U.S. patent application number 12/646703 was filed with the patent office on 2010-07-01 for compositions and methods for wt1 specific immunotherapy.
Invention is credited to Martin A. Cheever, Alexander Gaiger.
Application Number | 20100166738 12/646703 |
Document ID | / |
Family ID | 39031442 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100166738 |
Kind Code |
A1 |
Gaiger; Alexander ; et
al. |
July 1, 2010 |
COMPOSITIONS AND METHODS FOR WT1 SPECIFIC IMMUNOTHERAPY
Abstract
Compositions and methods for the therapy of malignant diseases,
such as leukemia and cancer, are disclosed. The compositions
comprise one or more of a WT1 polynucleotide, a WT1 polypeptide, an
antigen-presenting cell presenting a WT1 polypeptide, an antibody
that specifically binds to a WT1 polypeptide; or a T cell that
specifically reacts with a WT1 polypeptide. Such compositions may
be used, for example, for the prevention and treatment of
metastatic diseases.
Inventors: |
Gaiger; Alexander; (Vienna,
AT) ; Cheever; Martin A.; (Mercer Island,
WA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Family ID: |
39031442 |
Appl. No.: |
12/646703 |
Filed: |
December 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11956201 |
Dec 13, 2007 |
7662386 |
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12646703 |
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09685830 |
Oct 9, 2000 |
7329410 |
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11956201 |
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09684361 |
Oct 6, 2000 |
7115272 |
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09685830 |
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09276484 |
Mar 25, 1999 |
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09684361 |
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09164223 |
Sep 30, 1998 |
7063854 |
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09276484 |
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Current U.S.
Class: |
514/1.1 ;
424/184.1; 424/93.71; 435/375; 514/44R; 530/300; 530/326; 530/327;
530/328; 530/329; 530/330; 536/23.5 |
Current CPC
Class: |
A61K 2039/55572
20130101; A61K 39/0011 20130101; Y10S 514/885 20130101; A61K
39/001153 20180801; A61K 2039/55566 20130101; A61K 2039/55522
20130101; Y10S 530/828 20130101; Y10S 530/806 20130101; A61P 37/04
20180101 |
Class at
Publication: |
424/130.1 ;
424/184.1; 424/93.71; 435/375; 514/2; 514/44.R; 530/300; 530/326;
530/327; 530/328; 530/329; 530/330; 536/23.5 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; A61K 35/26 20060101
A61K035/26; C12N 5/00 20060101 C12N005/00; A61K 38/02 20060101
A61K038/02; A61K 31/7088 20060101 A61K031/7088; C07K 2/00 20060101
C07K002/00; C07K 7/06 20060101 C07K007/06; C07H 21/00 20060101
C07H021/00 |
Claims
1. A polypeptide comprising an immunogenic portion of a native WT1,
or a variant thereof that differs in one or more substitutions,
deletions, additions and/or insertions such that the ability of the
variant to react with WT1-specific antisera and/or T-cell lines or
clones is not substantially diminished, wherein the polypeptide
comprises no more than 16 consecutive amino acid residues present
within a native WT1 polypeptide.
2. A polypeptide according to claim 1, wherein the immunogenic
portion binds to a MHC class I molecule.
3. A polypeptide according to claim 1, wherein the immunogenic
portion binds to a MHC class II molecule.
4. A polypeptide according to claim 1, wherein the polypeptide
comprises a sequence selected from the group consisting of: (a)
sequences recited in one or more of Tables II-XLVI; (b) SEQ ID
NO:34, 35, 88, 49, 147, 185, 198 and 199; (c) variants of the
foregoing sequences that differ in one or more substitutions,
deletions, additions and/or insertions such that the ability of the
variant to react with antigen-specific antisera and/or T-cell lines
or clones is not substantially diminished; and (d) mimetics of the
foregoing sequences, wherein the ability of the mimetic to react
with antigen-specific antisera and/or T-cell lines or clones is not
substantially diminished.
5. A polypeptide according to claim 1, wherein the polypeptide
comprises 4-16 consecutive amino acids of a native WT1
polypeptide.
6. A polypeptide according to claim 1, wherein the polypeptide
comprises 8-10 consecutive amino acids of a native WT1
polypeptide.
7. A polypeptide comprising an immunogenic portion of amino acid
residues 1-174 of a native WT1 polypeptide, or a variant thereof
that differs in one or more substitutions, deletions, additions
and/or insertions such that the ability of the variant to react
with WT1 specific T-cell lines or clones is not substantially
diminished, wherein the polypeptide comprises no more than 16
consecutive amino acid residues present within amino acids 175-449
of the native WT1 polypeptide.
8. A polynucleotide encoding a polypeptide according to claim
1.
9. A pharmaceutical composition, comprising: (a) a first component
selected from: a. a polynucleotide encoding a WT1 polypeptide,
wherein the polypeptide comprises an immunogenic portion of a
native WT1 or a variant thereof that differs in one or more
substitutions, deletions, additions and/or insertions such that the
ability of the variant to react with antigen-specific antibodies
and/or T cell lines or clones is not substantially diminished; b.
an antibody or antigen-binding fragment thereof that specifically
binds to a WT1 polypeptide; c. a T cell that specifically reacts
with a WT1 polypeptide; d. a WT1 polypeptide that comprises an
immunogenic portion of a native WT1 or variant thereof that differs
in one or more substitutions, deletions, additions, and/or
insertions such that the ability of the variant to react with
antigen-specific antibodies and/or T cell lines or clones is not
substantially diminished; and (b) a pharmaceutically acceptable
carrier or excipient.
10. A method for enhancing or inducing an immune response in a
human patient, comprising administering to a patient a
pharmaceutical composition according to claim 9.
11. A pharmaceutical composition according to claim 9, further
comprising a non-specific immune response enhancer.
12. A method for stimulating and/or expanding T cells, comprising
contacting T cells with a WT1 polypeptide, a polynucleotide
encoding a WT1 polypeptide and/or an antigen presenting cell that
expresses a WT1 polypeptide under conditions and for a time
sufficient to permit the stimulation and/or expansion of the T
cells.
13. A method according to claim 12, wherein the T cells are present
within bone marrow, peripheral blood or a fraction of bone marrow
or peripheral blood.
14. A method according to claim 12, wherein the bone marrow,
peripheral blood or fraction is obtained from a patient afflicted
with a malignant disease associated with WT1 expression.
15. A method for stimulating and or expanding T cells in a mammal,
comprising administering to a mammal a pharmaceutical composition
comprising: (a) one or more of: (i) a WT1 polypeptide; (ii) a
polynucleotide encoding a WT1 polypeptide; or (iii) an
antigen-presenting cell that expresses a WT1 polypeptide; and (b) a
physiologically acceptable carrier or excipient; and thereby
stimulating and/or expanding T cells in a mammal.
16. A method for inhibiting the development of a malignant disease
associated with WT1 expression in a patient, comprising
administering to a patient cells expressing T cell receptors for T
cells specific for WT1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 11/956,201, filed Dec. 13, 2007 (now allowed); which is a
Continuation of U.S. application Ser. No. 09/685,830, filed Oct. 9,
2000 (now U.S. Pat. No. 7,329,410, issued Feb. 12, 2008); which is
a Continuation-in-Part of U.S. application Ser. No. 09/684,361,
filed Oct. 6, 2000 (now U.S. Pat. No. 7,115,272, issued Oct. 3,
2006); which is a Continuation-in-Part of U.S. application Ser. No.
09/276,484, filed Mar. 25, 1999 (now abandoned); which is a
Continuation-in-Part of U.S. application Ser. No. 09/164,223, filed
Sep. 30, 1998 (now U.S. Pat. No. 7,063,854, issued Jun. 20, 2006);
where these applications are incorporated herein by reference in
their entireties.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
210121.sub.--465C16_SEQUENCE_LISTING.txt. The text file is 61 KB,
was created on Dec. 23, 2009, and is being submitted electronically
via EFS-Web, concurrent with the filing of the specification.
TECHNICAL FIELD
[0003] The present invention relates generally to the immunotherapy
of malignant diseases such as leukemia and cancers. The invention
is more specifically related to compositions for generating or
enhancing an immune response to WT1, and to the use of such
compositions for preventing and/or treating malignant diseases.
BACKGROUND OF THE INVENTION
[0004] Cancer and leukemia are significant health problems in the
United States and throughout the world. Although advances have been
made in detection and treatment of such diseases, no vaccine or
other universally successful method for prevention or treatment of
cancer and leukemia is currently available. Management of the
diseases currently relies on a combination of early diagnosis and
aggressive treatment, which may include one or more of a variety of
treatments such as surgery, radiotherapy, chemotherapy and hormone
therapy. The course of treatment for a particular cancer is often
selected based on a variety of prognostic parameters, including an
analysis of specific tumor markers. However, the use of established
markers often leads to a result that is difficult to interpret, and
the high mortality continues to be observed in many cancer
patients.
[0005] Immunotherapies have the potential to substantially improve
cancer and leukemia treatment and survival. Recent data demonstrate
that leukemia can be cured by immunotherapy in the context of bone
marrow transplantation (e.g., donor lymphocyte infusions). Such
therapies may involve the generation or enhancement of an immune
response to a tumor-associated antigen (TAA). However, to date
relatively few TAAs are known and the generation of an immune
response against such antigens has, with rare exception, not been
shown to be therapeutically beneficial.
[0006] Accordingly, there is a need in the art for improved methods
for leukemia and cancer prevention and therapy. The present
invention fulfills these needs and further provides other related
advantages.
SUMMARY OF THE INVENTION
[0007] Briefly stated, this invention provides compositions and
methods for the diagnosis and therapy of diseases such as leukemia
and cancer. In one aspect, the present invention provides
polypeptides comprising an immunogenic portion of a native WT1, or
a variant thereof that differs in one or more substitutions,
deletions, additions and/or insertions such that the ability of the
variant to react with antigen-specific antisera and/or T-cell lines
or clones is not substantially diminished. Within certain
embodiments, the polypeptide comprises no more than 16 consecutive
amino acid residues of a native WT1 polypeptide. Within other
embodiments, the polypeptide comprises an immunogenic portion of
amino acid residues 1-174 of a native WT1 polypeptide or a variant
thereof, wherein the polypeptide comprises no more than 16
consecutive amino acid residues present within amino acids 175 to
449 of the native WT1 polypeptide. The immunogenic portion
preferably binds to an MHC class I and/or class II molecule. Within
certain embodiments, the polypeptide comprises a sequence selected
from the group consisting of (a) sequences recited in any one or
more of Tables II-XLVI, (b) variants of the foregoing sequences
that differ in one or more substitutions, deletions, additions
and/or insertions such that the ability of the variant to react
with antigen-specific antisera and/or T-cell lines or clones is not
substantially diminished and (c) mimetics of the polypeptides
recited above, such that the ability of the mimetic to react with
antigen-specific antisera and/or T cell lines or clones is not
substantially diminished.
[0008] Within other embodiments, the polypeptide comprises a
sequence selected from the group consisting of (a) ALLPAVPSL (SEQ
ID NO:34), GATLKGVAA (SEQ ID NO:88), CMTWNQMNL (SEQ ID NOs: 49 and
258), SCLESQPTI (SEQ ID NOs: 199 and 296), SCLESQPAI (SEQ ID
NO:198), NLYQMTSQL (SEQ ID NOs: 147 and 284), ALLPAVSSL (SEQ ID
NOs: 35 and 255), RMFPNAPYL (SEQ ID NOs: 185 and 293), (b) variants
of the foregoing sequences that differ in one or more
substitutions, deletions, additions and/or insertions such that the
ability of the variant to react with antigen-specific antisera
and/or T-cell lines or clones is not substantially diminished and
(c) mimetics of the polypeptides recited above, such that the
ability of the mimetic to react with antigen-specific antisera
and/or T cell lines or clones is not substantially diminished.
Mimetics may comprises amino acids in combination with one or more
amino acid mimetics or may be entirely nonpeptide mimetics.
[0009] Within further aspects, the present invention provides
polypeptides comprising a variant of an immunogenic portion of a
WT1 protein, wherein the variant differs from the immunogenic
portion due to substitutions at between 1 and 3 amino acid
positions within the immunogenic portion such that the ability of
the variant to react with antigen-specific antisera and/or T-cell
lines or clones is enhanced relative to a native WT1 protein.
[0010] The present invention further provides WT1 polynucleotides
that encode a WT1 polypeptide as described above.
[0011] Within other aspects, the present invention provides
pharmaceutical compositions and vaccines. Pharmaceutical
compositions may comprise a polypeptide or mimetic as described
above and/or one or more of (i) a WT1 polynucleotide; (ii) an
antibody or antigen-binding fragment thereof that specifically
binds to a WT1 polypeptide; (iii) a T cell that specifically reacts
with a WT1 polypeptide or (iv) an antigen-presenting cell that
expresses a WT1 polypeptide, in combination with a pharmaceutically
acceptable carrier or excipient. Vaccines comprise a polypeptide as
described above and/or one or more of (i) a WT1 polynucleotide,
(ii) an antigen-presenting cell that expresses a WT1 polypeptide or
(iii) an anti-idiotypic antibody, and a non-specific immune
response enhancer. Within certain embodiments, less than 23
consecutive amino acid residues, preferably less than 17 amino acid
residues, of a native WT1 polypeptide are present within a WT1
polypeptide employed within such pharmaceutical compositions and
vaccines. The immune response enhancer may be an adjuvant.
Preferably, an immune response enhancer enhances a T cell
response.
[0012] The present invention further provides methods for enhancing
or inducing an immune response in a patient, comprising
administering to a patient a pharmaceutical composition or vaccine
as described above. In certain embodiments, the patient is a
human.
[0013] The present invention further provides methods for
inhibiting the development of a malignant disease in a patient,
comprising administering to a patient a pharmaceutical composition
or vaccine as described above. Malignant diseases include, but are
not limited to leukemias (e.g., acute myeloid, acute lymphocytic
and chronic myeloid) and cancers (e.g., breast, lung, thyroid or
gastrointestinal cancer or a melanoma). The patient may, but need
not, be afflicted with the malignant disease, and the
administration of the pharmaceutical composition or vaccine may
inhibit the onset of such a disease, or may inhibit progression
and/or metastasis of an existing disease.
[0014] The present invention further provides, within other
aspects, methods for removing cells expressing WT1 from bone marrow
and/or peripheral blood or fractions thereof, comprising contacting
bone marrow, peripheral blood or a fraction of bone marrow or
peripheral blood with T cells that specifically react with a WT1
polypeptide, wherein the step of contacting is performed under
conditions and for a time sufficient to permit the removal of WT1
positive cells to less than 10%, preferably less than 5% and more
preferably less than 1%, of the number of myeloid or lymphatic
cells in the bone marrow, peripheral blood or fraction. Bone
marrow, peripheral blood and fractions may be obtained from a
patient afflicted with a disease associated with WT1 expression, or
may be obtained from a human or non-human mammal not afflicted with
such a disease.
[0015] Within related aspects, the present invention provides
methods for inhibiting the development of a malignant disease in a
patient, comprising administering to a patient bone marrow,
peripheral blood or a fraction of bone marrow or peripheral blood
prepared as described above. Such bone marrow, peripheral blood or
fractions may be autologous, or may be derived from a related or
unrelated human or non-human animal (e.g., syngeneic or
allogeneic).
[0016] In other aspects, the present invention provides methods for
stimulating (or priming) and/or expanding T cells, comprising
contacting T cells with a WT1 polypeptide under conditions and for
a time sufficient to permit the stimulation and/or expansion of T
cells. Such T cells may be autologous, allogeneic, syngeneic or
unrelated WT1-specific T cells, and may be stimulated in vitro or
in vivo. Expanded T cells may, within certain embodiments, be
present within bone marrow, peripheral blood or a fraction of bone
marrow or peripheral blood, and may (but need not) be clonal.
Within certain embodiments, T cells may be present in a mammal
during stimulation and/or expansion. WT1-specific T cells may be
used, for example, within donor lymphocyte infusions.
[0017] Within related aspects, methods are provided for inhibiting
the development of a malignant disease in a patient, comprising
administering to a patient T cells prepared as described above.
Such T cells may, within certain embodiments, be autologous,
syngeneic or allogeneic.
[0018] The present invention further provides, within other
aspects, methods for monitoring the effectiveness of an
immunization or therapy for a malignant disease associated with WT1
expression in a patient. Such methods are based on monitoring
antibody, CD4+ T cell and/or CD8+ T cell responses in the patient.
Within certain such aspects, a method may comprise the steps of:
(a) incubating a first biological sample with one or more of: (i) a
WT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide;
or (iii) an antigen presenting cell that expresses a WT1
polypeptide, wherein the first biological sample is obtained from a
patient prior to a therapy or immunization, and wherein the
incubation is performed under conditions and for a time sufficient
to allow immunocomplexes to form; (b) detecting immunocomplexes
formed between the WT1 polypeptide and antibodies in the biological
sample that specifically bind to the WT1 polypeptide; (c) repeating
steps (a) and (b) using a second biological sample obtained from
the same patient following therapy or immunization; and (d)
comparing the number of immunocomplexes detected in the first and
second biological samples, and therefrom monitoring the
effectiveness of the therapy or immunization in the patient.
[0019] Within certain embodiments of the above methods, the step of
detecting comprises (a) incubating the immunocomplexes with a
detection reagent that is capable of binding to the
immunocomplexes, wherein the detection reagent comprises a reporter
group, (b) removing unbound detection reagent, and (c) detecting
the presence or absence of the reporter group. The detection
reagent may comprise, for example, a second antibody, or
antigen-binding fragment thereof, capable of binding to the
antibodies that specifically bind to the WT1 polypeptide or a
molecule such as Protein A. Within other embodiments, a reporter
group is bound to the WT1 polypeptide, and the step of detecting
comprises removing unbound WT1 polypeptide and subsequently
detecting the presence or absence of the reporter group.
[0020] Within further aspects, methods for monitoring the
effectiveness of an immunization or therapy for a malignant disease
associated with WT1 expression in a patient may comprise the steps
of: (a) incubating a first biological sample with one or more of:
(i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1
polypeptide; or (iii) an antigen presenting cell that expresses a
WT1 polypeptide, wherein the biological sample comprises CD4+
and/or CD8+ T cells and is obtained from a patient prior to a
therapy or immunization, and wherein the incubation is performed
under conditions and for a time sufficient to allow specific
activation, proliferation and/or lysis of T cells; (b) detecting an
amount of activation, proliferation and/or lysis of the T cells;
(c) repeating steps (a) and (b) using a second biological sample
comprising CD4+ and/or CD8+ T cells, wherein the second biological
sample is obtained from the same patient following therapy or
immunization; and (d) comparing the amount of activation,
proliferation and/or lysis of T cells in the first and second
biological samples, and therefrom monitoring the effectiveness of
the therapy or immunization in the patient.
[0021] The present invention further provides methods for
inhibiting the development of a malignant disease associated with
WT1 expression in a patient, comprising the steps of: (a)
incubating CD4+ and/or CD8+ T cells isolated from a patient with
one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide
encoding a WT1 polypeptide; or (iii) an antigen presenting cell
that expresses a WT1 polypeptide, such that the T cells
proliferate; and (b) administering to the patient an effective
amount of the proliferated T cells, and therefrom inhibiting the
development of a malignant disease in the patient. Within certain
embodiments, the step of incubating the T cells may be repeated one
or more times.
[0022] Within other aspects, the present invention provides methods
for inhibiting the development of a malignant disease associated
with WT1 expression in a patient, comprising the steps of: (a)
incubating CD4+ and/or CD8+ T cells isolated from a patient with
one or more of: (i) a WT1 polypeptide; (ii) a polynucleotide
encoding a WT1 polypeptide; or (iii) an antigen presenting cell
that expresses a WT1 polypeptide, such that the T cells
proliferate; (b) cloning one or more cells that proliferated; and
(c) administering to the patient an effective amount of the cloned
T cells.
[0023] Within other aspects, methods are provided for determining
the presence or absence of a malignant disease associated with WT1
expression in a patient, comprising the steps of: (a) incubating
CD4.sup.+ and/or CD8+ T cells isolated from a patient with one or
more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a
WT1 polypeptide; or (iii) an antigen presenting cell that expresses
a WT1 polypeptide; and (b) detecting the presence or absence of
specific activation of the T cells, therefrom determining the
presence or absence of a malignant disease associated with WT1
expression. Within certain embodiments, the step of detecting
comprises detecting the presence or absence of proliferation of the
T cells.
[0024] Within further aspects, the present invention provides
methods for determining the presence or absence of a malignant
disease associated with WT1 expression in a patient, comprising the
steps of: (a) incubating a biological sample obtained from a
patient with one or more of: (i) a WT1 polypeptide; (ii) a
polynucleotide encoding a WT1 polypeptide; or (iii) an antigen
presenting cell that expresses a WT1 polypeptide, wherein the
incubation is performed under conditions and for a time sufficient
to allow immunocomplexes to form; and (b) detecting immunocomplexes
formed between the WT1 polypeptide and antibodies in the biological
sample that specifically bind to the WT1 polypeptide; and therefrom
determining the presence or absence of a malignant disease
associated with WT1 expression.
[0025] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entirety as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 depicts a comparison of the mouse (MO) and human (HU)
WT1 protein sequences (SEQ ID NOS: 320 and 319 respectively).
[0027] FIG. 2 is a Western blot illustrating the detection of WT1
specific antibodies in patients with hematological malignancy
(AML). Lane 1 shows molecular weight markers; lane 2 shows a
positive control (WT1 positive human leukemia cell line
immunoprecipitated with a WT1 specific antibody); lane 3 shows a
negative control (WT1 positive cell line immunoprecipitated with
mouse sera); and lane 4 shows a WT1 positive cell line
immunoprecipitated with sera of a patient with AML. For lanes 2-4,
the immunoprecipitate was separated by gel electrophoresis and
probed with a WT1 specific antibody.
[0028] FIG. 3 is a Western blot illustrating the detection of a WT1
specific antibody response in B6 mice immunized with TRAMP-C, a WT1
positive tumor cell line. Lanes 1, 3 and 5 show molecular weight
markers, and lanes 2, 4 and 6 show a WT1 specific positive control
(N180, Santa Cruz Biotechnology, polypeptide spanning 180 amino
acids of the N-terminal region of the WT1 protein, migrating on the
Western blot at 52 kD). The primary antibody used was WT180 in lane
2, sera of non-immunized B6 mice in lane 4 and sera of the
immunized B6 mice in lane 6.
[0029] FIG. 4 is a Western blot illustrating the detection of WT1
specific antibodies in mice immunized with representative WT1
peptides. Lanes 1, 3 and 5 show molecular weight markers and lanes
2, 4 and 6 show a WT1 specific positive control (N180, Santa Cruz
Biotechnology, polypeptide spanning 180 amino acids of the
N-terminal region of the WT1 protein, migrating on the Western blot
at 52 kD). The primary antibody used was WT180 in lane 2, sera of
non-immunized B6 mice in lane 4 and sera of the immunized B6 mice
in lane 6.
[0030] FIGS. 5A to 5C are graphs illustrating the stimulation of
proliferative T cell responses in mice immunized with
representative WT1 peptides. Thymidine incorporation assays were
performed using one T cell line and two different clones, as
indicated, and results were expressed as cpm. Controls indicated on
the x axis were no antigen (No Ag) and B6/media; antigens used were
p6-22 human (p1), p117-139 (p2) or p244-262 human (p3).
[0031] FIGS. 6A and 6B are histograms illustrating the stimulation
of proliferative T cell responses in mice immunized with
representative WT1 peptides. Three weeks after the third
immunization, spleen cells of mice that had been inoculated with
Vaccine A or Vaccine B were cultured with medium alone (medium) or
spleen cells and medium (B6/no antigen), B6 spleen cells pulsed
with the peptides p6-22 (p6), p117-139 (p117), p244-262 (p244)
(Vaccine A; FIG. 6A) or p287-301 (p287), p299-313 (p299), p421-435
(p421) (Vaccine B; FIG. 6B) and spleen cells pulsed with an
irrelevant control peptide (irrelevant peptide) at 25 ug/ml and
were assayed after 96 hr for proliferation by (3H) thymidine
incorporation. Bars represent the stimulation index (SI), which is
calculated as the mean of the experimental wells divided by the
mean of the control (B6 spleen cells with no antigen).
[0032] FIGS. 7A-7D are histograms illustrating the generation of
proliferative T-cell lines and clones specific for p117-139 and
p6-22. Following in vivo immunization, the initial three in vitro
stimulations (IVS) were carried out using all three peptides of
Vaccine A or B, respectively. Subsequent IVS were carried out as
single peptide stimulations using only the two relevant peptides
p117-139 and p6-22. Clones were derived from both the p6-22 and
p117-139 specific T cell lines, as indicated. T cells were cultured
with medium alone (medium) or spleen cells and medium (B6/no
antigen), B6 spleen cells pulsed with the peptides p6-22 (p6),
p117-139 (p117) or an irrelevant control peptide (irrelevant
peptide) at 25 ug/ml and were assayed after 96 hr for proliferation
by (3H) thymidine incorporation. Bars represent the stimulation
index (SI), which is calculated as the mean of the experimental
wells divided by the mean of the control (B6 spleen cells with no
antigen).
[0033] FIGS. 8A and 8B present the results of TSITES Analysis of
human WT1 (SEQ ID NO:319) for peptides that have the potential to
elicit Th responses. Regions indicated by "A" are AMPHI midpoints
of blocks, "R" indicates residues matching the Rothbard/'Taylor
motif, "D" indicates residues matching the IAd motif, and `d`
indicates residues matching the IEd motif.
[0034] FIGS. 9A and 9B are graphs illustrating the elicitation of
WT1 peptide-specific CTL in mice immunized with WT1 peptides. FIG.
9A illustrates the lysis of target cells by allogeneic cell lines
and FIG. 9B shows the lysis of peptide coated cell lines. In each
case, the % lysis (as determined by standard chromium release
assays) is shown at three indicated effector:target ratios. Results
are provided for lymphoma cells (LSTRA and E10), as well as
E10+p235-243 (E10+P235). E10 cells are also referred to herein as
EL-4 cells.
[0035] FIGS. 10A-10D are graphs illustrating the elicitation of WT1
specific CTL, which kill WT1 positive tumor cell lines but do not
kill WT1 negative cell lines, following vaccination of B6 mice with
WT1 peptide P117. FIG. 10A illustrates that T-cells of
non-immunized B6 mice do not kill WT1 positive tumor cell lines.
FIG. 10B illustrates the lysis of the target cells by allogeneic
cell lines. FIGS. 10C and 10D demonstrate the lysis of WT1 positive
tumor cell lines, as compared to WT1 negative cell lines in two
different experiments. In addition, FIGS. 10C and 10D show the
lysis of peptide-coated cell lines (WT1 negative cell line E10
coated with the relevant WT1 peptide P117) In each case, the %
lysis (as determined by standard chromium release assays) is shown
at three indicated effector:target ratios. Results are provided for
lymphoma cells (E10), prostate cancer cells (TRAMP-C), a
transformed fibroblast cell line (BLK-SV40), as well as
E10+p117.
[0036] FIGS. 11A and 11B are histograms illustrating the ability of
representative peptide P117-139 specific CTL to lyse WT1 positive
tumor cells. Three weeks after the third immunization, spleen cells
of mice that had been inoculated with the peptides p235-243 or
p117-139 were stimulated in vitro with the relevant peptide and
tested for ability to lyse targets incubated with WT1 peptides as
well as WT1 positive and negative tumor cells. The bars represent
the mean % specific lysis in chromium release assays performed in
triplicate with an E:T ratio of 25:1. FIG. 11A shows the cytotoxic
activity of the p235-243 specific T cell line against the WT1
negative cell line EL-4 (EL-4, WT1 negative); EL-4 pulsed with the
relevant (used for immunization as well as for restimulation)
peptide p235-243 (EL-4+p235); EL-4 pulsed with the irrelevant
peptides p117-139 (EL-4+p117), p126-134 (EL-4+p126) or p130-138
(EL-4+p130) and the WT1 positive tumor cells BLK-SV40 (BLK-SV40,
WT1 positive) and TRAMP-C (TRAMP-C, WT1 positive), as indicated.
FIG. 11B shows cytotoxic activity of the p117-139 specific T cell
line against EL-4; EL-4 pulsed with the relevant peptide P117-139
(EL-4+p117) and EL-4 pulsed with the irrelevant peptides p123-131
(EL-4+p123), or p128-136 (EL-4+p128); BLK-SV40 and TRAMP-C, as
indicated.
[0037] FIGS. 12A and 12B are histograms illustrating the
specificity of lysis of WT1 positive tumor cells, as demonstrated
by cold target inhibition. The bars represent the mean % specific
lysis in chromium release assays performed in triplicate with an
E:T ratio of 25:1. FIG. 12A shows the cytotoxic activity of the
p117-139 specific T cell line against the WT1 negative cell line
EL-4 (EL-4, WT1 negative); the WT1 positive tumor cell line TRAMP-C
(TRAMP-C, WT1 positive); TRAMP-C cells incubated with a ten-fold
excess (compared to the hot target) of EL-4 cells pulsed with the
relevant peptide p117-139 (TRAMP-C+p117 cold target) without
.sup.51Cr labeling and TRAMP-C cells incubated with EL-4 pulsed
with an irrelevant peptide without .sup.51Cr labeling
(TRAMP-C+irrelevant cold target), as indicated. FIG. 12B shows the
cytotoxic activity of the p117-139 specific T cell line against the
WT1 negative cell line EL-4 (EL-4, WT1 negative); the WT1 positive
tumor cell line BLK-SV40 (BLK-SV40, WT1 positive); BLK-SV40 cells
incubated with the relevant cold target (BLK-SV40+p117 cold target)
and BLK-SV40 cells incubated with the irrelevant cold target
(BLK-SV40+irrelevant cold target), as indicated.
[0038] FIGS. 13A-13C are histograms depicting an evaluation of the
9mer CTL epitope within p117-139. The p117-139 tumor specific CTL
line was tested against peptides within aa117-139 containing or
lacking an appropriate H-2.sup.b class I binding motif and
following restimulation with p126-134 or p130-138. The bars
represent the mean % specific lysis in chromium release assays
performed in triplicate with an E:T ratio of 25:1. FIG. 13A shows
the cytotoxic activity of the p117-139 specific T cell line against
the WT1 negative cell line EL-4 (EL-4, WT1 negative) and EL-4 cells
pulsed with the peptides p117-139 (EL-4+p117), p119-127
(EL-4+p119), p120-128 (EL-4+p120), p123-131 (EL-4+p123), p126-134
(EL-4+p126), p128-136 (EL-4+p128), and p130-138 (EL-4+p130). FIG.
13B shows the cytotoxic activity of the CTL line after
restimulation with p126-134 against the WT1 negative cell line
EL-4, EL-4 cells pulsed with p117-139 (EL-4+p117), p126-134
(EL-4+p126) and the WT1 positive tumor cell line TRAMP-C. FIG. 13C
shows the cytotoxic activity of the CTL line after restimulation
with p130-138 against EL-4, EL-4 cells pulsed with p117-139
(EL-4+p117), p130-138 (EL-4+p130) and the WT1 positive tumor cell
line TRAMP-C.
