NZ723130B2 - Cyclin a1-targeted t-cell immunotherapy for cancer - Google Patents
Cyclin a1-targeted t-cell immunotherapy for cancer Download PDFInfo
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- A61K39/001149—Cell cycle regulated proteins, e.g. cyclin, CDC, CDK or INK-CCR
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Abstract
Discloses isolated human cyclin A1 (CCNA1)-specific T cell comprising at least one recombinant expression vector encoding a T-cell receptor polypeptide that specifically binds in a human class I HLA-restricted manner to a CCNA1 polypeptide epitope of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 amino acids comprising the amino acid sequence set forth in SEQ ID NO:1, 3, 4, 5, 6, or 8. Further discloses compositinos suitable for use in the ex vivo manufacture of a medicament for treating a condition characterized by CCNA1 overexpression in cells of a subject. 11, 10, or 9 amino acids comprising the amino acid sequence set forth in SEQ ID NO:1, 3, 4, 5, 6, or 8. Further discloses compositinos suitable for use in the ex vivo manufacture of a medicament for treating a condition characterized by CCNA1 overexpression in cells of a subject.
Description
CYCLIN A1-TARGETED T-CELL IMMUNOTHERAPY FOR CANCER
STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under Grant
No. P01 CA018029 awarded by the National Institutes of Health/ National
Cancer Institute. The government has certain rights in this invention.
STATEMENT REGARDING SEQUENCE LISTING
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
360056_407WO_SEQUENCE_LISTING.txt. The text file is about 9 KB, was
created on November 9, 2012, and is being submitted electronically via EFS-
Web.
BACKGROUND
Technical Field
The present disclosure relates generally to methods for eliciting
antigen-specific T-cell immune responses to a cancer-associated antigen.
More specifically, the human cyclin A1 (CCNA1) isoform c polypeptide is herein
identified as containing epitopes useful for the elicitation of specific T-cell
responses against leukemic cells that overexpress CCNA1, including leukemic
stem cells (LSC) and acute myeloid leukemia (AML) cells.
Description of the Related Art
In higher vertebrates the immune system distinguishes “self” from
“non-self” molecular structures in cells and tissues, and provides a host
organism with the means to quickly and specifically mount protective
responses, such as destruction of pathogenic microorganisms and rejection of
malignant tumors. Immune responses have been generally described as
including humoral responses, in which antibodies specific for antigens are
produced by differentiated B lymphocytes, and cell-mediated responses, in
which various types of T lymphocytes eliminate antigens by a variety of
mechanisms. For example, CD4 (also called CD4+) helper T-cells that are
capable of recognizing specific antigens may respond by releasing soluble
mediators such as cytokines to recruit additional cells of the immune system to
participate in an immune response by a variety of mechanisms. CD8 (also
called CD8+) cytotoxic T lymphocytes (CTL) are also capable of recognizing
specific antigens and may bind to, and destroy or damage, an antigen-bearing
cell or particle. In particular, cell mediated immune responses that include a
cytotoxic T lymphocyte (CTL) response can be important for elimination of
tumor cells and also for elimination of cells infected by pathogens, such as
viruses, bacteria, or microbial parasites.
It is well established that acute myeloid leukemia (AML) is
organized hierarchically, initiated and maintained by a small population of cells
referred to as leukemia stem cells (LSCs) that are characterized not only by
unlimited reproductive capacity but also by enhanced resistance to
chemotherapy and radiation. This primitive cell population, which has been
found to be negative for the expression of lineage markers and CD38 but
positive for CD34, is essential for long ‐term engraftment of primary AML cells in
NOD/SCID transplantation models (Bonnet et al., 1997 Nat Med 3 (7):730 ‐737;
Lapidot et al., 1994 Nature 367 (6464):645 ‐648. doi:10.1038/367645a0; Blair et
al., 1998 Blood 92 (11):4325 ‐4335). The leukemia stem cell hypothesis
suggests that for a therapeutic anti ‐AML effect to be curative in patients,
beneficial strategies would include those that efficiently eliminate the LSC
compartment, which is resistant to conventional therapy approaches.
In patients with intermediate ‐ and high ‐risk and/or relapsed AML,
the allogeneic T ‐cell mediated graft ‐versus ‐leukemia effect detected in some
individuals following hematopoietic stem cell transplantation (HSCT) or after
infusion of donor ‐derived lymphocytes in the post ‐transplant period has been
shown to be essential for the achievement of long ‐term complete remissions
(Cornelissen et al., 2007 Blood 109 (9):3658 ‐3666. doi:blood ‐2006 ‐06 ‐ 025627
[pii] 10.1182/ blood ‐2006 ‐06 ‐025627; Yanada et al., 2005 Cancer 103
(8):1652 ‐1658. doi:10.1002/ cncr.20945; Breems et al., 2005 J Clin Oncol 23
(9):1969 ‐1978. doi:JCO. 2005.06.027 [pii] 10.1200/ JCO.2005.06.027; Levine et
al., 2002 J Clin Oncol 20 (2):405 ‐412). However, allogeneic HSCT and
unselected donor lymphocyte infusions are associated with significant toxicity
due to both the conditioning regimen and the graft versus ‐host activity of donor
lymphocytes. An alternative strategy for providing an anti ‐LSC cytotoxic
T ‐lymphocyte (CTL) component to the treatment of AML patients would be to
engage more targeted T ‐cell therapy, consisting of either the adoptive transfer
of T ‐cells specific for, or the vaccination against, leukemia associated antigens
(LAA) (Van Driessche et al., 2005 Leukemia 19 (11):1863 ‐1871. doi: 2403930
[pii] 10.1038/ sj.leu.2403930; Rezvani et al., 2008 Blood 111 (1):236 ‐242. doi:
blood ‐2007 ‐ 08 ‐108241 [pii] 10.1182/ blood ‐2007 ‐08 ‐108241). The ability of
antigen ‐specific T ‐cells to mediate elimination of AML LSCs has already been
demonstrated in NOD/SCID transplantation models (Bonnet et al., 1999 Proc
Natl Acad Sci USA 96 (15):8639 ‐8644; Rosinski et al., 2008 Blood 111
(9):4817 ‐4826. doi:blood ‐2007 ‐06 ‐ 096313 [pii] 10.1182/
blood ‐2007 ‐06 ‐096313; Xue et al., 2005 Blood 106 (9):3062 ‐3067. doi:2005 ‐01 ‐
0146 [pii] 10.1182/ blood ‐2005 ‐01 ‐0146).
Targeted T ‐cell therapy represents a potentially less toxic strategy
than allogeneic hematopoietic stem cell transplantation to provide a cytotoxic
anti ‐leukemia effect for eliminating the leukemic stem cell (LSC) compartment
in acute myeloid leukemia (AML) patients. However, this strategy requires the
identification of leukemia ‐associated antigens (LAA) that exhibit selective high
expression in AML LSCs to maximize the anti ‐leukemic effect and minimize
immune ‐mediated toxicities in normal tissues.
A precondition for targeted T ‐cell therapy achieving a maximal
anti ‐AML effect that would be accompanied by minimal immunological toxicity is
therefore to identify LAAs with high expression in and presentation by the
malignant cell compartment, but without significant expression in healthy
tissues. Although several AML LAAs have been described, only Wilms tumor
protein 1 (WT1) has been shown to be expressed in the LSC compartment of
the majority of AML patients at levels significantly higher than in physiological
hematopoietic stem cells (HSCs). WT1 is currently being targeted in clinical
trials both with adoptive T ‐cell transfer and peptide vaccination (e.g., U.S. Pat.
