U.S. patent application number 12/574555 was filed with the patent office on 2010-09-23 for synthetic hla binding peptide analogues and uses thereof.
Invention is credited to Javier Pinilla-Ibarz, David A. Scheinberg.
Application Number | 20100239547 12/574555 |
Document ID | / |
Family ID | 34652399 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100239547 |
Kind Code |
A1 |
Scheinberg; David A. ; et
al. |
September 23, 2010 |
SYNTHETIC HLA BINDING PEPTIDE ANALOGUES AND USES THEREOF
Abstract
The present invention is directed to a synthetic peptide
comprising a sequence of amino acids containing at least a segment
that is an analogue of a native peptide that specifically binds to
HLA A0201 or HLA A0301 molecules on a cell characteristic of a
pathophysiologic state in a mammal. The synthetic peptide may be
derived form native peptides comprising a breakpoint region of the
WT1 protein.
Inventors: |
Scheinberg; David A.; (New
York, NY) ; Pinilla-Ibarz; Javier; (New York,
NY) |
Correspondence
Address: |
Pearl Cohen Zedek Latzer, LLP
1500 Broadway, 12th Floor
New York
NY
10036
US
|
Family ID: |
34652399 |
Appl. No.: |
12/574555 |
Filed: |
October 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11223139 |
Sep 12, 2005 |
7598221 |
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12574555 |
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10999425 |
Nov 30, 2004 |
7488718 |
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11223139 |
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60525955 |
Dec 1, 2003 |
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Current U.S.
Class: |
424/93.71 ;
435/325; 435/375; 514/19.3; 514/44R; 530/327; 530/328; 530/350;
530/362 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4702 20130101; C07K 14/82 20130101; A61P 35/00 20180101;
A61P 35/02 20180101; C07K 14/4748 20130101 |
Class at
Publication: |
424/93.71 ;
435/325; 435/375; 514/19.3; 514/44.R; 530/327; 530/328; 530/350;
530/362 |
International
Class: |
A61K 35/26 20060101
A61K035/26; C12N 5/00 20060101 C12N005/00; C12N 5/02 20060101
C12N005/02; A61K 38/08 20060101 A61K038/08; A61K 31/7088 20060101
A61K031/7088; C07K 7/08 20060101 C07K007/08; C07K 7/06 20060101
C07K007/06; C07K 14/00 20060101 C07K014/00; C07K 14/76 20060101
C07K014/76; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
[0002] This invention was produced in part using funds obtained
through National Cancer Institute Core Grant No. 08748 and National
Institutes of Health Grant Nos. PO1 33049 and PO1 2376,
consequently, the federal government has certain rights in this
invention.
Claims
1. A synthetic WT-1 peptide comprising a sequence of amino acids
selected from the group consisting of YMFPNAPYL (SEQ ID NO: 18),
YLGEQQYSV (SEQ ID NO: 20), YLLPAVPSL (SEQ ID NO: 22), YLGATLKGV
(SEQ ID NO: 24), YLNALLPAV (SEQ ID NO: 26), GLRRGIQDV (SEQ ID NO:
28), KLYFKLSHL (SEQ ID NO: 30), ALLLRTPYV (SEQ ID NO: 32),
YMTWNQMNL (SEQ ID NO: 34), NMYQRNMTK (SEQ ID NO: 36), NMHQRVMTK
(SEQ ID NO: 37), NMYQRVMTK (SEQ ID NO: 38), QMYLGATLK (SEQ ID NO:
40), QMNLGVTLK (SEQ ID NO: 41), QMYLGVTLK (SEQ ID NO: 42),
FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK (SEQ ID NO: 45), FMYAYPFCNK
(SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQ ID NO:
49), and KLYHLQMHSK (SEQ ID NO: 50), that is an analogue peptide of
a native WT-1 peptide selected from the group consisting of
RMFPNAPYL (SEQ ID NO: 17), SLGEQQYSV (SEQ ID NO: 19), ALLPAVPSL
(SEQ ID NO: 21), NLGATLKGV (SEQ ID NO: 23), DLNALLPAV (SEQ ID NO:
25), GVFRGIQDV (SEQ ID NO: 27), KRYFKLSHL (SEQ ID NO: 29),
ALLLRTPYS (SEQ ID NO: 31), CMTWNQMNL (SEQ ID NO: 33), NMHQRNMTK
(SEQ ID NO: 35), QMNLGATLK (SEQ ID NO: 39), FMCAYPGCNK (SEQ ID NO:
43), and KLSHLQMHSR (SEQ ID NO: 47), wherein said analogue
specifically binds to HLA A0201 or HLA A0301 molecules on a human
cell.
2. The synthetic WT-1 peptide of claim 1, wherein said analogue
peptide is a degradation product of said synthetic WT-1
peptide.
3. The synthetic WT-1 peptide of claim 1, further comprising: an
immunogenic carrier linked thereto.
4. The synthetic WT-1 peptide of claim 3, wherein said immunogenic
carrier is a carrier protein, a carrier peptide or an
antigen-presenting cell.
5. The synthetic WT-1 peptide of claim 4, wherein said carrier
protein or carrier peptide is keyhole limpet hemocyanin, an albumin
or a polyamino acid.
6. The synthetic WT-1 peptide of claim 4, wherein said antigen
presenting cell is a dendritic cell.
7. The synthetic WT-1 peptide of claim 1, wherein a total number of
amino acids in said analogue peptide is about 70% to about 130% of
a total number of amino acids in said native WT-1 peptide.
8. The synthetic WT-1 peptide of claim 8, wherein said analogue
peptide has about 8 to about 12 amino acids.
9.-11. (canceled)
12. The synthetic WT-1 peptide of claim 1, wherein said human cell
is characteristic of a pathophysiologic state.
13. The synthetic WT-1 peptide of claim 12, wherein said
pathophysiologic state is a cancer selected from the group
consisting of a leukemia, a breast cancer, a lymphoma, a
mesothelioma, a lung cancer, a testicular cancer, and an ovarian
cancer.
14. The synthetic WT-1 peptide of claim 13, wherein said leukemia
is a chronic myelogenous leukemia.
15. A synthetic WT-1 peptide having an amino acid sequence selected
from the group consisting of NMYQRNMTK (SEQ ID NO: 36), NMHQRVMTK
(SEQ ID NO: 37), NMYQRVMTK (SEQ ID NO: 38), QMYLGATLK (SEQ ID NO:
40), 25 QMNLGVTLK (SEQ ID NO: 41), QMYLGVTLK (SEQ ID NO: 42),
FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK (SEQ ID NO: 45), FMYAYPFCNK
(SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQ ID NO:
49), or KLYHLQMHSK (SEQ ID NO: 50).
16. A method of inducing formation and proliferation of human
cytotoxic T cells that recognize a cancer cell, wherein said cancer
cell presents the native WT-1 peptide of claim 1 on a major
histocompatibility complex (MHC) class I molecule thereof, said
method comprising contacting human immune cells in vivo or ex vivo
with the synthetic WT-1 peptide of claim 1 or DNA encoding said
synthetic WT-1 peptide, whereby said synthetic WT-1 peptide or DNA
encoding said synthetic peptide induces formation and proliferation
of said human cytotoxic T cells.
17. The method of claim 16, whereby said human cytotoxic T cells
are capable of mounting a heteroclitic immune response against said
cancer cell.
18. The method of claim 16, wherein the step of contacting said
human immune cells are is performed in vivo in an individual having
a cancer.
19. A method of treating a subject having a cancer, wherein a
malignant cell of said cancer presents the native WT-1 peptide of
claim 16 on a major histocompatibility complex (MHC) class I
molecule thereof, comprising a. inducing in vivo in a donor or ex
vivo using immune cells from a donor formation and proliferation of
human cytotoxic T cells that recognize said malignant cell by the
method of claim 16; and b. infusing said human cytotoxic T cells
into said subject, thereby treating a subject having a cancer.
20.-21. (canceled)
22. The method of claim 16, whereby said DNA molecule is in a
vector or an antigen presenting cell.
23. The method of claim 16, wherein said human immune cells are
peripheral blood mononuclear cells, bone marrow cells, dendritic
cells, or macrophages.
24. The method of claim 16, wherein said cancer cell is selected
from the group consisting of a leukemia cell, a breast cancer cell,
a lymphoma cell, a mesothelioma cell, a lung cancer cell, a
testicular cancer cell, and an ovarian cancer cell.
25.-29. (canceled)
30. A pharmaceutical composition, comprising: a therapeutically
effective amount of the synthetic WT-1 peptide of claim 1 or a DNA
encoding said synthetic WT-1 peptide; and a pharmaceutically
acceptable carrier.
31. The pharmaceutical composition of claim 30, wherein said DNA is
in a vector or an antigen presenting cell.
32.-33. (canceled)
34. The pharmaceutical composition of claim 30, further comprising
an adjuvant, a diluent or a combination thereof.
35. The pharmaceutical composition of claim 34, wherein said
adjuvant is a protein, a peptide or an antigen presenting cell
linked to said synthetic peptide.
36. The pharmaceutical composition of claim 35, wherein said
protein or peptide is keyhole limpet hemocyanin, an albumin or a
polyamino acid.
37. The pharmaceutical composition of claim 35, wherein said
antigen presenting cell is a dendritic cell.
38.-42. (canceled)
43. The method of claim 16 wherein said synthetic WT-1 peptide
further comprises an adjuvant.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/223,139, filed Sep. 12, 2005, issued as U.S. Pat. No.
7,598,221, which is a continuation of Ser. No. 10/999,425, filed
Nov. 30, 2004, issued as U.S. Pat. No. 7,488,718, which claims
benefit of priority of U.S. Provisional Application Ser. No.
60/525,955, filed Dec. 1, 2003. These applications are hereby
incorporated in their entirety by reference herein.
FIELD OF THE INVENTION
[0003] This invention relates to the fields of immunology and
leukemia therapy. More specifically, this invention relates to the
use of synthetic analogue peptides to induce heteroclitic human T
cell responses against native peptides of the synthetic
analogues.