[0039] FIG. 14 depicts serum antibody reactivity to WT1 in 63
patients with AML. Reactivity of serum antibody to WT1/N-terminus
protein was evaluated by ELISA in patients with AML. The first and
second lanes represent the positive and negative controls,
respectively. The first and second lanes represent the positive and
negative controls, respectively. Commercially obtained WT1 specific
antibody WT180 was used for the positive control. The next 63 lanes
represent results using sera from each individual patient. The OD
values depicted were from ELISA using a 1:500 serum dilution. The
figure includes cumulative data from 3 separate experiments.
[0040] FIG. 15 depicts serum antibody reactivity to WT1 proteins
and control proteins in 2 patients with AML. Reactivity of serum
antibody to WT1/full-length, WT1N-terminus, TRX and Ra12 proteins
was evaluated by ELISA in 2 patients with AML. The OD values
depicted were from ELISA using a 1:500 serum dilution. AML-1 and
AML-2 denote serum from 2 of the individual patients in FIG. 1 with
demonstrated antibody reactivity to WT1/full-length. The WT1
full-length protein was expressed as a fusion protein with Ra12.
The WT1/N-terminus protein was expressed as a fusion protein with
TRX. The control Ra12 and TRX proteins were purified in a similar
manner. The results confirm that the serum antibody reactivity
against the WT1 fusion proteins is directed against the WT1
portions of the protein.
[0041] FIG. 16 depicts serum antibody reactivity to WT1 in 81
patients with CML. Reactivity of serum antibody to WT1/full-length
protein was evaluated by ELISA in patients with AML. The first and
second lanes represent the positive and negative controls,
respectively. Commercially obtained WT1 specific antibody WT180 was
used for the positive control. The next 81 lanes represent results
using sera from each individual patient. The OD values depicted
were from ELISA using a 1:500 serum dilution. The figure includes
cumulative data from 3 separate experiments.
[0042] FIG. 17 depicts serum antibody reactivity to WT1 proteins
and control proteins in 2 patients with CML. Reactivity of serum
antibody to WT1/full-length, WT1/N-terminus, TRX and Ra12 proteins
was evaluated by ELISA in 2 patients with CML. The OD values
depicted were from ELISA using a 1:500 serum dilution. CML-1 and
CML-2 denote serum from 2 of the individual patients in FIG. 3 with
demonstrated antibody reactivity to WT1/full-length. The
WT1/full-length protein was expressed as a fusion protein with
Ra12. The WT1/N-terminus protein was expressed as a fusion protein
with TRX. The control Ra12 and TRX proteins were purified in a
similar manner. The results confirm that the serum antibody
reactivity against the WT1 fusion proteins is directed against the
WT1 portions of the protein.
[0043] FIG. 18 provides the characteristics of the recombinant WT1
proteins used for serological analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0044] As noted above, the present invention is generally directed
to compositions and methods for the immunotherapy and diagnosis of
malignant diseases. The compositions described herein may include
WT1 polypeptides, WT1 polynucleotides, antigen-presenting cells
(APC, e.g., dendritic cells) that express a WT1 polypeptide, agents
such as antibodies that bind to a WT1 polypeptide and/or immune
system cells (e.g., T cells) specific for WT1. WT1 Polypeptides of
the present invention generally comprise at least a portion of a
Wilms Tumor gene product (WT1) or a variant thereof. Nucleic acid
sequences of the subject invention generally comprise a DNA or RNA
sequence that encodes all or a portion of such a polypeptide, or
that is complementary to such a sequence. Antibodies are generally
immune system proteins, or antigen-binding fragments thereof, that
are capable of binding to a portion of a WT1 polypeptide. T cells
that may be employed within such compositions are generally T cells
(e.g., CD4.sup.+ and/or CD8.sup.+) that are specific for a WT1
polypeptide. Certain methods described herein further employ
antigen-presenting cells that express a WT1 polypeptide as provided
herein.
[0045] The present invention is based on the discovery that an
immune response raised against a Wilms Tumor (WT) gene product
(e.g., WT1) can provide prophylactic and/or therapeutic benefit for
patients afflicted with malignant diseases characterized by
increased WT1 gene expression. Such diseases include, but are not
limited to, leukemias (e.g., acute myeloid leukemia (AML), chronic
myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and
childhood ALL), as well as many cancers such as lung, breast,
thyroid and gastrointestinal cancers and melanomas. The WT1 gene
was originally identified and isolated on the basis of a
cytogenetic deletion at chromosome 11p13 in patients with Wilms'
tumor (see Call et al., U.S. Pat. No. 5,350,840). The gene consists
of 10 exons and encodes a zinc finger transcription factor, and
sequences of mouse and human WT1 proteins are provided in FIG. 1
and SEQ ID NOs: 319 and 320.
WT1 Polypeptides
[0046] Within the context of the present invention, a WT1
polypeptide is a polypeptide that comprises at least an immunogenic
portion of a native WT1 (i.e., a WT1 protein expressed by an
organism that is not genetically modified), or a variant thereof,
as described herein. A WT1 polypeptide may be of any length,
provided that it comprises at least an immunogenic portion of a
native protein or a variant thereof. In other words, a WT1
polypeptide may be an oligopeptide (i.e., consisting of a
relatively small number of amino acid residues, such as 8-10
residues, joined by peptide bonds), a full length WT1 protein
(e.g., present within a human or non-human animal, such as a mouse)
or a polypeptide of intermediate size. Within certain embodiments,
the use of WT1 polypeptides that contain a small number of
consecutive amino acid residues of a native WT1 polypeptide is
preferred. Such polypeptides are preferred for certain uses in
which the generation of a T cell response is desired. For example,
such a WT1 polypeptide may contain less than 23, preferably no more
than 18, and more preferably no more than 15 consecutive amino acid
residues, of a native WT1 polypeptide. Polypeptides comprising nine
consecutive amino acid residues of a native WT1 polypeptide are
generally suitable for such purposes. Additional sequences derived
from the native protein and/or heterologous sequences may be
present within any WT1 polypeptide, and such sequences may (but
need not) possess further immunogenic or antigenic properties.
Polypeptides as provided herein may further be associated
(covalently or noncovalently) with other polypeptide or
non-polypeptide compounds.
[0047] An "immunogenic portion," as used herein is a portion of a
polypeptide that is recognized (i.e., specifically bound) by a
B-cell and/or T-cell surface antigen receptor. Certain preferred
immunogenic portions bind to an MHC class I or class II molecule.
As used herein, an immunogenic portion is said to "bind to" an MHC
class I or class II molecule if such binding is detectable using
any assay known in the art. For example, the ability of a
polypeptide to bind to MHC class I may be evaluated indirectly by
monitoring the ability to promote incorporation of .sup.125I
labeled .beta.2-microglobulin (.beta.2m) into MHC class
I/.beta.2m/peptide heterotrimeric complexes (see Parker et al., J.
Immunol. 152:163, 1994). Alternatively, functional peptide
competition assays that are known in the art may be employed.
Certain immunogenic portions have one or more of the sequences
recited within one or more of Tables II-XIV. Representative
immunogenic portions include, but are not limited to,
RDLNALLPAVPSLGGGG (human WT1 residues 6-22; SEQ ID NO:1),
PSQASSGQARMFPNAPYLPSCLE (human and mouse WT1 residues 117-139; SEQ
ID NOs: 2 and 3 respectively), GATLKGVAAGSSSSVKWTE (human WT1
residues 244-262; SEQ ID NO:4), GATLKGVAA (human WT1 residues
244-252; SEQ ID NO:88), CMTWNQMNL (human and mouse WT1 residues
235-243; SEQ ID NOs: 49 and 258 respectively), SCLESQPTI (mouse WT1
residues 136-144; SEQ ID NO:296), SCLESQPAI (human WT1 residues
136-144; SEQ ID NO:198), NLYQMTSQL (human and mouse WT1 residues
225-233; SEQ ID NOs: 147 and 284 respectively); ALLPAVSSL (mouse
WT1 residues 10-18; SEQ ID NO:255); or RMFPNAPYL (human and mouse
WT1 residues 126-134; SEQ ID NOs: 185 and 293 respectively).
Further immunogenic portions are provided herein, and others may
generally be identified using well known techniques, such as those
summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven
Press, 1993) and references cited therein. Representative
techniques for identifying immunogenic portions include screening
polypeptides for the ability to react with antigen-specific
antisera and/or T-cell lines or clones. An immunogenic portion of a
native WT1 polypeptide is a portion that reacts with such antisera
and/or T-cells at a level that is not substantially less than the
reactivity of the full length WT1 (e.g., in an ELISA and/or T-cell
reactivity assay). In other words, an immunogenic portion may react
within such assays at a level that is similar to or greater than
the reactivity of the full length polypeptide. Such screens may
generally be performed using methods well known to those of
ordinary skill in the art, such as those described in Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988.
[0048] Alternatively, immunogenic portions may be identified using
computer analysis, such as the Tsites program (see Rothbard and
Taylor, EMBO J. 7:93-100, 1988; Deavin et al., Mol. Immunol.
33:145-155, 1996), which searches for peptide motifs that have the
potential to elicit Th responses. CTL peptides with motifs
appropriate for binding to murine and human class I or class II MHC
may be identified according to BIMAS (Parker et al., J. Immunol.
152:163, 1994) and other HLA peptide binding prediction analyses.
To confirm immunogenicity, a peptide may be tested using an HLA A2
transgenic mouse model and/or an in vitro stimulation assay using
dendritic cells, fibroblasts or peripheral blood cells.
[0049] As noted above, a composition may comprise a variant of a
native WT1 protein. A polypeptide "variant," as used herein, is a
polypeptide that differs from a native polypeptide in one or more
substitutions, deletions, additions and/or insertions, such that
the immunogenicity of the polypeptide is retained (i.e., the
ability of the variant to react with antigen-specific antisera
and/or T-cell lines or clones is not substantially diminished
relative to the native polypeptide). In other words, the ability of
a variant to react with antigen-specific antisera and/or T-cell
lines or clones may be enhanced or unchanged, relative to the
native polypeptide, or may be diminished by less than 50%, and
preferably less than 20%, relative to the native polypeptide. Such
variants may generally be identified by modifying one of the above
polypeptide sequences and evaluating the reactivity of the modified
polypeptide with antisera and/or T-cells as described herein. It
has been found, within the context of the present invention, that a
relatively small number of substitutions (e.g., 1 to 3) within an
immunogenic portion of a WT1 polypeptide may serve to enhance the
ability of the polypeptide to elicit an immune response. Suitable
substitutions may generally be identified by using computer
programs, as described above, and the effect confirmed based on the
reactivity of the modified polypeptide with antisera and/or T-cells
as described herein. Accordingly, within certain preferred
embodiments, a WT1 polypeptide comprises a variant in which 1 to 3
amino acid resides within an immunogenic portion are substituted
such that the ability to react with antigen-specific antisera
and/or T-cell lines or clones is statistically greater than that
for the unmodified polypeptide. Such substitutions are preferably
located within an MHC binding site of the polypeptide, which may be
identified as described above. Preferred substitutions allow
increased binding to MHC class I or class II molecules.
[0050] Certain variants contain conservative substitutions. A
"conservative substitution" is one in which an amino acid is
substituted for another amino acid that has similar properties,
such that one skilled in the art of peptide chemistry would expect
the secondary structure and hydropathic nature of the polypeptide
to be substantially unchanged. Amino acid substitutions may
generally be made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity and/or the amphipathic
nature of the residues. For example, negatively charged amino acids
include aspartic acid and glutamic acid; positively charged amino
acids include lysine and arginine; and amino acids with uncharged
polar head groups having similar hydrophilicity values include
leucine, isoleucine and valine; glycine and alanine; asparagine and
glutamine; and serine, threonine, phenylalanine and tyrosine. Other
groups of amino acids that may represent conservative changes
include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys,
ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;
and (5) phe, tyr, trp, his. A variant may also, or alternatively,
contain nonconservative changes. Variants may also (or
alternatively) be modified by, for example, the deletion or
addition of amino acids that have minimal influence on the
immunogenicity, secondary structure and hydropathic nature of the
polypeptide.
[0051] In a preferred embodiment, a variant polypeptide of the WT1
N-terminus (amino acids 1-249) is constructed, wherein the variant
polypeptide is capable of binding to an antibody that recognizes
full-length WT1 and/or WT1 N-terminus polypeptide. A non-limiting
example of an antibody is anti WT-1 antibody WT180 (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.).
[0052] As noted above, WT1 polypeptides may be conjugated to a
signal (or leader) sequence at the N-terminal end of the protein
which co-translationally or post-translationally directs transfer
of the protein. A polypeptide may also, or alternatively, be
conjugated to a linker or other sequence for ease of synthesis,
purification or identification of the polypeptide (e.g., poly-His),
or to enhance binding of the polypeptide to a solid support. For
example, a polypeptide may be conjugated to an immunoglobulin Fc
region.
[0053] WT1 polypeptides may be prepared using any of a variety of
well known techniques. Recombinant polypeptides encoded by a WT1
polynucleotide as described herein may be readily prepared from the
polynucleotide. In general, any of a variety of expression vectors
known to those of ordinary skill in the art may be employed to
express recombinant WT1 polypeptides. Expression may be achieved in
any appropriate host cell that has been transformed or transfected
with an expression vector containing a DNA molecule that encodes a
recombinant polypeptide. Suitable host cells include prokaryotes,
yeast and higher eukaryotic cells. Preferably, the host cells
employed are E. coli, yeast or a mammalian cell line such as COS or
CHO. Supernatants from suitable host/vector systems which secrete
recombinant protein or polypeptide into culture media may be first
concentrated using a commercially available filter. The concentrate
may then be applied to a suitable purification matrix such as an
affinity matrix or an ion exchange resin. Finally, one or more
reverse phase HPLC steps can be employed to further purify a
recombinant polypeptide. Such techniques may be used to prepare
native polypeptides or variants thereof. For example,
polynucleotides that encode a variant of a native polypeptide may
generally be prepared using standard mutagenesis techniques, such
as oligonucleotide-directed site-specific mutagenesis, and sections
of the DNA sequence may be removed to permit preparation of
truncated polypeptides.
[0054] Certain portions and other variants may also be generated by
synthetic means, using techniques well known to those of ordinary
skill in the art. For example, polypeptides having fewer than about
500 amino acids, preferably fewer than about 100 amino acids, and
more preferably fewer than about 50 amino acids, may be
synthesized. Polypeptides may be synthesized using any of the
commercially available solid-phase techniques, such as the
Merrifield solid-phase synthesis method, where amino acids are
sequentially added to a growing amino acid chain. See Merrifield,
J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated
synthesis of polypeptides is commercially available from suppliers
such as Applied BioSystems, Inc. (Foster City, Calif.), and may be
operated according to the manufacturer's instructions.
[0055] In general, polypeptides and polynucleotides as described
herein are isolated. An "isolated" polypeptide or polynucleotide is
one that is removed from its original environment. For example, a
naturally-occurring protein is isolated if it is separated from
some or all of the coexisting materials in the natural system.
Preferably, such polypeptides are at least about 90% pure, more
preferably at least about 95% pure and most preferably at least
about 99% pure. A polynucleotide is considered to be isolated if,
for example, it is cloned into a vector that is not a part of the
natural environment.
[0056] Within further aspects, the present invention provides
mimetics of WT1 polypeptides. Such mimetics may comprise amino
acids linked to one or more amino acid mimetics (i.e., one or more
amino acids within the WT1 protein may be replaced by an amino acid
mimetic) or may be entirely nonpeptide mimetics. An amino acid
mimetic is a compound that is conformationally similar to an amino
acid such that it can be substituted for an amino acid within a WT1
polypeptide without substantially diminishing the ability to react
with antigen-specific antisera and/or T cell lines or clones. A
nonpeptide mimetic is a compound that does not contain amino acids,
and that has an overall conformation that is similar to a WT1
polypeptide such that the ability of the mimetic to react with
WT1-specific antisera and/or T cell lines or clones is not
substantially diminished relative to the ability of a WT1
polypeptide. Such mimetics may be designed based on standard
techniques (e.g., nuclear magnetic resonance and computational
techniques) that evaluate the three dimensional structure of a
peptide sequence. Mimetics may be designed where one or more of the
side chain functionalities of the WT1 polypeptide are replaced by
groups that do not necessarily have the same size or volume, but
have similar chemical and/or physical properties which produce
similar biological responses. It should be understood that, within
embodiments described herein, a mimetic may be substituted for a
WT1 polypeptide.
[0057] Within other illustrative embodiments, a polypeptide may be
a fusion polypeptide that comprises multiple polypeptides as
described herein, or that comprises at least one polypeptide as
described herein and an unrelated sequence, such as a known tumor
protein. A fusion partner may, for example, assist in providing T
helper epitopes (an immunological fusion partner), preferably T
helper epitopes recognized by humans, or may assist in expressing
the protein (an expression enhancer) at higher yields than the
native recombinant protein. Certain preferred fusion partners are
both immunological and expression enhancing fusion partners. Other
fusion partners may be selected so as to increase the solubility of
the polypeptide or to enable the polypeptide to be targeted to
desired intracellular compartments. Still further fusion partners
include affinity tags, which facilitate purification of the
polypeptide.
[0058] Fusion polypeptides may generally be prepared using standard
techniques, including chemical conjugation. Preferably, a fusion
polypeptide is expressed as a recombinant polypeptide, allowing the
production of increased levels, relative to a non-fused
polypeptide, in an expression system. Briefly, DNA sequences
encoding the polypeptide components may be assembled separately,
and ligated into an appropriate expression vector. The 3' end of
the DNA sequence encoding one polypeptide component is ligated,
with or without a peptide linker, to the 5' end of a DNA sequence
encoding the second polypeptide component so that the reading
frames of the sequences are in phase. This permits translation into
a single fusion polypeptide that retains the biological activity of
both component polypeptides.
[0059] A peptide linker sequence may be employed to separate the
first and second polypeptide components by a distance sufficient to
ensure that each polypeptide folds into its secondary and tertiary
structures. Such a peptide linker sequence is incorporated into the
fusion polypeptide using standard techniques well known in the art.
Suitable peptide linker sequences may be chosen based on the
following factors: (1) their ability to adopt a flexible extended
conformation; (2) their inability to adopt a secondary structure
that could interact with functional epitopes on the first and
second polypeptides; and (3) the lack of hydrophobic or charged
residues that might react with the polypeptide functional epitopes.
Preferred peptide linker sequences contain Gly, Asn and Ser
residues. Other near neutral amino acids, such as Thr and Ala may
also be used in the linker sequence. Amino acid sequences which may
be usefully employed as linkers include those disclosed in Maratea
et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci.
USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No.
4,751,180. The linker sequence may generally be from 1 to about 50
amino acids in length. Linker sequences are not required when the
first and second polypeptides have non-essential N-terminal amino
acid regions that can be used to separate the functional domains
and prevent steric interference.
[0060] The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The
regulatory elements responsible for expression of DNA are located
only 5' to the DNA sequence encoding the first polypeptides.
Similarly, stop codons required to end translation and
transcription termination signals are only present 3' to the DNA
sequence encoding the second polypeptide.
[0061] The fusion polypeptide can comprise a polypeptide as
described herein together with an unrelated immunogenic protein,
such as an immunogenic protein capable of eliciting a recall
response. Examples of such proteins include tetanus, tuberculosis
and hepatitis proteins (see, for example, Stoute et al. New Engl.
J. Med., 336:86-91, 1997).
[0062] In one preferred embodiment, the immunological fusion
partner is derived from a Mycobacterium sp., such as a
Mycobacterium tuberculosis-derived Ra12 fragment. Ra12 compositions
and methods for their use in enhancing the expression and/or
immunogenicity of heterologous polynucleotide/polypeptide sequences
is described in U.S. Patent Application 60/158,585, now lapsed, the
disclosure of which is incorporated herein by reference in its
entirety. Briefly, Ra12 refers to a polynucleotide region that is a
subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid.
MTB32A is a serine protease of 32 KD molecular weight encoded by a
gene in virulent and avirulent strains of M. tuberculosis. The
nucleotide sequence and amino acid sequence of MTB32A have been
described (for example, U.S. Patent Application 60/158,585, now
lapsed; see also, Skeiky et al., Infection and Immun. (1999)
67:3998-4007, incorporated herein by reference). C-terminal
fragments of the MTB32A coding sequence express at high levels and
remain as soluble polypeptides throughout the purification process.
Moreover, Ra12 may enhance the immunogenicity of heterologous
immunogenic polypeptides with which it is fused. One preferred Ra12
fusion polypeptide comprises a 14 KD C-terminal fragment
corresponding to amino acid residues 192 to 323 of MTB32A. Other
preferred Ra12 polynucleotides generally comprise at least about 15
consecutive nucleotides, at least about 30 nucleotides, at least
about 60 nucleotides, at least about 100 nucleotides, at least
about 200 nucleotides, or at least about 300 nucleotides that
encode a portion of a Ra12 polypeptide. Ra12 polynucleotides may
comprise a native sequence (i.e., an endogenous sequence that
encodes a Ra12 polypeptide or a portion thereof) or may comprise a
variant of such a sequence. Ra12 polynucleotide variants may
contain one or more substitutions, additions, deletions and/or
insertions such that the biological activity of the encoded fusion
polypeptide is not substantially diminished, relative to a fusion
polypeptide comprising a native Ra12 polypeptide. Variants
preferably exhibit at least about 70% identity, more preferably at
least about 80% identity and most preferably at least about 90%
identity to a polynucleotide sequence that encodes a native Ra12
polypeptide or a portion thereof.
[0063] Within other preferred embodiments, an immunological fusion
partner is derived from protein D, a surface protein of the
gram-negative bacterium Haemophilus influenza B (WO 91/18926).
Preferably, a protein D derivative comprises approximately the
first third of the protein (e.g., the first N-terminal 100-110
amino acids), and a protein D derivative may be lipidated. Within
certain preferred embodiments, the first 109 residues of a
Lipoprotein D fusion partner is included on the N-terminus to
provide the polypeptide with additional exogenous T-cell epitopes
and to increase the expression level in E. coli (thus functioning
as an expression enhancer). The lipid tail ensures optimal
presentation of the antigen to antigen presenting cells. Other
fusion partners include the non-structural protein from influenzae
virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids
are used, although different fragments that include T-helper
epitopes may be used.
[0064] In another embodiment, the immunological fusion partner is
the protein known as LYTA, or a portion thereof (preferably a
C-terminal portion). LYTA is derived from Streptococcus pneumoniae,
which synthesizes an N-acetyl-L-alanine amidase known as amidase
LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an
autolysin that specifically degrades certain bonds in the
peptidoglycan backbone. The C-terminal domain of the LYTA protein
is responsible for the affinity to the choline or to some choline
analogues such as DEAE. This property has been exploited for the
development of E. coli C-LYTA expressing plasmids useful for
expression of fusion proteins. Purification of hybrid proteins
containing the C-LYTA fragment at the amino terminus has been
described (see Biotechnology 10:795-798, 1992). Within a preferred
embodiment, a repeat portion of LYTA may be incorporated into a
fusion polypeptide. A repeat portion is found in the C-terminal
region starting at residue 178. A particularly preferred repeat
portion incorporates residues 188-305.
[0065] Yet another illustrative embodiment involves fusion
polypeptides, and the polynucleotides encoding them, wherein the
fusion partner comprises a targeting signal capable of directing a
polypeptide to the endosomal/lysosomal compartment, as described in
U.S. Pat. No. 5,633,234. An immunogenic polypeptide of the
invention, when fused with this targeting signal, will associate
more efficiently with MHC class II molecules and thereby provide
enhanced in vivo stimulation of CD4.sup.+ T-cells specific for the
polypeptide.
WT1 Polynucleotides
[0066] Any polynucleotide that encodes a WT1 polypeptide as
described herein is a WT1 polynucleotide encompassed by the present
invention. Such polynucleotides may be single-stranded (coding or
antisense) or double-stranded, and may be DNA (genomic, cDNA or
synthetic) or RNA molecules. Additional coding or non-coding
sequences may, but need not, be present within a polynucleotide of
the present invention, and a polynucleotide may, but need not, be
linked to other molecules and/or support materials.
[0067] WT1 polynucleotides may encode a native WT1 protein, or may
encode a variant of WT1 as described herein. Polynucleotide
variants may contain one or more substitutions, additions,
deletions and/or insertions such that the immunogenicity of the
encoded polypeptide is not diminished, relative to a native WT1
protein. The effect on the immunogenicity of the encoded
polypeptide may generally be assessed as described herein.
Preferred variants contain nucleotide substitutions, deletions,
insertions and/or additions at no more than 20%, preferably at no
more than 10%, of the nucleotide positions that encode an
immunogenic portion of a native WT1 sequence. Certain variants are
substantially homologous to a native gene, or a portion thereof.
Such polynucleotide variants are capable of hybridizing under
moderately stringent conditions to a naturally occurring DNA
sequence encoding a WT1 polypeptide (or a complementary sequence).
Suitable moderately stringent conditions include prewashing in a
solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);
hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC, overnight;
followed by washing twice at 65.degree. C. for 20 minutes with each
of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS).
Such hybridizing DNA sequences are also within the scope of this
invention.
[0068] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a WT1 polypeptide. Some of
these polynucleotides bear minimal homology to the nucleotide
sequence of any native gene. Nonetheless, polynucleotides that vary
due to differences in codon usage are specifically contemplated by
the present invention.
[0069] Once an immunogenic portion of WT1 is identified, as
described above, a WT1 polynucleotide may be prepared using any of
a variety of techniques. For example, a WT1 polynucleotide may be
amplified from cDNA prepared from cells that express WT1. Such
polynucleotides may be amplified via polymerase chain reaction
(PCR). For this approach, sequence-specific primers may be designed
based on the sequence of the immunogenic portion and may be
purchased or synthesized. For example, suitable primers for PCR
amplification of a human WT1 gene include: first step--P118:
1434-1414: 5' GAG AGT CAG ACT TGA AAG CAGT 3' (SEQ ID NO:5) and
P135: 5' CTG AGC CTC AGC AAA TGG GC 3' (SEQ ID NO:6); second
step--P136: 5' GAG CAT GCA TGG GCT CCG ACG TGC GGG 3' (SEQ ID NO:7)
and P137: 5' GGG GTA CCC ACT GAA CGG TCC CCG A 3' (SEQ ID NO:8).
Primers for PCR amplification of a mouse WT1 gene include: first
step--P138: 5' TCC GAG CCG CAC CTC ATG 3' (SEQ ID NO:9) and P139:
5' GCC TGG GAT GCT GGA CTG 3' (SEQ ID NO:10), second step--P140: 5'
GAG CAT GCG ATG GGT TCC GAC GTG CGG 3' (SEQ ID NO:11) and P141: 5'
GGG GTA CCT CAA AGC GCC ACG TGG AGT TT 3' (SEQ ID NO:12).
[0070] An amplified portion may then be used to isolate a full
length gene from a human genomic DNA library or from a suitable
cDNA library, using well known techniques. Alternatively, a full
length gene can be constructed from multiple PCR fragments. WT1
polynucleotides may also be prepared by synthesizing
oligonucleotide components, and ligating components together to
generate the complete polynucleotide.
[0071] WT1 polynucleotides may also be synthesized by any method
known in the art, including chemical synthesis (e.g., solid phase
phosphoramidite chemical synthesis). Modifications in a
polynucleotide sequence may also be introduced using standard
mutagenesis techniques, such as oligonucleotide-directed
site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983).
Alternatively, RNA molecules may be generated by in vitro or in
vivo transcription of DNA sequences encoding a WT1 polypeptide,
provided that the DNA is incorporated into a vector with a suitable
RNA polymerase promoter (such as T7 or SP6). Certain portions may
be used to prepare an encoded polypeptide, as described herein. In
addition, or alternatively, a portion may be administered to a
patient such that the encoded polypeptide is generated in vivo
(e.g., by transfecting antigen-presenting cells such as dendritic
cells with a cDNA construct encoding a WT1 polypeptide, and
administering the transfected cells to the patient).
[0072] Polynucleotides that encode a WT1 polypeptide may generally
be used for production of the polypeptide, in vitro or in vivo. WT1
polynucleotides that are complementary to a coding sequence (i.e.,
antisense polynucleotides) may also be used as a probe or to
inhibit WT1 expression. cDNA constructs that can be transcribed
into antisense RNA may also be introduced into cells of tissues to
facilitate the production of antisense RNA.
[0073] Any polynucleotide may be further modified to increase
stability in vivo. Possible modifications include, but are not
limited to, the addition of flanking sequences at the 5' and/or 3'
ends; the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages in the backbone; and/or the inclusion of
nontraditional bases such as inosine, queosine and wybutosine, as
well as acetyl- methyl-, thio- and other modified forms of adenine,
cytidine, guanine, thymine and uridine.
[0074] Nucleotide sequences as described herein may be joined to a
variety of other nucleotide sequences using established recombinant
DNA techniques. For example, a polynucleotide may be cloned into
any of a variety of cloning vectors, including plasmids, phagemids,
lambda phage derivatives and cosmids. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors and sequencing vectors. In general, a vector
will contain an origin of replication functional in at least one
organism, convenient restriction endonuclease sites and one or more
selectable markers. Other elements will depend upon the desired
use, and will be apparent to those of ordinary skill in the
art.
[0075] Within certain embodiments, polynucleotides may be
formulated so as to permit entry into a cell of a mammal, and
expression therein. Such formulations are particularly useful for
therapeutic purposes, as described below. Those of ordinary skill
in the art will appreciate that there are many ways to achieve
expression of a polynucleotide in a target cell, and any suitable
method may be employed. For example, a polynucleotide may be
incorporated into a viral vector such as, but not limited to,
adenovirus, adeno-associated virus, retrovirus, or vaccinia or
other pox virus (e.g., avian pox virus). Techniques for
incorporating DNA into such vectors are well known to those of
ordinary skill in the art. A retroviral vector may additionally
transfer or incorporate a gene for a selectable marker (to aid in
the identification or selection of transduced cells) and/or a
targeting moiety, such as a gene that encodes a ligand for a
receptor on a specific target cell, to render the vector target
specific. Targeting may also be accomplished using an antibody, by
methods known to those of ordinary skill in the art. cDNA
constructs within such a vector may be used, for example, to
transfect human or animal cell lines for use in establishing WT1
positive tumor models which may be used to perform tumor protection
and adoptive immunotherapy experiments to demonstrate tumor or
leukemia-growth inhibition or lysis of such cells.
[0076] Other therapeutic formulations for polynucleotides include
colloidal dispersion systems, such as macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. A preferred colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (i.e., an artificial
membrane vesicle). The preparation and use of such systems is well
known in the art.
Antibodies and Fragments Thereof
[0077] The present invention further provides binding agents, such
as antibodies and antigen-binding fragments thereof, that
specifically bind to a WT1 polypeptide. As used herein, an agent is
said to "specifically bind" to a WT1 polypeptide if it reacts at a
detectable level (within, for example, an ELISA) with a WT1
polypeptide, and does not react detectably with unrelated proteins
under similar conditions. As used herein, "binding" refers to a
noncovalent association between two separate molecules such that a
"complex" is formed. The ability to bind may be evaluated by, for
example, determining a binding constant for the formation of the
complex. The binding constant is the value obtained when the
concentration of the complex is divided by the product of the
component concentrations. In general, two compounds are said to
"bind," in the context of the present invention, when the binding
constant for complex formation exceeds about 10.sup.3 L/mol. The
binding constant maybe determined using methods well known in the
art.
