly Gly 65 70
75 80 Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr 85
90 90 147 PRT Homo sapien 90 Cys Thr Phe Asp Asn Leu Ser Pro Gly
Leu Glu Tyr Asn Val Ser Val 1 5 10 15 Tyr Thr Val Lys Asp Asp Lys
Glu Ser Val Pro Ile Ser Asp Thr Ile 20 25 30 Ile Pro Glu Val Pro
Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr 35 40 45 Asp Ser Ser
Ile Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser Thr Ile 50 55 60 Ile
Gly Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile 65 70
75 80 Phe Glu Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr Thr Val Thr
Gly 85 90 95 Leu Glu Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr
Leu Ile Asn 100 105 110 Gly Gly Glu Ser Ala Pro Thr Thr Leu Thr Gln
Gln Thr Ala Val Pro 115 120 125 Pro Pro Thr Asp Leu Arg Phe Thr Asn
Ile Gly Pro Asp Thr Met Arg 130 135 140 Val Thr Trp 145 91 2823 DNA
Homo sapien 91 ctgcactttt gataacctga gtcccggcct ggagtacaat
gtcagtgttt acactgtcaa 60 ggatgacaag gaaagtgtcc ctatctctga
taccatcatc ccaggtaata gaaaataagc 120 tgctatcctg agagtgacat
tccaataaga gtggggatta gcatcttaat ccccagatgc 180 ttaagggtgt
caactatatt tgggatttaa ttccgatctc ccagctgcac tttccaaaac 240
caagaagtca aagcagcgat ttggacaaaa tgcttgctgt taacactgct ttactgtctg
300 tgcttcactg ggatgctgtg tgttgcagcg agtatgtaat ggagtggcag
ccatggcttt 360 aactctgtat tgtctgctca catggaagta tgactaaaac
actgtcacgt gtctgtactc 420 agtactgata ggctcaaagt aatatggtaa
atgcatccca tcagtacatt tctgcccgat 480 tttacaatcc atatcaattt
ccaacagctg cctatttcat cttgcagttt caaatccttc 540 tttttgaaaa
ttggatttta aaaaaaagtt aagtaaaagt cacaccttca gggttgttct 600
ttcttgtggc cttgaaagac aacattgcaa aggcctgtcc taaggatagg cttgtttgtc
660 cattgggtta taacataatg aaagcattgg acagatcgtg tccccctttg
gactcttcag 720 tagaatgctt ttactaacgc taattacatg ttttgattat
gaatgaacct aaaatagtgg 780 caatggcctt aacctaggcc tgtctttcct
cagcctgaat gtgcttttga atggcacatt 840 tcacaccata cattcataat
gcattagcgt tatggccatg atgttgtcat gagttttgta 900 tgggagaaaa
aaaatcaatt tatcacccat ttattatttt ttccggttgt tcatgcaagc 960
ttattttcta ctaaaacagt tttggaatta ttaaaagcat tgctgatact tacttcagat
1020 attatgtcta ggctctaaga atggtttcga catcctaaac agccatatga
tttttaggaa 1080 tctgaacagt tcaaattgta ccctttaagg atgttttcaa
aatgtaaaaa atatatatat 1140 atatatatat tccctaaaag aatattcctg
tttattcttc tagggaagca aactgttcat 1200 gatgcttagg aagtcttttc
agagaattta aaacagattg catattacca tcattgcttt 1260 aacattccac
caattttact actagtaacc tgatatacac tgctttattt tttcctcttt 1320
ttttccctct attttccttt tgcctccccc tccctttgct ttgtaactca atagaggtgc
1380 cccaactcac tgacctaagc tttgttgata taaccgattc aagcatcggc
ctgaggtgga 1440 ccccgctaaa ctcttccacc attattgggt accgcatcac
agtagttgcg gcaggagaag 1500 gtatccctat ttttgaagat tttgtggact