Nos. 7,342,092; 7,608,685; 7,622,119), and objective remissions have been
observed in some patients (Cheever et al., 2009 Clin Cancer Res 15 (17):5323 ‐
5337. doi:15/17/ 5323 [pii] 10.1158/ 1078 ‐0432.CCR ‐09 ‐0737; Majeti et al.,
2009 Proc Natl Acad Sci USA 106 (9): 3396 ‐3401. doi: 0900089106 [pii]
10.1073/ pnas.0900089106; Xue et al., 2005 Blood 106 (9):3062 ‐3067; Keilholz
et al., 2009 Blood 113 (26):6541 ‐6548. doi:blood ‐ 2009 ‐02 ‐202598 [pii]
.1182/ blood ‐2009 ‐02 ‐ 202598). In some AML patients, however, WT1 is not
expressed, or is not detected at levels sufficiently distinct from those in HSC, or
no anti ‐WT1 T ‐cell response can be elicited. WT1 expression has also been
detected in several non ‐hematopoietic organs such as spleen, ovary and
kidney, at levels that can be as high or higher than in leukemic blasts, raising
concerns that WT1-targeted immunotherapy would produce toxicities in these
tissues as undesirable and potentially harmful side-effects.
Clearly there is a need for additional candidate leukemia-
associated antigens that are expressed in malignant cells including AML cells,
and in particular in AML leukemic stem cells, to be used as immunogens for the
development of highly specific, targeted immunotherapies for the treatment of
cancers, including leukemias such as AML. The presently disclosed invention
embodiments address this need and provide other related advantages.
BRIEF SUMMARY
The present invention provides, according to certain
embodiments, an isolated peptide capable of eliciting an antigen-specific T-cell
response to human cyclin A1 (CCNA1), comprising a polypeptide of no more
than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids wherein the
polypeptide comprises a sequence of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20 contiguous amino acids from the CCNA1 amino acid sequence
set forth in SEQ ID NO:9.
In another embodiment there is provided an isolated peptide
capable of eliciting an antigen-specific T-cell response to human cyclin A1
(CCNA1), comprising a polypeptide of general formula I: N-X-C, [I] wherein: (a)
N-X-C is a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
or 9 amino acids in which X comprises an amino acid sequence that is
selected from: CCNA1(120 ‐131) VDTGTLKSDLHF [SEQ ID NO:1],
CCNA1(218 ‐226) AETLYLAVN [SEQ ID NO:2], CCNA1(227 ‐235) FLDRFLSCM
[SEQ ID NO:3], CCNA1(253 ‐261) ASKYEEIYP [SEQ ID NO:4],
CCNA1(118 ‐127) YEVDTGTLKS [SEQ ID NO:5], CCNA1(167 ‐175)
YAEEIYQYL [SEQ ID NO:6], CCNA1(330 ‐339) LEADPFLKYL [SEQ ID NO:7],
and CCNA1(341 ‐351) SLIAAAAFCLA [SEQ IN NO:8], (b) N is an amino
terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino
acids that are independently selected from natural amino acids and non-natural
amino acids, and (c) C is a carboxy terminus of the peptide and consists of 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are independently selected from
natural amino acids and non-natural amino acids.
In certain further embodiments the antigen-specific T-cell
response comprises major histocompatibility complex (MHC)-restricted T-cell
recognition of the peptide. In certain other further embodiments the isolated
peptide is capable of eliciting an antigen-specific CD8 T-cell response to
human cyclin A1 (CCNA1) in a class I human leukocyte antigen (HLA)-
restricted manner. In a still further embodiment the class I HLA antigen is HLA-
A*201. In certain other further embodiments the isolated peptide is capable of
eliciting an antigen-specific CD4 T-cell response to human cyclin A1 (CCNA1)
in a class II human leukocyte antigen (HLA)-restricted manner. In certain other
further embodiments the antigen-specific T-cell response comprises an
interferon-gamma (IFN- ) response. In certain other further embodiments the
antigen-specific T-cell response comprises at least one of a CD4 helper T
lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response.
In certain further embodiments the CTL response is directed against a CCNA1-
overexpressing cell. In certain still further embodiments the CCNA1-
overexpressing cell is an acute myeloid leukemia (AML) cell or a leukemic stem
cell (LSC).
Turning to another embodiment, there is provided an isolated
polynucleotide that encodes a peptide that is capable of eliciting an antigen-
specific T-cell response to human cyclin A1 (CCNA1), the peptide comprising a
polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7
amino acids wherein the polypeptide comprises a sequence of at least 7, 8, 9,
, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids from the
CCNA1 amino acid sequence set forth in SEQ ID NO:9.
In another embodiment there is provided an isolated
polynucleotide that encodes a peptide that is capable of eliciting an antigen-
specific T-cell response to human cyclin A1 (CCNA1), the peptide comprising a
polypeptide of general formula I: N-X-C, [I] wherein: (a) N-X-C is a polypeptide
of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 amino acids in
which X comprises an amino acid sequence that is selected from the group
consisting of: CCNA1(120 ‐131) VDTGTLKSDLHF [SEQ ID NO:1],
CCNA1(218 ‐226) AETLYLAVN [SEQ ID NO:2], CCNA1(227 ‐235) FLDRFLSCM
[SEQ ID NO:3], CCNA1(253 ‐261) ASKYEEIYP [SEQ ID NO:4],
CCNA1(118 ‐127) YEVDTGTLKS [SEQ ID NO:5], CCNA1(167 ‐175)
YAEEIYQYL [SEQ ID NO:6], CCNA1(330 ‐339) LEADPFLKYL [SEQ ID NO:7],
and CCNA1(341 ‐351) SLIAAAAFCLA [SEQ IN NO:8], (b) N is an amino
terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino
acids that are independently selected from natural amino acids and non-natural
amino acids, and (c) C is a carboxy terminus of the peptide and consists of 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are independently selected from
natural amino acids and non-natural amino acids.
In certain other embodiments there is provided an immunogenic
composition comprising a recombinant expression vector comprising either of
the polynucleotides just described operably linked to an expression control
sequence. In a further embodiment the vector is capable of delivering the
polynucleotide to an antigen-presenting cell. In a still further embodiment the
antigen-presenting cell is a dendritic cell. In certain other further embodiments
the antigen-specific T-cell response comprises major histocompatibility complex
(MHC)-restricted T-cell recognition of the peptide. In certain other further
embodiments the immunogenic composition is capable of eliciting an antigen-
specific CD8 T-cell response to human cyclin A1 (CCNA1) in a class I human
leukocyte antigen (HLA)-restricted manner. In a still further embodiment the
class I HLA antigen is HLA-A*201. In certain other further embodiments the
immunogenic composition is capable of eliciting an antigen-specific CD4 T-cell
response to human cyclin A1 (CCNA1) in a class II human leukocyte antigen
(HLA)-restricted manner. In certain other further embodiments the antigen-
specific T-cell response comprises an interferon-gamma (IFN- ) response. In
certain other further embodiments the antigen-specific T-cell response
comprises at least one of a CD4 helper T lymphocyte (Th) response and a
CD8+ cytotoxic T lymphocyte (CTL) response. In certain further embodiments
the CTL response is directed against a CCNA1-overexpressing cell, which in
certain still further embodiments is an acute myeloid leukemia (AML) cell or a
leukemic stem cell (LSC).