[0078] Any agent that satisfies the above requirements may be a
binding agent. In a preferred embodiment, a binding agent is an
antibody or an antigen-binding fragment thereof. Certain antibodies
are commercially available from, for example, Santa Cruz
Biotechnology (Santa Cruz, Calif.). Alternatively, antibodies may
be prepared by any of a variety of techniques known to those of
ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In
general, antibodies can be produced by cell culture techniques,
including the generation of monoclonal antibodies as described
herein, or via transfection of antibody genes into suitable
bacterial or mammalian cell hosts, in order to allow for the
production of recombinant antibodies. In one technique, an
immunogen comprising the polypeptide is initially injected into any
of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or
goats). In this step, the polypeptides of this invention may serve
as the immunogen without modification. Alternatively, particularly
for relatively short polypeptides, a superior immune response may
be elicited if the polypeptide is joined to a carrier protein, such
as bovine serum albumin or keyhole limpet hemocyanin. The immunogen
is injected into the animal host, preferably according to a
predetermined schedule incorporating one or more booster
immunizations, and the animals are bled periodically. Polyclonal
antibodies specific for the polypeptide may then be purified from
such antisera by, for example, affinity chromatography using the
polypeptide coupled to a suitable solid support.
[0079] Monoclonal antibodies specific for the antigenic polypeptide
of interest may be prepared, for example, using the technique of
Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and
improvements thereto. Briefly, these methods involve the
preparation of immortal cell lines capable of producing antibodies
having the desired specificity (i.e., reactivity with the
polypeptide of interest). Such cell lines may be produced, for
example, from spleen cells obtained from an animal immunized as
described above. The spleen cells are then immortalized by, for
example, fusion with a myeloma cell fusion partner, preferably one
that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and
myeloma cells may be combined with a nonionic detergent for a few
minutes and then plated at low density on a selective medium that
supports the growth of hybrid cells, but not myeloma cells. A
preferred selection technique uses HAT (hypoxanthine, aminopterin,
thymidine) selection. After a sufficient time, usually about 1 to 2
weeks, colonies of hybrids are observed. Single colonies are
selected and their culture supernatants tested for binding activity
against the polypeptide. Hybridomas having high reactivity and
specificity are preferred.
[0080] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be
harvested from the ascites fluid or the blood. Contaminants may be
removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation, and extraction. The
polypeptides of this invention may be used in the purification
process in, for example, an affinity chromatography step.
[0081] Within certain embodiments, the use of antigen-binding
fragments of antibodies may be preferred. Such fragments include
Fab fragments, which may be prepared using standard techniques.
Briefly, immunoglobulins may be purified from rabbit serum by
affinity chromatography on Protein A bead columns (Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988) and digested by papain to yield Fab and Fc fragments. The Fab
and Fc fragments may be separated by affinity chromatography on
protein A bead columns.
[0082] Monoclonal antibodies and fragments thereof may be coupled
to one or more therapeutic agents. Suitable agents in this regard
include radioactive tracers and chemotherapeutic agents, which may
be used, for example, to purge autologous bone marrow in vitro).
Representative therapeutic agents include radionuclides,
differentiation inducers, drugs, toxins, and derivatives thereof.
Preferred radionuclides include .sup.90Y, .sup.123I, .sup.125I,
.sup.131I, .sup.186Re, .sup.188Re, .sup.211At, and .sup.212Bi.
Preferred drugs include methotrexate, and pyrimidine and purine
analogs. Preferred differentiation inducers include phorbol esters
and butyric acid. Preferred toxins include ricin, abrin, diptheria
toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella
toxin, and pokeweed antiviral protein. For diagnostic purposes,
coupling of radioactive agents may be used to facilitate tracing of
metastases or to determine the location of WT1-positive tumors.
[0083] A therapeutic agent may be coupled (e.g., covalently bonded)
to a suitable monoclonal antibody either directly or indirectly
(e.g., via a linker group). A direct reaction between an agent and
an antibody is possible when each possesses a substituent capable
of reacting with the other. For example, a nucleophilic group, such
as an amino or sulfhydryl group, on one may be capable of reacting
with a carbonyl-containing group, such as an anhydride or an acid
halide, or with an alkyl group containing a good leaving group
(e.g., a halide) on the other.
[0084] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0085] It will be evident to those skilled in the art that a
variety of bifunctional or polyfunctional reagents, both homo- and
hetero-functional (such as those described in the catalog of the
Pierce Chemical Co., Rockford, Ill.), may be employed as the linker
group.
[0086] Coupling may be effected, for example, through amino groups,
carboxyl groups, sulfhydryl groups or oxidized carbohydrate
residues. There are numerous references describing such
methodology, e.g., U.S. Pat. No. 4,671,958, to Rodwell et al.
[0087] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710, to Spitler), by irradiation of a photolabile bond (e.g.,
U.S. Pat. No. 4,625,014, to Senter et al.), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045,
to Kohn et al.), by serum complement-mediated hydrolysis (e.g.,
U.S. Pat. No. 4,671,958, to Rodwell et al.), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789, to Blattler et al.).
[0088] It may be desirable to couple more than one agent to an
antibody. In one embodiment, multiple molecules of an agent are
coupled to one antibody molecule. In another embodiment, more than
one type of agent may be coupled to one antibody. Regardless of the
particular embodiment, immunoconjugates with more than one agent
may be prepared in a variety of ways. For example, more than one
agent may be coupled directly to an antibody molecule, or linkers
which provide multiple sites for attachment can be used.
Alternatively, a carrier can be used. A carrier may bear the agents
in a variety of ways, including covalent bonding either directly or
via a linker group. Suitable carriers include proteins such as
albumins (e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides
and polysaccharides such as aminodextran (e.g., U.S. Pat. No.
4,699,784, to Shih et al.). A carrier may also bear an agent by
noncovalent bonding or by encapsulation, such as within a liposome
vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers
specific for radionuclide agents include radiohalogenated small
molecules and chelating compounds. For example, U.S. Pat. No.
4,735,792 discloses representative radiohalogenated small molecules
and their synthesis. A radionuclide chelate may be formed from
chelating compounds that include those containing nitrogen and
sulfur atoms as the donor atoms for binding the metal, or metal
oxide, radionuclide. For example, U.S. Pat. No. 4,673,562, to
Davison et al. discloses representative chelating compounds and
their synthesis.
[0089] A variety of routes of administration for the antibodies and
immunoconjugates may be used. Typically, administration will be
intravenous, intramuscular, subcutaneous or in the bed of a
resected tumor. It will be evident that the precise dose of the
antibody/immunoconjugate will vary depending upon the antibody
used, the antigen density on the tumor, and the rate of clearance
of the antibody.
[0090] Also provided herein are anti-idiotypic antibodies that
mimic an immunogenic portion of WT1. Such antibodies may be raised
against an antibody, or antigen-binding fragment thereof, that
specifically binds to an immunogenic portion of WT1, using well
known techniques. Anti-idiotypic antibodies that mimic an
immunogenic portion of WT1 are those antibodies that bind to an
antibody, or antigen-binding fragment thereof, that specifically
binds to an immunogenic portion of WT1, as described herein.
T Cells
[0091] Immunotherapeutic compositions may also, or alternatively,
comprise T cells specific for WT1. Such cells may generally be
prepared in vitro or ex vivo, using standard procedures. For
example, T cells may be present within (or isolated from) bone
marrow, peripheral blood or a fraction of bone marrow or peripheral
blood of a mammal, such as a patient, using a commercially
available cell separation system, such as the CEPRATE.TM. system,
available from CellPro Inc., Bothell WA (see also U.S. Pat. No.
5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO
92/07243). Alternatively, T cells may be derived from related or
unrelated humans, non-human animals, cell lines or cultures.
[0092] T cells may be stimulated with WT1 polypeptide,
polynucleotide encoding a WT1 polypeptide and/or an antigen
presenting cell (APC) that expresses a WT1 polypeptide. Such
stimulation is performed under conditions and for a time sufficient
to permit the generation of T cells that are specific for the WT1
polypeptide. Preferably, a WT1 polypeptide or polynucleotide is
present within a delivery vehicle, such as a microsphere, to
facilitate the generation of antigen-specific T cells. Briefly, T
cells, which may be isolated from a patient or a related or
unrelated donor by routine techniques (such as by
FICOLL.RTM./HYPAQUE.RTM. density gradient centrifugation of
peripheral blood lymphocytes), are incubated with WT1 polypeptide.
For example, T cells may be incubated in vitro for 2-9 days
(typically 4 days) at 37.degree. C. with WT1 polypeptide (e.g., 5
to 25 .mu.g/ml) or cells synthesizing a comparable amount of WT1
polypeptide. It may be desirable to incubate a separate aliquot of
a T cell sample in the absence of WT1 polypeptide to serve as a
control.
[0093] T cells are considered to be specific for a WT1 polypeptide
if the T cells kill target cells coated with a WT1 polypeptide or
expressing a gene encoding such a polypeptide. T cell specificity
may be evaluated using any of a variety of standard techniques. For
example, within a chromium release assay or proliferation assay, a
stimulation index of more than two fold increase in lysis and/or
proliferation, compared to negative controls, indicates T cell
specificity. Such assays may be performed, for example, as
described in Chen et al., Cancer Res. 54:1065-1070, 1994.
Alternatively, detection of the proliferation of T cells may be
accomplished by a variety of known techniques. For example, T cell
proliferation can be detected by measuring an increased rate of DNA
synthesis (e.g., by pulse-labeling cultures of T cells with
tritiated thymidine and measuring the amount of tritiated thymidine
incorporated into DNA). Other ways to detect T cell proliferation
include measuring increases in interleukin-2 (IL-2) production,
Ca.sup.2+ flux, or dye uptake, such as
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
Alternatively, synthesis of lymphokines (such as interferon-gamma)
can be measured or the relative number of T cells that can respond
to a WT1 polypeptide may be quantified. Contact with a WT1
polypeptide (200 ng/ml-100 .mu.g/ml, preferably 100 ng/ml-25
.mu.g/ml) for 3-7 days should result in at least a two fold
increase in proliferation of the T cells and/or contact as
described above for 2-3 hours should result in activation of the T
cells, as measured using standard cytokine assays in which a two
fold increase in the level of cytokine release (e.g., TNF or
IFN-.gamma.) is indicative of T cell activation (see Coligan et
al., Current Protocols in Immunology, vol. 1, Wiley Interscience
(Greene 1998). WT1 specific T cells may be expanded using standard
techniques. Within preferred embodiments, the T cells are derived
from a patient or a related or unrelated donor and are administered
to the patient following stimulation and expansion.
[0094] T cells that have been activated in response to a WT1
polypeptide, polynucleotide or WT1-expressing APC may be CD4.sup.+
and/or CD8.sup.+. Specific activation of CD4.sup.+ or CD8.sup.+ T
cells may be detected in a variety of ways. Methods for detecting
specific T cell activation include detecting the proliferation of T
cells, the production of cytokines (e.g., lymphokines), or the
generation of cytolytic activity (i.e., generation of cytotoxic T
cells specific for WT1). For CD4.sup.+ T cells, a preferred method
for detecting specific T cell activation is the detection of the
proliferation of T cells. For CD8.sup.+ T cells, a preferred method
for detecting specific T cell activation is the detection of the
generation of cytolytic activity.
[0095] For therapeutic purposes, CD4.sup.+ or CD8.sup.+ T cells
that proliferate in response to the WT1 polypeptide, polynucleotide
or APC can be expanded in number either in vitro or in vivo.
Proliferation of such T cells in vitro may be accomplished in a
variety of ways. For example, the T cells can be re-exposed to WT1
polypeptide, with or without the addition of T cell growth factors,
such as interleukin-2, and/or stimulator cells that synthesize a
WT1 polypeptide. The addition of stimulator cells is preferred
where generating CD8.sup.+ T cell responses. T cells can be grown
to large numbers in vitro with retention of specificity in response
to intermittent restimulation with WT1 polypeptide. Briefly, for
the primary in vitro stimulation (IVS), large numbers of
lymphocytes (e.g., greater than 4.times.10.sup.7) may be placed in
flasks with media containing human serum. WT1 polypeptide (e.g.,
peptide at 10 .mu.g/ml) may be added directly, along with tetanus
toxoid (e.g., 5 .mu.g/ml). The flasks may then be incubated (e.g.,
37.degree. C. for 7 days). For a second IVS, T cells are then
harvested and placed in new flasks with 2-3.times.10.sup.7
irradiated peripheral blood mononuclear cells. WT1 polypeptide
(e.g., 10 .mu.g/ml) is added directly. The flasks are incubated at
37.degree. C. for 7 days. On day 2 and day 4 after the second IVS,
2-5 units of interleukin-2 (IL-2) may be added. For a third IVS,
the T cells may be placed in wells and stimulated with the
individual's own EBV transformed B cells coated with the peptide.
IL-2 may be added on days 2 and 4 of each cycle. As soon as the
cells are shown to be specific cytotoxic T cells, they may be
expanded using a 10 day stimulation cycle with higher IL-2 (20
units) on days 2, 4 and 6.
[0096] Alternatively, one or more T cells that proliferate in the
presence of WT1 polypeptide can be expanded in number by cloning.
Methods for cloning cells are well known in the art, and include
limiting dilution. Responder T cells may be purified from the
peripheral blood of sensitized patients by density gradient
centrifugation and sheep red cell rosetting and established in
culture by stimulating with the nominal antigen in the presence of
irradiated autologous filler cells. In order to generate CD4.sup.+
T cell lines, WT1 polypeptide is used as the antigenic stimulus and
autologous peripheral blood lymphocytes (PBL) or lymphoblastoid
cell lines (LCL) immortalized by infection with Epstein Barr virus
are used as antigen presenting cells. In order to generate
CD8.sup.+ T cell lines, autologous antigen-presenting cells
transfected with an expression vector which produces WT1
polypeptide may be used as stimulator cells. Established T cell
lines may be cloned 2-4 days following antigen stimulation by
plating stimulated T cells at a frequency of 0.5 cells per well in
96-well flat-bottom plates with 1.times.10.sup.6 irradiated PBL or
LCL cells and recombinant interleukin-2 (rIL2) (50 U/ml). Wells
with established clonal growth may be identified at approximately
2-3 weeks after initial plating and restimulated with appropriate
antigen in the presence of autologous antigen-presenting cells,
then subsequently expanded by the addition of low doses of rIL2 (10
U/ml) 2-3 days following antigen stimulation. T cell clones may be
maintained in 24-well plates by periodic restimulation with antigen
and rIL2 approximately every two weeks.
[0097] Within certain embodiments, allogeneic T-cells may be primed
(i.e., sensitized to WT1) in vivo and/or in vitro. Such priming may
be achieved by contacting T cells with a WT1 polypeptide, a
polynucleotide encoding such a polypeptide or a cell producing such
a polypeptide under conditions and for a time sufficient to permit
the priming of T cells. In general, T cells are considered to be
primed if, for example, contact with a WT1 polypeptide results in
proliferation and/or activation of the T cells, as measured by
standard proliferation, chromium release and/or cytokine release
assays as described herein. A stimulation index of more than two
fold increase in proliferation or lysis, and more than three fold
increase in the level of cytokine, compared to negative controls,
indicates T-cell specificity. Cells primed in vitro may be
employed, for example, within a bone marrow transplantation or as
donor lymphocyte infusion.
[0098] T cells specific for WT1 can kill cells that express WT1
protein. Introduction of genes encoding T-cell receptor (TCR)
chains for WT1 are used as a means to quantitatively and
qualitatively improve responses to WT1 bearing leukemia and cancer
cells. Vaccines to increase the number of T cells that can react to
WT1 positive cells are one method of targeting WT1 bearing cells. T
cell therapy with T cells specific for WT1 is another method. An
alternative method is to introduce the TCR chains specific for WT1
into T cells or other cells with lytic potential. In a suitable
embodiment, the TCR alpha and beta chains are cloned out from a WT1
specific T cell line and used for adoptive T cell therapy, such as
described in WO96/30516, incorporated herein by reference.
Pharmaceutical Compositions and Vaccines
[0099] Within certain aspects, polypeptides, polynucleotides,
antibodies and/or T cells may be incorporated into pharmaceutical
compositions or vaccines. Alternatively, a pharmaceutical
composition may comprise an antigen-presenting cell (e.g., a
dendritic cell) transfected with a WT1 polynucleotide such that the
antigen presenting cell expresses a WT1 polypeptide. Pharmaceutical
compositions comprise one or more such compounds or cells and a
physiologically acceptable carrier or excipient. Certain vaccines
may comprise one or more such compounds or cells and a non-specific
immune response enhancer, such as an adjuvant or a liposome (into
which the compound is incorporated). Pharmaceutical compositions
and vaccines may additionally contain a delivery system, such as
biodegradable microspheres which are disclosed, for example, in
U.S. Pat. Nos. 4,897,268 and 5,075,109. Pharmaceutical compositions
and vaccines within the scope of the present invention may also
contain other compounds, which may be biologically active or
inactive.
[0100] Within certain embodiments, pharmaceutical compositions and
vaccines are designed to elicit T cell responses specific for a WT1
polypeptide in a patient, such as a human. In general, T cell
responses may be favored through the use of relatively short
polypeptides (e.g., comprising less than 23 consecutive amino acid
residues of a native WT1 polypeptide, preferably 4-16 consecutive
residues, more preferably 8-16 consecutive residues and still more
preferably 8-10 consecutive residues. Alternatively, or in
addition, a vaccine may comprise a non-specific immune response
enhancer that preferentially enhances a T cell response. In other
words, the immune response enhancer may enhance the level of a T
cell response to a WT1 polypeptide by an amount that is
proportionally greater than the amount by which an antibody
response is enhanced. For example, when compared to a standard oil
based adjuvant, such as CFA, an immune response enhancer that
preferentially enhances a T cell response may enhance a
proliferative T cell response by at least two fold, a lytic
response by at least 10%, and/or T cell activation by at least two
fold compared to WT1-negative control cell lines, while not
detectably enhancing an antibody response. The amount by which a T
cell or antibody response to a WT1 polypeptide is enhanced may
generally be determined using any representative technique known in
the art, such as the techniques provided herein.
[0101] A pharmaceutical composition or vaccine may contain DNA
encoding one or more of the polypeptides as described above, such
that the polypeptide is generated in situ. As noted above, the DNA
may be present within any of a variety of delivery systems known to
those of ordinary skill in the art, including nucleic acid
expression systems, bacterial and viral expression systems and
mammalian expression systems. Appropriate nucleic acid expression
systems contain the necessary DNA, cDNA or RNA sequences for
expression in the patient (such as a suitable promoter and
terminating signal). Bacterial delivery systems involve the
administration of a bacterium (such as Bacillus-Calmette-Guerrin)
that expresses an immunogenic portion of the polypeptide on its
cell surface. In a preferred embodiment, the DNA may be introduced
using a viral expression system (e.g., vaccinia or other pox virus,
retrovirus, or adenovirus), which may involve the use of a
non-pathogenic (defective), replication competent virus. Techniques
for incorporating DNA into such expression systems are well known
to those of ordinary skill in the art. The DNA may also be "naked,"
as described, for example, in Ulmer et al., Science 259:1745-1749,
1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake
of naked DNA may be increased by coating the DNA onto biodegradable
beads, which are efficiently transported into the cells.
[0102] As noted above, a pharmaceutical composition or vaccine may
comprise an antigen-presenting cell that expresses a WT1
polypeptide. For therapeutic purposes, as described herein, the
antigen presenting cell is preferably an autologous dendritic cell.
Such cells may be prepared and transfected using standard
techniques, such as those described by Reeves et al., Cancer Res.
56:5672-5677, 1996; Tuting et al., J. Immunol. 160:1139-1147, 1998;
and Nair et al., Nature Biotechnol. 16:364-369, 1998). Expression
of a WT1 polypeptide on the surface of an antigen-presenting cell
may be confirmed by in vitro stimulation and standard proliferation
as well as chromium release assays, as described herein.
[0103] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration. Compositions of the present invention may be
formulated for any appropriate manner of administration, including
for example, topical, oral, nasal, intravenous, intracranial,
intraperitoneal, subcutaneous or intramuscular administration. For
parenteral administration, such as subcutaneous injection, the
carrier preferably comprises water, saline, alcohol, a fat, a wax
or a buffer. For oral administration, any of the above carriers or
a solid carrier, such as mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, sucrose,
and magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactate polyglycolate) may also be employed
as carriers for the pharmaceutical compositions of this invention.
For certain topical applications, formulation as a cream or lotion,
using well known components, is preferred.
[0104] Such compositions may also comprise buffers (e.g., neutral
buffered saline or phosphate buffered saline), carbohydrates (e.g.,
glucose, mannose, sucrose or dextrans), mannitol, proteins,
polypeptides or amino acids such as glycine, antioxidants,
chelating agents such as EDTA or glutathione, adjuvants (e.g.,
aluminum hydroxide) and/or preservatives. Alternatively,
compositions of the present invention may be formulated as a
lyophilizate. Compounds may also be encapsulated within liposomes
using well known technology.
[0105] Any of a variety of non-specific immune response enhancers,
such as adjuvants, may be employed in the vaccines of this
invention. Most adjuvants contain a substance designed to protect
the antigen from rapid catabolism, such as aluminum hydroxide or
mineral oil, and a stimulator of immune responses, such as lipid A,
Bortadella pertussis or Mycobacterium tuberculosis derived
proteins. Suitable non-specific immune response enhancers include
alum-based adjuvants (e.g., Alhydrogel, Rehydragel, aluminum
phosphate, Algammulin, aluminum hydroxide); oil based adjuvants
(Freund's adjuvant (FA), Specol, RIBI, TiterMax, MONTANIDE.RTM.
ISA50 or Seppic MONTANIDE.RTM. ISA 720; cytokines (e.g., GM-CSF or
Flt3-ligand); microspheres; nonionic block copolymer-based
adjuvants; dimethyl dioctadecyl ammoniumbromide (DDA) based
adjuvants AS-1, AS-2 (Smith Kline Beecham); Ribi Adjuvant system
based adjuvants; QS21 (Aquila); saponin based adjuvants (crude
saponin, the saponin Quil A); muramyl dipeptide (MDP) based
adjuvants such as SAF (Syntex adjuvant in its microfluidized form
(SAF-m)); dimethyl-dioctadecyl ammonium bromide (DDA); human
complement based adjuvants M. vaccae and derivatives; immune
stimulating complex (ISCOM.RTM.) based adjuvants; inactivated
toxins; and attenuated infectious agents (such as M.
tuberculosis).
[0106] As noted above, within certain embodiments, immune response
enhancers are chosen for their ability to preferentially elicit or
enhance a T cell response (e.g., CD4.sup.+ and/or CD8.sup.+) to a
WT1 polypeptide. Such immune response enhancers are well known in
the art, and include (but are not limited to) MONTANIDE.RTM. ISA50,
Seppic MONTANIDE.RTM. ISA 720, cytokines (e.g., GM-CSF,
Flat3-ligand), microspheres, dimethyl dioctadecyl ammoniumbromide
(DDA) based adjuvants, AS-1 (Smith Kline Beecham), AS-2 (Smith
Kline Beecham), Ribi Adjuvant system based adjuvants, QS21
(Aquila), saponin based adjuvants (crude saponin, the saponin Quil
A), Syntex adjuvant in its microfluidized form (SAF-m), MV, ddMV
(Genesis), immune stimulating complex (ISCOM.RTM.) based adjuvants
and inactivated toxins.
[0107] The compositions and vaccines described herein may be
administered as part of a sustained release formulation (i.e., a
formulation such as a capsule or sponge that effects a slow release
of compound following administration). Such formulations may
generally be prepared using well known technology and administered
by, for example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release
formulations may contain a polypeptide, polynucleotide, antibody or
cell dispersed in a carrier matrix and/or contained within a
reservoir surrounded by a rate controlling membrane. Carriers for
use within such formulations are biocompatible, and may also be
biodegradable; preferably the formulation provides a relatively
constant level of active component release. The amount of active
compound contained within a sustained release formulation depends
upon the site of implantation, the rate and expected duration of
release and the nature of the condition to be treated or
prevented.
Therapy of Malignant Diseases
[0108] In further aspects of the present invention, the
compositions and vaccines described herein may be used to inhibit
the development of malignant diseases (e.g., progressive or
metastatic diseases or diseases characterized by small tumor burden
such as minimal residual disease). In general, such methods may be
used to prevent, delay or treat a disease associated with WT1
expression. In other words, therapeutic methods provided herein may
be used to treat an existing WT1-associated disease, or may be used
to prevent or delay the onset of such a disease in a patient who is
free of disease or who is afflicted with a disease that is not yet
associated with WT1 expression.
[0109] As used herein, a disease is "associated with WT1
expression" if diseased cells (e.g., tumor cells) at some time
during the course of the disease generate detectably higher levels
of a WT1 polypeptide than normal cells of the same tissue.
Association of WT1 expression with a malignant disease does not
require that WT1 be present on a tumor. For example, overexpression
of WT1 may be involved with initiation of a tumor, but the protein
expression may subsequently be lost. Alternatively, a malignant
disease that is not characterized by an increase in WT1 expression
may, at a later time, progress to a disease that is characterized
by increased WT1 expression. Accordingly, any malignant disease in
which diseased cells formerly expressed, currently express or are
expected to subsequently express increased levels of WT1 is
considered to be "associated with WT1 expression."
[0110] Immunotherapy may be performed using any of a variety of
techniques, in which compounds or cells provided herein function to
remove WT1-expressing cells from a patient. Such removal may take
place as a result of enhancing or inducing an immune response in a
patient specific for WT1 or a cell expressing WT1. Alternatively,
WT1-expressing cells may be removed ex vivo (e.g., by treatment of
autologous bone marrow, peripheral blood or a fraction of bone
marrow or peripheral blood). Fractions of bone marrow or peripheral
blood may be obtained using any standard technique in the art.
[0111] Within such methods, pharmaceutical compositions and
vaccines may be administered to a patient. As used herein, a
"patient" refers to any warm-blooded animal, preferably a human. A
patient may or may not be afflicted with a malignant disease.
Accordingly, the above pharmaceutical compositions and vaccines may
be used to prevent the onset of a disease (i.e., prophylactically)
or to treat a patient afflicted with a disease (e.g., to prevent or
delay progression and/or metastasis of an existing disease). A
patient afflicted with a disease may have a minimal residual
disease (e.g., a low tumor burden in a leukemia patient in complete
or partial remission or a cancer patient following reduction of the
tumor burden after surgery radiotherapy and/or chemotherapy). Such
a patient may be immunized to inhibit a relapse (i.e., prevent or
delay the relapse, or decrease the severity of a relapse). Within
certain preferred embodiments, the patient is afflicted with a
leukemia (e.g., AML, CML, ALL or childhood ALL), a myelodysplastic
syndrome (MDS) or a cancer (e.g., gastrointestinal, lung, thyroid
or breast cancer or a melanoma), where the cancer or leukemia is
WT1 positive (i.e., reacts detectably with an anti-WT1 antibody, as
provided herein or expresses WT1 mRNA at a level detectable by
RT-PCR, as described herein) or suffers from an autoimmune disease
directed against WT1-expressing cells.
[0112] Other diseases associated with WT1 overexpression include
kidney cancer (such as renal cell carcinoma, or Wilms tumor), as
described in Satoh F., et al., Pathol. Int. 50(6):458-71 (2000),
and Campbell C. E. et al., Int. J. Cancer 78(2):182-8 (1998); and
mesothelioma, as described in Amin, K. M. et al., Am. J. Pathol.
146(2):344-56 (1995). Harada et al. (Mol. Urol. 3(4):357-364 (1999)
describe WT1 gene expression in human testicular germ-cell tumors.
Nonomura et al. Hinyokika Kiyo 45(8):593-7 (1999) describe
molecular staging of testicular cancer using polymerase chain
reaction of the testicular cancer-specific genes. Shimizu et al.,
Int. J. Gynecol. Pathol. 19(2):158-63 (2000) describe the
immunohistochemical detection of the Wilms' tumor gene (WT1) in
epithelial ovarian tumors.
[0113] WT1 overexpression was also described in desmoplastic small
round cell tumors, by Barnoud, R. et al., Am. J. Surg. Pathol.
24(6):830-6 (2000); and Pathol. Res. Pract. 194(10):693-700 (1998).
WT1 overexpression in glioblastoma and other cancer was described
by Menssen, H. D. et al., J. Cancer Res. Clin. Oncol. 126(4):226-32
(2000), "Wilms' tumor gene (WT1) expression in lung cancer, colon
cancer and glioblastoma cell lines compared to freshly isolated
tumor specimens." Other diseases showing WT1 overexpression include
EBV associated diseases, such as Burkitt's lymphoma and
nasopharyngeal cancer (Spinsanti P. et al., Leuk. Lymphoma
38(5-6):611-9 (2000), "Wilms' tumor gene expression by normal and
malignant human B lymphocytes."
[0114] In Leukemia 14(9):1634-4 (2000), Pan et al., describe in
vitro IL-12 treatment of peripheral blood mononuclear cells from
patients with leukemia or myelodysplastic syndromes, and reported
an increase in cytotoxicity and reduction in WT1 gene expression.
In Leukemia 13(6):891-900 (1999), Patmasiriwat et al. reported WT1
and GATA1 expression in myelodysplastic syndrome and acute
leukemia. In Leukemia 13(3):393-9 (1999), Tamaki et al. reported
that the Wilms' tumor gene WT1 is a good marker for diagnosis of
disease progression of myelodysplastic syndromes. Expression of the
Wilms' tumor gene WT1 in solid tumors, and its involvement in tumor
cell growth, was discussed in relation to gastric cancer, colon
cancer, lung cancer, breast cancer cell lines, germ cell tumor cell
line, ovarian cancer, the uterine cancer, thyroid cancer cell line,
hepatocellular carcinoma, in Oji et al., Jpn. J. Cancer Res.
90(2):194-204 (1999).
[0115] The compositions provided herein may be used alone or in
combination with conventional therapeutic regimens such as surgery,
irradiation, chemotherapy and/or bone marrow transplantation
(autologous, syngeneic, allogeneic or unrelated). As discussed in
greater detail below, binding agents and T cells as provided herein
may be used for purging of autologous stem cells. Such purging may
be beneficial prior to, for example, bone marrow transplantation or
transfusion of blood or components thereof. Binding agents, T
cells, antigen presenting cells (APC) and compositions provided
herein may further be used for expanding and stimulating (or
priming) autologous, allogeneic, syngeneic or unrelated
WT1-specific T-cells in vitro and/or in vivo. Such WT1-specific T
cells may be used, for example, within donor lymphocyte
infusions.