DESCRIPTION OF THE RELATED ART
[0004] Chronic myelogenous leukemia (CML) is a pluripotent stem
cell disorder characterized by the presence of the Philadelphia
chromosome (Ph). The Philadelphia chromosome represents a
translocation in which the c-abl oncogene has moved from chromosome
9 into the breakpoint cluster region (bcr) within the bcr gene on
chromosome 22, resulting in a chimeric bcr-abl gene. The fused
genes encode an 8.5 kb chimeric mRNA which is usually translated
into a 210-kDa or 190-kDa protein. This bcr-abl protein is a
tyrosine kinase which is uniquely present in the leukemia cells of
chronic myelogenous leukemia patients and is necessary and
sufficient for transformation. In chronic myelogenous leukemia, the
breakpoint in the bcr gene occurs either between bcr exon 2 (b2)
and 3 (b3) or between bcr exon 3(b3) and 4(b4). Although aberrant
bcr-abl fusion genes and bcr-abl mRNA splicing can occur, the
majority of patients with chronic myelogenous leukemia therefore
express p210-b.3a2 or p.210-b2a2; often both p210 and pi90 proteins
are expressed together with low levels of p190-ela2 bcr-abl
proteins. In Phi positive acute lymphocytic leukemia (ALL), the
predominant breakpoint is at the ela2 site.
[0005] The chimeric fusion proteins are potential antigens. First,
the proteins are uniquely expressed in chronic myelogenous leukemia
cells in which the functional regions contain a sequence of amino
acids that is not expressed on any normal protein. Secondly, as a
result of the codon split on the fused message, a new amino acid,
lysine in b3a2, and a conserved amino acid, glutamic acid in b2a2,
is present at the exact fusion point in each of the proteins.
Therefore, the unique amino acid sequences encompassing the b3a2
and b2a.2 breakpoint region can be considered truly tumor specific
antigens. Despite the intracellular location of these proteins,
short peptides produced by cellular processing of the products of
the fusion proteins can be presented on the cell surface within the
cleft of HLA molecules and in this form they can be recognized by T
cells.
[0006] Recent clinical trials demonstrated that a rumor specific,
bcr-abl derived multivalent vaccine may be safely administered to
patients with chronic phase chronic myelogenous leukemia The
vaccine reliably elicits a bcr-abl peptide-specific CD4 immune
response as measured by DTH in vivo, by CD4+T cell proliferation ex
vivo and by gamma interferon secretion in an ELISPOT assay.
However, no CD8 responses in HLA A0201 patients and only weak
responses in HLA A0301 patients were detected using a sensitive
gamma interferon ELISPOT assay.
[0007] Wilms tumor protein I (WT1) is a zinc finger transcription
factor expressed during normal ontogenesis such as in fetal kidney,
testis and ovary. In adults, WTI expression is limited to low
levels on hematopoietic stem cells, myoepithelial progenitor cells,
renal podocytes and some cells in testis and ovary. Recent
demonstration that WTI is over expressed in several types of
leukemia suggested that WTI would be an attractive target for
immunotherapy; three peptide nonamers from WTI have been identified
to generate a WTI specific cytotoxic response in the context of HLA
0201 and HLA 2402. However, as WTI protein is a self-antigen,
breaking tolerance is a potential concern. For stimulation of
responses the strength of CD8 responses depends upon the binding
affinity of the target peptide to class I MHC molecules, the
peptide-HLA complex stability, and the avidity of the T cell
receptor binding for the peptide complex. Killing of native CML
cells also requires adequate processing and presentation of the
natural antigen. Therefore the lack of reproducible CD8 responses
in these clinical trials could be the result of the biochemistry of
these class I peptide-HLA interactions, which results in their weak
immunogenicity to cytotoxic CDS cells, None of the native CML
peptides reported to bind to human MHC bound the HLA pocket with
high affinity. This may explain, in part, the lack of a detectable
immune response to bcr-abl peptides as proteins seen in patients
with chronic myelogenous leukemia despite the appearance of this
antigen in the CML cells.
[0008] In some antigenic systems peptide analogues are used to
circumvent a poor immunogenic response. A high correlation has been
found between overall analogue peptide affinities for MHC class I
molecules and in vivo peptide immunogenicity in HLA-A2 Kb
transgenic mice. A better correlation with a peptide's ability to
form stable HLA-A0201 complexes and immunogenicity has been
reported. Improved immunogenicity in HLA-A020I/Kb transgenic mice
also has been reported for analogues of a self-peptide, gp
100154-162, displaying both higher affinity and more prolonged
complex stability than the natural peptide.
[0009] To design peptide analogues several successful algorithms
have been utilized in which large protein sequences are scanned for
the presence of suitable binding motifs, leading to the
identification of predicted antigens that have subsequently been
experimentally validated. Analogs of antigenic peptides have been
formulated by direct modifications of MHC anchor positions, which
are referred to as "MHC anchor-modified ligands", or modifications
of TCR contact sites, which generally are termed "altered peptide
ligands". The identification of peptide epitope analogues that
strengthen the stability of the MHC-peptide complex in vivo and in
vitro is thought to enhance the potency of intrinsically weak
immunogenic peptides for the activation and amplification of
relevant T-cell subsets, This concept was originally described in a
murine CD4.sup.+T cell model using HIV peptides (1), and now has
been extended to a variety of viral and tumor immunological
systems.
[0010] Artificial variants of MHC class I-binding self-peptides
have been designed (2). Since these variant peptides were foreign
to the host immune system, a strong CTL response was induced.
Unlike weak T cell responses to self-peptide-MHC complexes, CTL
responses to variant peptides can be sustained for a longer period
without causing annihilation of the clones due to insufficient
signals for cell division or survival. Since a substantial fraction
of such CTLs cross-react with non-mutated self-peptides expressed
in tumor cells in much smaller amounts, immunization with variant
peptides may be a more efficient method to induce CTLs against
tumors. The scoring system for MHC class I-binding peptides should
provide a convenient method for design of cross-reactive
self-mimicking peptides for immunization.
[0011] The improved immunogenicity in vivo and relevance of MHC
anchor-modified ligands was first shown formally in human
neoplastic disease in a controlled study of patients with malignant
melanoma using a melanoma-associated A0201 restricted peptide
derived from gpl. It has been shown recently with HLA-tetramer
based detection methods that the parental Melan-A antigenic
peptides are weak agonists which activate antigen-specific T cells
suboptimally (3). In contrast, melan A peptide analogues were
identified that behaved as full agonists and induced full T cell
activation leading to strong tumor antigen-specific CTL responses
(4).
[0012] Simple motifs and the statistical binding matrices can be
used to perform a crude search for MHC-binding peptides.
Unfortunately, the presence of a simple sequence motif does not
correlate well with binding. Therefore these simple motifs are not
always necessary or sufficient for binding. Only 30% of the
peptides that carry such simple motifs bind well when examined in a
biochemical binding assay. Predictions of binding can be improved
considerably when extended motifs are used, rather than the simple
motifs. About 70% of the peptides carrying an extended motif bind
well.
[0013] Assuming that each amino acid in each position contributes a
certain binding energy independent of the neighboring residues and
that the binding of a given peptide is the result of combining the
contributions from the different residues, multiplying the relevant
matrix values should give an indication of the binding of the
corresponding peptide. Such statistical matrix-driven predictions
have been somewhat more successful, thereby suggesting that MHC
binding is to some extent the result of a combinatorial
specificity. The identification of analogues peptides based on
these methods has been applied recently to the identification of
CTL epitopes deduced from proteinase 3, melanoma antigen 3, mucin I
and telomerase.
[0014] The weak immunogenicity of native bcr-abl fusion peptides,
as demonstrated by poor lysis of the cells, or the problem of
tolerance using native peptides from a self-antigen, such as WT1,
has prevented use of these native peptides as an effective vaccine
against CML. A need exists in the art to develop therapeutic
strategies using vaccination against a truly tumor specific antigen
that is also the oncogenic protein requited for neoplasia. There is
a need for improved synthetic peptide analogues designed to elicit
a greater immunogenic response. The prior art is deficient in the
lack of synthetic analogue peptides that could generate an immune
response that not only recognizes the immunizing epitopes, but that
also cross reacts with the original native peptides. Specifically,
the prior art is deficient in synthetic peptide analogs with both
improved HLA binding and improved ability to elicit a greater
immunogenic response against cancer cells. The present invention
fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
[0015] The present invention is directed to a synthetic peptide
comprising a sequence of amino acids containing at least a segment
that is an analogue of a native peptide that specifically binds to
HLA A0201 or HLA A0301 molecules on a cell characteristic of a
pathophysiologic state in a mammal. The synthetic peptide may be
derived from native peptides comprising a breakpoint region of the
bcr-abl fusion protein or of the WT1 protein.
[0016] The present invention also is directed to a related
synthetic peptide with an amino acid sequence selected from
YLKALQRPV (SEQ ID NO: 2), KQSSKALQV (SEQ ID NO: 4), KLSSKALQV (SEQ
ID NO: 5), KLLQRPVAV (SEQ ID NO: 7), TLFKQSSKV (SEQ ID NO: 9),
YLFKQSSKV (SEQ ID NO: 10), LLINKEEAL (SEQ ID NO: 12), LTINKVEAL
(SEQ ID NO: 13), YLINKEEAL (SEQ ID NO: 14), YLINKEEAV (SEQ ID NO:
15), or YLINKVEAL (SEQ ID NO: 16), NMYQRNMTK (SEQ ID NO: 36),
NMHQRVMTK (SEQ ID NO: 37), NMYQRVMTK (SEQ ID NO: 38), MYLGATLK (SEQ
ID NO: 40), QMNLGVTLK (SEQ ID NO: 41), QMYLGVTLK. (SEQ ID NO: 42),
FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK (SEQ ID NO: 45), FMYAYPFCNK
(SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQ ID NO:
49), or KLYHLQMHSK (SEQ ID NO: 50).