According to certain other embodiments there is provided a
method of treating a condition characterized by CCNA1 overexpression in cells
of a subject, comprising administering to the subject an effective amount of a
composition that comprises one or more isolated peptides that are capable of
eliciting an antigen-specific T-cell response to human cyclin A1 (CCNA1), each
of said isolated peptides comprising a polypeptide of no more than 20, 19, 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids wherein the polypeptide
comprises a sequence of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
or 20 contiguous amino acids from the CCNA1 amino acid sequence set forth in
SEQ ID NO:9.
In another embodiment there is provided a method of treating a
condition characterized by CCNA1 overexpression in cells of a subject,
comprising administering to the subject an effective amount of a composition
that comprises one or more isolated peptides that are capable of eliciting an
antigen-specific T-cell response to human cyclin A1 (CCNA1), each of said
isolated peptides comprising a polypeptide of general formula I: N-X-C, [I]
wherein: (a) N-X-C is a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10 or 9 amino acids in which X comprises an amino acid sequence
that is selected from: CCNA1(120 ‐131) VDTGTLKSDLHF [SEQ ID NO:1],
CCNA1(218 ‐226) AETLYLAVN [SEQ ID NO:2], CCNA1(227 ‐235) FLDRFLSCM
[SEQ ID NO:3], CCNA1(253 ‐261) ASKYEEIYP [SEQ ID NO:4],
CCNA1(118 ‐127) YEVDTGTLKS [SEQ ID NO:5], CCNA1(167 ‐175)
YAEEIYQYL [SEQ ID NO:6], CCNA1(330 ‐339) LEADPFLKYL [SEQ ID NO:7],
and CCNA1(341 ‐351) SLIAAAAFCLA [SEQ IN NO:8], (b) N is an amino
terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino
acids that are independently selected from natural amino acids and non-natural
amino acids, and (c) C is a carboxy terminus of the peptide and consists of 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are independently selected from
natural amino acids and non-natural amino acids.
In another embodiment there is provided a method of treating a
condition characterized by CCNA1 overexpression in cells of a subject,
comprising administering to the subject an effective amount of a composition
that comprises one or more isolated polynucleotides that each encodes a
peptide that is capable of eliciting an antigen-specific T-cell response to human
cyclin A1 (CCNA1), each of said peptides comprising a polypeptide of no more
than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids wherein the
polypeptide comprises a sequence of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20 contiguous amino acids from the CCNA1 amino acid sequence
set forth in SEQ ID NO:9.
In another embodiment there is provided a method of treating a
condition characterized by CCNA1 overexpression in cells of a subject,
comprising administering to the subject an effective amount of a composition
that comprises one or more isolated polynucleotides that each encodes a
peptide that is capable of eliciting an antigen-specific T-cell response to human
cyclin A1 (CCNA1), each of said peptides comprising a polypeptide of general
formula I: N-X-C, [I] wherein: (a) N-X-C is a polypeptide of no more than 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 amino acids in which X comprises an
amino acid sequence that is selected from the group consisting of:
CCNA1(120 ‐131) VDTGTLKSDLHF [SEQ ID NO:1], CCNA1(218 ‐226)
AETLYLAVN [SEQ ID NO:2], CCNA1(227 ‐235) FLDRFLSCM [SEQ ID NO:3],
CCNA1(253 ‐261) ASKYEEIYP [SEQ ID NO:4], CCNA1(118 ‐127)
YEVDTGTLKS [SEQ ID NO:5], CCNA1(167 ‐175) YAEEIYQYL [SEQ ID NO:6],
CCNA1(330 ‐339) LEADPFLKYL [SEQ ID NO:7], and CCNA1(341 ‐351)
SLIAAAAFCLA [SEQ IN NO:8], (b) N is an amino terminus of the peptide and
consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are
independently selected from natural amino acids and non-natural amino acids,
and (c) C is a carboxy terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or 11 amino acids that are independently selected from natural
amino acids and non-natural amino acids.
In certain further embodiments of the above described methods
the step of administering comprises administering an immunogenic composition
comprising one or more recombinant expression vectors that comprise the one
or more isolated polynucleotides, each of said isolated polynucleotide being
operably linked to an expression control sequence. In a further embodiment
the vector is capable of delivering the polynucleotide to an antigen-presenting
cell. In a still further embodiment the antigen-presenting cell is a dendritic cell.
In certain other further embodiments of the above described methods, the
condition characterized by CCNA1 overexpression is a leukemia, which in
certain further embodiments is acute myeloid leukemia.
Turning to another embodiment, there is provided a method for
treating a condition characterized by CCNA1 overexpression in cells of a
subject, comprising: (A) contacting in vitro, under conditions and for a time
sufficient for antigen processing and presentation by antigen-presenting cells to
take place, (i) a population of antigen-presenting cells that are
immunocompatible with the subject, and (ii) an isolated peptide capable of
eliciting an antigen-specific T-cell response to human cyclin A1 (CCNA1),
comprising a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
, 9, 8 or 7 amino acids wherein the polypeptide comprises a sequence of at
least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids
from the CCNA1 amino acid sequence set forth in SEQ ID NO:9; and (B)
administering one or a plurality of said antigen-pulsed antigen-presenting cells
to the subject in an amount effective to elicit said antigen-specific T-cell
response to human cyclin A1 (CCNA1).
In another embodiment there is provided a method for treating a
condition characterized by CCNA1 overexpression in cells of a subject,
comprising: (A) contacting in vitro, under conditions and for a time sufficient for
antigen processing and presentation by antigen-presenting cells to take place,
(i) a population of antigen-presenting cells that are immunocompatible with the
subject, and (ii) an isolated peptide capable of eliciting an antigen-specific T-cell
response to human cyclin A1 (CCNA1), comprising a polypeptide of general
formula I: N-X-C, [I] wherein: (a) N-X-C is a polypeptide of no more than 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 amino acids in which X comprises an
amino acid sequence that is selected from: CCNA1(120 ‐131) VDTGTLKSDLHF
[SEQ ID NO:1], CCNA1(218 ‐226) AETLYLAVN [SEQ ID NO:2],
CCNA1(227 ‐235) FLDRFLSCM [SEQ ID NO:3], CCNA1(253 ‐261) ASKYEEIYP
[SEQ ID NO:4], CCNA1(118 ‐127) YEVDTGTLKS [SEQ ID NO:5],
CCNA1(167 ‐175) YAEEIYQYL [SEQ ID NO:6], CCNA1(330 ‐339)
LEADPFLKYL [SEQ ID NO:7], and CCNA1(341 ‐351) SLIAAAAFCLA [SEQ IN
NO:8], (b) N is an amino terminus of the peptide and consists of 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 or 11 amino acids that are independently selected from natural
amino acids and non-natural amino acids, and (c) C is a carboxy terminus of
the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that
are independently selected from natural amino acids and non-natural amino
acids, and thereby obtaining a population of antigen-pulsed antigen-presenting
cells; and (B) administering one or a plurality of said antigen-pulsed antigen-
presenting cells to the subject in an amount effective to elicit said antigen-
specific T-cell response to human cyclin A1 (CCNA1).