[0116] Routes and frequency of administration, as well as dosage,
will vary from individual to individual, and may be readily
established using standard techniques. In general, the
pharmaceutical compositions and vaccines may be administered by
injection (e.g., intracutaneous, intramuscular, intravenous or
subcutaneous), intranasally (e.g., by aspiration) or orally. In
some tumors, pharmaceutical compositions or vaccines may be
administered locally (by, for example, rectocoloscopy, gastroscopy,
videoendoscopy, angiography or other methods known in the art).
Preferably, between 1 and 10 doses may be administered over a 52
week period. Preferably, 6 doses are administered, at intervals of
1 month, and booster vaccinations may be given periodically
thereafter. Alternate protocols may be appropriate for individual
patients. A suitable dose is an amount of a compound that, when
administered as described above, is capable of promoting an
anti-tumor immune response that is at least 10-50% above the basal
(i.e., untreated) level. Such response can be monitored by
measuring the anti-tumor antibodies in a patient or by
vaccine-dependent generation of cytolytic effector cells capable of
killing the patient's tumor cells in vitro. Such vaccines should
also be capable of causing an immune response that leads to an
improved clinical outcome (e.g., more frequent complete or partial
remissions, or longer disease-free and/or overall survival) in
vaccinated patients as compared to non-vaccinated patients. In
general, for pharmaceutical compositions and vaccines comprising
one or more polypeptides, the amount of each polypeptide present in
a dose ranges from about 100 .mu.g to 5 mg. Suitable dose sizes
will vary with the size of the patient, but will typically range
from about 0.1 mL to about 5 mL.
[0117] In general, an appropriate dosage and treatment regimen
provides the active compound(s) in an amount sufficient to provide
therapeutic and/or prophylactic benefit. Such a response can be
monitored by establishing an improved clinical outcome (e.g., more
frequent complete or partial remissions, or longer disease-free
and/or overall survival) in treated patients as compared to
non-treated patients. Increases in preexisting immune responses to
WT1 generally correlate with an improved clinical outcome. Such
immune responses may generally be evaluated using standard
proliferation, cytotoxicity or cytokine assays, which may be
performed using samples obtained from a patient before and after
treatment.
[0118] Within further aspects, methods for inhibiting the
development of a malignant disease associated with WT1 expression
involve the administration of autologous T cells that have been
activated in response to a WT1 polypeptide or WT1-expressing APC,
as described above. Such T cells may be CD4.sup.+ and/or CD8.sup.+,
and may be proliferated as described above. The T cells may be
administered to the individual in an amount effective to inhibit
the development of a malignant disease. Typically, about
1.times.10.sup.9 to 1.times.10.sup.11 T cells/M.sup.2 are
administered intravenously, intracavitary or in the bed of a
resected tumor. It will be evident to those skilled in the art that
the number of cells and the frequency of administration will be
dependent upon the response of the patient.
[0119] Within certain embodiments, T cells may be stimulated prior
to an autologous bone marrow transplantation. Such stimulation may
take place in vivo or in vitro. For in vitro stimulation, bone
marrow and/or peripheral blood (or a fraction of bone marrow or
peripheral blood) obtained from a patient may be contacted with a
WT1 polypeptide, a polynucleotide encoding a WT1 polypeptide and/or
an APC that expresses a WT1 polypeptide under conditions and for a
time sufficient to permit the stimulation of T cells as described
above. Bone marrow, peripheral blood stem cells and/or WT1-specific
T cells may then be administered to a patient using standard
techniques.
[0120] Within related embodiments, T cells of a related or
unrelated donor may be stimulated prior to a syngeneic or
allogeneic (related or unrelated) bone marrow transplantation. Such
stimulation may take place in vivo or in vitro. For in vitro
stimulation, bone marrow and/or peripheral blood (or a fraction of
bone marrow or peripheral blood) obtained from a related or
unrelated donor may be contacted with a WT1 polypeptide, WT1
polynucleotide and/or APC that expresses a WT1 polypeptide under
conditions and for a time sufficient to permit the stimulation of T
cells as described above. Bone marrow, peripheral blood stem cells
and/or WT1-specific T cells may then be administered to a patient
using standard techniques.
[0121] Within other embodiments, WT1-specific T cells as described
herein may be used to remove cells expressing WT1 from autologous
bone marrow, peripheral blood or a fraction of bone marrow or
peripheral blood (e.g., CD34.sup.+ enriched peripheral blood (PB)
prior to administration to a patient). Such methods may be
performed by contacting bone marrow or PB with such T cells under
conditions and for a time sufficient to permit the reduction of WT1
expressing cells to less than 10%, preferably less than 5% and more
preferably less than 1%, of the total number of myeloid or
lymphatic cells in the bone marrow or peripheral blood. The extent
to which such cells have been removed may be readily determined by
standard methods such as, for example, qualitative and quantitative
PCR analysis, morphology, immunohistochemistry and FACS analysis.
Bone marrow or PB (or a fraction thereof) may then be administered
to a patient using standard techniques.
Diagnostic Methods
[0122] The present invention further provides methods for detecting
a malignant disease associated with WT1 expression, and for
monitoring the effectiveness of an immunization or therapy for such
a disease. Such methods are based on the discovery, within the
present invention, that an immune response specific for WT1 protein
can be detected in patients afflicted with such diseases, and that
methods which enhance such immune responses may provide a
preventive or therapeutic benefit.
[0123] To determine the presence or absence of a malignant disease
associated with WT1 expression, a patient may be tested for the
level of T cells specific for WT1. Within certain methods, a
biological sample comprising CD4.sup.+ and/or CD8.sup.+ T cells
isolated from a patient is incubated with a WT1 polypeptide, a
polynucleotide encoding a WT1 polypeptide and/or an APC that
expresses a WT1 polypeptide, and the presence or absence of
specific activation of the T cells is detected, as described
herein. Suitable biological samples include, but are not limited
to, isolated T cells. For example, T cells may be isolated from a
patient by routine techniques (such as by FICOLL.RTM./HYPAQUE.RTM.
density gradient centrifugation of peripheral blood lymphocytes). T
cells may be incubated in vitro for 2-9 days (typically 4 days) at
37.degree. C. with WT1 polypeptide (e.g., 5-25 .mu.g/ml). It may be
desirable to incubate another aliquot of a T cell sample in the
absence of WT1 polypeptide to serve as a control. For CD4' T cells,
activation is preferably detected by evaluating proliferation of
the T cells. For CD8' T cells, activation is preferably detected by
evaluating cytolytic activity. A level of proliferation that is at
least two fold greater and/or a level of cytolytic activity that is
at least 20% greater than in disease-free patients indicates the
presence of a malignant disease associated with WT1 expression.
Further correlation may be made, using methods well known in the
art, between the level of proliferation and/or cytolytic activity
and the predicted response to therapy. In particular, patients that
display a higher antibody, proliferative and/or lytic response may
be expected to show a greater response to therapy.
[0124] Within other methods, a biological sample obtained from a
patient is tested for the level of antibody specific for WT1. The
biological sample is incubated with a WT1 polypeptide, a
polynucleotide encoding a WT1 polypeptide and/or an APC that
expresses a WT1 polypeptide under conditions and for a time
sufficient to allow immunocomplexes to form. Immunocomplexes formed
between the WT1 polypeptide and antibodies in the biological sample
that specifically bind to the WT1 polypeptide are then detected. A
biological sample for use within such methods may be any sample
obtained from a patient that would be expected to contain
antibodies. Suitable biological samples include blood, sera,
ascites, bone marrow, pleural effusion, and cerebrospinal
fluid.
[0125] The biological sample is incubated with the WT1 polypeptide
in a reaction mixture under conditions and for a time sufficient to
permit immunocomplexes to form between the polypeptide and
antibodies specific for WT1. For example, a biological sample and
WT1 polypeptide may be incubated at 4.degree. C. for 24-48
hours.
[0126] Following the incubation, the reaction mixture is tested for
the presence of immunocomplexes. Detection of immunocomplexes
formed between the WT1 polypeptide and antibodies present in the
biological sample may be accomplished by a variety of known
techniques, such as radioimmunoassays (RIA) and enzyme linked
immunosorbent assays (ELISA). Suitable assays are well known in the
art and are amply described in the scientific and patent literature
(e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988). Assays that may be used include,
but are not limited to, the double monoclonal antibody sandwich
immunoassay technique of David et al. (U.S. Pat. No. 4,376,110);
monoclonal-polyclonal antibody sandwich assays (Wide et al., in
Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S.
Livingstone, Edinburgh, 1970); the "western blot" method of Gordon
et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled
ligand (Brown et al., J. Biol. Chem. 255:4980-4983, 1980);
enzyme-linked immunosorbent assays as described by, for example,
Raines and Ross (J. Biol. Chem. 257:5154-5160, 1982);
immunocytochemical techniques, including the use of fluorochromes
(Brooks et al., Clin. Exp. Immunol. 39: 477, 1980); and
neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.
Sci. USA 81:2396-2400, 1984). Other immunoassays include, but are
not limited to, those described in U.S. Pat. Nos. 3,817,827;
3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074;
and 4,098,876.
[0127] For detection purposes, WT1 polypeptide may either be
labeled or unlabeled. Unlabeled WT1 polypeptide may be used in
agglutination assays or in combination with labeled detection
reagents that bind to the immunocomplexes (e.g.,
anti-immunoglobulin, protein G, protein A or a lectin and secondary
antibodies, or antigen-binding fragments thereof, capable of
binding to the antibodies that specifically bind to the WT1
polypeptide). If the WT1 polypeptide is labeled, the reporter group
may be any suitable reporter group known in the art, including
radioisotopes, fluorescent groups, luminescent groups, enzymes,
biotin and dye particles.
[0128] Within certain assays, unlabeled WT1 polypeptide is
immobilized on a solid support. The solid support may be any
material known to those of ordinary skill in the art to which the
polypeptide may be attached. For example, the solid support may be
a test well in a microtiter plate or a nitrocellulose or other
suitable membrane. Alternatively, the support may be a bead or
disc, such as glass, fiberglass, latex or a plastic material such
as polystyrene or polyvinylchloride. The support may also be a
magnetic particle or a fiber optic sensor, such as those disclosed,
for example, in U.S. Pat. No. 5,359,681. The polypeptide may be
immobilized on the solid support using a variety of techniques
known to those of skill in the art, which are amply described in
the patent and scientific literature. In the context of the present
invention, the term "immobilization" refers to both noncovalent
association, such as adsorption, and covalent attachment (which may
be a direct linkage between the antigen and functional groups on
the support or may be a linkage by way of a cross-linking agent).
Immobilization by adsorption to a well in a microtiter plate or to
a membrane is preferred. In such cases, adsorption may be achieved
by contacting the WT1 polypeptide, in a suitable buffer, with the
solid support for a suitable amount of time. The contact time
varies with temperature, but is typically between about 1 hour and
about 1 day. In general, contacting a well of a plastic microtiter
plate (such as polystyrene or polyvinylchloride) with an amount of
polypeptide ranging from about 10 ng to about 10 .mu.g, and
preferably about 100 ng to about 1 .mu.g, is sufficient to
immobilize an adequate amount of polypeptide.
[0129] Following immobilization, the remaining protein binding
sites on the support are typically blocked. Any suitable blocking
agent known to those of ordinary skill in the art, such as bovine
serum albumin, TWEEN.RTM. 20.TM. (Sigma Chemical Co., St. Louis,
Mo.), heat-inactivated normal goat serum (NGS), or BLOTTO (buffered
solution of nonfat dry milk which also contains a preservative,
salts, and an antifoaming agent). The support is then incubated
with a biological sample suspected of containing specific antibody.
The sample can be applied neat, or, more often, it can be diluted,
usually in a buffered solution which contains a small amount
(0.1%-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. In
general, an appropriate contact time (i.e., incubation time) is a
period of time that is sufficient to detect the presence of
antibody that specifically binds WT1 within a sample containing
such an antibody. Preferably, the contact time is sufficient to
achieve a level of binding that is at least about 95% of that
achieved at equilibrium between bound and unbound antibody. Those
of ordinary skill in the art will recognize that the time necessary
to achieve equilibrium may be readily determined by assaying the
level of binding that occurs over a period of time. At room
temperature, an incubation time of about 30 minutes is generally
sufficient.
[0130] Unbound sample may then be removed by washing the solid
support with an appropriate buffer, such as PBS containing 0.1%
TWEEN.RTM. 20.TM.. A detection reagent that binds to the
immunocomplexes and that comprises a reporter group may then be
added. The detection reagent is incubated with the immunocomplex
for an amount of time sufficient to detect the bound antibody. An
appropriate amount of time may generally be determined by assaying
the level of binding that occurs over a period of time. Unbound
detection reagent is then removed and bound detection reagent is
detected using the reporter group. The method employed for
detecting the reporter group depends upon the nature of the
reporter group. For radioactive groups, scintillation counting or
autoradiographic methods are generally appropriate. Spectroscopic
methods may be used to detect dyes, luminescent groups and
fluorescent groups. Biotin may be detected using avidin, coupled to
a different reporter group (commonly a radioactive or fluorescent
group or an enzyme). Enzyme reporter groups (e.g., horseradish
peroxidase, beta-galactosidase, alkaline phosphatase and glucose
oxidase) may generally be detected by the addition of substrate
(generally for a specific period of time), followed by
spectroscopic or other analysis of the reaction products.
Regardless of the specific method employed, a level of bound
detection reagent that is at least two fold greater than background
(i.e., the level observed for a biological sample obtained from a
disease-free individual) indicates the presence of a malignant
disease associated with WT1 expression.
[0131] In general, methods for monitoring the effectiveness of an
immunization or therapy involve monitoring changes in the level of
antibodies or T cells specific for WT1 in the patient. Methods in
which antibody levels are monitored may comprise the steps of: (a)
incubating a first biological sample, obtained from a patient prior
to a therapy or immunization, with a WT1 polypeptide, wherein the
incubation is performed under conditions and for a time sufficient
to allow immunocomplexes to form; (b) detecting immunocomplexes
formed between the WT1 polypeptide and antibodies in the biological
sample that specifically bind to the WT1 polypeptide; (c) repeating
steps (a) and (b) using a second biological sample taken from the
patient following therapy or immunization; and (d) comparing the
number of immunocomplexes detected in the first and second
biological samples. Alternatively, a polynucleotide encoding a WT1
polypeptide, or an APC expressing a WT1 polypeptide may be employed
in place of the WT1 polypeptide. Within such methods,
immunocomplexes between the WT1 polypeptide encoded by the
polynucleotide, or expressed by the APC, and antibodies in the
biological sample are detected.
[0132] Methods in which T cell activation and/or the number of WT1
specific precursors are monitored may comprise the steps of: (a)
incubating a first biological sample comprising CD4+ and/or CD8+
cells (e.g., bone marrow, peripheral blood or a fraction thereof),
obtained from a patient prior to a therapy or immunization, with a
WT1 polypeptide, wherein the incubation is performed under
conditions and for a time sufficient to allow specific activation,
proliferation and/or lysis of T cells; (b) detecting an amount of
activation, proliferation and/or lysis of the T cells; (c)
repeating steps (a) and (b) using a second biological sample
comprising CD4+ and/or CD8+ T cells, and taken from the same
patient following therapy or immunization; and (d) comparing the
amount of activation, proliferation and/or lysis of T cells in the
first and second biological samples. Alternatively, a
polynucleotide encoding a WT1 polypeptide, or an APC expressing a
WT1 polypeptide may be employed in place of the WT1
polypeptide.
[0133] A biological sample for use within such methods may be any
sample obtained from a patient that would be expected to contain
antibodies, CD4+ T cells and/or CD8+ T cells. Suitable biological
samples include blood, sera, ascites, bone marrow, pleural effusion
and cerebrospinal fluid. A first biological sample may be obtained
prior to initiation of therapy or immunization or part way through
a therapy or vaccination regime. The second biological sample
should be obtained in a similar manner, but at a time following
additional therapy or immunization. The second biological sample
may be obtained at the completion of, or part way through, therapy
or immunization, provided that at least a portion of therapy or
immunization takes place between the isolation of the first and
second biological samples.
[0134] Incubation and detection steps for both samples may
generally be performed as described above. A statistically
significant increase in the number of immunocomplexes in the second
sample relative to the first sample reflects successful therapy or
immunization.
[0135] The following Examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Identification of an Immune Response to WT1 in Patients with
Hematological Malignancies
[0136] This Example illustrates the identification of an existent
immune response in patients with a hematological malignancy.
[0137] To evaluate the presence of preexisting WT1 specific
antibody responses in patients, sera of patients with acute
myelogenous leukemia (AML), acute lymphocytic leukemia (ALL),
chronic myelogenous leukemia (CML) and severe aplastic anemia were
analyzed using Western blot analysis. Sera were tested for the
ability to immunoprecipitate WT1 from the human leukemic cell line
K562 (American Type Culture Collection, Manassas, Va.). In each
case, immunoprecipitates were separated by gel electrophoresis,
transferred to membrane and probed with the anti WT-1 antibody
WT180 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). This
Western blot analysis identified potential WT1 specific antibodies
in patients with hematological malignancy. A representative Western
blot showing the results for a patient with AML is shown in FIG. 2.
A 52 kD protein in the immunoprecipitate generated using the
patient sera was recognized by the WT1 specific antibody. The 52 kD
protein migrated at the same size as the positive control.
[0138] Additional studies analyzed the sera of patients with AML
and CML for the presence of antibodies to full-length and truncated
WT1 proteins. cDNA constructs representing the human
WT1/full-length (aa 1-449), the N-terminus (aa 1-249)
(WT1/N-terminus) and C-terminus (aa 267-449) (WT1/C-terminus)
region were subcloned into modified pET28 vectors. The
WT1/full-length and WT1/N-terminus proteins were expressed as Ra12
fusion proteins. Ra12 is the C-terminal fragment of a secreted
Mycobacterium tuberculosis protein, denoted as MTB32B. (Skeiky et
al., Infect Immun. 67; 3998, 1999). The Ra12-WT1/full-length fusion
region was cloned 3' to a histidine-tag in a histidine-tag modified
pET28 vector. The WT1/N-terminus region was subcloned into a
modified pET28 vector that has a 5' histidine-tag followed by the
thioredoxin (TRX)-WT1/N-terminus fusion region followed by a 3'
histidine-tag. The WT1/C-terminus coding region was subcloned into
a modified pET28 vector without a fusion partner containing only
the 5' and 3' histidine-tag, followed by a Thrombin and EK
site.
[0139] BL21 pLysS E. coli (Stratagene, La Jolla, Calif.) were
transformed with the three WT1 expression constructs, grown
overnight and induced with isopropyl-.beta.-D-thiogalactoside
(IPTG). WT1 proteins were purified as follows: Cells were harvested
and lysed by incubation in 10 mM Tris, pH 8.0 with Complete
Protease Inhibitor Tablets (Boehringer Mannheim Biochemicals,
Indianapolis, Ind.) at 37.degree. C. followed by repeated rounds of
sonication. Inclusion bodies were washed twice with 10 mM Tris, pH
8.0. Proteins were then purified by metal chelate affinity
chromatography over nickel-nitrilotriacetic acid resin (QIAGEN
Inc., Valencia, Calif.; Hochuli et al., Biologically Active
Molecules: 217, 1989) followed by chromatography on a Source Q
anion exchange resin (Amersham Pharmacia Biotech, Upsala, Sweden).
The identity of the WT1 proteins was confirmed by N-terminal
sequencing.
[0140] Sera from adult patients with de nova AML or CML were
studied for the presence of WT1 specific Ab. Recombinant proteins
were adsorbed to TC microwell plates (Nunc, Roskilde, Denmark).
Plates were washed with PBS/0.5% TWEEN.RTM. 20 and blocked with 1%
BSA/PBS/0.1% TWEEN.RTM. 20. After washing, serum dilutions were
added and incubated overnight at 4.degree. C. Plates were washed
and Donkey anti-human IgG-HRP secondary antibody was added
(Jackson-Immunochem, West Grove, Pa.) and incubated for 2 h at room
temperature. Plates were washed, incubated with TMB Peroxidase
substrate solution (Kirkegaard and Perry Laboratories, MA),
quenched with 1N H.sub.2SO.sub.4, and immediately read (Cyto-Fluor
2350; Millipore, Bedford, Mass.).
[0141] For the serological survey, human sera were tested by ELISA
over a range of serial dilutions from 1:50 to 1:20,000. A positive
reaction was defined as an OD value of a 1:500 diluted serum that
exceeded the mean OD value of sera from normal donors (n=96) by
three (WT1/full-length, WT1C-terminus) standard deviations. Due to
a higher background in normal donors to the WT1/N-terminus protein
a positive reaction to WT1/N-terminus was defined as an OD value of
1:500 diluted serum that exceeded the mean OD value of sera from
normal donors by four standard deviations. To verify that the
patient Ab response was directed against WT1 and not to the Ra12 or
TRX fusion part of the protein or possible E. coli contaminant
proteins, controls included the Ra12 and TRX protein alone purified
in a similar manner. Samples that showed reactivity against the
Ra12 and/or TRX proteins were excluded from the analysis.
[0142] To evaluate for the presence of immunity to WT1, Ab to
recombinant full-length and truncated WT1 proteins in the sera of
normal individuals and patients with leukemia were determined.
Antibody reactivity was analyzed by ELISA reactivity to
WT1/full-length protein, WT1/N-terminus protein and WT1/C-terminus
protein.
[0143] Only 2 of 96 normal donors had serum antibodies reactive
with WT1/full-length protein (FIG. 18). One of those individuals
had antibody to WT1/N-terminus protein and one had antibody to
WT1/C-terminus protein. In contrast, 16 of 63 patients (25%) with
AML had serum antibodies reactive with WT1/full-length protein. By
marked contrast, only 2 of 63 patients (3%) had reactivity to
WT1/C-terminus protein. Fifteen of 81 patients (19%) with CML had
serum antibodies reactive with WT1/full-length protein and 12 of 81
patients (15%) had serum antibodies reactive with WT1/N-terminus.
Only 3 of 81 patients (3%) had reactivity to WT1/C-terminus
protein. (FIGS. 16 and 17.)
[0144] These data demonstrate that Ab responses to WT1 are
detectable in some patients with AML and CML. The greater incidence
of antibody in leukemia patients provides strong evidence that
immunization to the WT1 protein occurred as a result of patients
bearing malignancy that expresses or at some time expressed WT1.
Without being limited to a specific theory, it is believed that the
observed antibody responses to WT1 most probably result from
patients becoming immune to WT1 on their own leukemia cells and
provide direct evidence that WT1 can be immunogenic despite being a
"self" protein.
[0145] The presence of antibody to WT1 strongly implies that
concurrent helper T cell responses are also present in the same
patients. WT1 is an internal protein. Thus, CTL responses are
likely to be the most effective in terms of leukemia therapy and
the most toxic arm of immunity. Thus, these data provide evidence
that therapeutic vaccines directed against WT1 will be able to
elicit an immune response to WT1.
[0146] The majority of the antibodies detected were reactive with
epitopes within the N-terminus while only a small subgroup of
patients showed a weak antibody response to the C-terminus. This is
consistent with observations in the animal model, where
immunization with peptides derived from the N-terminus elicited
antibody, helper T cell and CTL responses, whereas none of the
peptides tested from the C-terminus elicited antibody or T cell
responses (Gaiger et al., Blood 96:1334, 2000).
Example 2
Induction of Antibodies to WT1 in Mice Immunized with Cell Lines
Expressing WT1
[0147] This Example illustrates the use of cells expressing WT1 to
induce a WT1 specific antibody response in vivo.
[0148] Detection of existent antibodies to WT1 in patients with
leukemia strongly implied that it is possible to immunize to WT1
protein to elicit immunity to WT1. To test whether immunity to WT1
can be generated by vaccination, mice were injected with TRAMP-C, a
WT1 positive tumor cell line of B6 origin. Briefly, male B6 mice
were immunized with 5.times.10.sup.6 TRAMP-C cells subcutaneously
and boosted twice with 5.times.10.sup.6 cells at three week
intervals. Three weeks after the final immunization, sera were
obtained and single cell suspensions of spleens were prepared in
RPMI 1640 medium (GIBCO.RTM.) with 25 .mu.M
.beta.-2-mercaptoethanol, 200 units of penicillin per ml, 10 mM
L-glutamine, and 10% fetal bovine serum.
[0149] Following immunization to TRAMP-C, a WT1 specific antibody
response in the immunized animals was detectable. A representative
Western blot is shown in FIG. 3. These results show that
immunization to WT1 protein can elicit an immune response to WT1
protein.
Example 3
Induction of Th and Antibody Responses in Mice Immunized with WT1
Peptides
[0150] This Example illustrates the ability of immunization with
WT1 peptides to elicit an immune response specific for WT1.
[0151] Peptides suitable for eliciting Ab and proliferative T cell
responses were identified according to the Tsites program (Rothbard
and Taylor, EMBO J. 7:93-100, 1988; Deavin et al., Mol. Immunol.
33:145-155, 1996), which searches for peptide motifs that have the
potential to elicit Th responses. Peptides shown in Table I were
synthesized and sequenced.
TABLE-US-00001 TABLE I WT1 Peptides Peptide Sequence Comments
Mouse: p6-22 RDLNALLPAVSSLGGGG 1 mismatch relative to (SEQ ID NO:
13) human WT1 sequence Human: p6-22 RDLNALLPAVPSLGGGG (SEQ ID NO:
1) Human/mouse: PSQASSGQARMFPNAPYLPSCLE p117-139 (SEQ ID NOs: 2 and
3) Mouse: p244-262 GATLKGMAAGSSSSVKWTE 1 mismatch relative to (SEQ
ID NO: 14) human WT1 sequence Human: p244-262 GATLKGVAAGSSSSVKWTE
(SEQ ID NO: 4) Human/mouse: RIHTHGVFRGIQDVR p287-301 (SEQ ID NOs:
15 and 16) Mouse: p299-313 VRRVSGVAPTLVRS 1 mismatch relative to
(SEQ ID NO: 17) human WT1 sequence Human/mouse: CQKKFARSDELVRHH
p421-435 (SEQ ID NOs: 19 and 20)
[0152] For immunization, peptides were grouped as follows: [0153]
Group A: p6-22 human: 10.9 mg in 1 ml (10 .mu.l=100 .mu.g) [0154]
p117-139 human/mouse: 7.6 mg in 1 ml (14 .mu.l=100 .mu.g) [0155]
p244-262 human: 4.6 mg in 1 ml (22 .mu.l=100 .mu.g) [0156] Group B:
p287-301 human/mouse: 7.2 mg in 1 ml (14 .mu.l=100 .mu.g) [0157]
mouse p299-313: 6.6 mg in 1 ml (15 .mu.l=100 .mu.g) [0158] p421-435
human/mouse: 3.3 mg in 1 ml (30 .mu.l=100 .mu.g) [0159] Control:
(FBL peptide 100 .mu.g)+CFA/IFA [0160] Control: (CD45 peptide 100
.mu.g)+CFA/IFA
[0161] Group A contained peptides present within the amino terminus
portion of WT1 (exon 1) and Group B contained peptides present
within the carboxy terminus, which contains a four zinc finger
region with sequence homology to other DNA-binding proteins. Within
group B, p287-301 and p299-313 were derived from exon 7, zinc
finger 1, and p421-435 was derived from exon 10, zinc finger
IV.
[0162] B6 mice were immunized with a group of WT1 peptides or with
a control peptide. Peptides were dissolved in 1 ml sterile water
for injection, and B6 mice were immunized 3 times at time intervals
of three weeks. Adjuvants used were CFA/IFA, GM-CSF, and
MONTANIDE.RTM.. The presence of antibodies specific for WT1 was
then determined as described in Examples 1 and 2, and proliferative
T cell responses were evaluated using a standard thymidine
incorporation assay, in which cells were cultured in the presence
of antigen and proliferation was evaluated by measuring
incorporated radioactivity (Chen et al., Cancer Res. 54:1065-1070,
1994). In particular, lymphocytes were cultured in 96-well plates
at 2.times.10.sup.5 cells per well with 4.times.10.sup.5 irradiated
(3000 rads) syngeneic spleen cells and the designated peptide.
[0163] Immunization of mice with the group of peptides designated
as Group A elicited an antibody response to WT1 (FIG. 4). No
antibodies were detected following immunization to Vaccine B, which
is consistent with a lack of helper T cell response from
immunization with Vaccine B. P117-139 elicited proliferative T cell
responses (FIGS. 5A-5C). The stimulation indices (SI) varied
between 8 and 72. Other peptides (P6-22 and P299-313) also were
shown to elicit proliferative T cell responses. Immunization with
P6-22 resulted in a stimulation index (SI) of 2.3 and immunization
with P299-313 resulted in a SI of 3.3. Positive controls included
ConA stimulated T cells, as well as T cells stimulated with known
antigens, such as CD45 and FBL, and allogeneic T cell lines
(DeBruijn et al., Eur. J. Immunol. 21:2963-2970, 1991).
[0164] FIGS. 6A and 6B show the proliferative response observed for
each of the three peptides within vaccine A (FIG. 6A) and vaccine B
(FIG. 6B). Vaccine A elicited proliferative T cell responses to the
immunizing peptides p6-22 and p117-139, with stimulation indices
(SI) varying between 3 and 8 (bulk lines). No proliferative
response to p244-262 was detected (FIG. 6A).
[0165] Subsequent in vitro stimulations were carried out as single
peptide stimulations using only p6-22 and p117-139. Stimulation of
the Vaccine A specific T cell line with p117-139 resulted in
proliferation to p117-139 with no response to p6-22 (FIG. 7A).
Clones derived from the line were specific for p117-139 (FIG. 7B).
By contrast, stimulation of the Vaccine A specific T cell line with
p6-22 resulted in proliferation to p6-22 with no response to
p117-139 (FIG. 7C). Clones derived from the line were specific for
p6-22 (FIG. 7D).
[0166] These results show that vaccination with WT1 peptides can
elicit antibody responses to WT1 protein and proliferative T cell
responses to the immunizing peptides.
Example 4
Induction of CTL Responses in Mice Immunized with WT1 Peptides
[0167] This Example illustrates the ability of WT1 peptides to
elicit CTL immunity.
[0168] Peptides (9-mers) with motifs appropriate for binding to
class I MHC were identified using a BIMAS HLA peptide binding
prediction analysis (Parker et al., J. Immunol. 152:163, 1994).
Peptides identified within such analyses are shown in Tables
II-XLIV. In each of these tables, the score reflects the
theoretical binding affinity (half-time of dissociation) of the
peptide to the MHC molecule indicated.
[0169] Peptides identified using the Tsites program (Rothbard and
Taylor, EMBO J. 7:93-100, 1988; Deavin et al., Mol. Immunol.
33:145-155, 1996), which searches for peptide motifs that have the
potential to elicit Th responses are further shown in FIGS. 8A and
8B, and Table XLV.