[0017] The present invention also is directed to pharmaceutical
composition comprising a therapeutically effective amount of the
synthetic peptides described herein or a DNA encoding the synthetic
peptide and a suitable carrier.
[0018] The present invention is directed further to an immunogenic
composition comprising an immunogenically effective amount of the
synthetic peptide described herein and a pharmaceutically
acceptable carrier, adjuvant or diluents or a combination
thereof.
[0019] The present invention is directed further still to a method
of treating a cancer in a human. The pharmaceutical compositions
described herein are administered to the human. A heteroclitic
response is induced by cytotoxic T-cells that recognize at least
the analogue segment of the synthetic peptides described herein
against cancer cells presenting a native peptide from which the
analogue segment is derived. Thus, the cytotoxic T-cells recognize
or kill the cancer cells thereby treating the cancer. In a related
method the present invention is directed to a method of treating
leukemia in a human using the pharmaceutical compositions
comprising the synthetic peptide containing at least the analogue
segment derived from a native peptide of a WT1 protein or a DNA
encoding the synthetic peptide.
[0020] The present invention is directed further still to a method
of inducing formation and proliferation of human cytotoxic T cells
that produce a heteroclitic immune response against cancer cells.
Human immune cells are contacted with a synthetic peptide
containing at least the analogue segment described herein. Thereby
the formation and proliferation of human cytotoxic T cells reactive
against the activated cells presenting the analogue segment of the
synthetic peptide is induced. The proliferating T cells will cross
react with the cancer cells presenting a native peptide from which
the analogue segment is derived such that the human cytotoxic T
cells are thereby produce a heteroclitic immune response against
the cancer cells. The present invention also is directed further to
a related method of inducing formation and proliferation of human
cytotoxic T cells that produce a heteroclitic immune response
against leukemic cells in a human as described in the method for
inducing formation and proliferation of cytotoxic T cells against a
cancer cell.
[0021] The present invention is directed to a related method of
inducing a heteroclitic response in a human. The immunogenic
compositions described herein are administered to a human to
activate human immune cells. The formation and proliferation of
cytotoxic T cells against the activated cells presenting the
analogue segment of the synthetic peptide described herein that
comprises the immunogenic composition is induced thereby. The
cytotoxic T cells will cross-react with a cancer cell presenting a
native peptide from which the analogue segment is derived to induce
the heteroclitic response The present invention is directed further
to a related method of inducing a heteroclitic response in a human
against leukemic cells as described in the method for inducing such
heteroclitic response against a cancer cell.
[0022] Other and further aspects, features, benefits, and
advantages of the present invention will be apparent from the
following description of the presently preferred embodiments of the
invention given for the purpose of disclosure
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention are briefly
summarized. Details of the above may be had by reference to certain
embodiments thereof which are illustrated in the appended drawings.
These drawings form a part of the specification. It is to be noted;
however, that the appended drawings illustrate preferred
embodiments of the invention and therefore are not to be considered
limiting in their scope.
[0024] FIGS. 1A-1B show T2 stabilization assays using peptides
derived from b3a2 translocation (FIG. 1A) and b2a2 translocation
(FIG. 1B).
[0025] FIGS. 2A-2B show the results of a CD8+ gamma interferon
ELISPOT from a healthy HLA A0201 donor (FIG. 2A) using p210C and
p210F peptides and from a CML patient in chronic phase HLA A0201
(FIG. 2B) using the p210C peptide.
[0026] FIGS. 3A-3B show the results of a CD3+ gamma interferon
ELISPOT from a healthy HLA A0201 donor using the b2a2 A3-A5
peptides (FIG. 3A) and from a chronic myelogenous leukemia patient
in chronic phase HLA A0201 (FIG. 3B) using the b2a2 A3 peptide.
[0027] FIGS. 4A-4B show the results of cytotoxicity assays with T
cells from a healthy HLA A0201 donor using p2IOC, p21 OF and p!90B
peptides (FIG. 4A) and the b2a2 A3 peptide (FIG. 4B).
[0028] FIGS. 5A-5E show binding of native and synthetic WT-1
peptides toHLA A0201 cells (FIG. 5A) and to HLA A0301 cells (FIGS.
5B-5E).
[0029] FIGS. 6A-6B show the results of a CD3.sup.+ gamma interferon
ELISPOT (FIG. 6A) and cytotoxicity (FIG. 6B) from a healthy HLA
A0201 donor against native and synthetic peptide pulsed T2
cells.
[0030] FIGS. 7A-7D shows the results of a CD8.sup.+ (FIG. 7A) and
CD3.sup.+ (FIGS. 7B-7D) gamma interferon ELISPOT from healthy HLA
A0201 donors using native and synthetic WT-1 peptides.
[0031] FIGS. 8A-8B show the results of cytotoxicity assays using
CD8.sup.+T cells stimulated with synthetic WT-1 AI peptides from a
HLA A0201 donor against HLA matched CML blasts presenting native
peptide sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In one embodiment of the present invention, there is
provided a synthetic peptide comprising a sequence of amino acids
containing at least a segment that is analogue of a native peptide
that specifically binds HLA A0201 or HLA A0301 molecules on a cell
characteristic of a pathophysiologic state in a mammal. In this
embodiment the analogue segment may have a number of amino acids
that is about 70% to about 130% of the number of amino acids in the
native peptide. The number of amino acids may be about 8 to about
12.
[0033] In all aspects of this invention the pathophysiologic state
may be a cancer.
[0034] The cancer may be a leukemia such as a chronic myelogenous
leukemic. Alternatively, the cancer may breast cancer, lymphoma,
mesothelioma, lung cancer, testicular cancer, or ovarian cancer.
Additionally, in all aspects the mammal may be a human.
[0035] Furthermore, in all aspects of this embodiment the synthetic
peptide may comprise an immunogenic carrier linked thereto.
Examples of carriers are a protein, a peptide or an
antigen-presenting cell. Representative examples of a protein or
peptide are keyhole limpet hemocyanin, an albumin or a polyamino
acid. A representative example of an antigen-presenting cell is a
dendritic cell.
[0036] In one aspect of this embodiment the amino acids comprise a
precursor to the analogue segment which is a degradation product
thereof. In this aspect the precursor may be a bcr-abl fusion
protein where the analogue segment spans the breakpoint region of
the fusion protein. Alternatively, the precursor may be WT1 where
the analogue segment replaces a native peptide of WTL.
[0037] In a related aspect, the analogue segment is derived from a
native peptide comprising a breakpoint region of a bcr-abl fusion
protein. The native peptide may be a native p190-31a2 peptide. The
native peptide may be a native p210-b3a2 peptide and the amino acid
sequence of the analogue segment may be YLKALQRPV (SEQ ID NO: 2),
KQSSKALQV (SEQ ID NO: 4), KLSSKALQV (SEQ ID NO: 5), KLLQRPVAV (SEQ
ID NO: 7), TLFKQSSKV (SEQ ID NO: 9), or YLFKQSSKV (SEQ ID NO: 10).
Preferably, the amino acid sequence is YLKALQRPV (SEQ ID NO: 2),
KLLQRPVAV (SEQ ID NO: 7) or YLFKQSSKV (SEQ ID NO: 10).
[0038] Alternatively, the native peptide may be a native p210-b2a2
peptide and the amino acid sequence of the analogue segment may be
LLINKEEAL (SEQ ID NO: 12), LTINKVEAL (SEQ ID NO: 13), YLINKEEAL
(SEQ ID NO: 14), YLINKEEAV (SEQ ID NO: 15), and YLINKVEAL (SEQ ID
NO: 16). Preferably the amino acid sequence is YLINKEEAL (SEQ ID
NO: 14).
[0039] In another aspect of this embodiment, the analogue segment
is derived from a native peptide comprising a WT1 protein. The
amino acid sequence of the WT1 derived analogue segment may be
YMFPNAPYL (SEQ ID NO: 18), YLGEQQYSV (SEQ ID NO: 20), YLLPAVPSL
(SEQ ID NO: 22), YLGATLKGV (SEQ ID NO: 24), YLNALLPAV (SEQ ID NO:
26), GLRRG1QDV (SEQ ID NO: 28), KLYFKLSHL (SEQ ID NO: 30),
ALLLRTPYV (SEQ ID NO: 32), YMTWNQMNL (SEQ ID NO: 34), NMYQRNMTK
(SEQ ID NO: 36), NMHQRVMTK (SEQ ID NO: 37), NMYQRVMTK (SEQ ID NO:
38), QMYLGATLK (SEQ ID NO: 40), QMNLGVTLK (SEQ ID NO: 41),
QMYLGVTLK (SEQ ID NO: 42), FMYAYPGCNK (SEQ ID NO: 44), FMCAYPFCNK
(SEQ ID NO: 45), FMYAYPFCNK (SEQ ID NO: 46), KLYHLQMHSR (SEQ ID NO:
48), KLSHLQMHSK (SEQ ID NO: 49), and KLYHLQMHSK (SEQ ID NO:
50).
[0040] In a related embodiment of this invention there is provided
a synthetic peptide with an amino acid sequence that may be one or
more of YLKALQRPV (SEQ ID NO; 2), KQSSKALQV {SEQ ID NO: 4),
KLSSKALQV (SEQ ID NO: 5), KLLQRPVAV (SEQ ID NO: 7), TLFKQSSKV (SEQ
ID NO: 9), YLFKQSSKY (SEQ ID NO: 10), 5 LLINKEEAL (SEQ ID NO: 12),
LTINKVEAL (SEQ ID NO: 13), YLINKEEAL (SEQ ID NO: 14), YLINKEEAV
(SEQ ID NO: 15), or YLINKVEAL (SEQ ID NO: 16). Alternatively, a
synthetic peptide is provided with an amino acid sequence that may
be one or more of NMYQRNMTK (SEQ ID NO: 36), NMHQRVMTK (SEQ ID NO:
37), NMYQRVMTK (SEQ ID NO: 38), QMYLGATLK (SEQ ID NO: 40),
QMNLGVTLK10 (SEQ ID NO: 41), QMYLGVTLK (SEQ ID NO: 42), FMYAYPGCNK
(SEQ ID NO: 44), FMCAYPFCNK {SEQ ID NO: 45), FMYAYPFCNK (SEQ ID NO:
46), KLYHLQMHSR (SEQ ID NO: 48), KLSHLQMHSK (SEQ ID NO: 49), or
KLYHLQMHSK (SEQ ID NO: 50).