In another embodiment there is provided a method for treating a
condition characterized by CCNA1 overexpression in cells of a subject,
comprising: (A) contacting in vitro, under conditions and for a time sufficient for
antigen processing and presentation by antigen-presenting cells to take place,
(i) a population of antigen-presenting cells that are immunocompatible with the
subject, and (ii) a composition that comprises an isolated polynucleotide that
can be expressed by said antigen-presenting cells and that encodes a peptide
that is capable of eliciting an antigen-specific T-cell response to human cyclin
A1 (CCNA1), the peptide comprising a polypeptide of no more than 20, 19, 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids wherein the polypeptide
comprises a sequence of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
or 20 contiguous amino acids from the CCNA1 amino acid sequence set forth in
SEQ ID NO:9; and (B) administering one or a plurality of said antigen-pulsed
antigen-presenting cells to the subject in an amount effective to elicit said
antigen-specific T-cell response to human cyclin A1 (CCNA1).
In another embodiment there is provided a method for treating a
condition characterized by CCNA1 overexpression in cells of a subject,
comprising: (A) contacting in vitro, under conditions and for a time sufficient for
antigen processing and presentation by antigen-presenting cells to take place,
(i) a population of antigen-presenting cells that are immunocompatible with the
subject, and (ii) a composition that comprises an isolated polynucleotide that
can be expressed by said antigen-presenting cells and that encodes a peptide
that is capable of eliciting an antigen-specific T-cell response to human cyclin
A1 (CCNA1), the peptide comprising a polypeptide of general formula I: N-X-C,
[I] wherein: (a) N-X-C is a polypeptide of no more than 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10 or 9 amino acids in which X comprises an amino acid
sequence that is selected from: CCNA1(120 ‐131) VDTGTLKSDLHF [SEQ ID
NO:1], CCNA1(218 ‐226) AETLYLAVN [SEQ ID NO:2], CCNA1(227 ‐235)
FLDRFLSCM [SEQ ID NO:3], CCNA1(253 ‐261) ASKYEEIYP [SEQ ID NO:4],
CCNA1(118 ‐127) YEVDTGTLKS [SEQ ID NO:5], CCNA1(167 ‐175)
YAEEIYQYL [SEQ ID NO:6], CCNA1(330 ‐339) LEADPFLKYL [SEQ ID NO:7],
and CCNA1(341 ‐351) SLIAAAAFCLA [SEQ IN NO:8], (b) N is an amino
terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino
acids that are independently selected from natural amino acids and non-natural
amino acids, and (c) C is a carboxy terminus of the peptide and consists of 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are independently selected from
natural amino acids and non-natural amino acids, and thereby obtaining a
population of antigen-pulsed antigen-presenting cells; and (B) administering
one or a plurality of said antigen-pulsed antigen-presenting cells to the subject
in an amount effective to elicit said antigen-specific T-cell response to human
cyclin A1 (CCNA1).
In certain further embodiments of the just-described methods, the
method further comprises a step of expanding the number of antigen-
presenting cells by culturing the antigen-presenting cells after the step of
contacting and prior to the step of administering. In certain other further
embodiments of the just-described methods, the method further comprises (C)
(1) contacting the antigen-pulsed antigen-presenting cells with one or a plurality
of immunocompatible T-cells after step (A), under conditions and for a time
sufficient to generate CCNA1-specific T-cells, and (2) adoptively transferring
the CCNA1-specific T-cells to the subject. In certain still further embodiments,
step (B) is omitted.
In certain other further embodiments of the just-described
methods, the method further comprises (C) (1) contacting the antigen-pulsed
antigen-presenting cells with one or a plurality of immunocompatible T-cells
after step (A), under conditions and for a time sufficient to generate CCNA1-
specific T-cells, (2) expanding the CCNA1-specific T-cells to obtain one or more
clones of said CCNA1-specific T-cells in amounts sufficient for T-cell receptor
structural characterization, (3) determining a T-cell receptor polypeptide
encoding nucleic acid sequence for one or more of said CCNA1-specific T-
cells, (4) transfecting an adoptive transfer T-cell population with at least one T-
cell receptor polypeptide encoding nucleic acid having a sequence determined
in (3) to obtain engineered CCNA1-specific adoptive transfer T-cells, and 4)
adoptively transferring the engineered CCNA1-specific adoptive transfer T-cells
to the subject. In certain still further embodiments, step (B) is omitted.
In certain further embodiments there is provided an isolated
human cyclin A1 (CCNA1)-specific T cell comprising at least one recombinant
expression vector encoding a T-cell receptor polypeptide that specifically binds
in a human class I HLA-restricted manner to a CCNA1 polypeptide epitope of
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 amino acids
comprising the amino acid sequence set forth in SEQ ID NO:1, 3, 4, 5, 6, or 8.
In certain other embodiments there is provided a composition for
use in the ex vivo manufacture of a medicament for treating a condition
characterized by CCNA1 overexpression in cells of a subject, comprising the
CCNA1-specific T cell as described herein in an amount that is therapeutically
effective following adoptive transfer to the subject.
These and other aspects and embodiments of the herein
described invention will be evident upon reference to the following detailed
description and attached drawings. All of the U.S. patents, U.S. patent
application publications, U.S. patent applications, foreign patents, foreign patent
applications and non-patent publications referred to in this specification and/or
listed in the Application Data Sheet are incorporated herein by reference in their
entirety, as if each was incorporated individually. Aspects and embodiments of
the invention can be modified, if necessary, to employ concepts of the various
patents, applications and publications to provide yet further embodiments.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Figure 1 shows model ‐based expression of probe set 205899_at
representing CCNA1: (A) Expression in AML LSC compared to HSCs/CD34+
BM mononuclear cells, PBMCs and non ‐hematopoietic tissues, * p<0.001
(explorative), (B) expression in AML LSCs and corresponding blasts.
Figure 2 shows CCNA1 expression quantified by qRT PCR: (A) in
AML, healthy subsets of hematopoietic cells and tissues, (B) in AML FAB
subtypes and BM of MDS and CML patients, (C) in healthy tissues.
Figure 3 shows HLA A*0201 ‐restricted epitopes CCNA1
227 ‐235
(A ‐C) and CCNA1 (D ‐F): (A, D) Mapping of the minimal immunogenic AA
341 ‐351
sequence using IFNγ intracellular staining (ICS). +/ ‐ refers to positivity of IFNγ
after coincubation of the respective T ‐cell line with peptide pulsed autologous
lymphoblastic cell lines (LCLs), (B, E) the immunogenic peptides stabilized HLA
A*0201 on T2 cells. Negative controls were T2 cells pulsed with an irrelevant
‐mer (shaded), (C, F) activation of specific clones was dependent on peptide
and expression of HLA A*0201. IFNγ ICS with autologous LCLs, 721.211 cells,
and 721.221 stably transfected with HLA A*0201 as APCs.