TABLE-US-00002 TABLE II Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A1 Subsequence Score (Estimate of Half Time of Start Residue
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) 1 137 CLESQPAIR 18.000 (SEQ ID NO: 47) 2 80 GAEPHEEQC
9.000 (SEQ ID NO: 87) 3 40 FAPPGASAY 5.000 (SEQ ID NO: 74) 4 354
QCDFKDCER 5.000 (SEQ ID NO: 162) 5 2 GSDVRDLNA 3.750 (SEQ ID NO:
101) 6 152 VTFDGTPSY 2.500 (SEQ ID NO: 244) 7 260 WTEGQSNHS 2.250
(SEQ ID NO: 247) 8 409 TSEKPFSCR 1.350 (SEQ ID NO: 232) 9 73
KQEPSWGGA 1.350 (SEQ ID NO: 125) 10 386 KTCQRKFSR 1.250 (SEQ ID NO:
128) 11 37 VLDFAPPGA 1.000 (SEQ ID NO: 241) 12 325 CAYPGCNKR 1.000
(SEQ ID NO: 44) 13 232 QLECMTWNQ 0.900 (SEQ ID NO: 167) 14 272
ESDNHTTPI 0.750 (SEQ ID NO: 71) 15 366 RSDQLKRHQ 0.750 (SEQ ID NO:
193) 16 222 SSDNLYQMT 0.750 (SEQ ID NO: 217) 17 427 RSDELVRHH 0.750
(SEQ ID NO: 191) 18 394 RSDHLKTHT 0.750 (SEQ ID NO: 192) 19 317
TSEKRPFMC 0.675 (SEQ ID NO: 233) 20 213 QALLLRTPY 0.500 (SEQ ID NO:
160)
TABLE-US-00003 TABLE III Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A 0201 Subsequence Score (Estimate of Half Time of Start Residue
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) 1 126 RMFPNAPYL 313.968 (SEQ ID NO: 185) 2 187
SLGEQQYSV 285.163 (SEQ ID NO: 214) 3 10 ALLPAVPSL 181.794 (SEQ ID
NO: 34) 4 242 NLGATLKGV 159.970 (SEQ ID NO: 146) 5 225 NLYQMTSQL
68.360 (SEQ ID NO: 147) 6 292 GVFRGIQDV 51.790 (SEQ ID NO: 103) 7
191 QQYSVPPPV 22.566 (SEQ ID NO: 171) 8 280 ILCGAQYRI 17.736 (SEQ
ID NO: 116) 9 235 CMTWNQMNL 15.428 (SEQ ID NO: 49) 10 441 NMTKLQLAL
15.428 (SEQ ID NO: 149) 11 7 DLNALLPAV 11.998 (SEQ ID NO: 58) 12
227 YQMTSQLEC 8.573 (SEQ ID NO: 251) 13 239 NQMNLGATL 8.014 (SEQ ID
NO: 151) 14 309 TLVRSASET 7.452 (SEQ ID NO :226) 15 408 KTSEKPFSC
5.743 (SEQ ID NO: 129) 16 340 LQMHSRKHT 4.752 (SEQ ID NO: 139) 17
228 QMTSQLECM 4.044 (SEQ ID NO: 169) 18 93 TVHFSGQFT 3.586 (SEQ ID
NO: 235) 19 37 VLDFAPPGA 3.378 (SEQ ID NO: 241) 20 86 EQCLSAFTV
3.068 (SEQ ID NO: 69)
TABLE-US-00004 TABLE IV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A 0205 Subsequence Score (Estimate of Half Time of Start Residue
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) 1 10 ALLPAVPSL 42.000 (SEQ ID NO: 34) 2 292 GVFRGIQDV
24.000 (SEQ ID NO: 103) 3 126 RMFPNAPYL 21.000 (SEQ ID NO: 185) 4
225 NLYQMTSQL 21.000 (SEQ ID NO: 147) 5 239 NQMNLGATL 16.800 (SEQ
ID NO: 151) 6 302 RVPGVAPTL 14.000 (SEQ ID NO: 195) 7 441 NMTKLQLAL
7.000 (SEQ ID NO: 149) 8 235 CMTWNQMNL 7.000 (SEQ ID NO:49) 9 187
SLGEQQYSV 6.000 (SEQ ID NO: 214) 10 191 QQYSVPPPV 4.800 (SEQ ID NO:
171) 11 340 LQMHSRKHT 4.080 (SEQ ID NO: 139) 12 242 NLGATLKGV 4.000
(SEQ ID NO: 146) 13 227 YQMTSQLEC 3.600 (SEQ ID NO: 251) 14 194
SVPPPVYGC 2.000 (SEQ ID NO: 218) 15 93 TVHFSGQFT 2.000 (SEQ ID NO:
235) 16 280 ILCGAQYRI 1.700 (SEQ ID NO: 116) 17 98 GQFTGTAGA 1.200
(SEQ ID NO: 99) 18 309 ILYRSASET 1.000 (SEQ ID NO: 226) 19 81
AEPHEEQCL 0.980 (SEQ ID NO: 30) 20 73 KQEPSWGGA 0.960 (SEQ ID NO:
125)
TABLE-US-00005 TABLE V Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A24 Subsequence Score (Estimate of Half Time of Start Residue
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) 1 302 RVPGVAPTL 16.800 (SEQ ID NO: 195) 2 218
RTPYSSDNL 12.000 (SEQ ID NO: 194) 3 356 DFKDCERRF 12.000 (SEQ ID
NO:55) 4 126 RMFPNAPYL 9.600 (SEQ ID NO: 185) 5 326 AYPGCNKRY 7.500
(SEQ ID NO: 42) 6 270 GYESDNHTT 7.500 (SEQ ID NO: 106) 7 239
NQMNLGATL 7.200 (SEQ ID NO: 151) 8 10 ALLPAVPSL 7.200 (SEQ ID NO:
34) 9 130 NAPYLPSCL 7.200 (SEQ ID NO: 144) 10 329 GCNKRYFKL 6.600
(SEQ ID NO: 90) 11 417 RWPSCQKKF 6.600 (SEQ ID NO: 196) 12 47
AYGSLGGPA 6.000 (SEQ ID NO: 41) 13 180 DPMGQQGSL 6.000 (SEQ ID NO:
59) 14 4 DVRDLNALL 5.760 (SEQ ID NO: 62) 15 285 QYRIHTHGV 5.000
(SEQ ID NO: 175) 16 192 QYSVPPPVY 5.000 (SEQ ID NO: 176) 17 207
DSCTGSQAL 4.800 (SEQ ID NO: 61) 18 441 NMTKLQLAL 4.800 (SEQ ID NO:
149) 19 225 NLYQMTSQL 4.000 (SEQ ID NO: 147) 20 235 CMTWNQMNL 4.000
(SEQ ID NO: 49)
TABLE-US-00006 TABLE VI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WI1 Peptides to Human HLA
A3 Subsequence Score (Estimate of Half Time of Start Residue
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) 1 436 NMHQRNMTK 40.000 (SEQ ID NO: 148) 2 240
QMNLGATLK 20.000 (SEQ ID NO: 168) 3 88 CLSAFTVHF 6.000 (SEQ ID NO:
48) 4 126 RMFPNAPYL 4.500 (SEQ ID NO: 185) 5 169 AQFPNHSFK 4.500
(SEQ ID NO: 36) 6 10 ALLPAVPSL 4.050 (SEQ ID NO: 34) 7 137
CLESQPAIR 4.000 (SEQ ID NO: 47) 8 225 NLYQMTSQL 3.000 (SEQ ID NO:
147) 9 32 AQWAPVLDF 2.700 (SEQ ID NO: 37) 10 280 ILCGAQYRI 2.700
(SEQ ID NO: 116) 11 386 KTCQRKFSR 1.800 (SEQ ID NO: 128) 12 235
CMTWNQMNL 1.200 (SEQ ID NO: 49) 13 441 NMTKLQLAL 1.200 (SEQ ID NO:
149) 14 152 VTFDGTPSY 1.000 (SEQ ID NO: 244) 15 187 SLGEQQYSV 0.900
(SEQ ID NO: 214) 16 383 FQCKTCQRK 0.600 (SEQ ID NO: 80) 17 292
GVFRGIQDV 0.450 (SEQ ID NO: 103) 18 194 SVPPPVYGC 0.405 (SEQ ID NO:
218) 19 287 RIHTHGVFR 0.400 (SEQ ID NO: 182) 20 263 GQSNHSTGY 0.360
(SEQ ID NO: 100)
TABLE-US-00007 TABLE VII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A68.1 Subsequence Score (Estimate of Half Time of Start Residue
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) 1 100 FTGTAGACR 100.000 (SSEQ ID NO: 84) 2 386
KTCQRKFSR 50.000 (SEQ ID NO: 128) 3 368 DQLKRHQRR 30.000 (SEQ ID
NO: 60) 4 312 RSASETSEK 18.000 (SEQ ID NO: 190) 5 337 LSHLQMHSR
15.000 (SEQ ID NO: 141) 6 364 FSRSDQLKR 15.000 (SEQ ID NO :83) 7
409 TSEKPFSCR 15.000 (SEQ ID NO: 232) 8 299 DVRRVPGVA 12.000 (SEQ
ID NO: 63) 9 4 DVRDLNALL 12.000 (SEQ ID NO:62) 10 118 SQASSGQAR
10.000 (SEQ ID NO:216) 11 343 HSRKHTGEK 9.000 (SEQ ID NO: 111) 12
169 AQFPNHSFK 9.000 (SEQ ID NO: 36) 13 292 GVFRGIQDV 8.000 (SEQ ID
NO: 103) 14 325 CAYPGCNKIR 7.500 (SEQ ID NO: 44) 15 425 FARSDELVR
7.500 (SEQ ID NO: 75) 16 354 QCDFKDCER 7.500 (SEQ ID NO: 162) 17
324 MCAYPGCNK 6.000 (SEQ ID NO: 142) 18 251 AAGSSSSVK 6.000 (SEQ ID
NO: 28) 19 379 GVKPFQCKT 6.000 (SEQ ID NO: 104) 20 137 CLESQPAIR
5.000 (SEQ ID NO: 47)
TABLE-US-00008 TABLE VIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A 1101 Score (Estimate of Half Time of Disas- Subsequence sociation
of a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 386 KTCQRKFSR 1.800 (SEQ ID NO: 128) 2 169 AQFPNHSFK
1.200 (SEQ ID NO: 36) 3 436 NMHQRNMTK 0.800 (SEQ ID NO: 148) 4 391
KFSRSDHLK 0.600 (SEQ ID NO: 120) 5 373 HQRRHTGVK 0.600 (SEQ ID NO:
109) 6 383 FQCKTCQRK 0.600 (SEQ ID NO: 80) 7 363 RFSRSDQLK 0.600
(SEQ ID NO: 178) 8 240 QMNLGATLK 0.400 (SEQ ID NO: 168) 9 287
RIHTHGVFR 0.240 (SEQ ID NO: 182) 10 100 FTGTAGACR 0.200 (SEQ ID NO:
84) 11 324 MCAYPGCNK 0.200 (SEQ ID NO: 142) 12 251 AAGSSSSVK 0.200
(SEQ ID NO: 28) 13 415 SCRWPSCQK 0.200 (SEQ ID NO: 201) 14 118
SQASSGQAR 0.120 (SEQ ID NO: 216) 15 292 GVFRGIQDV 0.120 (SEQ ID NO:
103) 16 137 CLESQPAIR 0.080 (SEQ ID NO: 47) 17 425 FARSDELVR 0.080
(SEQ ID NO: 75) 18 325 CAYPGCNKR 0.080 (SEQ ID NO: 44) 19 312
RSASETSEK 0.060 (SEQ ID NO: 190) 20 65 PPPPHSFI 0.060 (SEQ ID NO:
156) K
TABLE-US-00009 TABLE IX Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A 3101 Score (Estimate of Half Time of Disas- Subsequence sociation
of a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 386 KTCQRKFSR 9.000 (SEQ ID NO: 128) 2 287 RIHTHGVFR
6.000 (SEQ ID NO: 182) 3 137 CLESQPAIR 2.000 (SEQ ID NO: 47) 4 118
SQASSGQAR 2.000 (SEQ ID NO: 216) 5 368 DQLKRHQRR 1.200 (SEQ ID NO:
60) 6 100 FTGTAGACR 1.000 (SEQ ID NO: 84) 7 293 VFRGIQDVR 0.600
(SEQ ID NO: 238) 8 325 CAYPGCNKR 0.600 (SEQ ID NO: 44) 9 169
AQFPNHSFK 0.600 (SEQ ID NO: 36) 10 279 PILCGAQYR 0.400 (SEQ ID NO:
155) 11 436 NMHQRNMTK 0.400 (SEQ ID NO: 148) 12 425 FARSDELVR 0.400
(SEQ ID NO: 75) 13 32 AQWAPVLDF 0.240 (SEQ ID NO: 37) 14 240
QMNLGATLK 0.200 (SEQ ID NO: 168) 15 354 QCDFKDCER 0.200 (SEQ ID NO:
162) 16 373 HQRRHTGVK 0.200 (SEQ ID NO: 109) 17 383 FQCKTCQRK 0.200
(SEQ ID NO: 80) 18 313 SASETSEKR 0.200 (SEQ ID NO: 197) 19 358
KDCERRFSR 0.180 (SEQ ID NO: 118) 20 391 KFSRSDHLK 0.180 (SEQ ID NO:
120)
TABLE-US-00010 TABLE X Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
A 3302 Score (Estimate of Half Time of Disas- Subsequence sociation
of a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 337 LSHLQMHSR 15.000 (SEQ ID NO: 141) 2 409
TSEKPFSCR 15.000 (SEQ ID NO: 232) 3 364 FSRSDQLKR 15.000 (SEQ ID
NO: 83) 4 137 CLESQPAIR 9.000 (SEQ ID NO: 47) 5 368 DQLKIRHQRR
9.000 (SEQ ID NO: 60) 6 287 RIHTHGVFR 4.500 (SEQ ID NO: 182) 7 210
TGSQALLLR 3.000 (SEQ ID NO: 223) 8 425 FARSDELVR 3.000 (SEQ ID NO:
75) 9 313 SASETSEKR 3.000 (SEQ ID NO: 197) 10 293 VFRGIQDVR 3.000
(SEQ ID NO: 238) 11 354 QCDFKDCER 3.000 (SEQ ID NO: 162) 12 100
FTGTAGACR 3.000 (SEQ ID NO: 84) 13 118 SQASSGQAR 3.000 (SEQ ID NO:
216) 14 325 CAYPGCNKR 3.000 (SEQ ID NO: 44) 15 207 DSCTGSQAL 1.500
(SEQ ID NO: 61) 16 139 ESQPAIRNQ 1.500 (SEQ ID NO: 72) 17 299
DVRRVPGVA 1.500 (SEQ ID NO: 63) 18 419 PSCQKKFAR 1.500 (SEQ ID NO:
159) 19 272 ESDNHTTPI 1.500 (SEQ ID NO: 71) 20 4 DVRDLNALL 1.500
(SEQ ID NO: 62)
TABLE-US-00011 TABLE XI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B14 Score (Estimate of Half Time of Disas- Subsequence sociation of
a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 362 RRFSRSDQL 1000.000 (SEQ ID NO: 187) 2 332
KRYFKLSHL 300.000 (SEQ ID NO: 127) 3 423 KKFARSDEL 150.000 (SEQ ID
NO: 122) 4 390 RKFSRSDHL 150.000 (SEQ ID NO: 183) 5 439 QRNMTKLQL
20.000 (SEQ ID NO: 173) 6 329 GCNKRYFKL 10.000 (SEQ ID NO: 90) 7 10
ALLPAVPSL 10.000 (SEQ ID NO: 34) 8 180 DPMGQQGSL 9.000 (SEQ ID NO:
59) 9 301 RRVPGVAPT 6.000 (SEQ ID NO: 189) 10 126 RMFPNAPYL 5.000
(SEQ ID NO: 185) 11 371 KRHQRRHTG 5.000 (SEQ ID NO: 126) 12 225
NLYQMTSQL 5.000 (SEQ ID NO: 147) 13 144 IRNQGYSTV 4.000 (SEQ ID NO:
117) 14 429 DELVRHHNM 3.000 (SEQ ID NO: 53) 15 437 MHQRNMTKL 3.000
(SEQ ID NO: 143) 16 125 ARMFPNAPY 3.000 (SEQ ID NO: 38) 17 239
NQMNLGATL 3.000 (SEQ ID NO: 151) 18 286 YRIHTHGVF 3.000 (SEQ ID NO:
252) 19 174 HSFKHEDPM 3.000 (SEQ ID NO: 110) 20 372 RHQRRHTGV 3.000
(SEQ ID NO: 181)
TABLE-US-00012 TABLE XII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B40 Score (Estimate of Half Time of Disas- Subsequence sociation of
a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 81 AEPHEEQCL 40.000 (SEQ ID NO: 30) 2 429 DELVRHHNM
24.000 (SEQ ID NO: 53) 3 410 SEKPFSCRW 20.000 (SEQ ID NO: 207) 4
318 SEKRPFMCA 15.000 (SEQ ID NO: 208) 5 233 LECMTWNQM 12.000 (SEQ
ID NO: 131) 6 3 SDVRDLNAL 10.000 (SEQ ID NO: 206) 7 349 GEKPYQCDF
8.000 (SEQ ID NO: 91) 8 6 RDLNALLPA 5.000 (SEQ ID NO: 177) 9 85
EEQCLSAFT 4.000 (SEQ ID NO: 65) 10 315 SETSEKRPF 4.000 (SEQ ID NO:
209) 11 261 TEGQSNHST 4.000 (SEQ ID NO: 221) 12 23 GCALPVSGA 3.000
(SEQ ID NO: 89) 13 38 LDFAPPGAS 3.000 (SEQ ID NO: 130) 14 273
SDNHTTPIL 2.500 (SEQ ID NO: 204) 15 206 TDSCTGSQA 2.500 (SEQ ID NO:
220) 16 24 CALPVSGAA 2.000 (SEQ ID NO: 43) 17 98 GQFTGTAGA 2.000
(SEQ ID NO: 99) 18 30 GAAQWAPVL 2.000 (SEQ ID NO: 86) 19 84
HEEQCLSAF 2.000 (SEQ ID NO: 107) 20 26 LPVSGAAQW 2.000 (SEQ ID NO:
138)
TABLE-US-00013 TABLE XIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B60 Score (Estimate of Half Time of Disas- Subsequence sociation of
a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 81 AEPHEEQCL 160.000 (SEQ ID NO: 30) 2 3 SDVRDLNAL
40.000 (SEQ ID NO: 206) 3 429 DELVRHHNM 40.000 (SEQ ID NO: 53) 4
233 LECMTWNQM 22.000 (SEQ ID NO: 131) 5 273 SDNHTTPIL 20.000 (SEQ
ID NO: 204) 6 209 CTGSQALLL 8.000 (SEQ ID NO: 52) 7 30 GAAQWAPVL
8.000 (SEQ ID NO: 86) 8 318 SEKRPFMCA 8.000 (SEQ ID NO: 208) 9 180
DPMGQQGSL 8.000 (SEQ ID NO: 59) 10 138 LESQPAIRN 5.280 (SEQ ID NO:
132) 11 239 NQMNLGATL 4.400 (SEQ ID NO: 151) 12 329 GCNKRYFKL 4.400
(SEQ ID NO: 90) 13 130 NAPYLPSCL 4.400 (SEQ ID NO: 144) 14 85
EEQCLSAFT 4.400 (SEQ ID NO: 65) 15 208 SCTGSQALL 4.000 (SEQ ID NO:
202) 16 207 DSCTGSQAL 4.000 (SEQ ID NO: 61) 17 218 RTPYSSDNL 4.000
(SEQ ID NO: 194) 18 261 TEGQSNHST 4.000 (SEQ ID NO: 221) 19 18
LGGGGGCAL 4.000 (SEQ ID NO: 134) 20 221 YSSDNLYQM 2.200 (SEQ ID NO:
253)
TABLE-US-00014 TABLE XIV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B61 Score (Estimate of Half Time of Disas- Subsequence sociation of
a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 318 SEKRPFMCA 20.000 (SEQ ID NO: 208) 2 429
DELVRHHNM 16.000 (SEQ ID NO: 53) 3 298 QDVRRVPGV 10.000 (SEQ ID NO:
164) 4 81 AEPHEEQCL 8.000 (SEQ ID NO: 30) 5 233 LECMTWNQM 8.000
(SEQ ID NO: 131) 6 6 RDLNALLPA 5.500 (SEQ ID NO: 177) 7 85
EEQCLSAFT 4.000 (SEQ ID NO: 65) 8 261 TEGQSNHST 4.000 (SEQ ID NO:
221) 9 206 TDSCTGSQA 2.500 (SEQ ID NO: 220) 10 295 RGIQDVRRV 2.200
(SEQ ID NO: 179) 11 3 SDVRDLNAL 2.000 (SEQ ID NO: 206) 12 250
VAAGSSSSV 2.000 (SEQ ID NO: 236) 13 29 SGAAQWAPV 2.000 (SEQ ID NO:
211) 14 315 SETSEKRPF 1.600 (SEQ ID NO: 209) 15 138 LESQPAIRN 1.200
(SEQ ID NO: 132) 16 244 GATLKGVAA 1.100 (SEQ ID NO: 88) 17 20
GGGGCALPV 1.100 (SEQ ID NO: 92) 18 440 RNMTKLQLA 1.100 (SEQ ID NO:
186) 19 23 GCALPVSGA 1.100 (SEQ ID NO: 89) 20 191 QQYSVPPPV 1.000
(SEQ ID NO: 171)
TABLE-US-00015 TABLE XV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B62 Score (Estimate of Half Time of Disas- Subsequence sociation of
a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 146 NQGYSTVTF 211.200 (SEQ ID NO: 150) 2 32
AQWAPVLDF 96.000 (SEQ ID NO: 37) 3 263 GQSNHSTGY 96.000 (SEQ ID NO:
100) 4 88 CLSAFTVHF 96.000 (SEQ ID NO: 48) 5 17 SLGGGGGCA 9.600
(SEQ ID NO: 215) 6 239 NQMNLGATL 8.800 (SEQ ID NO: 151) 7 191
QQYSVPPPV 8.000 (SEQ ID NO: 171) 8 98 GQFTGTAGA 8.000 (SEQ ID NO:
99) 9 384 QCKTCQRKF 6.000 (SEQ ID NO: 163) 10 40 FAPPGASAY 4.800
(SEQ ID NO: 74) 11 227 YQMTSQLEC 4.800 (SEQ ID NO: 251) 12 187
SLGEQQYSV 4.400 (SEQ ID NO: 214) 13 86 EQCLSAFTV 4.400 (SEQ ID NO:
69) 14 152 VTFDGTPSY 4.400 (SEQ ID NO: 244) 15 101 TGTAGACRY 4.000
(SEQ ID NO: 224) 16 242 NLGATLKGV 4.000 (SEQ ID NO: 146) 17 92
FTVHFSGQF 4.000 (SEQ ID NO: 85) 18 7 DLNALLPAV 4.000 (SEQ ID NO:
58) 19 123 GQARMFPNA 4.000 (SEQ ID NO: 98) 20 280 ILCGAQYRI 3.120
(SEQ ID NO: 116)
TABLE-US-00016 TABLE XVI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B7 Score (Estimate of Half Time of Disas- Subsequence sociation of
a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 180 DPMGQQGSL 240.000 (SEQ ID NO: 59) 2 4 DVRDLNALL
200.000 (SEQ ID NO: 62) 3 302 RVPGVAPTL 20.000 (SEQ ID NO: 195) 4
30 GAAQWAPVL 12.000 (SEQ ID NO: 86) 5 239 NQMNLGATL 12.000 (SEQ ID
NO: 151) 6 130 NAPYLPSCL 12.000 (SEQ ID NO: 144) 7 10 ALLPAVPSL
12.000 (SEQ ID NO: 34) 8 299 DVRRVPGVA 5.000 (SEQ ID NO: 63) 9 208
SCTGSQALL 4.000 (SEQ ID NO: 202) 10 303 VPGVAPTLV 4.000 (SEQ ID NO:
242) 11 18 LGGGGGCAL 4.000 (SEQ ID NO: 134) 12 218 RTPYSSDNL 4.000
(SEQ ID NO: 194) 13 207 DSCTGSQAL 4.000 (SEQ ID NO: 61) 14 209
CTGSQALLL 4.000 (SEQ ID NO: 52) 15 329 GCNKRYFKL 4.000 (SEQ ID NO:
90) 16 235 CMTWNQMNL 4.000 (SEQ ID NO: 49) 17 441 NMTKLQLAL 4.000
(SEQ ID NO: 149) 18 126 RMFPNAPYL 4.000 (SEQ ID NO: 185) 19 225
NLYQMTSQL 4.000 (SEQ ID NO: 147) 20 143 AIRNQGYST 3.000 (SEQ ID NO:
33)
TABLE-US-00017 TABLE XVII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B8 Score (Estimate of Half Time of Disas- Subsequence sociation of
a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 329 GCNKRYFKL 16.000 (SEQ ID NO: 90) 2 4 DVRDLNALL
12.000 (SEQ ID NO: 62) 3 316 ETSEKRPFM 3.000 (SEQ ID NO: 73) 4 180
DPMGQQGSL 1.600 (SEQ ID NO: 59) 5 208 SCTGSQALL 0.800 (SEQ ID NO:
202) 6 130 NAPYLPSCL 0.800 (SEQ ID NO: 144) 7 244 GATLKGVAA 0.800
(SEQ ID NO: 88) 8 30 GAAQWAPVL 0.800 (SEQ ID NO: 86) 9 299
DVRRVPGVA 0.400 (SEQ ID NO: 63) 10 420 SCQKKFARS 0.400 (SEQ ID NO:
200) 11 387 TCQRKFSRS 0.400 (SEQ ID NO: 219) 12 225 NLYQMTSQL 0.400
(SEQ ID NO: 147) 13 141 QPAIRNQGY 0.400 (SEQ ID NO: 170) 14 10
ALLPAVPSL 0.400 (SEQ ID NO: 34) 15 207 DSCTGSQAL 0.400 (SEQ ID NO:
61) 16 384 QCKTCQRKF 0.400 (SEQ ID NO: 163) 17 136 SCLESQPAI 0.300
(SEQ ID NO: 198) 18 347 HTGEKPYQC 0.300 (SEQ ID NO: 112) 19 401
HTRTHTGKT 0.200 (SEQ ID NO: 114) 20 332 KRYFKLSHL 0.200 (SEQ ID NO:
127)
TABLE-US-00018 TABLE XVIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 2702 Score (Estimate of Half Time of Disas- Subsequence sociation
of a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 332 KRYFKLSHL 900.000 (SEQ ID NO: 127) 2 362
RRFSRSDQL 900.000 (SEQ ID NO: 187) 3 286 YRIHTHGVF 200.000 (SEQ ID
NO: 252) 4 125 ARMFPNAPY 200.000 (SEQ ID NO: 38) 5 375 RRHTGVKPF
180.000 (SEQ ID NO: 188) 6 32 AQWAPVLDF 100.000 (SEQ ID NO: 37) 7
301 RRVPGVAPT 60.000 (SEQ ID NO: 189) 8 439 QRNMTKLQL 60.000 (SEQ
ID NO: 173) 9 126 RMFPNAPYL 22.500 (SEQ ID NO: 185) 10 426
ARSDELVRH 20.000 (SEQ ID NO: 39) 11 146 NQGYSTVTF 20.000 (SEQ ID
NO: 150) 12 144 IRNQGYSTV 20.000 (SEQ ID NO: 117) 13 389 QRKFSRSDH
20.000 (SEQ ID NO: 172) 14 263 GQSNHSTGY 20.000 (SEQ ID NO: 100) 15
416 CRWPSCQKK 20.000 (SEQ ID NO: 50) 16 191 QQYSVPPPV 10.000 (SEQ
ID NO: 171) 17 217 LRTPYSSDN 10.000 (SEQ ID NO: 140) 18 107
CRYGPFGPP 10.000 (SEQ ID NO: 51) 19 98 GQFTGTAGA 10.000 (SEQ ID NO:
99) 20 239 NQMNLGATL 6.000 (SEQ ID NO: 151)
TABLE-US-00019 TABLE XIX Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 2705 Score (Estimate of Half Time of Disas- Subsequence sociation
of a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 332 KRYFKLSHL 30000.000 (SEQ ID NO: 127) 2 362
RRFSRSDQL 30000.000 (SEQ ID NO: 187) 3 416 CRWPSCQKK 10000.000 (SEQ
ID NO: 50) 4 439 QRNMTKLQL 2000.000 (SEQ ID NO: 173) 5 286
YRIHTHGVF 1000.000 (SEQ ID NO: 252) 6 125 ARMFPNAPY 1000.000 (SEQ
ID NO: 38) 7 294 FRGIQDVRR 1000.000 (SEQ ID NO: 81) 8 432 VRHHNMHQR
1000.000 (SEQ ID NO: 243) 9 169 AQFPNHSFK 1000.000 (SEQ ID NO: 36)
10 375 RRHTGVKPF 900.000 (SEQ ID NO: 188) 11 126 RMFPNAPYL 750.000
(SEQ ID NO: 185) 12 144 IRNQGYSTV 600.000 (SEQ ID NO: 117) 13 301
RRVPGVAPT 600.000 (SEQ ID NO: 189) 14 32 AQWAPVLDF 500.000 (SEQ ID
NO: 37) 15 191 QQYSVPPPV 300.000 (SEQ ID NO: 171) 16 373 HQRRHTGVK
200.000 (SEQ ID NO: 109) 17 426 ARSDELVRH 200.000 (SEQ ID NO: 39)
18 383 FQCKTCQRK 200.000 (SEQ ID NO: 80) 19 239 NQMNLGATL 200.000
(SEQ ID NO: 151) 20 389 QRKFSRSDH 200.000 (SEQ ID NO: 172)
TABLE-US-00020 TABLE XX Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 3501 Score (Estimate of Half Time of Disas- Subsequence sociation
of a Mole- Start Residue cule Containing This Rank Position Listing
Subsequence) 1 278 TPILCGAQY 40.000 (SEQ ID NO: 227) 2 141
QPAIRNQGY 40.000 (SEQ ID NO: 170) 3 219 TPYSSDNLY 40.000 (SEQ ID
NO: 231) 4 327 YPGCNKRYF 20.000 (SEQ ID NO: 250) 5 163 TPSHHAAQF
20.000 (SEQ ID NO: 228) 6 180 DPMGQQGSL 20.000 (SEQ ID NO: 59) 7
221 YSSDNLYQM 20.000 (SEQ ID NO: 253) 8 26 LPVSGAAQW 10.000 (SEQ ID