[0041] In another related embodiment there is provided a
pharmaceutical composition comprising a therapeutically effective
amount of the synthetic peptide of claim 1 or a DNA encoding the
synthetic peptide; and a pharmaceutically acceptable carrier. In
aspects of this embodiment where the pharmaceutical composition
comprises a DNA encoding the synthetic peptide, the DNA may be
inserted into a vector or into an antigen-presenting cell. An
example of an antigen presenting cell is a dendritic cell.
[0042] In one aspect of this embodiment an analogue segment
comprising the synthetic peptide is derived from a native
p210-b.3a2 peptide, a native p210-b2a2 peptide or a native
p190-ela2 peptide. The amino acid sequences for these analogue
segments are as identified supra for p210.about.b3a2 and p210-b2a2
derived analogues. In a related aspect of this embodiment an
analogue segment comprising the synthetic peptide is derived from a
native peptide comprising the WT-1 protein. These amino acid
sequences for these WTI-derived analogue segments are as identified
supra for WT-1 derived analogues.
[0043] In still another related embodiment of the present
invention, there is provided an immunogenic composition comprising
an immunogenically effective amount of the synthetic peptides
described supra and a pharmaceutically acceptable carrier, adjuvant
or diluent or a combination thereof. The carrier may be a protein,
a peptide or an antigen-presenting cell linked to the synthetic
peptide Examples of a protein or peptide carrier are keyhole limpet
hemocyanin, an albumin or a polyamino acid. An example of an
antigen-presenting cell is a dendritic cell. The synthetic peptides
and analogue segments comprising the synthetic peptides are as
described supra. In aspects of this embodiment, the analogue
segments may be derived from those native peptides and the amino
acid sequences may be those sequences described supra for a
pharmaceutical composition. In another embodiment of the present
invention, there is provided a method of inducing formation and
proliferation of hum an cytotoxic T cells that produce a
heteroclitic immune response against cancer cells, comprising
contacting human immune cells with the synthetic peptides described
supra to activate the immune cells; and inducing formation and
proliferation of human cytotoxic T cells reactive against the
activated cells presenting at least the analogue segment of the
synthetic peptide, where the proliferating T cells will cross react
with the cancer cells presenting a native peptide from which said
analogue segment is derived such that the human cytotoxic T cells
are capable of producing a heteroclitic immune response against the
cancer cells.
[0044] In this embodiment the method further comprises providing a
DNA encoding the synthetic peptide and expressing the DNA. The DNA
may be inserted into a suitable vector. Alternatively, the DNA may
be inserted into an antigen-presenting cell. An example of an
antigen-presenting cell is a dendritic cell. In one aspect of this
embodiment the human immune cells are contacted in vivo in an
individual having a cancer. In a related aspect the human immune
cells are contacted in vivo in a donor and the method further
comprises obtaining the cytotoxic T cells from the donor and
infusing the cytotoxic T cells into a recipient having a
cancer.
[0045] In another aspect the cells are contacted ex vivo and the
method further comprises obtaining the human immune cells from a
donor prior to the contacting the human immune cells with the
pharmaceutical composition and infusing the activated immune cells
into an individual having a cancer prior to the inducing formation
and proliferation of cytotoxic T cells. In a related aspect the
cells are contacted ex vivo and the method further comprises
obtaining human immune cells from a donor prior to contacting the
human immune cells. In this aspect, both contacting the human
immune cells and formation and proliferation of the cytotoxic
T-cells occurs ex vivo, infusing the cytotoxic T-cells into an
individual having a cancer.
[0046] In all aspects of this embodiment, representative human
immune cells may be peripheral blood mononuclear cells, bone marrow
cells, dendritic cells, or macrophages. The synthetic peptides and
analogue segments comprising the synthetic peptides are as
described supra. In aspects of this embodiment the analogue
segments comprising the synthetic peptide may be derived from a
native p210.about.b3a2 peptide, a native p210-b2a2 peptide, a
native p190-ela2 peptide or from a native WT-1 peptide as described
supra, Furthermore, the amino acid sequences of these synthetic
peptides or the analogue segments comprising the synthetic peptides
may have an amino acid sequence as identified supra for p210-b3a2-,
p210-b2a2- or WT-1-derived analogues.
[0047] In a related aspect WT-1 analogue segments and p210-b3a2 and
p210-b2a2 derived analogue segments may induce a heteroclitic
response against Seukemic cells. Representative leukemic cells are
chronic myelogenous leukemic cells. In another related aspect WT-1
analogue segments may induce a heteroclitic response against from
breast cancer, lymphoma, mesothelioma, lung cancer, testicular
cancer, or ovarian cancer.
[0048] In yet another embodiment of the present invention there is
provided a method of treating a cancer in a human, comprising
administering the pharmaceutical compositions described herein to
the human; and inducing a heteroclitic response by cytotoxic
T-cells that recognize at least the analogue segment of said
synthetic peptide against cancer cells presenting a native peptide
from which said analogue segment is derived, said cytotoxic T cells
recognizing or killing said cancer cells thereby treating the
cancer.
[0049] The synthetic peptides or DNAs encoding the synthetic
peptides are as described supra. In aspects of this embodiment the
analogue segment comprising the synthetic peptides may be derived
from a native p190 ela2 peptide, a native p210-b3a2 peptide, a
native p210.about.b2a2 peptide, or from a native WT-1 peptide as
described supra. Furthermore, the amino acid sequences of these
synthetic peptides or the analogue segments comprising the
synthetic peptides are as identified supra for p210-b3a2-,
p210-b2a2- or WT-1-derived analogue segments.
[0050] In one aspect WT-1 at least the analogue segments comprising
the synthetic peptides and p210-b3a2 and p210-b2a2 derived
synthetic peptides may treat a leukemia. A representative leukemia
is chronic myelogenous leukemia. In another aspect WT-1 at least
the analogue segments comprising the synthetic peptides may treat
breast cancer, lymphoma, mesothelioma, lung cancer, testicular
cancer, or ovarian cancer.
[0051] In a related embodiment, there is provided a method of
inducing a heteroclitic immune response in a human, comprising
administering to the human an effective amount of the immunogenic
compositions described supra; activating human immune cells with
the immunogenic composition; and inducing formation and
proliferation of human cytotoxic T cells against the activated
cells presenting at least the analogue segment of the synthetic
peptide comprising the immunogenic composition. In this method, the
human cytotoxic T cells will cross-react with a cell comprising a
cancer presenting a native peptide from which said analogue segment
is derived, thereby inducing the heteroclitic immune response.
[0052] In one aspect of this embodiment, the human may have an
active cancer, may be in remission from cancer or may be at risk of
developing a cancer. In an alternative aspect of this embodiment
human donates the cytotoxic T-cells to an individual having an
active cancer, is in remission from cancer or is at risk of
developing a cancer.
[0053] In aspects of this embodiment, the immunogenic compositions,
the human immune cells and the synthetic peptides and the analogue
segments comprising the synthetic peptides are as described supra.
In one aspect at least WT-1 analogue segments and p210-b3a2 and
p210-b2a2 derived analogue segment comprising synthetic peptides
may induce a heteroclitic response in the human against
leukemia.
[0054] A representative leukemia is chronic myelogenous leukemia.
In another aspect at least WT-1 analogue segments may induce a
heteroclitic response in the human against breast cancer, lymphoma,
mesothelioma, lung cancer, testicular cancer, or ovarian
cancer.
[0055] Provided herein are synthetic immunogenic peptides with an
amino acid sequence containing at least an analogue segment of a
native peptide that demonstrates improved binding over the native
peptides to HLA A0201 or to HLA A0301 complexes. These synthetic
peptides or analogue segments can stimulate T-cells to cross-react
with the native peptides thus eliciting a heteroclitic immune
response that will recognize or kill cells presenting the native
peptides. Such cells are characteristic of a pathophysiological
state, for example, but not limited to, a cancer. At least the
analogue segments comprising the synthetic peptides will bind with
more affinity to the HLA class I and class II molecules that are
instrumental in presenting the analogue segments to the T-cells
than the native peptide itself.
[0056] The synthetic peptide analogue segments are designed by
making one or two amino acid substitutions in anchor or auxiliary
residues. Although the native peptides particularly described
herein are nonamers encompassing the anchor or auxiliary residues,
analogues may be designed having about 70% to about 130% of the
amino acids in the native peptide. In the instant invention the
synthetic peptide analogues may have about 8-12 amino acids. Such
substitutions are determined by a bioinformatic model system
(BIMAS) which uses a matrix approach to predict binding and ranks
the peptides based on predicted binding to the HLA molecule. The
amino acid sequences and predicted score for binding to HLA A0201
and HLA A0301 are generated by online software BIMAS available at
http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken parker comboform and
SYFPEITHI available at http://syfpeimi-bmi-heidelberg.com/. The
synthetic peptide may be a precursor to the analogue segment which
may be a degradation product of the synthetic peptide. Such
precursor may be a bcr-able fusion protein such that the synthetic
analogue spans the breakpoint region of the fusion protein.
Alternatively, the precursor may be a WT1 protein such that the
analogue segments replace a native peptide sequence within WT1.
[0057] Additionally, the synthetic peptide is or comprises analogue
segments that may be analogues of the breakpoint region of the
bcr-abl fusion protein, which is the oncogenic protein required for
neoplasia in chronic myelogenous leukemia. The synthetic peptides
are or comprise analogue segments derived from the junctional
sequences of p210-b3a2, p210-b2a2 and p19Q-ela2. More preferably,
the synthetic peptides or the analogue segments are derived from
p210-b3a2 and p210-b2a2 in which single or double amino acid
substitutions were introduced into the peptides at key HLA A0201
binding positions. These high affinity peptide analogues were able
to generate specific CD8+T cells far more efficiently than the
native peptides and are capable of stimulating human CD8+ CTL
heteroclitic responses that cross-react with the native sequences
presented on leukemic cells.