Figure 4 shows T ‐cell clones against CCNA1 [SEQ ID NO:3]
227 ‐235
and CCNA1341 ‐351 [SEQ ID NO:8] displayed cytotoxic activity. (A) Expression of
CCNA1 in several myeloid cell lines quantified by qRT PCR, (B) IFNγ ICS: high
avidity clones 2196.D9 and D11 produced IFNγ in the presence of
CCNA1+/HLA A*0201+ cell line THP ‐1 independent of exogenous peptide; low
avidity clone 2196.E1 only recognized peptide-pulsed cell lines, (C) 6h Cr
release assay. Clone 2196.D11 caused specific lysis in THP ‐1. Low avidity
clone 2196.E1 is shown for comparison, (D) Caspase ‐3 assay. Clones against
both epitopes induced apoptosis in THP ‐1. Negative control: targets alone
(shaded) and clone 2264.A1 specific for epitope CCNA1 [SEQ ID NO:5],
118 ‐127
which was HLA B*4001 restricted (data not shown, THP ‐1 was
B*4001 ‐negative), positive control: targets in presence of 4 M camptothecine.
2196.D9, 2196.D11 were specific for epitope CCNA1 [SEQ ID NO:3],
227 ‐235
clone 2264.E30 was specific for epitope CCNA1 [SEQ ID NO:8].
341 ‐351
Figure 5 shows human cyclin A1 (CCNA1) isoform c amino acid
(Fig. 5A)(SEQ ID NO:9) and encoding polynucleotide(Fig. 5B) (SEQ ID NO:10)
sequences.
Figure 6 shows activity of T ‐cell clone 2196.D11 specific for
CCNA1 [SEQ ID NO:3] in an apoptosis induction (caspase-3) assay (Fig.
227 ‐235
6A) and in a Cr release cytolysis assay (Fig. 6B).
DETAILED DESCRIPTION
Embodiments of the present invention as disclosed herein relate
to the unexpected discoveries that the intracellular protein human cyclin A1
(CCNA1, e.g., NCBI reference sequence (isoform c) NP_001104517.1,
GI:161377472; NM_001111047.1 GI:161377471) is a leukemia-associated
antigen (LAA), and that certain specific short peptides of at least 9, 10, 11 or 12
contiguous amino acids from the CCNA1 sequence contain immunogenic
epitopes that are recognized by T-cells in a major histocompatibility complex
(MHC) antigen-restricted (e.g., HLA-restricted) manner. Surprisingly, despite
the occurrence of CCNA1 as an intracellular protein with a limited cell type
expression pattern and tissue distribution, as disclosed herein CCNA1 is a
cancer associated antigen and CCNA1-derived peptides are capable of eliciting
CCNA1-specific T-cell responses. In certain preferred embodiments the herein
described CCNA1-derived peptides are capable of eliciting CCNA1-specific
cytotoxic lymphocyte (CTL) responses by class I HLA-restricted CD8 T-cells.
As described in greater detail below, the intracellular protein
CCNA1, which has been previously shown in murine studies to contribute to
leukemogenesis and to promote cell proliferation and survival, has been
detected in the LSC compartment of approximately 50% of all AML patients,
and is not detectable in other tissues with the exception of the testis. Using
dendritic cells pulsed with a peptide library spanning the entire CCNA1 isoform
c that is found in LSC, T ‐cells were generated that were capable of responding
to many different CCNA1-derived oligopeptides. Eight CCNA1-derived
peptides were identified that were immunogenic for T-cells, two of which were
more fully characterized as immunogenic, HLA A*0201-restricted epitopes of
CCNA1. T ‐cell clones specific for these epitopes recognized peptide-pulsed
target cells and also exhibited cytotoxicity against an HLA A*0201 ‐positive AML
line, THP ‐1, which endogenously expresses CCNA1.
The compositions and methods described herein will in certain
embodiments have therapeutic utility for the treatment of diseases and
conditions associated with CCNA1 overexpression (e.g., detectable CCNA1
expression at a level that is greater in magnitude, in a statistically significant
manner, than the level of CCNA1 expression that is detectable in a normal or
disease-free cell). Such diseases include various forms of cancer and include
without limitation hematologic malignancies that arise from CCNA1
overexpressing leukemia stem cells (LSC), for instance, acute myeloid
leukemia (AML). Non-limiting examples of these and related uses are
described herein and include in vitro and in vivo stimulation of CCNA1 antigen-
specific T-cell responses, such as by the use of immunogenic CCNA1 peptides
in peptide-based vaccines, the use of vaccines that are based on engineered
polynucleotides that encode such immunogenic CCNA1 peptides or additional
immunogenic peptides present in CCNA1, or the use of larger fragments or the
whole CCNA1 protein to induce T-cell responses.
Also contemplated, by way of illustration and not limitation, are
immunotherapeutic protocols involving the adoptive transfer to a subject (e.g.,
an AML patient) of antigen-presenting cells that have been pulsed in vitro with
immunogenic CCNA1 peptides or with CCNA1 protein or that have been
modified to express immunogenic CCNA1 peptides, and/or adoptive transfer to
the subject of CCNAspecific T-cells that have been induced in vitro by
exposure to antigen-presenting cells that have been pulsed in vitro with
immunogenic CCNA1 peptides. Principles of antigen processing by antigen
presenting cells (APC) such as dendritic cells, macrophages, lymphocytes and
other cell types, and of antigen presentation by APC to T-cells, including major
histocompatibility complex- (MHC) restricted presentation between
immunocompatible (e.g., sharing at least one allelic form of an MHC gene that
is relevant for antigen presentation) APC and T-cells, are well established (see,
e.g., Murphy, Janeway’s Immunobiology (8 Ed.) 2011 Garland Science, NY;
chapters 6, 9 and 16). Adoptive transfer protocols using unselected or selected
T-cells are known in the art (e.g., US2011/0052530, US2010/0310534; Ho et
al., 2006 J. Imm. Meth. 310:40; Ho et al., 2003 Canc. Cell 3:431) and may be
modified according to the teachings herein for use with transfer cell populations
containing T-cells that are specifically induced by one or more immunogenic
CCNA1-derived T-cell epitope-containing peptides.
As another non-limiting example, certain presently disclosed
embodiments contemplate cloning CCNA1-reactive T-cells that have been
induced in vitro by exposure to antigen-presenting cells that have been pulsed
in vitro with immunogenic CCNA1 peptides, and from such T-cells identifying
and cloning the functional (e.g., productively rearranged) T-cell receptor (TCR)
encoding genes, which may then be used to transfect/transduce a T-cell
population for adoptive transfer into subjects. Recent advances in TCR
sequencing have been described (e.g., Robins et al., 2009 Blood 114:4099;
Robins et al., 2010 Sci. Translat. Med. 2:47ra64, PMID: 20811043; Robins et
al. 2011 (Sept. 10) J. Imm. Meth. Epub ahead of print, PMID: 21945395;
Warren et al., 2011 Genome Res. 21:790) and may be employed in the course
of practicing these embodiments according to the present disclosure. Similarly,
methods for transfecting/transducing T-cells with desired nucleic acids have
been described (e.g., US2004/0087025) as have adoptive transfer procedures
using T-cells of desired antigen-specificity (e.g., Schmitt et al., 2009 Hum. Gen.
:1240; Dossett et al., 2009 Mol. Ther. 17:742; Till et al., 2008 Blood
112:2261; Wang et al., 2007 Hum. Gene Ther. 18:712; Kuball et al., 2007 Blood
109:2331; US2011/0243972; US2011/0189141; Leen et al., 2007 Ann. Rev.
Immunol. 25:243), such that adaptation of these methodologies to the presently
disclosed embodiments is contemplated, based on the teachings herein,
including those that are directed to specific CCNA1-derived peptides that are
capable of eliciting antigen-specific T-cell responses.