NO: 138) 9 174 HSFKHEDPM 10.000 (SEQ ID NO: 110) 10 82 EPHEEQCLS
6.000 (SEQ ID NO: 68) 11 213 QALLLRTPY 6.000 (SEQ ID NO: 160) 12
119 QASSGQARM 6.000 (SEQ ID NO: 161) 13 4 DVRDLNALL 6.000 (SEQ ID
NO: 62) 14 40 FAPPGASAY 6.000 (SEQ ID NO: 74) 15 120 ASSGQARMF
5.000 (SEQ ID NO: 40) 16 207 DSCTGSQAL 5.000 (SEQ ID NO: 61) 17 303
VPGVAPTLV 4.000 (SEQ ID NO: 242) 18 316 ETSEKRPFM 4.000 (SEQ ID NO:
73) 19 152 VTFDGTPSY 4.000 (SEQ ID NO: 244) 20 412 KPFSCRWPS 4.000
(SEQ ID NO: 123)
TABLE-US-00021 TABLE XXI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 3701 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 3 SDVRDLNAL 40.000 (SEQ ID NO: 206) 2 273 SDNHTTPIL
40.000 (SEQ ID NO: 204) 3 81 AEPHEEQCL 10.000 (SEQ ID NO: 30) 4 298
QDVRRVPGV 8.000 (SEQ ID NO: 164) 5 428 SDELVRHHN 6.000 (SEQ ID NO:
203) 6 85 EEQCLSAFT 5.000 (SEQ ID NO: 65) 7 208 SCTGSQALL 5.000
(SEQ ID NO: 202) 8 4 DVRDLNALL 5.000 (SEQ ID NO: 62) 9 209
CTGSQALLL 5.000 (SEQ ID NO: 52) 10 38 LDFAPPGAS 4.000 (SEQ ID NO:
130) 11 223 SDNLYQMTS 4.000 (SEQ ID NO: 205) 12 179 EDPMGQQGS 4.000
(SEQ ID NO: 64) 13 206 TDSCTGSQA 4.000 (SEQ ID NO: 220) 14 6
RDLNALLPA 4.000 (SEQ ID NO: 177) 15 84 HEEQCLSAF 2.000 (SEQ ID NO:
107) 16 233 LECMTWNQM 2.000 (SEQ ID NO: 131) 17 429 DELVRHHNM 2.000
(SEQ ID NO: 53) 18 315 SETSEKRPF 2.000 (SEQ ID NO: 209) 19 349
GEKPYQCDF 2.000 (SEQ ID NO: 91) 20 302 RVPGVAPTL 1.500 (SEQ ID NO:
195)
TABLE-US-00022 TABLE XXII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 3801 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 437 MHQRNMTKIL 36.000 (SEQ ID NO: 143) 2 434
HHNMHQRNM 6.000 (SEQ ID NO: 108) 3 372 RHQRRHTGV 6.000 (SEQ ID NO:
181) 4 180 DPMGQQGSL 4.000 (SEQ ID NO: 59) 5 433 RHHNMHQRN 3.900
(SEQ ID NO: 180) 6 165 SHHAAQFPN 3.900 (SEQ ID NO: 213) 7 202
CHTPTDSCT 3.000 (SEQ ID NO: 45) 8 396 DHLKTHTRT 3.000 (SEQ ID NO:
57) 9 161 GHTPSHHAA 3.000 (SEQ ID NO: 94) 10 302 RVPGVAPTL 2.600
(SEQ ID NO: 195) 11 417 RWPSCQKKF 2.400 (SEQ ID NO: 196) 12 327
YPGCNKRYF 2.400 (SEQ ID NO: 250) 13 208 SCTGSQALL 2.000 (SEQ ID NO:
202) 14 163 TPSHHAAQF 2.000 (SEQ ID NO: 228) 15 120 ASSGQARMF 2.000
(SEQ ID NO: 40) 16 18 LGGGGGCAL 2.000 (SEQ ID NO: 134) 17 177
KHEDPMGQQ 1.800 (SEQ ID NO: 121) 18 83 PHEEQCLSA 1.800 (SEQ ID NO:
154) 19 10 ALLPAVPSL 1.300 (SEQ ID NO: 34) 20 225 NLYQMTSQL 1.300
(SEQ ID NO: 147)
TABLE-US-00023 TABLE XXIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 3901 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 437 MHQRNMTKIL 135.000 (SEQ ID NO: 143) 2 332
KRYFKLSHL 45.000 (SEQ ID NO: 127) 3 434 HHNMHQRNM 30.000 (SEQ ID
NO: 108) 4 362 RRFSRSDQL 30.000 (SEQ ID NO: 187) 5 372 RHQRRHTGV
30.000 (SEQ ID NO: 181) 6 10 ALLPAVPSL 9.000 (SEQ ID NO: 34) 7 439
QRNMTKLQL 7.500 (SEQ ID NO: 173) 8 390 RKFSRSDHL 6.000 (SEQ ID NO:
183) 9 396 DHLKTHTRT 6.000 (SEQ ID NO: 57) 10 239 NQMNLGATL 6.000
(SEQ ID NO: 151) 11 423 KKFARSDEL 6.000 (SEQ ID NO: 122) 12 126
RMFPNAPYL 6.000 (SEQ ID NO: 185) 13 225 NLYQMTSQL 6.000 (SEQ ID NO:
147) 14 180 DPMGQQGSL 6.000 (SEQ ID NO: 59) 15 144 IRNQGYSTV 5.000
(SEQ ID NO: 117) 16 136 SCLESQPAI 4.000 (SEQ ID NO: 198) 17 292
GVFRGIQDV 3.000 (SEQ ID NO: 103) 18 302 RVPGVAPTL 3.000 (SEQ ID NO:
195) 19 208 SCTGSQALL 3.000 (SEQ ID NO: 202) 20 207 DSCTGSQAL 3.000
(SEQ ID NO: 61)
TABLE-US-00024 TABLE XXIV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 3902 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 239 NQMNLGATL 24.000 (SEQ ID NO: 151) 2 390
RKFSRSDHL 20.000 (SEQ ID NO: 183) 3 423 KKFARSDEL 20.000 (SEQ ID
NO: 122) 4 32 AQWAPVLDF 5.000 (SEQ ID NO: 37) 5 146 NQGYSTVTF 5.000
(SEQ ID NO: 150) 6 130 NAPYLPSCL 2.400 (SEQ ID NO: 144) 7 225
NLYQMTSQL 2.400 (SEQ ID NO: 147) 8 30 GAAQWAPVL 2.400 (SEQ ID NO:
86) 9 441 NMTKLQLAL 2.400 (SEQ ID NO: 149) 10 302 RVPGVAPTL 2.400
(SEQ ID NO: 195) 11 126 RMFPNAPYL 2.000 (SEQ ID NO: 185) 12 218
RTPYSSDNL 2.000 (SEQ ID NO: 194) 13 209 CTGSQALLL 2.000 (SEQ ID NO:
52) 14 332 KRYFKLSHL 2.000 (SEQ ID NO: 127) 15 180 DPMGQQGSL 2.000
(SEQ ID NO: 59) 16 437 MHQRNMTKL 2.000 (SEQ ID NO: 143) 17 207
DSCTGSQAL 2.000 (SEQ ID NO: 61) 18 208 SCTGSQALL 2.000 (SEQ ID NO:
202) 19 329 GCNKRYFKL 2.000 (SEQ ID NO: 90) 20 10 ALLPAVPSL 2.000
(SEQ ID NO: 34)
TABLE-US-00025 TABLE XXV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 4403 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 315 SETSEKRPF 80.000 (SEQ ID NO: 209) 2 349
GEKPYQCDF 80.000 (SEQ ID NO: 91) 3 84 HEEQCLSAF 60.000 (SEQ ID NO:
107) 4 410 SEKPFSCRW 48.000 (SEQ ID NO: 207) 5 429 DELVRHHNM 24.000
(SEQ ID NO: 53) 6 278 TPILCGAQY 15.000 (SEQ ID NO: 227) 7 141
QPAIRNQGY 9.000 (SEQ ID NO: 170) 8 40 FAPPGASAY 9.000 (SEQ ID NO:
74) 9 213 QALLLRTPY 9.000 (SEQ ID NO: 160) 10 318 SEKRPFMCA 8.000
(SEQ ID NO: 208) 11 81 AEPHEEQCL 8.000 (SEQ ID NO: 30) 12 152
VTFDGTPSY 4.500 (SEQ ID NO: 244) 13 101 TGTAGACRY 4.500 (SEQ ID NO:
224) 14 120 ASSGQARMF 4.500 (SEQ ID NO: 40) 15 261 TEGQSNHST 4.000
(SEQ ID NO: 221) 16 85 EEQCLSAFT 4.000 (SEQ ID NO: 65) 17 233
LECMTWNQM 4.000 (SEQ ID NO: 131) 18 104 AGACRYGPF 4.000 (SEQ ID NO:
31) 19 3 SDVRDLNAL 3.000 (SEQ ID NO: 206) 20 185 QGSLGEQQY 3.000
(SEQ ID NO: 166)
TABLE-US-00026 TABLE XXVI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 5101 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 303 VPGVAPTLV 314.600 (SEQ ID NO: 242) 2 180
DPMGQQGSL 242.000 (SEQ ID NO: 59) 3 250 VAAGSSSSV 157.300 (SEQ ID
NO: 236) 4 130 NAPYLPSCL 50.000 (SEQ ID NO: 144) 5 30 GAAQWAPVL
50.000 (SEQ ID NO: 86) 6 20 GGGGCALPV 44.000 (SEQ ID NO: 92) 7 64
PPPPPHSFI 40.000 (SEQ ID NO: 157) 8 29 SGAAQWAPV 40.000 (SEQ ID NO:
211) 9 18 LGGGGGCAL 31.460 (SEQ ID NO: 134) 10 295 RGIQDVRRV 22.000
(SEQ ID NO: 179) 11 119 QASSGQARM 18.150 (SEQ ID NO: 161) 12 418
WPSCQKKFA 12.100 (SEQ ID NO: 246) 13 82 EPHEEQCLS 12.100 (SEQ ID
NO: 68) 14 110 GPFGPPPPS 11.000 (SEQ ID NO: 96) 15 272 ESDNHTTPI
8.000 (SEQ ID NO: 71) 16 306 VAPTLVRSA 7.150 (SEQ ID NO: 237) 17
280 ILCGAQYRI 6.921 (SEQ ID NO: 116) 18 219 TPYSSDNLY 6.600 (SEQ ID
NO: 231) 19 128 FPNAPYLPS 6.500 (SEQ ID NO: 79) 20 204 TPTDSCTGS
6.050 (SEQ ID NO: 230)
TABLE-US-00027 TABLE XXVII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 5102 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 295 RGIQDVRRV 290.400 (SEQ ID NO: 179) 2 303
VPGVAPTLV 200.000 (SEQ ID NO: 242) 3 180 DPMGQQGSL 133.100 (SEQ ID
NO: 59) 4 250 VAAGSSSSV 110.000 (SEQ ID NO: 236) 5 30 GAAQWAPVL
55.000 (SEQ ID NO: 86) 6 130 NAPYLPSCL 50.000 (SEQ ID NO: 144) 7 20
GGGGCALPV 44.000 (SEQ ID NO: 92) 8 29 SGAAQWAPV 44.000 (SEQ ID NO:
211) 9 64 PPPPPHSFI 40.000 (SEQ ID NO: 157) 10 119 QASSGQARM 36.300
(SEQ ID NO: 161) 11 110 GPFGPPPPS 27.500 (SEQ ID NO: 96) 12 412
KPFSCRWPS 25.000 (SEQ ID NO: 123) 13 18 LGGGGGCAL 24.200 (SEQ ID
NO: 134) 14 24 CALPVSGAA 16.500 (SEQ ID NO: 43) 15 219 TPYSSDNLY
15.000 (SEQ ID NO: 231) 16 292 GVFRGIQDV 14.641 (SEQ ID NO: 103) 17
136 SCLESQPAI 14.520 (SEQ ID NO: 198) 18 418 WPSCQKKFA 12.100 (SEQ
ID NO: 246) 19 269 TGYESDNHT 11.000 (SEQ ID NO: 225) 20 351
KPYQCDFKD 11.000 (SEQ ID NO: 124)
TABLE-US-00028 TABLE XXVIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 5201 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 191 QQYSVPPPV 100.000 (SEQ ID NO: 171) 2 32
AQWAPVLDF 30.000 (SEQ ID NO: 37) 3 243 LGATLKGVA 16.500 (SEQ ID NO:
133) 4 303 VPGVAPTLV 13.500 (SEQ ID NO: 242) 5 86 EQCLSAFTV 12.000
(SEQ ID NO: 69) 6 295 RGIQDVRRV 10.000 (SEQ ID NO: 179) 7 98
GQFTGTAGA 8.250 (SEQ ID NO: 99) 8 292 GVFRGIQDV 8.250 (SEQ ID NO:
103) 9 29 SGAAQWAPV 6.000 (SEQ ID NO: 211) 10 146 NQGYSTVTF 5.500
(SEQ ID NO: 150) 11 20 GGGGCALPV 5.000 (SEQ ID NO: 92) 12 239
NQMNLGATL 4.000 (SEQ ID NO: 151) 13 64 PPPPPHSFI 3.600 (SEQ ID NO:
157) 14 273 SDNHTTPIL 3.300 (SEQ ID NO: 204) 15 286 YRIHTHGVF 3.000
(SEQ ID NO: 252) 16 269 TGYESDNHT 3.000 (SEQ ID NO: 225) 17 406
TGKTSEKPF 2.750 (SEQ ID NO: 222) 18 327 YPGCNKRYF 2.750 (SEQ ID NO:
250) 19 7 DLNALLPAV 2.640 (SEQ ID NO: 58) 20 104 AGACRYGPF 2.500
(SEQ ID NO: 31)
TABLE-US-00029 TABLE XXIX Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
B 5801 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 230 TSQLECMTW 96.800 (SEQ ID NO: 234) 2 92 FTVHFSGQF
60.000 (SEQ ID NO: 85) 3 120 ASSGQARMF 40.000 (SEQ ID NO: 40) 4 168
AAQFPNHSF 20.000 (SEQ ID NO: 29) 5 408 KTSEKPFSC 12.000 (SEQ ID NO:
129) 6 394 RSDHLKTHT 9.900 (SEQ ID NO: 192) 7 276 HTTPILCGA 7.200
(SEQ ID NO: 115) 8 218 RTPYSSDNL 6.600 (SEQ ID NO: 194) 9 152
VTFDGTPSY 6.000 (SEQ ID NO: 244) 10 40 FAPPGASAY 6.000 (SEQ ID NO:
74) 11 213 QALLLRTPY 4.500 (SEQ ID NO: 160) 12 347 HTGEKPYQC 4.400
(SEQ ID NO: 112) 13 252 AGSSSSVKW 4.400 (SEQ ID NO: 32) 14 211
GSQALLLRT 4.356 (SEQ ID NO: 102) 15 174 HSFKHEDPM 4.000 (SEQ ID NO:
110) 16 317 TSEKRPFMC 4.000 (SEQ ID NO: 233) 17 26 LPVSGAAQW 4.000
(SEQ ID NO: 138) 18 289 HTHGVFRGI 3.600 (SEQ ID NO: 113) 19 222
SSDNLYQMT 3.300 (SEQ ID NO: 217) 20 96 FSGQFTGTA 3.300 (SEQ ID NO:
82)
TABLE-US-00030 TABLE XXX Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
CW0301 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 10 ALLPAVPSL 100.000 (SEQ ID NO: 34) 2 332 KRYFKLSHL
48.000 (SEQ ID NO: 127) 3 126 RMFPNAPYL 36.000 (SEQ ID NO: 185) 4 3
SDVRDLNAL 30.000 (SEQ ID NO: 206) 5 239 NQMNLGATL 24.000 (SEQ ID
NO: 151) 6 225 NLYQMTSQL 24.000 (SEQ ID NO: 147) 7 180 DPMGQQGSL
20.000 (SEQ ID NO: 59) 8 362 RRFSRSDQL 12.000 (SEQ ID NO: 187) 9
329 GCNKRYFKL 10.000 (SEQ ID NO: 90) 10 286 YRIHTHGVF 10.000 (SEQ
ID NO: 252) 11 301 RRVPGVAPT 10.000 (SEQ ID NO: 189) 12 24
CALPVSGAA 10.000 (SEQ ID NO: 43) 13 136 SCLESQPAI 7.500 (SEQ ID NO:
198) 14 437 MHQRNMTKL 7.200 (SEQ ID NO: 143) 15 390 RKFSRSDHL 6.000
(SEQ ID NO: 183) 16 423 KKFARSDEL 6.000 (SEQ ID NO: 122) 17 92
FTVHFSGQF 5.000 (SEQ ID NO: 85) 18 429 DELVRHHNM 5.000 (SEQ ID NO:
53) 19 130 NAPYLPSCL 4.800 (SEQ ID NO: 144) 20 30 GAAQWAPVL 4.000
(SEQ ID NO: 86)
TABLE-US-00031 TABLE XXXI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
CW0401 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 356 DFKDCERRF 120.000 (SEQ ID NO: 55) 2 334
YFKLSHLQM 100.000 (SEQ ID NO: 248) 3 180 DPMGQQGSL 88.000 (SEQ ID
NO: 59) 4 163 TPSHHAAQF 52.800 (SEQ ID NO: 228) 5 327 YPGCNKRYF
40.000 (SEQ ID NO: 250) 6 285 QYRIHTHGV 27.500 (SEQ ID NO: 175) 7
424 KFARSDELV 25.000 (SEQ ID NO: 119) 8 326 AYPGCNKRY 25.000 (SEQ
ID NO: 42) 9 192 QYSVPPPVY 25.000 (SEQ ID NO: 176) 10 417 RWPSCQKKF
22.000 (SEQ ID NO: 196) 11 278 TPILCGAQY 12.000 (SEQ ID NO: 227) 12
10 ALLPAVPSL 11.616 (SEQ ID NO: 34) 13 141 QPAIRNQGY 11.000 (SEQ ID
NO: 170) 14 303 VPGVAPTLV 11.000 (SEQ ID NO: 242) 15 219 TPYSSDNLY
10.000 (SEQ ID NO: 231) 16 39 DFAPPGASA 7.920 (SEQ ID NO: 54) 17 99
QFTGTAGAC 6.000 (SEQ ID NO: 165) 18 4 DVRDLNALL 5.760 (SEQ ID NO:
62) 19 70 SFIKQEPSW 5.500 (SEQ ID NO: 210) 20 63 PPPPPPHSF 5.280
(SEQ ID NO: 158)
TABLE-US-00032 TABLE XXXII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
CW0602 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 332 KRYFKLSHL 9.680 (SEQ ID NO: 127) 2 239 NQMNLGATL
6.600 (SEQ ID NO: 151) 3 130 NAPYLPSCL 6.600 (SEQ ID NO: 144) 4 7
DLNALLPAV 6.000 (SEQ ID NO: 58) 5 441 NMTKLQLAL 6.000 (SEQ ID NO:
149) 6 225 NLYQMTSQL 6.000 (SEQ ID NO: 147) 7 4 DVRDLNALL 6.000
(SEQ ID NO: 62) 8 3 SDVRDLNAL 4.400 (SEQ ID NO: 206) 9 10 ALLPAVPSL
4.000 (SEQ ID NO: 34) 10 213 QALLLRTPY 3.300 (SEQ ID NO: 160) 11
319 EKRPFMCAY 3.000 (SEQ ID NO: 67) 12 30 GAAQWAPVL 2.200 (SEQ ID
NO: 86) 13 242 NLGATLKGV 2.200 (SEQ ID NO: 146) 14 292 GVFRGIQDV
2.200 (SEQ ID NO: 103) 15 207 DSCTGSQAL 2.200 (SEQ ID NO: 61) 16
362 RRFSRSDQL 2.200 (SEQ ID NO: 187) 17 439 QRNMTKLQL 2.200 (SEQ ID
NO: 173) 18 295 RGIQDVRRV 2.200 (SEQ ID NO: 179) 19 423 KKFARSDEL
2.200 (SEQ ID NO: 122) 20 180 DPMGQQGSL 2.200 (SEQ ID NO: 59)
TABLE-US-00033 TABLE XXXIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Human HLA
CW0702 Score (Estimate of Half Time of Disassociation Subsequence
of a Molecule Start Residue Containing This Rank Position Listing
Subsequence) 1 319 EKRPFMCAY 26.880 (SEQ ID NO: 67) 2 326 AYPGCNKRY
24.000 (SEQ ID NO: 42) 3 40 FAPPGASAY 14.784 (SEQ ID NO: 74) 4 192
QYSVPPPVY 12.000 (SEQ ID NO: 176) 5 278 TPILCGAQY 12.000 (SEQ ID
NO: 227) 6 219 TPYSSDNLY 12.000 (SEQ ID NO: 231) 7 213 QALLLRTPY
8.800 (SEQ ID NO: 160) 8 125 ARMFPNAPY 8.000 (SEQ ID NO: 38) 9 327
YPGCNKRYF 6.600 (SEQ ID NO: 250) 10 152 VTFDGTPSY 5.600 (SEQ ID NO:
244) 11 141 QPAIRNQGY 4.800 (SEQ ID NO: 170) 12 345 RKHTGEKPY 4.000
(SEQ ID NO: 184) 13 185 QGSLGEQQY 4.000 (SEQ ID NO: 166) 14 101
TGTAGACRY 4.000 (SEQ ID NO: 224) 15 375 RRHTGVKPF 4.000 (SEQ ID NO:
188) 16 263 GQSNHSTGY 4.000 (SEQ ID NO: 100) 17 163 TPSHHAAQF 3.000
(SEQ ID NO: 228) 18 33 QWAPVLDFA 2.688 (SEQ ID NO: 174) 19 130
NAPYLPSCL 2.640 (SEQ ID NO: 144) 20 84 HEEQCLSAF 2.400 (SEQ ID NO:
107)
TABLE-US-00034 TABLE XXXIV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC
Class I Db Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 235 CMTWNQMNL 5255.712 (SEQ ID NO:
49) 2 126 RMFPNAPYL 1990.800 (SEQ ID NO: 185) 3 221 YYSDNLYQM
9310.000 (SEQ ID NO: 253) 4 228 AMTSQLECM 33.701 (SEQ ID NO: 169) 5
239 NQMNLGATL 21.470 (SEQ ID NO: 151) 6 441 NMTKLQLAL 19.908 (SEQ
ID NO: 149) 7 437 MHQRNMTKL 19.837 (SEQ ID NO: 143) 8 136 SCLESQPAI
11.177 (SEQ ID NO: 198) 9 174 HSFKHEDPM 10.800 (SEQ ID NO: 110) 10
302 RVPGVAPTL 10.088 (SEQ ID NO: 195) 11 130 NAPYLPSCL 8.400 (SEQ
ID NO: 144) 12 10 ALLPAVPSL 5.988 (SEQ ID NO: 34) 13 208 SCTGSQALL
4.435 (SEQ ID NO: 202) 14 209 CTGSQALLL 3.548 (SEQ ID NO: 52) 15
238 WNQMNLGAT 3.300 (SEQ ID NO: 245) 16 218 RTPYSSDNL 3.185 (SEQ ID
NO: 194) 17 24 CALPVSGAA 2.851 (SEQ ID NO: 43) 18 18 LLGGGGGCAL
2.177 (SEQ ID NO: 134) 19 142 PAIRNQGYS 2.160 (SEQ ID NO: 152) 20
30 GAAQWAPVL 1.680 (SEQ ID NO: 86)
TABLE-US-00035 TABLE XXXV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC
Class I Dd Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 112 FGPPPPSQA 48.000 (SEQ ID NO: 76)
2 122 SGQARMFPN 36.000 (SEQ ID NO: 212) 3 104 AGACRYGPF 30.000 (SEQ
ID NO: 31) 4 218 RTPYSSDNL 28.800 (SEQ ID NO: 194) 5 130 NAPYLPSCL
20.000 (SEQ ID NO: 144) 6 302 RVPGVAPTL 20.000 (SEQ ID NO: 195) 7
18 LGGGGGCAL 20.000 (SEQ ID NO: 134) 8 81 AEPHEEQCL 10.000 (SEQ ID
NO: 30) 9 29 SGAAQWAPV 7.200 (SEQ ID NO: 211) 10 423 KKFARSDEL
7.200 (SEQ ID NO: 122) 11 295 RGIQDVRRV 7.200 (SEQ ID NO: 179) 12
390 RKFSRSDHL 6.000 (SEQ ID NO: 183) 13 332 KRYFKLSHL 6.000 (SEQ ID
NO: 127) 14 362 RRFSRSDQL 6.000 (SEQ ID NO: 187) 15 417 RWPSCQKKF
6.000 (SEQ ID NO: 196) 16 160 YGHTPSHHA 6.000 (SEQ ID NO: 249) 17
20 GGGGCALPV 6.000 (SEQ ID NO: 92) 18 329 GCNKRYFKL 5.000 (SEQ ID
NO: 90) 19 372 RHQRRHTGV 4.500 (SEQ ID NO: 181) 20 52 GGPAPPPAP
4.000 (SEQ ID NO: 93)
TABLE-US-00036 TABLE XXXVI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC
Class I Kb Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 329 GCNKRYFKIL 24.000 (SEQ ID NO:
90) 2 225 NLYQMTSQL 10.000 (SEQ ID NO: 147) 3 420 SCQKKFARS 3.960
(SEQ ID NO: 200) 4 218 RTPYSSDNL 3.630 (SEQ ID NO: 194) 5 437
MHQRNMTKL 3.600 (SEQ ID NO: 143) 6 387 TCQRKFSRS 3.600 (SEQ ID NO:
219) 7 302 RVPGVAPTL 3.300 (SEQ ID NO: 195) 8 130 NAPYLPSCL 3.000
(SEQ ID NO: 144) 9 289 HTHGVFRGI 3.000 (SEQ ID NO: 113) 10 43
PGASAYGSL 2.400 (SEQ ID NO: 153) 11 155 DGTPSYGHT 2.400 (SEQ ID NO:
56) 12 273 SDNHTTPIL 2.200 (SEQ ID NO: 204) 13 126 RMFPNAPYL 2.200
(SEQ ID NO: 185) 14 128 FPNAPYLPS 2.000 (SEQ ID NO: 79) 15 3
SDVRDLNAL 1.584 (SEQ ID NO: 206) 16 207 DSCTGSQAL 1.584 (SEQ ID NO:
61) 17 332 KRYFKLSHL 1.500 (SEQ ID NO: 127) 18 18 LGGGGGCAL 1.320
(SEQ ID NO: 134) 19 233 LECMTWNQM 1.320 (SEQ ID NO: 131) 20 441
NMTKLQLAL 1.200 (SEQ ID NO: 149)
TABLE-US-00037 TABLE XXXVII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC
Class I Kd Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 285 QYRIHTHGV 600.000 (SEQ ID NO:
175) 2 424 KFARSDELV 288.000 (SEQ ID NO: 119) 3 334 YFKLSHLQM
120.000 (SEQ ID NO: 248) 4 136 SCLESQPTI 115.200 (SEQ ID NO: 199) 5
239 NQMNLGATL 115.200 (SEQ ID NO: 151) 6 10 ALLPAVSSL 115.200 (SEQ
ID NO: 35) 7 47 AYGSLGGPA 86.400 (SEQ ID NO: 41) 8 180 DPMGQQGSL
80.000 (SEQ ID NO: 59) 9 270 GYESDNHTA 72.000 (SEQ ID NO: 105) 10
326 AYPGCNKRY 60.000 (SEQ ID NO: 42) 11 192 QYSVPPPVY 60.000 (SEQ
ID NO: 176) 12 272 ESDNHTAPI 57.600 (SEQ ID NO: 70) 13 289
HTHGVFRGI 57.600 (SEQ ID NO: 113) 14 126 DVRDLNALL 57.600 (SEQ ID
NO: 62) 15 4 CTGSQALLL 57.600 (SEQ ID NO: 52) 16 208 SCTGSQALL
48.000 (SEQ ID NO :202) 17 441 NMTKLQLAL 48.000 (SEQ ID NO: 149) 18
207 DSCTGSQAL 48.000 (SEQ ID NO: 61) 19 130 NAPYLPSCL 48.000 (SEQ
ID NO: 144) 20 235 CMTWNQMNL 48.000 (SEQ ID NO: 49)
TABLE-US-00038 TABLE XXXVIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC
Class I Kk Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 81 AEPHEEQCL 40.000 (SEQ ID NO: 30)
2 85 EEQCLSAFT 40.000 (SEQ ID NO: 65) 3 429 DELVRHHNM 20.000 (SEQ
ID NO: 53) 4 315 SETSEKRPF 20.000 (SEQ ID NO: 209) 5 261 TEGQSNHST
20.000 (SEQ ID NO: 221) 6 410 SEKPFSCRW 10.000 (SEQ ID NO: 207) 7
272 ESDNHTTPI 10.000 (SEQ ID NO: 71) 8 318 SEKRPFMCA 10.000 (SEQ ID
NO: 208) 9 138 LESQPAIRN 10.000 (SEQ ID NO: 132) 10 233 LECMTWNQM
10.000 (SEQ ID NO: 131) 11 298 QDVRRVPGV 10.000 (SEQ ID NO: 164) 12
84 HEEQCLSAF 10.000 (SEQ ID NO: 107) 13 349 GEKPYQCDF 10.000 (SEQ
ID NO: 91) 14 289 HTHGVFRGI 10.000 (SEQ ID NO: 113) 15 179
EDPMGQQGS 8.000 (SEQ ID NO: 64) 16 136 SCLESQPAI 5.000 (SEQ ID NO:
198) 17 280 ILCGAQYRI 5.000 (SEQ ID NO: 116) 18 273 SDNHTTPIL 4.000
(SEQ ID NO: 204) 19 428 SDELVRHHN 4.000 (SEQ ID NO: 203) 20 3
SDVRDLNAL 4.000 (SEQ ID NO: 206)
TABLE-US-00039 TABLE XXXIX Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WT1 Peptides to Mouse MHC
Class I Ld Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 163 TPSHHAAQF 360.000 (SEQ ID NO:
228) 2 327 YPGCNKRYF 300.000 (SEQ ID NO: 250) 3 180 DPMGQQGSL
150.000 (SEQ ID NO: 59) 4 26 LPVSGAAQW 93.600 (SEQ ID NO: 138) 5
278 TPILCGAQY 72.000 (SEQ ID NO: 227) 6 141 QPAIRNQGY 60.000 (SEQ
ID NO: 170) 7 219 TPYSSDNLY 60.000 (SEQ ID NO: 231) 8 303 VPGVAPTLV
60.000 (SEQ ID NO: 242) 9 120 ASSGQARMF 50.000 (SEQ ID NO: 40) 10
63 PPPPPPHSF 45.000 (SEQ ID NO: 158) 11 113 GPPPPSQAS 45.000 (SEQ
ID NO: 97) 12 157 TPSYGHTPS 39.000 (SEQ ID NO: 229) 13 207
DSCTGSQAL 32.500 (SEQ ID NO: 61) 14 110 GPFGPPPPS 30.000 (SEQ ID
NO: 96) 15 82 EPHEEQCLS 30.000 (SEQ ID NO: 68) 16 412 KPFSCRWPS
30.000 (SEQ ID NO: 123) 17 418 WPSCQKKFA 30.000 (SEQ ID NO: 246) 18
221 YSSDNLYQM 30.000 (SEQ ID NO: 253) 19 204 TPTDSCTGS 30.000 (SEQ
ID NO: 230) 20 128 FPNAPYLPS 30.000 (SEQ ID NO: 79)