[0058] Preferred synthetic peptides or analogue segments are the
p210-b.3a2 analogues p210C, p210D, p210E, and p210F more preferably
p210C, and the p210-b2a2 analogues b2a2 A3, b2a2 A4 and b2a2 A5,
more preferably b2a2 A3, Table I shows the amino acid sequences and
binding predictions of native and synthetic analogues. The
underlined K in the b3a2 and underlined E in b2a2 are the amino
acids in the breakpoint. Boldface, italic residues represent
modification from the native sequence.
TABLE-US-00001 TABLE 1 "Native Analogue BIMAS Name sequence
sequence score CMLA2 SSKALQRPV 0.003 SEQ ID NO: 1 p210F KALQRPV
2240 SEQ ID NO: 2 CMLA3 KQSSKALQR 0.005 SEQ ID NO: 3 p210A KQSSKALQ
24.681 SEQ ID NO: 4 p210B K SSKALQ 243.432 SEQ ID NO: 5 p210Cn
KALQRPVAS 0.013 SEQ ID NO: 6 p210C K LQRPVA 900.689 SEQ ID NO: 7
p210Dn TGFKQSSKA 0.120 SEQ ID NO: 8 p210D T FKQSSK 257.342 SEQ ID
NO: 9 p210E FKQSSK 1183.775 SEQ ID NO: 10 b3a2A LTINKEEAL 0.247 SEQ
ID NO: 11 b3a2 A1 L INKEEAL 17.795 SEQ ID NO: 12 b3a2 A2 LTINK EAL
21.996 SEQ ID NO: 13 b3a2 A3 INKEEAL 48.151 SEQ ID NO: 14 b3a2 A4
INKEEA 156.770 SEQ ID NO: 15 b3a2 A5 INK EAL 110.747 SEQ ID NO:
16
[0059] The synthetic immunogenic peptides may be analogue segments
or comprise analogue segments derived from WT1 protein. Computer
prediction analysis, as described herein, predicted synthetic
peptides analogues derived from nonamer sequences of the WT1
protein in which single amino-acid substitutions were introduced at
HLA A0201 binding and single or double amino acid substitutions
were introduced at A0301 binding positions. These synthetic peptide
analogues or analogue segments were able to generate specific
CD8.sup.+ or CD3.sup.+T cells far more efficiently than the native
peptides and are capable of stimulating human CD8.sup.+ or
CD3.sup.+CTL heteroclitic responses that cross-react with the
native sequences presented on leukemic cells or on other cells that
present these native WT-1 peptides. Tables 2 and 3 show the amino
acid sequences and binding predictions of native WT-1 and synthetic
WT-1 peptide analogues. Boldface, italic residues represent
modifications from the native sequence.
TABLE-US-00002 TABLE 2 HLA 0201 native peptides from WT-1 and
synthetic analogues Native Analogue BIMAS Name sequence sequence
score WT-1 A RMFPNAPYL 313 SEQ ID NO: 17 WT-1 A1 MFPNAPYL 1444 SEQ
ID NO: 18 WT-1 B SLGEQQYSV 285 SEQ ID NO: 19 WT-1 B1 LGEQQYSV 1311
SEQ ID NO: 20 WT-1 C ALLPAVPSL 181 SEQ ID NO: 21 WT-1 C1 LLPAVPSL
%36 SEQ ID NO: 22 WT-1 D NLGATLKGV 159 SEQ ID NO: 23 WT-1 D1
LGATLKGV 735 SEQ ID NO: 24 WT-1 E DLNALLPAV 11 SEQ ID NO: 25 WT-1
E1 LNALLPAV 735 SEQ ID NO: 26 WT-1 F GVFRGIQDV 51 SEQ ID NO: 21
WT-1 F1 G RGIQDV 591 SEQ ID NO: 28 WT-1 G KRYFKLSHL 1 SEQ ID NO: 29
WT-1 G1 K YFKLSHL 550 SEQ ID NO: 30 WT-1 H ALLLRTPYS 1 SEQ ID NO:
31 WT-1 H1 ALLLRTPY 1415 SEQ ID NO: 32 WT-1 J CMTWNQMNL 15 SEQ ID
NO: 33 WT-1 J1 MTWNQMNL 70 SEQ ID NO: 34
TABLE-US-00003 TABLE 3 HLA 0201 native peptides from WT-1 and
synthetic analogues Native Analogue BIMAS Name sequence sequence
score A3 WT-1 A NMHQRNMTK 40 SEQ ID NO: 35 A3 WT-1 A1 NM QRNMTK 200
SEQ ID NO: 36 A3 WT-1 A2 NMHQR MTK 120 SEQ ID NO: 37 A3 WT-1 A3 NM
QR MTK 600 SEQ ID NO: 38 A3 WT-1 B QMNLGATLK 20 SEQ ID NO: 39 A3
WT-1 B1 QM LGATLK 100 SEQ ID NO: 40 A3 WT-1 B2 QMNLG TLK 60 SEQ ID
NO: 41 A3 WT-1 B3 QM LG TLK 300 SEQ ID NO: 42 A3 WT-1 C FMCAYPGCNK
30 SEQ ID NO: 43 A3 WT-1 C1 FM AYPGCNK 150 SEQ ID NO: 44 A3 WT-1 C2
FMCAYP CNK 90 SEQ ID NO: 45 A3 WT-1 C1 FM AYP CNK 450 SEQ ID NO: 46
A3 WT-1 D KLSHLQMHSR 18 SEQ ID NO: 47 A3 WT-1 D1 KL HLQMHSR 90 SEQ
ID NO: 48 A3 WT-1 D2 KLSHLQMHS 90 SEQ ID NO: 49 A3 WT-1 D3 KL
HLQMHS 450 SEQ ID NO: 50
[0060] The present invention also provides a pharmaceutical
composition of a therapeutic amount of the synthetic peptides or
analogue segments or a genetic sequence or DNA encoding the same
and a pharmaceutical carrier, as is known in the art. The
pharmaceutical composition may be formulated with the
pharmaceutical carrier for administration by any of the many
techniques known to those of skill in the art. For example, the
pharmaceutical composition may be administered parenterally,
intravenously, subcutaneously, intradermally, intramucosally,
topically, orally, or by inhalation.
[0061] Therefore, it is contemplated that the synthetic peptides or
analogue segments or pharmaceutical compositions thereof may be
used in the preparation of an immunogenic composition suitable to
effect immunization of a subject. The immunogenic composition may
comprise a carrier or a suitable adjuvant to boost immune response
or a combination thereof, as are known in the art. The immunogenic
composition further may comprise a diluent standard in the art as
described herein. The immunogenic composition may comprise a
vaccine.
[0062] A carrier may comprise one or more proteins or peptides.
Examples of carriers are well known and may be, although not
limited to keyhole limpet hemocyanin, an albumin, such as human
serum albumin or a polyamino acid. Additionally, a carrier may
comprise a live antigen-presenting cell, such as a dendritic cell,
which presents the synthetic peptides described herein. A suitable
adjuvant may be Freund's adjuvant, aluminum phosphate, aluminum
hydroxide, alum, QS21, BCG. These compositions further may comprise
a physiologically acceptable diluent, e.g., water, phosphate
buffered saline or saline. Additionally, a genetic sequence
encoding a synthetic peptide or an analogue segment thereof may be
delivered as naked DNA to an individual via appropriate methods
known in the art. Alternatively, the genetic sequence may be
introduced or inserted into a suitable vector, such as for example,
but not limited to, attenuated viral or bacterial vectors, as are
standard in the art. Furthermore, the naked DNA or vectors
comprising the genetic sequence or DNA may be transduced into an
antigen-presenting cell, e.g., a dendritic cell. The genetic
sequence, DNA, vector or transduced antigen-presenting cell may be
introduced into an individual in need of the treatment or into a
healthy donor whereupon the DNA encoding the genetic sequence
expresses the synthetic peptide to elicit a cytotoxic T-cell
response. Donor T-cells may then be infused into a patient in need
thereof.
[0063] The pharmaceutical or immunogenic compositions may be used
to treat a disease or a condition such as cancer. Administration of
the synthetic peptides or analogue segments comprising the
pharmaceutical compositions induces a heteroclitic response against
native peptides expressed on the cancer cells thereby effecting a
therapeutic result. Native peptides of the breakpoint region of
bcr-abl proteins and native peptides of WT-1 protein are expressed
on leukemic cells in chronic myelogenous leukemia. Native WT-1
peptides are expressed on other leukemic cells and, additionally,
on cancerous cells of different solid tumors. Such cancers may be,
although not limited to, breast, lymphoma, mesothelioma, lung,
testicular, or ovarian cancers.
[0064] It is contemplated that the synthetic peptides or synthetic
analogue segments thereof or genetic sequences encoding the same or
the pharmaceutical or the immunogenic compositions thereof can
induce human cytotoxic T cells to produce a heteroclitic immune
response against cancerous cells, for example, leukemic cells.
Contacting human immune cells with at least the analogue segment
that is or comprises the synthetic peptides activates the immune
cells to induce formation and proliferation of human cytotoxic T
cells that will recognize or react against a cell presenting the
synthetic peptide. Such cytotoxic T cells cross react with human
cells presenting the native peptides from which the analogue
segment is derived thereby producing a heteroclitic response.
[0065] One of ordinary skill in this art would recognize the word
"contacting" in terms of activating target immune cells to elicit a
subsequent immune response as referring to any suitable delivery
method of bringing an immunogenic agent into contact with the
target cells. In vitro or ex vivo this is achieved by exposing the
target cells to the agent in a suitable medium. For in vivo
applications, any known method of administration is suitable as
described herein.