Presently disclosed T-cell immunogens, for use in inducing or
eliciting immune responses against inappropriately CCNA1-overexpressing
cells such as cancer cells, include isolated peptides that are capable of eliciting
an antigen-specific T-cell response to human cyclin A1 (CCNA1), each peptide
comprising at least one of a full length CCNA1 polypeptide or a CCNA1-derived
polypeptide of no more than 400, 350, 300, 250, 200, 150, 125, 100, 80, 70, 60,
50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acids
wherein the polypeptide comprises a sequence of at least 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20, or 400, 350, 300, 250, 200, 150, 125, 100, 80,
70, 60, 50, 40, 30, 25 contiguous amino acids from the CCNA1 amino acid
sequence set forth in SEQ ID NO:9. CCNA1-overexpressing cells cancer cells
include cells of hematologic malignancies such as lymphoma and leukemia,
and in particular, leukemia stem cells and/or acute myeloid leukemia cells.
According to certain presently disclosed embodiments, an isolated
peptide capable of eliciting an antigen-specific T-cell response to human cyclin
A1 (CCNA1), comprises a polypeptide of general formula I:
N-X-C [I]
wherein:
(a) N-X-C is a polypeptide of no more than 20, 19, 18, 17, 16,
, 14, 13, 12, 11, 10 or 9 amino acids in which X comprises an amino acid
sequence that is selected from:
CCNA1(120 ‐131) VDTGTLKSDLHF [SEQ ID NO:1],
CCNA1(218 ‐226) AETLYLAVN [SEQ ID NO:2],
CCNA1(227 ‐235) FLDRFLSCM [SEQ ID NO:3],
CCNA1(253 ‐261) ASKYEEIYP [SEQ ID NO:4],
CCNA1(118 ‐127) YEVDTGTLKS [SEQ ID NO:5],
CCNA1(167 ‐175) YAEEIYQYL [SEQ ID NO:6],
CCNA1(330 ‐339) LEADPFLKYL [SEQ ID NO:7], and
CCNA1(341 ‐351) SLIAAAAFCLA [SEQ IN NO:8],
and also wherein:
(b) N is an amino terminus of the peptide and consists of 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are independently selected from
natural and non-natural amino acids amino acids, and wherein (c) C is a
carboxy terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
11 amino acids that are independently selected from natural and non-natural
amino acids amino acids.
Accordingly in these and other embodiments it will be appreciated
that the amino terminus of certain CCNA1-derived peptides disclosed herein as
comprising T-cell immunogenic epitopes may consist of 1-11 independently
selected natural or non-natural amino acids, and/or that in certain embodiments
the carboxy terminus of certain such peptides may consist of 1-11
independently selected natural or non-natural amino acids, where such amino
and carboxy termini may have any sequence so long as the isolated peptide is
of no more than 9-20 amino acids and comprises N-X-C as recited herein, and
is capable of specifically eliciting an antigen-specific T-cell response to human
cyclin A1 (CCNA1).
Disclosed herein are a number of representative CCNA1-derived
peptides that comprise N-X-C according to formula [I] as recited herein, and
that are capable of specifically eliciting an antigen-specific T-cell response to
human cyclin A1 (CCNA1). The presently contemplated invention
embodiments, however, are not intended to be so limited such that in view of
the present disclosure those familiar with the art will be able readily to make
and use additional CCNA1 peptides (and variants thereof) that are
immunogenic for T-cells.
For example, determination of the three-dimensional structures of
representative immunogenic CCNA1-derived peptides bearing T-cell epitopes
as described herein may be made through routine methodologies such that
substitution of one or more amino acids with selected natural or non-natural
amino acids can be virtually modeled for purposes of determining whether a so
derived structural variant retains the space-filling, charge, hydrophilic and/or
hydrophobic properties of presently disclosed species, including modeling of
potential peptide affinity interactions with MHC peptide-binding grooves (e.g.,
BIMAS molecular modeling software, described by Parker et al., J. Immunol.
152:163, 1994; Tsites database, Feller et al. 1991 Nature 349:720; Rothbard et
al., 1988 EMBO J. 7:93-100; Deavin et al., 1996 Mol. Immunol. 33:145-155;
and other HLA peptide binding prediction analyses). See also, for instance,
Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science 309: 1868-1871
(2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat.
Acad. Sci. USA 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et
al., Nature 450:259 (2007); Raman et al. Science 327:1014-1018 (2010).
These and other references describe computer algorithms that may be used for
related embodiments, such as for rational design of variants of the CCNA1-
derived peptides bearing T-cell epitopes as provided herein (e.g., SEQ ID
NOS:1-8), for instance, by allowing for determination of atomic dimensions from
space-filling models (van der Waals radii) of energy-minimized conformations.
In view of the present disclosure that the CCNA1 polypeptide and
CCNA1-derived peptides contain immunogenic epitopes, e.g., the molecular
structures that are specifically recognized by T-cells via the T cell receptor
(TCR) including via MHC-restricted T-cell recognition, it is thus expressly
contemplated that alterations (e.g., increases or decreases that are detectable
with statistical significance) in the immunogenicity of any given epitope-bearing
CCNA1 peptide may be introduced by structural modification, for example, to
obtain immunogenic CCNA1 peptide-derived variants. Means for enhancing
the immunogenicity of a peptide-defined epitope are known in the art, and may
include the altered peptide ligand (APL) approach by which structural
modifications are made to a given peptide. Peptide variants of enhanced
immunogenicity have been generated as APLs, as described in other antigen
systems, for instance, by Abdul-Alim et al. (2010 J. Immunol. 184:6514); Douat-
Casassus et al. (2007 J. Med. Chem. 50:1598); Carrabba et al. (2003 Canc.
Res. 63:1560); and Shang et al. (2009 Eur. J. Immunol. 39:2248). Accordingly
it will be appreciated from the present disclosure that CCNA1 peptide
sequences include a large number of immunogenic epitopes for T-cells, such
that CCNA1 fragments (e.g., sequences of at least 7, 8, 9, 10, 11, 12, 13, 14,
, 16, 17, 18, 19 or 20, or 400, 350, 300, 250, 200, 150, 125, 100, 80, 70, 60,
50, 40, 30, 25 contiguous amino acids from the CCNA1 amino acid sequence
set forth in SEQ ID NO:9) and/or variants as provided herein (including APLs)
may be encompassed within certain embodiments.
Some additional non-limiting examples of computer algorithms
that may be used for these and related embodiments, such as for rational
design of variants of the herein described CCNA1 immunogenic peptide
epitopes (e.g., SEQ ID NOS:1-8), include NAMD, a parallel molecular dynamics
code designed for high-performance simulation of large biomolecular systems,
and VMD which is a molecular visualization program for displaying, animating,
and analyzing large biomolecular systems using 3-D graphics and built-in
scripting (see Phillips, et al., Journal of Computational Chemistry, 26:1781-
1802, 2005; Humphrey, et al., "VMD - Visual Molecular Dynamics", J. Molec.