TABLE-US-00040 TABLE XL Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Human WI1 Peptides to Cattle HLA
A20 Subsequence Score (Estimate of Half Time of Start Residue
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) 1 350 EKPYQCDFK 1000.00 (SEQ ID NO: 66) 2 319
EKRPFMCAY 500.000 (SEQ ID NO: 67) 3 423 KKFARSDEL 500.000 (SEQ ID
NO: 122) 4 345 RKHTGEKPY 500.000 (SEQ ID NO: 184) 5 390 RKFSRSDHL
500.000 (SEQ ID NO: 183) 6 137 CLESQPAIR 120.000 (SEQ ID NO: 47) 7
380 VKPFQCKTC 100.000 (SEQ ID NO: 239) 8 407 GKTSEKPFS 100.000 (SEQ
ID NO: 95) 9 335 FKLSHLQMH 100.000 (SEQ ID NO: 78) 10 247 LKGVAAGSS
100.000 (SEQ ID NO: 135) 11 370 LKRHQRRHT 100.000 (SEQ ID NO: 136)
12 258 VKWTEGQSN 100.000 (SEQ ID NO: 240) 13 398 LKTHTRTHT 100.000
(SEQ ID NO: 137) 14 331 NKRYFKLSH 100.000 (SEQ ID NO: 145) 15 357
FKDCERRFS 100.000 (SEQ ID NO: 77) 16 385 CKTCQRKFS 100.000 (SEQ ID
NO: 46) 17 294 FRGIQDVRR 80.000 (SEQ ID NO: 81) 18 368 DQLKRHQRR
80.000 (SEQ ID NO: 60) 19 432 VRHHNMHQR 80.000 (SEQ ID NO: 243) 20
118 SQASSGQAR 80.000 (SEQ ID NO: 216)
TABLE-US-00041 TABLE XLI Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC
Class I A_0201 Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 126 RMFPNAPYL 313.968 (SEQ ID NO:
293) 2 187 SLGEQQYSV 285.163 (SEQ ID NO: 299) 3 10 ALLPAVSSL
181.794 (SEQ ID NO: 255) 4 225 NLYQMTSQL 68.360 (SEQ ID NO: 284) 5
292 GVFRGIQDV 51.790 (SEQ ID NO: 270) 6 93 TLHFSGQFT 40.986 (SEQ ID
NO: 302) 7 191 QQYSVPPPV 22.566 (SEQ ID NO :290) 8 280 ILCGAQYRI
17.736 (SEQ ID NO :274) 9 441 NMTKLHVAL 15.428 (SEQ ID NO: 285) 10
235 CMIWNQMNL 15.428 (SEQ ID NO: 258) 11 7 DLNALLPAV 11.998 (SEQ ID
NO: 261) 12 242 NLGATLKGM 11.426 (SEQ ID NO: 283) 13 227 YQMTSQLEC
8.573 (SEQ ID NO: 307) 14 239 NQMNLGATL 8.014 (SEQ ID NO: 286) 15
309 ILYRSASET 7.452 (SEQ ID NO: 303) 16 408 KTSEKPFSC 5.743 (SEQ ID
NO :277) 17 340 LQMHSRKHT 4.752 (SEQ ID NO: 280) 18 228 QMTSQLECM
4.044 (SEQ ID NO: 289) 19 37 VLDFAPPGA 3.378 (SEQ ID NO: 304) 20
302 RYSGYAPIL 1.869 (SEQ ID NO: 295)
TABLE-US-00042 TABLE XLII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC
Class I Db Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 221 YSSDNLYQM 312.000 (SEQ ID NO:
308) 2 126 RMFPNAPYL 260.000 (SEQ ID NO: 293) 3 235 CMTWNQMNL
260.000 (SEQ ID NO: 258) 4 437 MHQRNMTKL 200.000 (SEQ ID NO: 281) 5
238 WNQMNLGAT 12.000 (SEQ ID NO: 305) 6 130 NAPYLPSCL 8.580 (SEQ ID
NO: 282) 7 3 SDVRDLNAL 7.920 (SEQ ID NO: 298) 8 136 SCLESQPTI 7.920
(SEQ ID NO: 296) 9 81 AEPHEEQCL 6.600 (SEQ ID NO: 254) 10 10
ALLPAVSSL 6.600 (SEQ ID NO: 255) 11 218 RTPYSSDNL 6.000 (SEQ ID NO:
294) 12 441 NMTKLHVAL 3.432 (SEQ ID NO: 285) 13 228 QMTSQLECM 3.120
(SEQ ID NO: 289) 14 174 HSFKHEDPM 3.120 (SEQ ID NO: 272) 15 242
NLGATLKGM 2.640 (SEQ ID NO: 283) 16 261 TEGQSNHGI 2.640 (SEQ ID NO:
301) 17 225 NLYQMTSQL 2.640 (SEQ ID NO: 284) 18 207 DSCTGSQAL 2.600
(SEQ ID NO: 263) 19 119 QASSGQARM 2.600 (SEQ ID NO: 288) 20 18
LGGGGGCGL 2.600 (SEQ ID NO: 279)
TABLE-US-00043 TABLE XLIII Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC
Class I Kb Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 329 GCNKRYFKIL 24.000 (SEQ ID NO:
268) 2 225 NLYQMTSQL 10.000 (SEQ ID NO: 284) 3 420 SCQKKFARS 3.960
(SEQ ID NO: 297) 4 218 RTPYSSDNL 3.630 (SEQ ID NO: 294) 5 437
MHQRNMTKL 3.600 (SEQ ID NO: 281) 6 387 TCQRKFSRS 3.600 (SEQ ID NO:
300) 7 289 HTHGVFRGI 3.000 (SEQ ID NO: 273) 8 130 NAPYLPSCL 3.000
(SEQ ID NO: 282) 9 43 PGASAYGSL 2.400 (SEQ ID NO: 287) 10 155
DGAPSYGHT 2.400 (SEQ ID NO: 260) 11 126 RMFPNAPYL 2.200 (SEQ ID NO:
293) 12 128 FPNAPYLPS 2.000 (SEQ IDNO: 267) 13 207 DSCTGSQAL 1.584
(SEQ ID NO: 263) 14 3 SDVRDLNAL 1.584 (SEQ ID NO: 298) 15 332
KRYFKLSHL 1.500 (SEQ ID NO: 276) 16 233 LECMTWNQM 1.320 (SEQ ID NO:
278) 17 18 LGGGGGCGL 1.320 (SEQ ID NO: 279) 18 242 NLGATLKGM 1.200
(SEQ ID NO: 283) 19 123 GQARMFPNA 1.200 (SEQ ID NO: 269) 20 441
NMTKLHVAL 1.200 (SEQ ID NO: 285)
TABLE-US-00044 TABLE XLIV Results of BIMAS HLA Peptide Binding
Prediction Analysis for Binding of Mouse WT1 Peptides to Mouse MHC
Class I Kd Subsequence Score (Estimate of Half Time of Start
Residue Disassociation of a Molecule Rank Position Listing
Containing This Subsequence) 1 285 QYRIHTHGV 600.000 (SEQ ID NO:
291) 2 424 KFARSDELV 288.000 (SEQ ID NO: 275) 3 334 YFKLSHLQM
120.000 (SEQ ID NO: 306) 4 136 SCLESQPTI 115.200 (SEQ ID NO: 296) 5
239 NQMNLGATL 115.200 (SEQ ID NO: 286) 6 10 ALLPAVSSL 115.200 (SEQ
ID NO: 255) 7 47 AYGSLGGPA 86.400 (SEQ ID NO: 256) 8 180 DPMGQQGSL
80.000 (SEQ ID NO: 262) 9 270 GYESDNHTA 72.000 (SEQ ID NO: 271) 10
192 QYSVPPPVY 60.000 (SEQ ID NO: 292) 11 326 AYPGCNKRY 60.000 (SEQ
ID NO: 257) 12 289 HTHGVFRGI 57.600 (SEQ ID NO: 273) 13 4 DVRDLNALL
57.600 (SEQ ID NO: 264) 14 126 RMFPNAPYL 57.600 (SEQ ID NO: 293) 15
209 CTGSQALLL 48.000 (SEQ ID NO: 259) 16 86 EQCLSAFTL 48.000 (SEQ
ID NO: 265) 17 302 RVSGVAPTL 48.000 (SEQ ID NO: 295) 18 218
RTPYSSDNL 48.000 (SEQ ID NO: 294) 19 272 ESDNHTAPI 48.000 (SEQ ID
NO: 266) 20 225 NLYQMTSQL 48.000 (SEQ ID NO: 284)
TABLE-US-00045 TABLE XLV Results of TSites Peptide Binding
Prediction Analysis for Human WT1 Peptides Capable of Eliciting a
Helper T cell Response Peptide Sequence p6-23 RDLNALLPAVPSLGGGG
(SEQ ID NO: 1) p30-35 GAAQWA (SEQ ID NO: 309) p45-56 ASAYGSLGGPAP
(SEQ ID NO: 310) p91-105 AFTVHFSGQFTGTAG (SEQ ID NO: 311) p117-139
PSQASSGQARMFPNAPYLPSCLE (SEQ ID NO: 2) p167-171 HAAQF (SEQ ID NO:
312) p202-233 CHTPTDSCTGSQALLLRTPYSSDNLYQMTSQL (SEQ ID NO: 313)
p244-262 GATLKGVAAGSSSSVKWTE (SEQ ID NO: 4) p287-318
RIHTHGVFRGIQDVRRVPGVAPTLVRSASETS (SEQ ID NO: 314) p333-336 RYFK
(SEQ ID NO: 315) p361-374 ERRFSRSDQLKRHQ (SEQ ID NO: 316) p389-410
QRKFSRSDHLKTHTRTHTGKTS (SEQ ID NO: 317) p421-441
CQKKFARSDELVRHHNMHQRN (SEQ ID NO: 318)
[0170] Certain CTL peptides (shown in Table XLVI) were selected for
further study. For each peptide in Table XLVI, scores obtained
using BIMAS HLA peptide binding prediction analysis are
provided.
TABLE-US-00046 TABLE XLVI WT1 Peptide Sequences and HLA Peptide
Binding Predictions Peptide Sequence Comments p329-337 GCNKRYFKL
Score 24,000 (SEQ ID NOs: 90 and 268) p225-233 NLYQMTSQL binds also
to class II (SEQ ID NOs: 147 and 284) and HLA A2, Kd, score 10,000
p235-243 CMTWNQMNL binds also to HLA A2, (SEQ ID NOs: 49 and 258)
score 5,255,712 p126-134 RMFPNAPYL binds also to Kd, (SEQ ID NOs:
185 and 293) class II and HLA A2, score 1,990,800 p221-229
YSSDNLYQM binds also to Ld, (SEQ ID NOs: 253 and 308) score 312,000
p228-236 QMTSQLECM score 3,120 (SEQ ID NOs: 169 and 289) p239-247
NQMNLGATL binds also to HLA A 0201, (SEQ ID NOs: 151 and 286) Kd,
score 8,015 mouse p136-144 SCLESQPTI binds also to Kd, (SEQ ID NO:
296) 1mismatch to human human p136-144 SCLESQPAI score 7,920 (SEQ
ID NO: 198) mouse p10-18 ALLPAVSSL binds also to Kd, HLA A2, (SEQ
ID NO: 255) 1mismatch to human human p10-18 ALLPAVPSL score 6,600
(SEQ ID NO: 34)
[0171] Peptide binding to C57Bl/6 murine MHC was confirmed using
the leukemia cell line RMA-S, as described by Ljunggren et al.,
Nature 346:476-480, 1990. In brief, RMA-S cells were cultured for 7
hours at 26.degree. C. in complete medium supplemented with 1% FCS.
A total of 10.sup.6 RMA-S cells were added into each well of a
24-well plate and incubated either alone or with the designated
peptide (25 ug/ml) for 16 hours at 26.degree. C. and additional 3
hours at 37.degree. C. in complete medium. Cells were then washed
three times and stained with fluorescein isothiocyanate-conjugated
anti D.sup.b or anti-K.sup.b antibody (PharMingen, San Diego,
Calif.). Labeled cells were washed twice, resuspended and fixed in
500 ul of PBS with 1% paraformaldehyde and analyzed for
fluorescence intensity in a flow cytometer (Becton-Dickinson
FACSCALIBUR.TM.). The percentage of increase of D.sup.b or K.sup.b
molecules on the surface of the RMA-S cells was measured by
increased mean fluorescent intensity of cells incubated with
peptide compared with that of cells incubated in medium alone.
[0172] Mice were immunized with the peptides capable of binding to
murine class I MHC. Following immunization, spleen cells were
stimulated in vitro and tested for the ability to lyse targets
incubated with WT1 peptides. CTL were evaluated with a standard
chromium release assay (Chen et al., Cancer Res. 54:1065-1070,
1994). 10.sup.6 target cells were incubated at 37.degree. C. with
150 .mu.Ci of sodium .sup.51Cr for 90 minutes, in the presence or
absence of specific peptides. Cells were washed three times and
resuspended in RPMI with 5% fetal bovine serum. For the assay,
10.sup.4 51Cr-labeled target cells were incubated with different
concentrations of effector cells in a final volume of 200 .mu.l in
U-bottomed 96-well plates. Supernatants were removed after 4 to 7
hours at 37.degree. C., and the percentage specific lysis was
determined by the formula:
% specific lysis=100.times.(experimental release-spontaneous
release)/(maximum release-spontaneous release).
[0173] The results, presented in Table XLVII, show that some WT1
peptides can bind to class I MHC molecules, which is essential for
generating CTL. Moreover, several of the peptides were able to
elicit peptide specific CTL (FIGS. 9A and 9B), as determined using
chromium release assays. Following immunization to CTL peptides
p10-18 human, p136-144 human, p136-144 mouse and p235-243, peptide
specific CTL lines were generated and clones were established.
These results indicate that peptide specific CTL can kill malignant
cells expressing WT1.
TABLE-US-00047 TABLE XLVII Binding of WT1 CTL Peptides to mouse B6
class I antigens Peptide Binding Affinity to Mouse MHC Class I
Positive control 91% negative control 0.5.-1.3% p235-243 33.6%
p136-144 mouse 27.9% p136-144 human 52% p10-18: human 2.2% p225-233
5.8% p329-337 1.2% p126-134 0.9% p221-229 0.8% p228-236 1.2%
p239-247 1%
Example 5
Use of a WT1 Polypeptide to Elicit WT1 Specific CTL in Mice
[0174] This Example illustrates the ability of a representative WT1
polypeptide to elicit CTL immunity capable of killing WT1 positive
tumor cell lines.
[0175] P117-139, a peptide with motifs appropriate for binding to
class I and class II MHC, was identified as described above using
TSITES and BIMAS HLA peptide binding prediction analyses. Mice were
immunized as described in Example 3. Following immunization, spleen
cells were stimulated in vitro and tested for the ability to lyse
targets incubated with WT1 peptides, as well as WT1 positive and
negative tumor cells. CTL were evaluated with a standard chromium
release assay. The results, presented in FIGS. 10A-10D, show that
P117 can elicit WT1 specific CTL capable of killing WT1 positive
tumor cells, whereas no killing of WT1 negative cells was observed.
These results demonstrate that peptide specific CTL in fact kill
malignant cells expressing WT1 and that vaccine and T cell therapy
are effective against malignancies that express WT1.
[0176] Similar immunizations were performed using the 9-mer class I
MHC binding peptides p136-144, p225-233, p235-243 as well as the
23-mer peptide p117-139. Following immunization, spleen cells were
stimulated in vitro with each of the 4 peptides and tested for
ability to lyse targets incubated with WT1 peptides. CTL were
generated specific for p136-144, p235-243 and p117-139, but not for
p225-233. CTL data for p235-243 and p117-139 are presented in FIGS.
11A and 11B. Data for peptides p136-144 and p225-233 are not
depicted.
[0177] CTL lysis demands that the target WT1 peptides are
endogenously processed and presented in association with tumor cell
class I MHC molecules. The above WT1 peptide specific CTL were
tested for ability to lyse WT1 positive versus negative tumor cell
lines. CTL specific for p235-243 lysed targets incubated with the
p235-243 peptides, but failed to lyse cell lines that expressed WT1
proteins (FIG. 11A). By marked contrast, CTL specific for p117-139
lysed targets incubated with p117-139 peptides and also lysed
malignant cells expressing WT1 (FIG. 11B). As a negative control,
CTL specific for p117-139 did not lyse WT1 negative EL-4 (also
referred to herein as E10).
[0178] Specificity of WT1 specific lysis was confirmed by cold
target inhibition (FIGS. 12A-12B). Effector cells were plated for
various effector: target ratios in 96-well U-bottom plates. A
ten-fold excess (compared to hot target) of the indicated
peptide-coated target without .sup.51Cr labeling was added.
Finally, 10.sup.4 51Cr-labeled target cells per well were added and
the plates incubated at 37.degree. C. for 4 hours. The total volume
per well was 200 .mu.l.
[0179] Lysis of TRAMP-C by p117-139 specific CTL was blocked from
58% to 36% by EL-4 incubated with the relevant peptide p117-139,
but not with EL-4 incubated with an irrelevant peptide (FIG. 12A).
Similarly, lysis of BLK-SV40 was blocked from 18% to 0% by EL-4
incubated with the relevant peptide p117-139 (FIG. 12B). Results
validate that WT1 peptide specific CTL specifically kill malignant
cells by recognition of processed WT1.
[0180] Several segments with putative CTL motifs are contained
within p117-139. To determine the precise sequence of the CTL
epitope all potential 9-mer peptides within p117-139 were
synthesized (Table XLVIII). Two of these peptides (p126-134 and
p130-138) were shown to bind to H-2.sup.b class I molecules (Table
XLVIII). CTL generated by immunization with p117-139 lysed targets
incubated with p126-134 and p130-138, but not the other 9-mer
peptides within p117-139 (FIG. 13A).
[0181] The p117-139 specific CTL line was restimulated with either
p126-134 or p130-138. Following restimulation with p126-134 or
p130-138, both T cell lines demonstrated peptide specific lysis,
but only p130-138 specific CTL showed lysis of a WT1 positive tumor
cell line (FIGS. 13B and 13C). Thus, p130-138 appears to be the
naturally processed epitope.
TABLE-US-00048 TABLE XLVIII Binding of WT1 CTL 9mer Peptides within
p117-139 to mouse B6 class I antigens Binding Affinity to Peptide
Mouse MHC Class I P117-125 PSQASSGQA 2% (SEQ ID NO: 221) P118-126
SQASSGQAR 2% (SEQ ID NO: 216) P119-127 QASSGQARM 2% (SEQ ID NOs:
161 and 288) P120-128 ASSGQARMF 1% (SEQ ID NO: 40 P121-129
SSGQARMFP 1% (SEQ ID NO :222) P122-130 SGQARMFPN 1% (SEQ ID NO:
212) P123-131 GQARMFPNA 1% (SEQ ID NOs: 98 and 269) P124-132
QARMFPNAP 1% (SEQ ID NO: 223) P125-133 ARMFPNAPY 1% (SEQ ID NO :38)
P126-134 RMFPNAPYL 79% (SEQ ID NOs: 185 and 293) P127-135 MFPNAPYLP
2% (SEQ ID NO :224) P128-136 FPNAPYLPS 1% (SEQ ID NOs: 79 and 267)
P129-137 PNAPYLPSC 1% (SEQ ID NO: 225) P130-138 NAPYLPSCL 79% (SEQ
ID NOs: 144 and 282) P131-139 APYLPSCLE 1% (SEQ ID NO: 226)
Example 6
Identification of WT1 Specific mRNA in Mouse Tumor Cell Lines
[0182] This Example illustrates the use of RT-PCR to detect WT1
specific mRNA in cells and cell lines.
[0183] Mononuclear cells were isolated by density gradient
centrifugation, and were immediately frozen and stored at
-80.degree. C. until analyzed by RT-PCR for the presence of WT1
specific mRNA. RT-PCR was generally performed as described by
Fraizer et al., Blood 86:4704-4706, 1995. Total RNA was extracted
from 10.sup.7 cells according to standard procedures. RNA pellets
were resuspended in 25 .mu.L diethylpyrocarbonate treated water and
used directly for reverse transcription. The zinc-finger region
(exons 7 to 10) was amplified by PCR as a 330 by mouse cDNA.
Amplification was performed in a thermocycler during one or, when
necessary, two sequential rounds of PCR. AMPLITAQ.RTM. DNA
Polymerase (Perkin Elmer Cetus, Norwalk, Conn.), 2.5 mM MgCl.sub.2
and 20 pmol of each primer in a total reaction volume of 50 .mu.l
were used. Twenty .mu.L aliquots of the PCR products were
electrophoresed on 2% agarose gels stained with ethidium bromide.
The gels were photographed with POLAROID.RTM. film (Polaroid 667,
Polaroid Ltd., Hertfordshire, England). Precautions against cross
contamination were taken following the recommendations of Kwok and
Higuchi, Nature 339:237-238, 1989. Negative controls included the
cDNA- and PCR-reagent mixes with water instead of cDNA in each
experiment. To avoid false negatives, the presence of intact RNA
and adequate cDNA generation was evaluated for each sample by a
control PCR using .beta.-actin primers. Samples that did not
amplify with these primers were excluded from analysis.
[0184] Primers for amplification of WT1 in mouse cell lines were:
P115: 1458-1478: 5' CCC AGG CTG CAA TAA GAG ATA 3' (forward primer;
SEQ ID NO:21); and P116: 1767-1787: 5' ATG TTG TGA TGG CGG ACC AAT
3' (reverse primer; SEQ ID NO:22) (see Inoue et al, Blood
88:2267-2278, 1996; Fraizer et al., Blood 86:4704-4706, 1995).
[0185] Beta Actin primers used in the control reactions were: 5'
GTG GGG CGC CCC AGG CAC CA 3' (sense primer; SEQ ID NO:23); and 5'
GTC CTT AAT GTC ACG CAC GAT TTC 3' (antisense primer; SEQ ID
NO:24)
[0186] Primers for use in amplifying human WT1 include: P117:
954-974: 5' GGC ATC TGA GAC CAG TGA GAA 3' (SEQ ID NO:25); and
P118: 1434-1414: 5' GAG AGT CAG ACT TGA AAG CAGT 3' (SEQ ID NO:5).
For nested RT-PCR, primers may be: P119: 1023-1043: 5' GCT GTC CCA
CTT ACA GAT GCA 3' (SEQ ID NO:26); and P120: 1345-1365: 5' TCA AAG
CGC CAG CTG GAG TTT 3' (SEQ ID NO:27).
[0187] Table XLVIII shows the results of WT1 PCR analysis of mouse
tumor cell lines. Within Table IV, (+++) indicates a strong WT1 PCR
amplification product in the first step RT PCR, (++) indicates a
WT1 amplification product that is detectable by first step WT1 RT
PCR, (+) indicates a product that is detectable only in the second
step of WT1 RT PCR, and (-) indicates WT1 PCR negative.
TABLE-US-00049 TABLE XLIX Detection of WT1 mRNA in Mouse Tumor Cell
Lines WT1 Cell Line mRNA K562 (human leukemia; ATCC): Positive
control; (Lozzio +++ and Lozzio, Blood 45: 321-334, 1975) TRAMPC
(SV40 transformed prostate, B6); Foster et al., +++ Cancer Res. 57:
3325-3330, 1997 BLK-SV40 HD2 (SV40-transf. fibroblast, B6; ATCC);
Nature ++ 276: 510-511, 1978 CTLL (T-cell, B6; ATCC); Gillis,
Nature 265: 154-156, + 1977) FM (FBL-3 subline, leukemia, B6);
Glynn and Fefer, Cancer + Res. 28: 434-439, 1968 BALB 3T3 (ATCC);
Aaroston and Todaro, J. Cell. Physiol. + 72: 141-148, 1968 S49.1
(Lymphoma, T-cell like, B/C; ATCC); Horibata and + Harris, Exp.
Cell. Res. 60: 61, 1970 BNL CL.2 (embryonic liver, B/C; ATCC);
Nature 276: 510- + 511, 1978 MethA (sarcoma, B/C); Old et al., Ann.