cctcagtagg atactacaca gtcacagggc 1560 tggagccggg cattgactat
gatatcagcg ttatcactct cattaatggc ggcgagagtg 1620 cccctactac
actgacacaa caaacgggtg aattttgaaa acttctgcgt ttgagacata 1680
gatggtgttg catgctgcca ccagttactc cggttaaata tggatgtttc atgggggaag
1740 tcagcaattg gccaaagatt cagataggtg gaattggggg gataaggaat
caaatgcatc 1800 tgctaaactg attggagaaa aacacatgca atatcttcag
tacactctca tttaaaccac 1860 aagtagatat aaagcctaga gaaatacaga
tgtctgctct gttaaatata aaatagcaaa 1920 tgttcattca atttgaagac
ctagaatttt tcttcttaaa taccaaacac gaataccaaa 1980 ttgcgtaagt
accaattgat aagaatatat caccaaaatg taccatcatg ctcttccttc 2040
taccctttga taaactctac catgctcctt ctttgtagct aaaaacccat caaaatttag
2100 ggtagagtgg atgggcattg ttttgaggta ggagaaaagt aaacttggga
ccattctagg 2160 ttttgttgct gtcactaggt aaagaaacac ctctttaacc
acagtctggg gacaagcatg 2220 caacatttta aaggttctct gctgtgcatg
ggaaaagaaa catgctgaga accaatttgc 2280 atgaacatgt tcacttgtaa
gtagaattca ctgaatggaa ctgtagctct agatatctca 2340 catgggggga
agtttaggac cctcttgtct ttttgtctgt gtgcatgtat ttctttgtaa 2400
agtactgcta tgtttctctt tgctgtgtgg caacttaagc ctcttcggcc tgggataaaa
2460 taatctgcag tggtattaat aatgtacata aagtcaacat atttgaaagt
agattaaaat 2520 cttttttaaa tatatcaatg atggcaaaaa ggttaaaggg
ggcctaacag tactgtgtgt 2580 agtgttttat ttttaacagt agtacactat
aacttaaaat agacttagat tagactgttt 2640 gcatgattat gattctgttt
cctttatgca tgaaatattg attttacctt tccagctact 2700 tcgttagctt
taattttaaa atacattaac tgagtcttcc ttcttgttcg aaaccagctg 2760
ttcctcctcc cactgacctg cgattcacca acattggtcc agacaccatg cgtgtcacct
2820 ggg 2823 92 9 PRT Homo sapien 92 Thr Ile Ile Pro Glu Val Pro
Gln Leu 1 5 93 10 PRT Homo sapien 93 Asp Thr Ile Ile Pro Glu Val
Pro Gln Leu 1 5 10 94 10 PRT Homo sapien 94 Glu Val Pro Gln Leu Thr
Asp Leu Ser Phe 1 5 10 95 8 PRT Homo sapien 95 Thr Pro Leu Asn Ser
Ser Thr Ile 1 5 96 8 PRT Homo sapien 96 Ile Gly Leu Arg Trp Thr Pro
Leu 1 5 97 9 PRT Homo sapien 97 Ser Ile Gly Leu Arg Trp Thr Pro Leu
1 5 98 9 PRT Homo sapien 98 Leu Asn Ser Ser Thr Ile Ile Gly Tyr 1 5
99 10 PRT Homo sapien 99 Pro Leu Asn Ser Ser Thr Ile Ile Gly Tyr 1
5 10 100 9 PRT Homo sapien 100 Thr Pro Leu Asn Ser Ser Thr Ile Ile
1 5 101 8 PRT Homo sapien 101 Ile Gly Tyr Arg Ile Thr Val Val 1 5
102 9 PRT Homo sapien 102 Ile Ile Gly Tyr Arg Ile Thr Val Val 1 5
103 10 PRT Homo sapien 103 Thr Ile Ile Gly Tyr Arg Ile Thr Val Val
1 5 10 104 9 PRT Homo sapien 104 Ile Gly Tyr Arg Ile Thr Val Val
Ala 1 5 105 10 PRT Homo sapien 105 Ile Ile Gly Tyr Arg Ile Thr Val
Val Ala 1 5 10 106 10 PRT Homo sapien 106 Glu Leu Ala Gly Ile Gly
Ile Leu Thr Val 1 5 10 107 10 PRT Homo sapien 107 Glu Ala Ala Gly
Ile Gly Ile Leu Thr Val 1 5 10 108 10 PRT Homo sapien 108 Phe Leu