Graphics, 1996, vol. 14, pp. 33-38; see also the website for the Theoretical and
Computational Biophysics Group, University of Illinois at Urbana-Champagne,
at ks.uiuc.edu/Research/vmd/). Many other computer programs are known in
the art and available to the skilled person and allow for determining atomic
dimensions from space-filling models (van der Waals radii) of energy-minimized
conformations; for example, GRID, which seeks to determine regions of high
affinity for different chemical groups, thereby enhancing binding; Monte Carlo
searches, which calculate mathematical alignment; and CHARMM (Brooks et
al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al (1981) J.
Comput. Chem. 106: 765), which assess force field calculations, and analysis
(see also, Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand
(1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644;
Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ. Health
Perspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488).
A variety of appropriate computational computer programs are also
commercially available, such as from Schrödinger (Munich, Germany).
"Natural or non-natural amino acid" includes any of the common
naturally occurring amino acids which serve as building blocks for the
biosynthesis of peptides, polypeptides and proteins (e.g., alanine, cysteine,
aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,
leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine,
valine, tryptophan, tyrosine) and also includes modified, derivatized,
enantiomeric, rare and/or unusual amino acids, whether naturally occurring or
synthetic, for instance, hydroxyproline, hydroxylysine, desmosine,
isodesmosine, -N-methyllysine, -N-trimethyllysine, methylhistidine,
dehydrobutyrine, dehydroalanine, -aminobutyric acid, -alanine, -
aminobutyric acid, homocysteine, homoserine, citrulline, ornithine and other
amino acids that may be isolated from a natural source and/or that may be
chemically synthesized, for instance, as may be found in Proteins, Peptides and
Amino Acids Sourcebook (White, J.S. and White, D.C., 2002 Humana Press,
Totowa, NJ) or in Amino Acid and Peptide Synthesis (Jones, J., 2002 Oxford
Univ. Press USA, New York) or in Unnatural Amino Acids, ChemFiles Vol. 1,
No. 5 (2001 Fluka Chemie GmbH; Sigma-Aldrich, St. Louis, MO) or in
Unnatural Amino Acids II, ChemFiles Vol. 2, No. 4 (2002 Fluka Chemie GmbH;
Sigma-Aldrich, St. Louis, MO). Additional descriptions of natural and/or non-
natural amino acids may be found, for example, in Kotha, 2003 Acc. Chem.
Res. 36:342; Maruoka et al., 2004 Proc. Nat. Acad. Sci. USA 101:5824;
Lundquist et al., 2001 Org. Lett. 3:781; Tang et al., 2002 J. Org. Chem.
67:7819; Rothman et al., 2003 J. Org. Chem. 68:6795; Krebs et al., 2004
Chemistry 10:544; Goodman et al., 2001 Biopolymers 60:229; Sabat et al.,
2000 Org. Lett. 2:1089; Fu et al., 2001 J. Org. Chem. 66:7118; and Hruby et al.,
1994 Meths. Mol. Biol. 35:249. The standard three-letter abbreviations and 1-
letter symbols are used herein to designate natural and non-natural amino
acids.
Other non-natural amino acids or amino acid analogues are
known in the art and include, but are not limited to, non-natural L or D
derivatives (such as D-amino acids present in peptides), fluorescent labeled
amino acids, as well as specific examples including O-methyl-L-tyrosine, L
(2-naphthyl)alanine, 3-methyl-phenylalanine, 3-idio-tyrosine, O-propargyl-
tyrosine, homoglutamine, an Oallyl-L-tyrosine, a 4-propyl-L-tyrosine, a 3-
nitro-L-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated
phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-
acyl-L-phenylalanine, a p-acetyl-L-phenylalanine, an m-acetyl-L-phenylalanine,
selenomethionine, telluromethionine, selenocysteine, an alkyne phenylalanine,
an O-allyl-L-tyrosine, an O-(2-propynyl)-L-tyrosine, a p-ethylthiocarbonyl-L-
phenylalanine, a p-(3-oxobutanoyl)-L-phenylalanine, a p-benzoyl-L-
phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine,
homoproparglyglycine, azidohomoalanine, a p-iodo-phenylalanine, a p-bromo-
L-phenylalanine, dihydroxy-phenylalanine, dihydroxyl-L-phenylalanine, a p-
nitro-L-phenylalanine, an m-methoxy-L-phenylalanine, a p-iodo-phenylalanine,
a p-bromophenylalanine, a p-amino-L-phenylalanine, and an isopropyl-L-
phenylalanine, trifluoroleucine, norleucine (“Nle”), D-norleucine (“dNle” or “D-
Nle”), 5-fluoro-tryptophan, para-halo-phenylalanine, homo-phenylalanine
(“homo-Phe”), seleno-methionine, ethionine, S-nitroso-homocysteine, thia-
proline, 3-thienyl-alanine, homo-allyl-glycine, trifluoroisoleucine, trans and cis
aminohexenoic acid, 2-butynyl-glycine, allyl-glycine, para-azido-
phenylalanine, para-cyano-phenylalanine, para-ethynyl-phenylalanine,
hexafluoroleucine, 1,2,4-triazolealanine, 2-fluoro-histidine, L-methyl histidine,
3-methyl-L-histidine, βthienyl-L-alanine, β-(2-thiazolyl)-DL-alanine,
homoproparglyglycine (HPG) and azidohomoalanine (AHA) and the like.
In certain embodiments a natural or non-natural amino acid may
be present that comprises an aromatic side chain, as found, for example, in
phenylalanine or tryptophan or analogues thereof including in other natural or
non-natural amino acids based on the structures of which the skilled person will
readily recognize when an aromatic ring system is present, typically in the form
of an aromatic monocyclic or multicyclic hydrocarbon ring system consisting
only of hydrogen and carbon and containing from 6 to 19 carbon atoms, where
the ring system may be partially or fully saturated, and which may be present as
a group that includes, but need not be limited to, groups such as fluorenyl,
phenyl and naphthyl.
In certain embodiments a natural or non-natural amino acid may
be present that comprises a hydrophobic side chain as found, for example, in
alanine, valine, isoleucine, leucine, proline, phenylalanine, tryptophan or
methionine or analogues thereof including in other natural or non-natural amino
acids based on the structures of which the skilled person will readily recognize
when a hydrophobic side chain (e.g., typically one that is non-polar when in a
physiological milieu) is present. In certain embodiments a natural or non-
natural amino acid may be present that comprises a basic side chain as found,
for example, in lysine, arginine or histidine or analogues thereof including in
other natural or non-natural amino acids based on the structures of which the
skilled person will readily recognize when a basic (e.g., typically polar and
having a positive charge when in a physiological milieu) is present.
Polypeptides disclosed herein may include L- and/or D- amino
acids so long as the biological activity (e.g., CCNA1-specific immunogenicity for
T-cells) of the polypeptide is maintained. The isolated CCNA1-derived
polypeptides may comprise in certain embodiments any of a variety of known
natural and artificial post-translational or post-synthetic covalent chemical
modifications by reactions that may include glycosylation (e.g., N-linked
oligosaccharide addition at asparagine residues, O-linked oligosaccharide
addition at serine or threonine residued, glycation, or the like), fatty acylation,
acetylation, PEGylation, and phosphorylation. Polypeptides herein disclosed
may further include analogs, alleles and allelic variants which may contain
amino acid deletions, or additions or substitutions of one or more amino acid
residues with other naturally occurring amino acid residues or non-natural
amino acid residues.