NY Acad. Sci. 101: - 80-106, 1962 P3.6.2.8.1 (myeloma, B/C; ATCC);
Proc. Natl. Acad. Sci. - USA 66: 344, 1970 P2N (leukemia, DBA/2;
ATCC); Melling et al., J. Immunol. - 117: 1267-1274, 1976 BCL1
(lymphoma, B/C; ATCC); Slavin and Strober, Nature - 272: 624-626,
1977 LSTRA (lymphoma, B/C); Glynn et al., Cancer Res. 28: 434- -
439, 1968 E10/EL-4 (lymphoma, B6); Glynn et al., Cancer Res. 28: -
434-439, 1968
[0188] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
326117PRTHomo sapiens 1Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro
Ser Leu Gly Gly Gly1 5 10 15Gly223PRTHomo sapiens 2Pro Ser Gln Ala
Ser Ser Gly Gln Ala Arg Met Phe Pro Asn Ala Pro1 5 10 15Tyr Leu Pro
Ser Cys Leu Glu 20323PRTMus musculus 3Pro Ser Gln Ala Ser Ser Gly
Gln Ala Arg Met Phe Pro Asn Ala Pro1 5 10 15Tyr Leu Pro Ser Cys Leu
Glu 20419PRTHomo sapiens 4Gly Ala Thr Leu Lys Gly Val Ala Ala Gly
Ser Ser Ser Ser Val Lys1 5 10 15Trp Thr Glu522DNAArtificial
SequencePrimer for use in amplifying human WT1 5gagagtcaga
cttgaaagca gt 22620DNAArtificial SequencePrimer for use in
amplifying human WT1 6ctgagcctca gcaaatgggc 20727DNAArtificial
SequencePrimer for use in amplifying human WT1 7gagcatgcat
gggctccgac gtgcggg 27825DNAArtificial SequencePrimer for use in
amplifying human WT1 8ggggtaccca ctgaacggtc cccga
25918DNAArtificial SequencePrimer for use in amplifying mouse WT1
9tccgagccgc acctcatg 181018DNAArtificial SequencePrimer for use in
amplifying mouse WT1 10gcctgggatg ctggactg 181127DNAArtificial
SequencePrimer for use in amplifying mouse WT1 11gagcatgcga
tgggttccga cgtgcgg 271229DNAArtificial SequencePrimer for use in
amplifying mouse WT1 12ggggtacctc aaagcgccac gtggagttt 291317PRTMus
musculus 13Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Ser Ser Leu Gly
Gly Gly1 5 10 15Gly1419PRTMus musculus 14Gly Ala Thr Leu Lys Gly
Met Ala Ala Gly Ser Ser Ser Ser Val Lys1 5 10 15Trp Thr
Glu1515PRTHomo sapiens 15Arg Ile His Thr His Gly Val Phe Arg Gly
Ile Gln Asp Val Arg1 5 10 151615PRTMus musculus 16Arg Ile His Thr
His Gly Val Phe Arg Gly Ile Gln Asp Val Arg1 5 10 151714PRTMus
musculus 17Val Arg Arg Val Ser Gly Val Ala Pro Thr Leu Val Arg Ser1
5 101814PRTHomo sapiens 18Val Arg Arg Val Pro Gly Val Ala Pro Thr
Leu Val Arg Ser1 5 101915PRTHomo sapiens 19Cys Gln Lys Lys Phe Ala
Arg Ser Asp Glu Leu Val Arg His His1 5 10 152015PRTMus musculus
20Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val Arg His His1 5 10
152121DNAArtificial Sequencesense primer for amplification of WT1
in mouse cell lines 21cccaggctgc aataagagat a 212221DNAArtificial
Sequenceantisense primer for amplification of WT1 in mouse cell
lines 22atgttgtgat ggcggaccaa t 212320DNAArtificial Sequencesense
Beta Actin primer used in the control reactions 23gtggggcgcc
ccaggcacca 202424DNAArtificial Sequenceantisense Beta Actin primer
used in the control reactions 24gtccttaatg ctacgcacga tttc
242521DNAArtificial SequencePrimer for use in amplifying human WT1
25ggcatctgag accagtgaga a 212621DNAArtificial SequencePrimer for
use in nested RT-PCR 26gctgtcccac ttacagatgc a 212721DNAArtificial
SequencePrimer for use in nested RT-PCR 27tcaaagcgcc agctggagtt t
21289PRTHomo sapiens 28Ala Ala Gly Ser Ser Ser Ser Val Lys1
5299PRTHomo sapiens 29Ala Ala Gln Phe Pro Asn His Ser Phe1
5309PRTHomo sapiens 30Ala Glu Pro His Glu Glu Gln Cys Leu1
5319PRTHomo sapiens 31Ala Gly Ala Cys Arg Tyr Gly Pro Phe1
5329PRTHomo sapiens 32Ala Gly Ser Ser Ser Ser Val Lys Trp1
5339PRTHomo sapiens 33Ala Ile Arg Asn Gln Gly Tyr Ser Thr1
5349PRTHomo sapiens 34Ala Leu Leu Pro Ala Val Pro Ser Leu1
5359PRTHomo sapiens 35Ala Leu Leu Pro Ala Val Ser Ser Leu1
5369PRTHomo sapiens 36Ala Gln Phe Pro Asn His Ser Phe Lys1
5379PRTHomo sapiens 37Ala Gln Trp Ala Pro Val Leu Asp Phe1
5389PRTHomo sapiens 38Ala Arg Met Phe Pro Asn Ala Pro Tyr1
5399PRTHomo sapiens 39Ala Arg Ser Asp Glu Leu Val Arg His1
5409PRTHomo sapiens 40Ala Ser Ser Gly Gln Ala Arg Met Phe1
5419PRTHomo sapiens 41Ala Tyr Gly Ser Leu Gly Gly Pro Ala1
5429PRTHomo sapiens 42Ala Tyr Pro Gly Cys Asn Lys Arg Tyr1
5439PRTHomo sapiens 43Cys Ala Leu Pro Val Ser Gly Ala Ala1
5449PRTHomo sapiens 44Cys Ala Tyr Pro Gly Cys Asn Lys Arg1
5459PRTHomo sapiens 45Cys His Thr Pro Thr Asp Ser Cys Thr1
5469PRTHomo sapiens 46Cys Lys Thr Cys Gln Arg Lys Phe Ser1
5479PRTHomo sapiens 47Cys Leu Glu Ser Gln Pro Ala Ile Arg1
5489PRTHomo sapiens 48Cys Leu Ser Ala Phe Thr Val His Phe1
5499PRTHomo sapiens 49Cys Met Thr Trp Asn Gln Met Asn Leu1
5509PRTHomo sapiens 50Cys Arg Trp Pro Ser Cys Gln Lys Lys1
5519PRTHomo sapiens 51Cys Arg Tyr Gly Pro Phe Gly Pro Pro1
5529PRTHomo sapiens 52Cys Thr Gly Ser Gln Ala Leu Leu Leu1
5539PRTHomo sapiens 53Asp Glu Leu Val Arg His His Asn Met1
5549PRTHomo sapiens 54Asp Phe Ala Pro Pro Gly Ala Ser Ala1
5559PRTHomo sapiens 55Asp Phe Lys Asp Cys Glu Arg Arg Phe1
5569PRTHomo sapiens 56Asp Gly Thr Pro Ser Tyr Gly His Thr1
5579PRTHomo sapiens 57Asp His Leu Lys Thr His Thr Arg Thr1
5589PRTHomo sapiens 58Asp Leu Asn Ala Leu Leu Pro Ala Val1
5599PRTHomo sapiens 59Asp Pro Met Gly Gln Gln Gly Ser Leu1
5609PRTHomo sapiens 60Asp Gln Leu Lys Arg His Gln Arg Arg1
5619PRTHomo sapiens 61Asp Ser Cys Thr Gly Ser Gln Ala Leu1
5629PRTHomo sapiens 62Asp Val Arg Asp Leu Asn Ala Leu Leu1
5639PRTHomo sapiens 63Asp Val Arg Arg Val Pro Gly Val Ala1
5649PRTHomo sapiens 64Glu Asp Pro Met Gly Gln Gln Gly Ser1
5659PRTHomo sapiens 65Glu Glu Gln Cys Leu Ser Ala Phe Thr1
5669PRTHomo sapiens 66Glu Lys Pro Tyr Gln Cys Asp Phe Lys1
5679PRTHomo sapiens 67Glu Lys Arg Pro Phe Met Cys Ala Tyr1
5689PRTHomo sapiens 68Glu Pro His Glu Glu Gln Cys Leu Ser1
5699PRTHomo sapiens 69Glu Gln Cys Leu Ser Ala Phe Thr Val1
5709PRTHomo sapiens 70Glu Ser Asp Asn His Thr Ala Pro Ile1
5719PRTHomo sapiens 71Glu Ser Asp Asn His Thr Thr Pro Ile1
5729PRTHomo sapiens 72Glu Ser Gln Pro Ala Ile Arg Asn Gln1
5739PRTHomo sapiens 73Glu Thr Ser Glu Lys Arg Pro Phe Met1
5749PRTHomo sapiens 74Phe Ala Pro Pro Gly Ala Ser Ala Tyr1
5759PRTHomo sapiens 75Phe Ala Arg Ser Asp Glu Leu Val Arg1
5769PRTHomo sapiens 76Phe Gly Pro Pro Pro Pro Ser Gln Ala1
5779PRTHomo sapiens 77Phe Lys Asp Cys Glu Arg Arg Phe Ser1
5789PRTHomo sapiens 78Phe Lys Leu Ser His Leu Gln Met His1
5799PRTHomo sapiens 79Phe Pro Asn Ala Pro Tyr Leu Pro Ser1
5809PRTHomo sapiens 80Phe Gln Cys Lys Thr Cys Gln Arg Lys1
5819PRTHomo sapiens 81Phe Arg Gly Ile Gln Asp Val Arg Arg1
5829PRTHomo sapiens 82Phe Ser Gly Gln Phe Thr Gly Thr Ala1
5839PRTHomo sapiens 83Phe Ser Arg Ser Asp Gln Leu Lys Arg1
5849PRTHomo sapiens 84Phe Thr Gly Thr Ala Gly Ala Cys Arg1
5859PRTHomo sapiens 85Phe Thr Val His Phe Ser Gly Gln Phe1
5869PRTHomo sapiens 86Gly Ala Ala Gln Trp Ala Pro Val Leu1
5879PRTHomo sapiens 87Gly Ala Glu Pro His Glu Glu Gln Cys1
5889PRTHomo sapiens 88Gly Ala Thr Leu Lys Gly Val Ala Ala1
5899PRTHomo sapiens 89Gly Cys Ala Leu Pro Val Ser Gly Ala1
5909PRTHomo sapiens 90Gly Cys Asn Lys Arg Tyr Phe Lys Leu1
5919PRTHomo sapiens 91Gly Glu Lys Pro Tyr Gln Cys Asp Phe1
5929PRTHomo sapiens 92Gly Gly Gly Gly Cys Ala Leu Pro Val1
5939PRTHomo sapiens 93Gly Gly Pro Ala Pro Pro Pro Ala Pro1
5949PRTHomo sapiens 94Gly His Thr Pro Ser His His Ala Ala1
5959PRTHomo sapiens 95Gly Lys Thr Ser Glu Lys Pro Phe Ser1
5969PRTHomo sapiens 96Gly Pro Phe Gly Pro Pro Pro Pro Ser1
5979PRTHomo sapiens 97Gly Pro Pro Pro Pro Ser Gln Ala Ser1
5989PRTHomo sapiens 98Gly Gln Ala Arg Met Phe Pro Asn Ala1
5999PRTHomo sapiens 99Gly Gln Phe Thr Gly Thr Ala Gly Ala1
51009PRTHomo sapiens 100Gly Gln Ser Asn His Ser Thr Gly Tyr1
51019PRTHomo sapiens 101Gly Ser Asp Val Arg Asp Leu Asn Ala1
51029PRTHomo sapiens 102Gly Ser Gln Ala Leu Leu Leu Arg Thr1
51039PRTHomo sapiens 103Gly Val Phe Arg Gly Ile Gln Asp Val1
51049PRTHomo sapiens 104Gly Val Lys Pro Phe Gln Cys Lys Thr1
51059PRTHomo sapiens 105Gly Tyr Glu Ser Asp Asn His Thr Ala1
51069PRTHomo sapiens 106Gly Tyr Glu Ser Asp Asn His Thr Thr1
51079PRTHomo sapiens 107His Glu Glu Gln Cys Leu Ser Ala Phe1
51089PRTHomo sapiens 108His His Asn Met His Gln Arg Asn Met1
51099PRTHomo sapiens 109His Gln Arg Arg His Thr Gly Val Lys1
51109PRTHomo sapiens 110His Ser Phe Lys His Glu Asp Pro Met1
51119PRTHomo sapiens 111His Ser Arg Lys His Thr Gly Glu Lys1
51129PRTHomo sapiens 112His Thr Gly Glu Lys Pro Tyr Gln Cys1
51139PRTHomo sapiens 113His Thr His Gly Val Phe Arg Gly Ile1
51149PRTHomo sapiens 114His Thr Arg Thr His Thr Gly Lys Thr1
51159PRTHomo sapiens 115His Thr Thr Pro Ile Leu Cys Gly Ala1
51169PRTHomo sapiens 116Ile Leu Cys Gly Ala Gln Tyr Arg Ile1
51179PRTHomo sapiens 117Ile Arg Asn Gln Gly Tyr Ser Thr Val1
51189PRTHomo sapiens 118Lys Asp Cys Glu Arg Arg Phe Ser Arg1
51199PRTHomo sapiens 119Lys Phe Ala Arg Ser Asp Glu Leu Val1
51209PRTHomo sapiens 120Lys Phe Ser Arg Ser Asp His Leu Lys1
51219PRTHomo sapiens 121Lys His Glu Asp Pro Met Gly Gln Gln1
51229PRTHomo sapiens 122Lys Lys Phe Ala Arg Ser Asp Glu Leu1
51239PRTHomo sapiens 123Lys Pro Phe Ser Cys Arg Trp Pro Ser1
51249PRTHomo sapiens 124Lys Pro Tyr Gln Cys Asp Phe Lys Asp1
51259PRTHomo sapiens 125Lys Gln Glu Pro Ser Trp Gly Gly Ala1
51269PRTHomo sapiens 126Lys Arg His Gln Arg Arg His Thr Gly1
51279PRTHomo sapiens 127Lys Arg Tyr Phe Lys Leu Ser His Leu1
51289PRTHomo sapiens 128Lys Thr Cys Gln Arg Lys Phe Ser Arg1
51299PRTHomo sapiens 129Lys Thr Ser Glu Lys Pro Phe Ser Cys1
51309PRTHomo sapiens 130Leu Asp Phe Ala Pro Pro Gly Ala Ser1
51319PRTHomo sapiens 131Leu Glu Cys Met Thr Trp Asn Gln Met1
51329PRTHomo sapiens 132Leu Glu Ser Gln Pro Ala Ile Arg Asn1
51339PRTHomo sapiens 133Leu Gly Ala Thr Leu Lys Gly Val Ala1
51349PRTHomo sapiens 134Leu Gly Gly Gly Gly Gly Cys Ala Leu1
51359PRTHomo sapiens 135Leu Lys Gly Val Ala Ala Gly Ser Ser1
51369PRTHomo sapiens 136Leu Lys Arg His Gln Arg Arg His Thr1
51379PRTHomo sapiens 137Leu Lys Thr His Thr Arg Thr His Thr1
51389PRTHomo sapiens 138Leu Pro Val Ser Gly Ala Ala Gln Trp1
51399PRTHomo sapiens 139Leu Gln Met His Ser Arg Lys His Thr1
51409PRTHomo sapiens 140Leu Arg Thr Pro Tyr Ser Ser Asp Asn1
51419PRTHomo sapiens 141Leu Ser His Leu Gln Met His Ser Arg1
51429PRTHomo sapiens 142Met Cys Ala Tyr Pro Gly Cys Asn Lys1
51439PRTHomo sapiens 143Met His Gln Arg Asn Met Thr Lys Leu1
51449PRTHomo sapiens 144Asn Ala Pro Tyr Leu Pro Ser Cys Leu1
51459PRTHomo sapiens 145Asn Lys Arg Tyr Phe Lys Leu Ser His1
51469PRTHomo sapiens 146Asn Leu Gly Ala Thr Leu Lys Gly Val1
51479PRTHomo sapiens 147Asn Leu Tyr Gln Met Thr Ser Gln Leu1
51489PRTHomo sapiens 148Asn Met His Gln Arg Asn Met Thr Lys1
51499PRTHomo sapiens 149Asn Met Thr Lys Leu Gln Leu Ala Leu1
51509PRTHomo sapiens 150Asn Gln Gly Tyr Ser Thr Val Thr Phe1
51519PRTHomo sapiens 151Asn Gln Met Asn Leu Gly Ala Thr Leu1
51529PRTHomo sapiens 152Pro Ala Ile Arg Asn Gln Gly Tyr Ser1
51539PRTHomo sapiens 153Pro Gly Ala Ser Ala Tyr Gly Ser Leu1
51549PRTHomo sapiens 154Pro His Glu Glu Gln Cys Leu Ser Ala1
51559PRTHomo sapiens 155Pro Ile Leu Cys Gly Ala Gln Tyr Arg1
51569PRTHomo sapiens 156Pro Pro Pro Pro His Ser Phe Ile Lys1
51579PRTHomo sapiens 157Pro Pro Pro Pro Pro His Ser Phe Ile1
51589PRTHomo sapiens 158Pro Pro Pro Pro Pro Pro His Ser Phe1
51599PRTHomo sapiens 159Pro Ser Cys Gln Lys Lys Phe Ala Arg1
51609PRTHomo sapiens 160Gln Ala Leu Leu Leu Arg Thr Pro Tyr1
51619PRTHomo sapiens 161Gln Ala Ser Ser Gly Gln Ala Arg Met1
51629PRTHomo sapiens 162Gln Cys Asp Phe Lys Asp Cys Glu Arg1
51639PRTHomo sapiens 163Gln Cys Lys Thr Cys Gln Arg Lys Phe1
51649PRTHomo sapiens 164Gln Asp Val Arg Arg Val Pro Gly Val1
51659PRTHomo sapiens 165Gln Phe Thr Gly Thr Ala Gly Ala Cys1
51669PRTHomo sapiens 166Gln Gly Ser Leu Gly Glu Gln Gln Tyr1
51679PRTHomo sapiens 167Gln Leu Glu Cys Met Thr Trp Asn Gln1
51689PRTHomo sapiens 168Gln Met Asn Leu Gly Ala Thr Leu Lys1
51699PRTHomo sapiens 169Gln Met Thr Ser Gln Leu Glu Cys Met1
51709PRTHomo sapiens 170Gln Pro Ala Ile Arg Asn Gln Gly Tyr1
51719PRTHomo sapiens 171Gln Gln Tyr Ser Val Pro Pro Pro Val1
51729PRTHomo sapiens 172Gln Arg Lys Phe Ser Arg Ser Asp His1
51739PRTHomo sapiens 173Gln Arg Asn Met Thr Lys Leu Gln Leu1
51749PRTHomo sapiens 174Gln Trp Ala Pro Val Leu Asp Phe Ala1
51759PRTHomo sapiens 175Gln Tyr Arg Ile His Thr His Gly Val1
51769PRTHomo sapiens 176Gln Tyr Ser Val Pro Pro Pro Val Tyr1
51779PRTHomo sapiens 177Arg Asp Leu Asn Ala Leu Leu Pro Ala1
51789PRTHomo sapiens 178Arg Phe Ser Arg Ser Asp Gln Leu Lys1
51799PRTHomo sapiens 179Arg Gly Ile Gln Asp Val Arg Arg Val1
51809PRTHomo sapiens 180Arg His His Asn Met His Gln Arg Asn1
51819PRTHomo sapiens 181Arg His Gln Arg Arg His Thr Gly Val1
51829PRTHomo sapiens 182Arg Ile His Thr His Gly Val Phe Arg1
51839PRTHomo sapiens 183Arg Lys Phe Ser Arg Ser Asp His Leu1
51849PRTHomo sapiens 184Arg Lys His Thr Gly Glu Lys Pro Tyr1
51859PRTHomo sapiens 185Arg Met Phe Pro Asn Ala Pro Tyr Leu1
51869PRTHomo sapiens 186Arg Asn Met Thr Lys Leu Gln Leu Ala1
51879PRTHomo sapiens 187Arg Arg Phe Ser Arg Ser Asp Gln Leu1
51889PRTHomo sapiens 188Arg Arg His Thr Gly Val Lys Pro Phe1
51899PRTHomo sapiens 189Arg Arg Val Pro Gly Val Ala Pro Thr1
51909PRTHomo sapiens 190Arg Ser Ala Ser Glu Thr Ser Glu Lys1
51919PRTHomo sapiens 191Arg Ser Asp Glu Leu Val Arg His His1
51929PRTHomo sapiens 192Arg Ser Asp His Leu Lys Thr His Thr1
51939PRTHomo sapiens 193Arg Ser Asp Gln Leu Lys Arg His Gln1
51949PRTHomo sapiens 194Arg Thr Pro Tyr Ser Ser Asp Asn Leu1
51959PRTHomo sapiens 195Arg Val Pro Gly Val Ala Pro Thr Leu1
51969PRTHomo sapiens 196Arg Trp Pro Ser Cys Gln Lys Lys Phe1
51979PRTHomo sapiens 197Ser Ala Ser Glu Thr Ser Glu Lys Arg1
51989PRTHomo sapiens 198Ser Cys Leu Glu Ser Gln Pro Ala Ile1
51999PRTHomo sapiens 199Ser Cys Leu Glu Ser Gln Pro Thr Ile1
52009PRTHomo sapiens 200Ser Cys Gln Lys Lys Phe Ala Arg Ser1
52019PRTHomo sapiens 201Ser Cys Arg Trp Pro Ser Cys Gln Lys1
52029PRTHomo sapiens 202Ser Cys Thr Gly Ser Gln Ala Leu Leu1
52039PRTHomo sapiens 203Ser Asp Glu Leu Val Arg His His Asn1
52049PRTHomo sapiens 204Ser Asp Asn His Thr Thr Pro Ile Leu1
52059PRTHomo sapiens 205Ser Asp Asn Leu Tyr Gln Met Thr Ser1
52069PRTHomo sapiens 206Ser Asp Val Arg Asp Leu Asn Ala Leu1
52079PRTHomo sapiens 207Ser Glu Lys Pro Phe Ser Cys Arg Trp1
52089PRTHomo sapiens 208Ser Glu Lys Arg Pro Phe Met Cys Ala1
52099PRTHomo sapiens 209Ser Glu Thr Ser Glu Lys Arg Pro Phe1
52109PRTHomo sapiens 210Ser Phe Ile Lys Gln Glu Pro Ser Trp1
52119PRTHomo sapiens 211Ser Gly Ala Ala Gln Trp Ala Pro Val1
52129PRTHomo sapiens 212Ser Gly Gln Ala Arg Met Phe Pro Asn1
52139PRTHomo sapiens 213Ser His His Ala Ala Gln Phe Pro Asn1
52149PRTHomo sapiens 214Ser Leu Gly Glu Gln Gln Tyr Ser Val1
52159PRTHomo sapiens 215Ser Leu Gly Gly Gly Gly Gly Cys Ala1
52169PRTHomo sapiens 216Ser Gln Ala Ser Ser Gly Gln Ala Arg1
52179PRTHomo sapiens 217Ser Ser Asp Asn Leu Tyr Gln Met Thr1
52189PRTHomo sapiens 218Ser Val Pro Pro Pro Val Tyr Gly Cys1
52199PRTHomo sapiens
219Thr Cys Gln Arg Lys Phe Ser Arg Ser1 52209PRTHomo sapiens 220Thr
Asp Ser Cys Thr Gly Ser Gln Ala1 52219PRTHomo sapiens 221Thr Glu
Gly Gln Ser Asn His Ser Thr1 52229PRTHomo sapiens 222Thr Gly Lys
Thr Ser Glu Lys Pro Phe1 52239PRTHomo sapiens 223Thr Gly Ser Gln
Ala Leu Leu Leu Arg1 52249PRTHomo sapiens 224Thr Gly Thr Ala Gly
Ala Cys Arg Tyr1 52259PRTHomo sapiens 225Thr Gly Tyr Glu Ser Asp
Asn His Thr1 52269PRTHomo sapiens 226Thr Leu Val Arg Ser Ala Ser
Glu Thr1 52279PRTHomo sapiens 227Thr Pro Ile Leu Cys Gly Ala Gln
Tyr1 52289PRTHomo sapiens 228Thr Pro Ser His His Ala Ala Gln Phe1
52299PRTHomo sapiens 229Thr Pro Ser Tyr Gly His Thr Pro Ser1
52309PRTHomo sapiens 230Thr Pro Thr Asp Ser Cys Thr Gly Ser1
52319PRTHomo sapiens 231Thr Pro Tyr Ser Ser Asp Asn Leu Tyr1
52329PRTHomo sapiens 232Thr Ser Glu Lys Pro Phe Ser Cys Arg1
52339PRTHomo sapiens 233Thr Ser Glu Lys Arg Pro Phe Met Cys1
52349PRTHomo sapiens 234Thr Ser Gln Leu Glu Cys Met Thr Trp1
52359PRTHomo sapiens 235Thr Val His Phe Ser Gly Gln Phe Thr1
52369PRTHomo sapiens 236Val Ala Ala Gly Ser Ser Ser Ser Val1
52379PRTHomo sapiens 237Val Ala Pro Thr Leu Val Arg Ser Ala1
52389PRTHomo sapiens 238Val Phe Arg Gly Ile Gln Asp Val Arg1
52399PRTHomo sapiens 239Val Lys Pro Phe Gln Cys Lys Thr Cys1
52409PRTHomo sapiens 240Val Lys Trp Thr Glu Gly Gln Ser Asn1
52419PRTHomo sapiens 241Val Leu Asp Phe Ala Pro Pro Gly Ala1
52429PRTHomo sapiens 242Val Pro Gly Val Ala Pro Thr Leu Val1
52439PRTHomo sapiens 243Val Arg His His Asn Met His Gln Arg1
52449PRTHomo sapiens 244Val Thr Phe Asp Gly Thr Pro Ser Tyr1
52459PRTHomo sapiens 245Trp Asn Gln Met Asn Leu Gly Ala Thr1
52469PRTHomo sapiens 246Trp Pro Ser Cys Gln Lys Lys Phe Ala1
52479PRTHomo sapiens 247Trp Thr Glu Gly Gln Ser Asn His Ser1
52489PRTHomo sapiens 248Tyr Phe Lys Leu Ser His Leu Gln Met1
52499PRTHomo sapiens 249Tyr Gly His Thr Pro Ser His His Ala1
52509PRTHomo sapiens 250Tyr Pro Gly Cys Asn Lys Arg Tyr Phe1
52519PRTHomo sapiens 251Tyr Gln Met Thr Ser Gln Leu Glu Cys1
52529PRTHomo sapiens 252Tyr Arg Ile His Thr His Gly Val Phe1
52539PRTHomo sapiens 253Tyr Ser Ser Asp Asn Leu Tyr Gln Met1
52549PRTMus musculus 254Ala Glu Pro His Glu Glu Gln Cys Leu1
52559PRTMus musculus 255Ala Leu Leu Pro Ala Val Ser Ser Leu1
52569PRTMus musculus 256Ala Tyr Gly Ser Leu Gly Gly Pro Ala1
52579PRTMus musculus 257Ala Tyr Pro Gly Cys Asn Lys Arg Tyr1
52589PRTMus musculus 258Cys Met Thr Trp Asn Gln Met Asn Leu1
52599PRTMus musculus 259Cys Thr Gly Ser Gln Ala Leu Leu Leu1
52609PRTMus musculus 260Asp Gly Ala Pro Ser Tyr Gly His Thr1
52619PRTMus musculus 261Asp Leu Asn Ala Leu Leu Pro Ala Val1
52629PRTMus musculus 262Asp Pro Met Gly Gln Gln Gly Ser Leu1
52639PRTMus musculus 263Asp Ser Cys Thr Gly Ser Gln Ala Leu1
52649PRTMus musculus 264Asp Val Arg Asp Leu Asn Ala Leu Leu1
52659PRTMus musculus 265Glu Gln Cys Leu Ser Ala Phe Thr Leu1
52669PRTMus musculus 266Glu Ser Asp Asn His Thr Ala Pro Ile1
52679PRTMus musculus 267Phe Pro Asn Ala Pro Tyr Leu Pro Ser1
52689PRTMus musculus 268Gly Cys Asn Lys Arg Tyr Phe Lys Leu1
52699PRTMus musculus 269Gly Gln Ala Arg Met Phe Pro Asn Ala1
52709PRTMus musculus 270Gly Val Phe Arg Gly Ile Gln Asp Val1
52719PRTMus musculus 271Gly Tyr Glu Ser Asp Asn His Thr Ala1
52729PRTMus musculus 272His Ser Phe Lys His Glu Asp Pro Met1
52739PRTMus musculus 273His Thr His Gly Val Phe Arg Gly Ile1
52749PRTMus musculus 274Ile Leu Cys Gly Ala Gln Tyr Arg Ile1
52759PRTMus musculus 275Lys Phe Ala Arg Ser Asp Glu Leu Val1
52769PRTMus musculus 276Lys Arg Tyr Phe Lys Leu Ser His Leu1
52779PRTMus musculus 277Lys Thr Ser Glu Lys Pro Phe Ser Cys1
52789PRTMus musculus 278Leu Glu Cys Met Thr Trp Asn Gln Met1
52799PRTMus musculus 279Leu Gly Gly Gly Gly Gly Cys Gly Leu1
52809PRTMus musculus 280Leu Gln Met His Ser Arg Lys His Thr1
52819PRTMus musculus 281Met His Gln Arg Asn Met Thr Lys Leu1
52829PRTMus musculus 282Asn Ala Pro Tyr Leu Pro Ser Cys Leu1
52839PRTMus musculus 283Asn Leu Gly Ala Thr Leu Lys Gly Met1
52849PRTMus musculus 284Asn Leu Tyr Gln Met Thr Ser Gln Leu1
52859PRTMus musculus 285Asn Met Thr Lys Leu His Val Ala Leu1
52869PRTMus musculus 286Asn Gln Met Asn Leu Gly Ala Thr Leu1
52879PRTMus musculus 287Pro Gly Ala Ser Ala Tyr Gly Ser Leu1
52889PRTMus musculus 288Gln Ala Ser Ser Gly Gln Ala Arg Met1
52899PRTMus musculus 289Gln Met Thr Ser Gln Leu Glu Cys Met1
52909PRTMus musculus 290Gln Gln Tyr Ser Val Pro Pro Pro Val1
52919PRTMus musculus 291Gln Tyr Arg Ile His Thr His Gly Val1
52929PRTMus musculus 292Gln Tyr Ser Val Pro Pro Pro Val Tyr1
52939PRTMus musculus 293Arg Met Phe Pro Asn Ala Pro Tyr Leu1
52949PRTMus musculus 294Arg Thr Pro Tyr Ser Ser Asp Asn Leu1
52959PRTMus musculus 295Arg Val Ser Gly Val Ala Pro Thr Leu1
52969PRTMus musculus 296Ser Cys Leu Glu Ser Gln Pro Thr Ile1
52979PRTMus musculus 297Ser Cys Gln Lys Lys Phe Ala Arg Ser1
52989PRTMus musculus 298Ser Asp Val Arg Asp Leu Asn Ala Leu1
52999PRTMus musculus 299Ser Leu Gly Glu Gln Gln Tyr Ser Val1
53009PRTMus musculus 300Thr Cys Gln Arg Lys Phe Ser Arg Ser1
53019PRTMus musculus 301Thr Glu Gly Gln Ser Asn His Gly Ile1
53029PRTMus musculus 302Thr Leu His Phe Ser Gly Gln Phe Thr1
53039PRTMus musculus 303Thr Leu Val Arg Ser Ala Ser Glu Thr1
53049PRTMus musculus 304Val Leu Asp Phe Ala Pro Pro Gly Ala1
53059PRTMus musculus 305Trp Asn Gln Met Asn Leu Gly Ala Thr1
53069PRTMus musculus 306Tyr Phe Lys Leu Ser His Leu Gln Met1
53079PRTMus musculus 307Tyr Gln Met Thr Ser Gln Leu Glu Cys1
53089PRTMus musculus 308Tyr Ser Ser Asp Asn Leu Tyr Gln Met1
53096PRTHomo sapiens 309Gly Ala Ala Gln Trp Ala1 531012PRTHomo
sapiens 310Ala Ser Ala Tyr Gly Ser Leu Gly Gly Pro Ala Pro1 5
1031115PRTHomo sapiens 311Ala Phe Thr Val His Phe Ser Gly Gln Phe
Thr Gly Thr Ala Gly1 5 10 153125PRTHomo sapiens 312His Ala Ala Gln
Phe1 531332PRTHomo sapiens 313Cys His Thr Pro Thr Asp Ser Cys Thr
Gly Ser Gln Ala Leu Leu Leu1 5 10 15Arg Thr Pro Tyr Ser Ser Asp Asn
Leu Tyr Gln Met Thr Ser Gln Leu 20 25 3031432PRTHomo sapiens 314Arg
Ile His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg1 5 10
15Val Pro Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser
20 25 303154PRTHomo sapiens 315Arg Tyr Phe Lys131614PRTHomo sapiens
316Glu Arg Arg Phe Ser Arg Ser Asp Gln Leu Lys Arg His Gln1 5
1031722PRTHomo sapiens 317Gln Arg Lys Phe Ser Arg Ser Asp His Leu
Lys Thr His Thr Arg Thr1 5 10 15His Thr Gly Lys Thr Ser
2031821PRTHomo sapiens 318Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu
Leu Val Arg His His Asn1 5 10 15Met His Gln Arg Asn 20319449PRTHomo
sapiens 319Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala
Val Pro1 5 10 15Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser
Gly Ala Ala 20 25 30Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly
Ala Ser Ala Tyr 35 40 45Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala
Pro Pro Pro Pro Pro 50 55 60Pro Pro Pro Pro His Ser Phe Ile Lys Gln
Glu Pro Ser Trp Gly Gly65 70 75 80Ala Glu Pro His Glu Glu Gln Cys
Leu Ser Ala Phe Thr Val His Phe 85 90 95Ser Gly Gln Phe Thr Gly Thr
Ala Gly Ala Cys Arg Tyr Gly Pro Phe 100 105 110Gly Pro Pro Pro Pro
Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe 115 120 125Pro Asn Ala
Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile 130 135 140Arg
Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr145 150
155 160Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser
Phe 165 170 175Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly
Glu Gln Gln 180 185 190Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His
Thr Pro Thr Asp Ser 195 200 205Cys Thr Gly Ser Gln Ala Leu Leu Leu
Arg Thr Pro Tyr Ser Ser Asp 210 215 220Asn Leu Tyr Gln Met Thr Ser
Gln Leu Glu Cys Met Thr Trp Asn Gln225 230 235 240Met Asn Leu Gly
Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser 245 250 255Ser Val
Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu 260 265
270Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile
275 280 285His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg
Val Pro 290 295 300Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu
Thr Ser Glu Lys305 310 315 320Arg Pro Phe Met Cys Ala Tyr Pro Gly
Cys Asn Lys Arg Tyr Phe Lys 325 330 335Leu Ser His Leu Gln Met His
Ser Arg Lys His Thr Gly Glu Lys Pro 340 345 350Tyr Gln Cys Asp Phe
Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp 355 360 365Gln Leu Lys
Arg His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln 370 375 380Cys
Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr385 390
395 400His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser
Cys 405 410 415Arg Trp Pro Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp
Glu Leu Val 420 425 430Arg His His Asn Met His Gln Arg Asn Met Thr
Lys Leu Gln Leu Ala 435 440 445Leu 320449PRTMus musculus 320Met Gly
Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Ser1 5 10 15Ser
Leu Gly Gly Gly Gly Gly Cys Gly Leu Pro Val Ser Gly Ala Ala 20 25
30Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr
35 40 45Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro
Pro 50 55 60Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp
Gly Gly65 70 75 80Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe
Thr Leu His Phe 85 90 95Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys
Arg Tyr Gly Pro Phe 100 105 110Gly Pro Pro Pro Pro Ser Gln Ala Ser
Ser Gly Gln Ala Arg Met Phe 115 120 125Pro Asn Ala Pro Tyr Leu Pro
Ser Cys Leu Glu Ser Gln Pro Thr Ile 130 135 140Arg Asn Gln Gly Tyr
Ser Thr Val Thr Phe Asp Gly Ala Pro Ser Tyr145 150 155 160Gly His
Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe 165 170
175Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln
180 185 190Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr
Asp Ser 195 200 205Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro
Tyr Ser Ser Asp 210 215 220Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu
Cys Met Thr Trp Asn Gln225 230 235 240Met Asn Leu Gly Ala Thr Leu
Lys Gly Met Ala Ala Gly Ser Ser Ser 245 250 255Ser Val Lys Trp Thr
Glu Gly Gln Ser Asn His Gly Ile Gly Tyr Glu 260 265 270Ser Asp Asn
His Thr Ala Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile 275 280 285His
Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Ser 290 295
300Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu
Lys305 310 315 320Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys
Arg Tyr Phe Lys 325 330 335Leu Ser His Leu Gln Met His Ser Arg Lys
His Thr Gly Glu Lys Pro 340 345 350Tyr Gln Cys Asp Phe Lys Asp Cys
Glu Arg Arg Phe Ser Arg Ser Asp 355 360 365Gln Leu Lys Arg His Gln
Arg Arg His Thr Gly Val Lys Pro Phe Gln 370 375 380Cys Lys Thr Cys
Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr385 390 395 400His
Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys 405 410
415Arg Trp His Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val
420 425 430Arg His His Asn Met His Gln Arg Asn Met Thr Lys Leu His
Val Ala 435 440 445Leu3219PRTHomo sapiens 321Pro Ser Gln Ala Ser
Ser Gly Gln Ala1 53229PRTHomo sapiens 322Ser Ser Gly Gln Ala Arg
Met Phe Pro1 53239PRTHomo sapiens 323Gln Ala Arg Met Phe Pro Asn
Ala Pro1 53249PRTHomo sapiens 324Met Phe Pro Asn Ala Pro Tyr Leu
Pro1 53259PRTHomo sapiens 325Pro Asn Ala Pro Tyr Leu Pro Ser Cys1
53269PRTHomo sapiens 326Ala Pro Tyr Leu Pro Ser Cys Leu Glu1 5
* * * * *