Pro Ser Asp Tyr Phe Pro Ser Val 1 5 10 109 9 PRT Homo sapien 109
Ala Glu Met Gly Lys Tyr Ser Phe Tyr 1 5 110 15 PRT Homo sapien 110
Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val Tyr Val 1 5 10
15 111 12 PRT Homo sapien 111 Asn Tyr Ala Arg Thr Glu Asp Phe Phe
Lys Leu Glu 1 5 10 112 20 PRT Homo sapien 112 Pro Glu Gly Asp Leu
Val Tyr Val Asn Tyr Ala Arg Thr Glu Asp Phe 1 5 10 15 Phe Lys Leu
Glu 20 113 14 PRT Homo sapien 113 Asn Tyr Ala Arg Thr Glu Asp Phe
Phe Lys Leu Glu Arg Asp 1 5 10 114 22 PRT Homo sapien 114 Pro Glu
Gly Asp Leu Val Tyr Val Asn Tyr Ala Arg Thr Glu Asp Phe 1 5 10 15
Phe Lys Leu Glu Arg Asp 20 115 15 PRT Homo sapien 115 Asn Tyr Ala
Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 1 5 10 15 116 14
PRT Homo sapien 116 Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu
Val Tyr 1 5 10 117 16 PRT Homo sapien 117 Val Asn Tyr Ala Arg Thr
Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 1 5 10 15 118 17 PRT Homo
sapien 118 Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val Tyr
Val Asn 1 5 10 15 Tyr 119 13 PRT Homo sapien 119 Ala Arg Thr Glu
Asp Phe Phe Lys Leu Glu Arg Asp Met 1 5 10 120 21 PRT Homo sapien
120 Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val Tyr Val Asn
1 5 10 15 Tyr Ala Arg Thr Glu 20 121 9 PRT Homo sapien 121 Asp Phe
Phe Lys Leu Glu Arg Asp Met 1 5 122 17 PRT Homo sapien 122 Tyr Val
Asn Tyr Ala Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp 1 5 10 15
Met 123 19 PRT Homo sapien 123 Gln Gly Met Pro Glu Gly Asp Leu Val
Tyr Val Asn Tyr Ala Arg Thr 1 5 10 15 Glu Asp Phe 124 9 PRT Homo
sapien 124 Asn Tyr Ala Arg Thr Glu Asp Phe Phe 1 5 125 17 PRT Homo
sapien 125 Arg Gly Ile Ala Glu Ala Val Gly Leu Pro Ser Ile Pro Val
His Pro 1 5 10 15 Ile 126 12 PRT Homo sapien 126 Ala Val Gly Leu
Pro Ser Ile Pro Val His Pro Ile 1 5 10 127 11 PRT Homo sapien 127
Val Gly Leu Pro Ser Ile Pro Val His Pro Ile 1 5 10 128 10 PRT Homo
sapien 128 Gly Leu Pro Ser Ile Pro Val His Pro Ile 1 5 10 129 13
PRT Homo sapien 129 Gly Tyr Tyr Asp Ala Gln Lys Leu Leu Glu Lys Met
Gly 1 5 10 130 8 PRT Homo sapien 130 Gly Tyr Tyr Asp Ala Gln Lys
Leu 1 5 131 25 PRT Homo sapien 131 Arg Gly Ile Ala Glu Ala Val Gly
Leu Pro Ser Ile Pro Val His Pro 1 5 10 15 Ile Gly Tyr Tyr Asp Ala
Gln Lys Leu 20 25 132 27 PRT Homo sapien 132 Arg Gly Ile Ala Glu
Ala Val Gly Leu Pro Ser Ile Pro Val His Pro 1 5 10 15 Ile Gly Tyr
Tyr Asp Ala Gln Lys Leu Leu Glu 20 25 133 22 PRT Homo sapien 133
Ala Val Gly Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp 1 5
10 15 Ala Gln Lys Leu Leu Glu 20 134 21 PRT Homo sapien 134 Val Gly
Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala 1 5 10 15