[0066] Thus, the synthetic peptides or analogue segments thereof
described herein may be used to activate T-cells ex vivo or in
vivo. In vivo, the synthetic peptides or analogue segments thereof
or DNA encoding the same may be administered to a patient or to a
healthy donor to induce cytotoxic T-cells If administered to a
donor these cytotoxic T-cells are obtained from the donor and
infused into an individual in need of them, such as an individual
with an active cancer, in remission from a cancer or at risk for
developing a cancer.
[0067] Ex vivo, the T cells are obtained from a patient or from a
healthy donor and are incubated in the presence of antigen
presenting cells and a synthetic peptide or at least an analogue
segment thereof to activate the T-cells. The activated T-cells
subsequently are infused back into the patient where they will
recognize and/or destroy cells presenting the native peptide.
Alternatively, human immune cells may be incubated with the
synthetic peptide or at least an analogue segment thereof whereupon
the activated immune cells are infused back into the patient to
induce T-cell production against both the activated cells and cell
presenting the native peptide. Examples of immune cells may be
peripheral blood mononuclear monocytic cells, bone marrow cells,
dendritic cells, or macrophages.
[0068] It is contemplated further that administration of the
synthetic peptide or at least an analogue segment thereof or
pharmaceutical compositions thereof induces an immune response in a
subject, preferably, although not limited to, a CD8/HLA A or
CD3/HLA A class I immune response. As such, the synthetic peptides
or at least an analogue segment thereof may be used in a method of
immunizing a subject against a pathophysiologic condition or
disease presenting HLA molecules, e.g., a leukemia, such as chronic
myelogenous leukemia- Additionally, WT-1 synthetic peptides or at
least analogue segments thereof may be used to induce an immune
response in a subject with other leukemias or cancers such as,
although not limited to, breast, lymphoma, mesothelioma, lung,
testicular, or ovarian cancers. As used herein, immunizing or
immunization of a subject encompasses full and partial immunization
whereby the subject becomes fully immune to the condition or
partially immune to the condition. The subject may be a mammal,
preferably a human. The subject may have a condition or disease
which may be active or in remission, prior to immunization.
Alternatively, if at risk for developing the disease or condition,
the subject may be immunized prior to such development. One of
ordinary skill in the art would be able to assess the risk factors,
such as environmental risk factors or personal risk factors, such
as family history, genetic makeup or behavior, to make a
determination of risk in the subject.
[0069] The pharmaceutical compositions and immunogenic compositions
may be administered one or more times to achieve a therapeutic or
an immunogenic effect. It is well within the skill of an artisan to
determine dosage or whether a suitable dosage comprises a single
administered dose or multiple administered doses. An appropriate
dosage depends on the subject's health, the progression or
remission of the disease, the route of administration and the
formulation used.
[0070] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
Example 1
Synthetic Peptides
[0071] Each of the peptides utilized in this study was purchased
and was synthesized by Genemed Synthesis Inc, CA using
fluorenylmethoxycarbonyl chemistry, solid phase synthesis and
purified by high pressure liquid chromatography. The quality of the
peptides was assessed by high-performance liquid chromatography
analysis and the expected molecular weight was observed using
matrix-assisted laser desorption mass spectrometry. Peptides were
sterile and >90% pure. The peptides were dissolved in DMSO and
diluted in phosphate-buffered saline (PBS) at pH 7.4 or saline to
give a concentration of 5 mg/ml and were stored at -80'C. For in
vitro experiments an irrelevant control peptide, HLA A24 consensus,
was used.
Example 2
Cell Lines
[0072] Cell lines were cultured in RPMI 1640 medium supplemented
with 5% FCS, penicillin, streptomycin, 2 mM glutamine and
2-mercaptoethanol at 37.degree. C. in humidifier air containing 5%
CO.sub.2. SKLY-16 is a human B cell lymphoma expressing HLA A0201
and T2 is a human cell line lacking TAP1 and TAP2 and therefore
unable to present peptides derived from cytosolic proteins.
Example 3
T2 Assay for Peptide Binding and Stabilization of HLA A0201
Molecules
[0073] T2 cells (TAP.sup.-, HLA-A0201+) were incubated overnight at
27.degree. C. at 1.times.10 E6 cells/ml in FCS-free RPMI medium
supplemented with 5 .mu.g/ml human .beta.2m (Sigma, St Louis, Mo.)
in the absence, i.e., negative control, or presence of either a
positive reference tyrosinase peptide or test peptides at various
final concentrations of 50, 10, 1, and 0.1 fig/ml Following a 4 h
incubation with 5 .mu.g/ml brefeldin A (Sigma), T2 cells were
labeled for 30 min at 4.degree. C. with a saturating concentration
of anti-HLA-A2.1 (BB7.2) mAb, then washed twice. The cells then
were incubated for 30 min at 4.degree. C. with a saturating
concentration of FITC-conjugated goat IgG F(ab').sub.2 anti-mouse
Ig (Caltag, South San Francisco, Calif.), washed twice, fixed in
PBS/1% paraformaldehyde and analyzed using a FACS Calibur
cytofluorometer (Becton Dickinson, Immunocytometry systems, San
Jose, Calif.).
[0074] The mean intensity of fluorescence (MIF) observed for each
peptide concentration, after subtraction of the MIF observed
without peptide, was used as an estimate of peptide binding.
Stabilization assays were performed similarly. Following initial
evaluation of peptide binding at time 0, cells were washed in RPMI
complete medium to remove free peptides and incubated in the
continuous presence of 0.5 .mu.g/ml brefeldin-A for 2, 4, 6, and 8
hours. The amount of stable peptide-HLA-A2.1 complexes was
estimated as described above by indirect immunofluorescence
analysis. The half time of complexes is an estimate of the time
required for a 50% reduction of the time mean intensity of
fluorescence value.
Example 4
Competition Radioimmunoassay
[0075] Target cells were washed two times in PBS with 1% bovine
serum albumin (Fisher Chemicals, Fairlawn, N.J.). Cells were
resuspended at 10.sup.7/ml on ice and the native cell surface bound
peptides were stripped for 2 minutes at 0.degree. C. using
citrate-phosphate buffer in the presence of beta.sub.2
microglobulin 3 mg/ml. The pellet was resuspended at 5 to
10.times.10.sup.6 cells/ml in PBS/1% BSA in the presence of 3 mg/ml
beta.sub.2 microglobulin and 30 mg/ml deoxyribonuclease and 200 ml
aliquots were incubated with HLA-specific peptides for 10 min at
20.degree. C.
[0076] Binding of .sup.125I-labeled peptide with or without
competitive unlabeled peptide was done for 30 min at 20.degree. C.
Total bound .sup.125I was determined after two washes using PBS/2%
BSA and one wash with PBS. Relative affinities were determined by
comparison of escalating concentrations of the test peptide versus
a known binding peptide. Peptides of affinities<500 nM were
chosen for use.
[0077] A specificity analysis of the binding of peptide to HLA on
the live cell surface (SKLY-16) was conducted to confirm that the
binding was to the appropriate HLA molecule and to characterize its
restriction. This included competition with excess unlabeled
peptides known to bind to the same or disparate HLA molecules and
use of target cells which expressed the same or disparate HLA
types. This assay was performed on live fresh or 0.25%
paraformaldehyde-fixed human peripheral blood mononuclear cells
(PBMC), leukemia cell lines and EBV-transformed T-cell lines of
specific HLA types. The relative avidity of the peptides found to
bind MHC molecules on the specific cells were assayed by
competition assays as described above against .sup.125I-labeled
peptides of known high affinity for the relevant HLA molecule,
e.g., tyrosinase or HBV peptide sequence.
Example 5
In Vitro Immunization and Human T Cell Cultures
[0078] After informed consent, peripheral blood mononuclear cells
from HLA-A0201 positive healthy donors and chronic myeloid leukemia
patients were obtained by Ficoll-density centrifugation. Peripheral
blood dendritic cells (DCs) were generated as follows:
Monocyte-enriched peripheral blood mononuclear cell fractions were
isolated, using a plastic adherence technique, from total
peripheral blood mononuclear cells. The plastic-adherent cells were
cultured further in RPMI 1640 medium supplemented with 1-5%
autologous plasma, 1000 U/mL recombinant human interleukin (IL)-4
(Schering-Plough, N.J.), and 1000 U/mL recombinant human
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Immunex,
Seattle).
[0079] On days 2 and 4 of incubation, part of the medium was
exchanged for fresh culture medium supplemented with IL-4 and
GM-CSF and culture was continued. On day 6, half of the medium was
exchanged for culture medium supplemented with IL-4, GM-CSF, and 10
ng/mL recombinant human tumor necrosis factor (TNF)-alpha (R&D
5 system) and 500 ng/ml of trimeric soluble CD40L (Immunex,
Seattle). On day 9, the cells were harvested and used as
monocyte-derived dendritic cells for antigen stimulation. The cells
generated expressed dendritic cell-associated antigens, such as
CD80, CD83, CD86, and HLA class 1 and class II on their cell
surfaces (data not shown).
[0080] T lymphocytes were isolated from the same donors by use of
negative selection by depletion with an anti-CD11b, anti-CD56 and
CD19 monoclonal antibody (Miltenyi, Calif.) A total of 1.times.10
E6 of pure T lymphocytes were cultured with 1.times.10 E5
autologous dendritic cells in RPMI1640 medium supplemented with 5%
heat-inactivated human autologous plasma with bcr-abl synthetic
peptides at a concentration of 10 .mu.g/mL and b2 microglobulin at
2 .mu.g/ml in 24 well plates in the presence of 5-10 ng/mL
recombinant human IL-7 (Genzyme) and 0.1 ng/ml of IL-12.
[0081] After culture for 3 days 20 U/ml of IL-2 was added. After 10
days, 1.times.10 E6 cells were stimulated again by adding
2.times.10 E5 autologous magnetically isolated CD 14.sup.+
monocytes together with 10 ng/ml of IL-7 and 20 U/ml of IL-2 and
peptide at a concentration of 10 .mu.g/mL. In some cases, after
culture for another 7 days, the cells were stimulated a third time
in the same manner. After the second or third stimulation, CD8 T
cells were isolated magnetically and cytotoxicity and gamma-IFN
secretion of these cells were examined.