Gln Lys Leu Leu Glu 20 135 20 PRT Homo sapien 135 Gly Leu Pro Ser
Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala Gln 1 5 10 15 Lys Leu
Leu Glu 20 136 19 PRT Homo sapien 136 Arg Gly Ile Ala Glu Ala Val
Gly Leu Pro Ser Ile Pro Val His Pro 1 5 10 15 Ile Gly Tyr 137 14
PRT Homo sapien 137 Ala Val Gly Leu Pro Ser Ile Pro Val His Pro Ile
Gly Tyr 1 5 10 138 13 PRT Homo sapien 138 Val Gly Leu Pro Ser Ile
Pro Val His Pro Ile Gly Tyr 1 5 10 139 12 PRT Homo sapien 139 Gly
Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr 1 5 10 140 24 PRT Homo
sapien 140 Val Gly Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr
Asp Ala 1 5 10 15 Gln Lys Leu Leu Glu Lys Met Gly 20 141 23 PRT
Homo sapien 141 Gly Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr
Asp Ala Gln 1 5 10 15 Lys Leu Leu Glu Lys Met Gly 20 142 11 PRT
Homo sapien 142 Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 1 5 10
143 16 PRT Homo sapien 143 Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg
Val Asp Cys Thr Pro Leu 1 5 10 15 144 17 PRT Homo sapien 144 Ser
Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val Asp Cys Thr Pro Leu 1 5 10
15 Met 145 9 PRT Homo sapien 145 Arg Val Asp Cys Thr Pro Leu Met
Tyr 1 5 146 18 PRT Homo sapien 146 Ser Ser Ile Glu Gly Asn Tyr Thr
Leu Arg Val Asp Cys Thr Pro Leu 1 5 10 15 Met Tyr 147 10 PRT Homo
sapien 147 Ser Leu Val His Asn Leu Thr Lys Glu Leu 1 5 10 148 12
PRT Homo sapien 148 Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu Leu
1 5 10 149 7 PRT Homo sapien 149 Leu Pro Asp Arg Pro Phe Tyr 1 5
150 11 PRT Homo sapien 150 Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe
Tyr 1 5 10 151 12 PRT Homo sapien 151 Ile Asp Pro Leu Gly Leu Pro
Asp Arg Pro Phe Tyr 1 5 10 152 13 PRT Homo sapien 152 Phe Ile Asp
Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr 1 5 10 153 14 PRT Homo
sapien 153 Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr
1 5 10 154 15 PRT Homo sapien 154 Arg Ala Phe Ile Asp Pro Leu Gly
Leu Pro Asp Arg Pro Phe Tyr 1 5 10 15 155 20 PRT Homo sapien 155
Phe Asp Lys Ser Asn Pro Ile Val Leu Arg Met Met Asn Asp Gln Leu 1 5
10 15 Met Phe Leu Glu 20 156 16 PRT Homo sapien 156 Glu Arg Ala Phe
Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr 1 5 10 15 157 19
PRT Homo sapien 157 Phe Asp Lys Ser Asn Pro Ile Val Leu Arg Met Met
Asn Asp Gln Leu 1 5 10 15 Met Phe Leu 158 17 PRT Homo sapien 158
Leu Glu Arg Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe 1 5
10 15 Tyr 159 18 PRT Homo sapien 159 Phe Asp Lys Ser Asn Pro Ile
Val Leu Arg Met Met Asn Asp Gln Leu 1 5 10 15 Met Phe
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