Example 6
Gamma Interferon ELISPOT
[0082] HA-Multiscreen plates (Millipore, Burlington, Mass.) were
coated with 100 .mu.l of mouse-anti-human IFN-gamma antibody (10
.mu.g/ml; clone 1-D1K, Mabtech, Sweden) in PBS, incubated overnight
at 4.degree. C., washed with PBS to remove unbound antibody and
blocked with RPMI/autologous plasma for 1 h at 37.degree. C.
Purified CD8.sup.+T cells, more than 95% pure, were plated at a
concentration of 1.times.10.sup.5/well. T cells were stimulated
with 1.times.10 E4 T2 cells per well, pulsed with 10 .mu.g/ml of
B2-microglobulin (Sigma, St. Louis) and either 50 .mu.g/ml of test
peptide, positive control influenza matrix peptide, or irrelevant
control peptide at a final volume of 100-200 .mu.l/well. Control
wells contained T2 cells with or without CD8.sup.+ cells.
Additional controls included medium or CD8.sup.+ alone plus PBS/5%
DMSO diluted according to the concentrations of peptides used for
pulsing T2 cells.
[0083] After incubation for 20 hours at 37.degree. C., plates were
washed extensively with PBS/0.05% Tween and 100 .mu.l/well
biotinylated detection antibody against human IFN-g at 2 .mu.g/ml
(clone 7-B6-1, Mabtech, Sweden) were added. Plates were incubated
for an additional 2 hours at 37.degree. C. and spot development was
performed. Spot numbers were automatically determined with the use
of a computer-assisted video image analyzer with KS EL1SPOT 4.0
software (Carl Zeiss Vision, Germany).
Example 7
Cytotoxicity Assay
[0084] The presence of specific CTLs was measured in a standard 4
h-chromium release assay. 4.times.10 E6 targets were labeled with
300 .mu.Ci of Na.sub.2.sup.51Cr0.sub.4 (NEN Life Science Products,
Inc. Boston, Mass.) for 1 hour at 37.degree. C. After washing,
cells at 2.times.10 E6/ml were incubated with or without synthetic
peptides at a concentration of 10 .mu.g/ml for 2 hours at
20.degree. C. in presence of B.sub.2 microglobulin at 3 .mu.g/ml.
After washing by centrifugation, target cells were resuspended in
complete media at 5.times.10 E4 cells per ml and plated in a 96
well U-bottom plate (Beeton Dickinson.RTM., NY) at 5.times.10 E3
cells per well with effector cells at effector to target ratios
(E/T) ranging from 100:1 to 10:1. Plates were incubated for 5 hours
at 37.degree. C. in 5% CO.sub.2.
[0085] Supernatant fluids were harvested and radioactivity was
measured in a gamma counter. Percent specific lysis was determined
from the following formula: 100.times.[(experimental release minus
spontaneous release)/(maximum release minus spontaneous release)].
Maximum release was determined by lysis of targets in 2.5% Triton
X-100.
Example 8
Identification and Generation of Peptides with a High Probability
to Bind to HLA 0201
[0086] Amino acid sequences of the human b3a2 and b2a2 fusion
proteins were scanned for peptides with potential binding capacity
for HLA A0201, a subtype encompassing 95% of the HLA-A02 allele.
HLA-A0201 is expressed in about 40% of the Caucasian population. No
peptides with high or intermediate affinity were identified in the
native b3a2 or b2a2 fusion proteins with > than 1 minute of
predicted half life. One peptide that does not exhibit the
consensus HLA 0201 binding motifs has been described but it has
weak avidity to MHC.
[0087] Based on this information and by using the software of the
Bioinformatics & Molecular Analysis Section (National
Institutes of Health, Washington, D.C.) available at
http://bimas.dcrtnih gov/cgi-bin/molbio/ken parker comboform This
software ranks 9-mer or 10-mer peptides on a predicted half-time
dissociation coefficient from HLA class I molecules (Pinilla, et al
Curr Opin Immunol, 11(2): p. 193-202 (1999)). Analogue peptides
were designed by changing one or both anchor amino acids or
additional amino acids adjacent to anchor amino acids. Single or
double amino acid substitutions were introduced at HLA A0201
preferred residues at positions 1, 2, 6 and 9 {Table 1) to yield
sequences that had comparatively high binding scores predicted for
HLA A0201 molecules.
[0088] The predicted half life for binding to HLA A0201 was greater
than 240 minutes in four synthetic peptides and less than 240 in
seven. All the native peptides were predicted to have less than an
hour of half life. Most of the substitutions affected the primary
or secondary anchor motifs, i.e., leucine in position 2 or valine
in position 9 or position 6, but in some cases, a tyrosine was
substituted in position 1. This substitution has been shown to
stabilize the binding of position 2 anchor residue.
Example 9
[0089] Binding of HLA-A0201 by synthetic peptides analogues of b2a2
and b3a2 native peptides. The immunogenicity of MHC class
I-restricted peptides requires the capacity to bind and stabilize
MHC class I molecules on the live cell surface. Moreover the
computer prediction models above have only 60-80% predictive
accuracy. Direct measurement of the strength of the interaction
between the peptides and the HLA-A0201 molecule was made using a
conventional binding and stabilization assay that uses the
antigen-transporting deficient (TAP2-) HLA-A0201 human T2 cells. T2
cells lack TAP function and consequently are defective in properly
loading class I molecules with antigenic peptides generated in the
cytosol. The association of exogenously added peptides with
thermolabile, empty HLA-A2 molecules stabilizes them and results in
an increase in the level of surface HLA-A0201 recognizable by
specific mAb such as BB7.2.
[0090] Seven out of the eleven peptides designed to have higher
binding scores exhibited a relatively high binding affinity for HLA
A0201 molecules as measured by the T2 assay (FIGS. 1A-1B). A rough
correlation between binding scores and binding affinity was
established, thus indicating the partial utility of the computer
generated binding scores for predicting peptides that will bind to
MHC class I molecules on live cells. Some of these peptides
demonstrated the same order of binding affinity as that of viral
antigen such as influenza, which are among the most potent known
antigens for CTL induction. In four cases a good correlation
between computer predicted half-life and T2 stabilization was not
found.
[0091] One of the peptides derived from b3a2, p210C, was mutated
from a peptide that did not have a good prediction score. However,
the native sequence is able to bind HLA A0201 weakly and at the
same level that the previously described CMLA2 peptide. To design
p210C, a neutral alanine in position two was substituted by a
leucine and a serine in position nine was substituted by a valine.
p210C has a high BIMAS score that correlated with T2 binding assay
data (FIG. 1A).
[0092] p210F is a peptide derived from the sequence previously
described by Yotonda et al, CM.LA2, shown to be a weak binder in
the T2 assay. In p210F the two serines in position one and two were
substituted by a tyro sine and a leucine. The BIMAS prediction
showed a 700 fold improvement and the binding to T2 cell revealed
an excellent avidity for HLA A0201 molecules (FIG. 1A).
[0093] Of the peptides derived from b2a2, all were generated from a
peptide that does not predict a good binding to HLA A0201. Three
peptides, b2a2 A3-A5 (Table 1) bound well to HLA A0201 molecules
(FIG. 1B). These three peptides have a tyrosine-leucine sequence
substitution at position 1 and 2 and also a valine substitution in
position 6 or 9.
Example 10
Assessment of the Dissociation Time of b2a2 and b3a2 Synthetic
Peptides Analogues from HLA A020J
[0094] The immunogenicity of peptide antigens depends on a low
dissociation rate of MHC/peptide complexes. The stability of
complexes formed between HLA-A0201 and the b3a2 analogue peptides
was assayed on T2 cells over time. Overnight incubation of T2 cells
with saturating amounts of HLA-A0201 binding peptides and human
.beta.2 microglobulin resulted in increased surface expression of
HLA-A0201 molecules. After peptide removal and addition of
Brefeldin A to inhibit protein synthesis, T2 cells were incubated
at 37.degree. C. and the amount of HLA-A0201 molecules remaining at
the cell surface was determined after various incubation times.
[0095] The stability of each peptide/HLA-A0201 complex was then
normalized relative to that observed for the tyrosinase D peptide
or HIV gag peptide which are peptides with known high affinity and
half life. HLA-A0201 complexes formed with peptides p210A and p210B
were unstable, reaching background levels in less than 1 h of 5
incubation at 37.degree. C. In contrast, peptides p210C, p210D,
p210E and p210F formed complexes that were relatively stable over
6-8 hours.
Example 11
Induction of CD8 Immune Response Against b2a2 and b3a2 Synthetic
Peptide Analogues
[0096] While affinity for MHC molecules is necessary for peptide
immunogenicity, there is also a requirement for presence of
reactive precursor T cells with appropriate T cell receptors. Using
an optimized T cell-expansion system, with monocyte derived DC, CD
14+ cells as APC, and purified T cells, the ability of the
synthetic b3a2 and b2a2 analogues to stimulate peptide-specific
CTLs is examined. Ten healthy HLA A0201 donors as well 5 patients
with CML were studied. Five out of the ten individuals responded to
immunization, generating T cells that secreted IFN gamma when
challenged with different peptide-pulsed T2 cells as targets. p210D
and p210E produced an immune response in some of the donors tested
although p210C and p210F generated a more consistent and higher
immune-responses (FIG. 2A), Responses were observed after the
second or third round of peptide stimulation after CD8.sup.+
isolation or in unpurified CD3.sup.+T cells.
[0097] The spot numbers were consistently higher with peptides that
bound with higher affinity to HLA 0201 molecules as determined by
T2 assay. More importantly, T cells generated in the presence of
the new synthetic analogues were able to recognize the native
sequences. p210C and p210F were able to stimulate T cells to
recognize their respective native sequences (FIG. 2A). CML A2, the
native sequence from p210F is a natural weak binder and there is
indirect evidence that it can be naturally expressed in the surface
of chronic myelogenous leukemia blasts. No immune response could be
generated against the p210A and p210B, despite attempts using
different donors. This result is consistent with their reduced
affinity for MHC.
[0098] A chronic myelogenous leukemia patient in chronic phase HLA
A0201 responded to p210C stimulation of T cells and demonstrated
T-cell cross-reactivity with native p210cn peptide. Response was
observed after the second round of T-cell stimulation in vitro
(FIG. 2B).
[0099] The peptides derived from b2a2 also generated a significant
immune response as measured by gamma interferon secretion CD3+T
cells. Peptides b2a2A3, A4 and A5 generated an immune response in
two healthy donors (FIG. 3A). The response against b2a2 A3 was more
consistent between donors. T cells generated in the presence of
b2a2 A3 were able to identify the original native sequence. This is
of special relevance because the native sequence is a
weak/intermediate binder to HLA. Again, a CML patient in chronic
phase HLA A0201 responded to b2a2 A3 stimulation of T cells and
demonstrated T-cell cross-reactivity with native b2a2 A peptide
(FIG. 3B).
[0100] Gamma interferon ELISPOT is not always associated with
functional killing. Therefore the T cell lines obtained after
several stimulations with the analogue peptide were tested in a
classic chromium-51 assay using peptide pulsed target cell lines. T
cells generated in vitro in the presence of p210C (FIG. 4A) and
b2a2 A3 (FIG. 4B) were able to kill T2 cell line pulsed with
specific peptides but not without peptide or with control peptide,
This experiment was also performed using HLA matched chronic
myelogenous leukemia cell lines or CML blasts expressing the
respective translocation b.3a.2 or b2a2. Significant cytotoxicity
was generated raising the possibility that the native peptides were
not naturally processed and/or sufficiently expressed in the
surface of the leukemic cells.
Example 12
Binding of HLA-A0201 and -A030 I by Synthetic Peptide Analogues
Derived from the WT1 Oncoprotein
[0101] Thermostabilization assays using a TAP 1/2 negative cell
line (T2) and a modified protocol using Raji A0301 cells showed
that several peptides that were predicted to be good binders to HLA
A0201 or A0301 molecules, could stabilize MHC class I A0201 or
A0301 molecules (Table 2), The synthetic analogues WT1-A1, -B1, C1,
and -D1 all predicted to bind HLA A0201 better than the respective
native WT-1 peptides demonstrated similar or increased binding
compared to WT1-A, -B, C, and D (FIG. 5A). WT1-D1 demonstrated a
significantly higher level of binding to HLA-A0201 over WT1-D which
was similar to control. A comparison of HLA A0301 binding of A3
WT1-A, -B, -C, and -D with each of their respective three analogues
demonstrated relatively similarly binding (FIGS. 5B-5E).
Example 13
Induction of CDS or CD3 Immune Response Against Synthetic Peptide
Analogues Derived from the WT1 Oncoprotein
[0102] Cells were primarily stimulated with autologous
monocyte-derived, peptide-pulsed dendritic cells generated in the
presence of GM-CSF, IL-4, TNF alpha, PGE2 and CD40L and
re-stimulated with peptide-pulsed CD 14.sup.+ monocytes in the
presence of IL-2 and IL-7. After two to four stimulations, the
CD8.sup.+CTL lines were assessed by either IFN alpha ELISPOT or a
chromium release assay using pulsed, HLA-matched leukemic cell
lines (FIGS. 6A-6B). Several analogue peptides generated greater
immune responses, i.e., increased CD8 T cell precursor frequency,
in comparison with the native peptides (FIGS. 7A-7D). CD8.sup.+T
cells stimulated with the new synthetic peptides cross-reacted with
the native WT1 peptide sequence and are able to kill HLA matched
chronic myelogenous leukemia blasts (FIGS. 8A-8B)
[0103] The following references are cited herein: [0104] 1. Kessler
et al. J Exp Med, 185(4): p. 629-40 (1997). [0105] 2. Dyall et al,
J Exp Med, 188(9): p. 1553-61 (1998). [0106] 3. Valmori et al, J
Immunol, 165(1): p. 5.3.3-8 (2000). [0107] 4. Valmori et al 0.1
Immunol, 164(2): p. 1125-31 (2000).
[0108] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was incorporated specifically and
individually by reference. One skilled in the art will readily
appreciate that the present invention is well adapted to carry out
the objects and obtain the ends and advantages mentioned, as well
as those inherent therein. The present examples along with the
methods, procedures, treatments, molecules, and specific compounds
described herein are presently representative of preferred
embodiments, are exemplary, and are not intended as limitations on
the scope of the invention. Changes therein and other uses will
occur to those skilled in the art which are encompassed within the
spirit of the invention as defined by the scope of the claims.
Sequence CWU 1
1
5019PRTHomo sapiens 1Ser Ser Lys Ala Leu Gln Arg Pro Val1
529PRTHomo sapiens 2Tyr Leu Lys Ala Leu Gln Arg Pro Val1 539PRTHomo
sapiens 3Lys Gln Ser Ser Lys Ala Leu Gln Arg1 549PRTHomo sapiens
4Lys Gln Ser Ser Lys Ala Leu Gln Val1 559PRTHomo sapiens 5Lys Leu
Ser Ser Lys Ala Leu Gln Val1 569PRTHomo sapiens 6Lys Ala Leu Gln
Arg Pro Val Ala Ser1 579PRTHomo sapiens 7Lys Leu Leu Gln Arg Pro
Val Ala Val1 589PRTHomo sapiens 8Thr Gly Phe Lys Gln Ser Ser Lys
Ala1 599PRTHomo sapiens 9Thr Leu Phe Lys Gln Ser Ser Lys Val1
5109PRTHomo sapiens 10Tyr Leu Phe Lys Gln Ser Ser Lys Val1
5119PRTHomo sapiens 11Leu Thr Ile Asn Lys Glu Glu Ala Leu1
5129PRTHomo sapiens 12Leu Leu Ile Asn Lys Glu Glu Ala Leu1
5139PRTHomo sapiens 13Leu Thr Ile Asn Lys Val Glu Ala Leu1
5149PRTHomo sapiens 14Tyr Leu Ile Asn Lys Glu Glu Ala Leu1
5159PRTHomo sapiens 15Tyr Leu Ile Asn Lys Glu Glu Ala Val1
5169PRTHomo sapiens 16Tyr Leu Ile Asn Lys Val Glu Ala Leu1
5179PRTHomo sapiens 17Arg Met Phe Pro Asn Ala Pro Tyr Leu1
5189PRTHomo sapiens 18Tyr Met Phe Pro Asn Ala Pro Tyr Leu1
5199PRTHomo sapiens 19Ser Leu Gly Glu Gln Gln Tyr Ser Val1
5209PRTHomo sapiens 20Tyr Leu Gly Glu Gln Gln Tyr Ser Val1
5219PRTHomo sapiens 21Ala Leu Leu Pro Ala Val Pro Ser Leu1
5229PRTHomo sapiens 22Tyr Leu Leu Pro Ala Val Pro Ser Leu1
5239PRTHomo sapiens 23Asn Leu Gly Ala Thr Leu Lys Gly Val1
5249PRTHomo sapiens 24Tyr Leu Gly Ala Thr Leu Lys Gly Val1
5259PRTHomo sapiens 25Asp Leu Asn Ala Leu Leu Pro Ala Val1
5269PRTHomo sapiens 26Tyr Leu Asn Ala Leu Leu Pro Ala Val1
5279PRTHomo sapiens 27Gly Val Phe Arg Gly Ile Gln Asp Val1
5289PRTHomo sapiens 28Gly Leu Arg Arg Gly Ile Gln Asp Val1
5299PRTHomo sapiens 29Lys Arg Tyr Phe Lys Leu Ser His Leu1
5309PRTHomo sapiens 30Lys Leu Tyr Phe Lys Leu Ser His Leu1
5319PRTHomo sapiens 31Ala Leu Leu Leu Arg Thr Pro Tyr Ser1
5329PRTHomo sapiens 32Ala Leu Leu Leu Arg Thr Pro Tyr Val1
5339PRTHomo sapiens 33Cys Met Thr Trp Asn Gln Met Asn Leu1
5349PRTHomo sapiens 34Tyr Met Thr Trp Asn Gln Met Asn Leu1
5359PRTHomo sapiens 35Asn Met His Gln Arg Asn Met Thr Lys1
5369PRTHomo sapiens 36Asn Met Tyr Gln Arg Asn Met Thr Lys1
5379PRTHomo sapiens 37Asn Met His Gln Arg Val Met Thr Lys1
5389PRTHomo sapiens 38Asn Met Tyr Gln Arg Val Met Thr Lys1
5399PRTHomo sapiens 39Gln Met Asn Leu Gly Ala Thr Leu Lys1
5409PRTHomo sapiens 40Gln Met Tyr Leu Gly Ala Thr Leu Lys1
5419PRTHomo sapiens 41Gln Met Asn Leu Gly Val Thr Leu Lys1
5429PRTHomo sapiens 42Gln Met Tyr Leu Gly Val Thr Leu Lys1
54310PRTHomo sapiens 43Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys1 5
104410PRTHomo sapiens 44Phe Met Tyr Ala Tyr Pro Gly Cys Asn Lys1 5
104510PRTHomo sapiens 45Phe Met Cys Ala Tyr Pro Phe Cys Asn Lys1 5
104610PRTHomo sapiens 46Phe Met Tyr Ala Tyr Pro Phe Cys Asn Lys1 5
104710PRTHomo sapiens 47Lys Leu Ser His Leu Gln Met His Ser Arg1 5
104810PRTHomo sapiens 48Lys Leu Tyr His Leu Gln Met His Ser Arg1 5
104910PRTHomo sapiens 49Lys Leu Ser His Leu Gln Met His Ser Lys1 5
105010PRTHomo sapiens 50Lys Leu Tyr His Leu Gln Met His Ser Lys1 5
10
* * * * *
References