Peptide and non-peptide analogs may be referred to as peptide
mimetics or peptidomimetics, and are known in the pharmaceutical industry
(Fauchere, J. Adv. Drug Res. 15:29 (1986); Evans et al. J. Med. Chem. 30:
1229 (1987)). These compounds may contain one or more non-natural amino
acid residue(s), one or more chemical modification moieties (for example,
glycosylation, pegylation, fluorescence, radioactivity, or other moiety), and/or
one or more non-natural peptide bond(s) (for example, a reduced peptide bond:
--CH -NH --). Peptidomimetics may be developed by a variety of methods,
including by computerized molecular modeling, random or site-directed
mutagenesis, PCR-based strategies, chemical mutagenesis, and others.
The term “isolated” means that the material is removed from its
original environment (e.g., the natural environment if it is naturally occurring).
For example, a naturally occurring nucleic acid or polypeptide present in a living
animal is not isolated, but the same nucleic acid or polypeptide, separated from
some or all of the co-existing materials in the natural system, is isolated. Such
nucleic acid could be part of a vector and/or such nucleic acid or polypeptide
could be part of a composition (e.g., a cell lysate), and still be isolated in that
such vector or composition is not part of the natural environment for the nucleic
acid or polypeptide. The term “gene” means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and following the
coding region “leader and trailer” as well as intervening sequences (introns)
between individual coding segments (exons).
Certain embodiments relate to nucleic acids that encode the
polypeptides contemplated herein, for instance, CCNA1-derived polypeptides
that contain epitopes recognized by, and immunogenic for, T-cells. As one of
skill in the art will recognize, a nucleic acid may refer to a single and/or a double
stranded DNA, cDNA or RNA in any form, and may include a positive and a
negative strand of the nucleic acid which complement each other, including
anti-sense DNA, cDNA and RNA. Also included are siRNA, microRNA, RNA—
DNA hybrids, ribozymes, and other various naturally occurring or synthetic
forms of DNA or RNA.
Certain embodiments include nucleic acids contained in a vector.
One of skill in the art can readily ascertain suitable vectors for use with certain
herein disclosed embodiments. A typical vector may comprise a nucleic acid
molecule capable of transporting another nucleic acid to which it has been
linked, or which is capable of replication in a host organism. Some examples of
vectors include plasmids, viral vectors, cosmids, and others. Some vectors
may be capable of autonomous replication in a host cell into which they are
introduced (e.g. bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors), whereas other vectors may be integrated into
the genome of a host cell upon introduction into the host cell and thereby
replicate along with the host genome. Additionally, some vectors are capable
of directing the expression of genes to which they are operatively linked (these
vectors may be referred to as “expression vectors”). According to related
embodiments, it is further understood that, if one or more agents (e.g.,
polynucleotides encoding CCNA1-derived immunogenic peptide epitopes, or
variants thereof, as described herein) is co-administered to a subject, that each
agent may reside in separate or the same vectors, and multiple vectors (each
containing a different agent the same agent) may be introduced to a cell or cell
population or administered to a subject.
In certain embodiments, the nucleic acid encoding the herein
described CCNA1-derived polypeptides that contain epitopes recognized by
and immunogenic for T-cells, may be operatively linked to certain elements of a
vector. For example, polynucleotide sequences that are needed to effect the
expression and processing of coding sequences to which they are ligated may
be operatively linked. Expression control sequences may include appropriate
transcription initiation, termination, promoter and enhancer sequences; efficient
RNA processing signals such as splicing and polyadenylation signals;
sequences that stabilize cytoplasmic mRNA; sequences that enhance
translation efficiency (i.e. Kozak consensus sequences); sequences that
enhance protein stability; and possibly sequences that enhance protein
secretion. Expression control sequences may be operatively linked if they are
contiguous with the gene of interest and expression control sequences that act
in trans or at a distance to control the gene of interest.
In particular embodiments, the recombinant expression vector is
delivered to an appropriate cell, for example, an antigen-presenting cell i.e., a
cell that displays a peptide/MHC complex on its cell surface (e.g., a dendritic
cell) that will induce the desired CCNA1-specific cell-mediated immune
response, such as CD8 T-cell response including a cytotoxic T lymphocyte
(CTL) response. The recombinant expression vectors may therefore also
include, for example, lymphoid tissue-specific transcriptional regulatory
elements (TRE) such as a B lymphocyte, T lymphocyte, or dendritic cell specific
TRE. Lymphoid tissue specific TRE are known in the art (see, e.g., Thompson
et al., Mol. Cell. Biol. 12, 1043-53 (1992); Todd et al., J. Exp. Med. 177, 1663-
74 (1993); Penix et al., J. Exp. Med. 178:1483-96 (1993)).
In certain configurations, recombinant expression vectors may
contain polynucleotide sequences that encode dendritic cell (DC) maturation /
stimulatory factors. Exemplary stimulatory molecules include GM-CSF, IL-2, IL-
4, IL-6, IL-7, IL-15, IL-21, IL-23, TNF , B7.1, B7.2, 4-1BB, CD40 ligand
(CD40L), drug-inducible CD40 (iCD40), and the like. These polynucleotides
are typically under the control of one or more regulatory elements that direct the
expression of the coding sequences in dendritic cells. Maturation of dendritic
cells contributes to successful vaccination (see, e.g., Banchereau et al., Nat.
Rev. Immunol. 5:296-306 (2005); Schuler et al., Curr. Opin. Immunol. 15:138-
147 (2003); Figdor et al., Nat. Med. 10:475-480 (2004)). Maturation can
transform DCs from cells actively involved in antigen capture into cells
specialized for T-cell priming. For example, engagement of CD40 by CD40L on
CD4-helper T-cells is an important signal for DC maturation, resulting in potent
activation of CD8+ T-cells. Such stimulatory molecules are also referred to as
maturation factors or maturation stimulatory factors.
In addition to vectors, certain embodiments relate to host cells
that comprise the vectors that are presently disclosed. One of skill in the art
readily understands that many suitable host cells are available in the art. A
host cell may include any individual cell or cell culture which may receive a
vector or the incorporation of nucleic acids and/or proteins, as well as any
progeny cells. The term also encompasses progeny of the host cell, whether
genetically or phenotypically the same or different. Suitable host cells may
depend on the vector and may include mammalian cells, animal cells, human
cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may
be induced to incorporate the vector or other material by use of a viral vector,
transformation via calcium phosphate precipitation, DEAE-dextran,
electroporation, microinjection, or other methods. For example, See Sambrook
et al. Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor
Laboratory, 1989).
In certain embodiments, immunogenic variants are provided of the
herein described CCNA1-derived polypeptides that contain epitopes recognized
by, and immunogenic for, T-cells; these variants include polypeptide species
that have one or more amino acid substitutions, insertions, or deletions in the
amino acid sequence relative to the sequences of formula (I) or SEQ ID NOS:1-
8 as presented herein. Conservative substitutions of amino acids are well
known and may occur naturally in the polypeptide or may be introduced when
the polypeptide is recombinantly produced. Amino acid substitutions, deletions,
and additions may be introduced into a polypeptide using well-known and
routinely practiced mutagenesis methods (see, e.g., Sambrook et al. Molecular
Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press,
NY 2001)). Oligonucleotide-directed site-specific (or segment specific)
mutagenesis procedures may be employed to provide an altered polynucleotide
that has particular codons altered according to the substitution, deletion, or
insertion desired. Deletion or truncation variants of specific peptides that may
be used as immunogens may also be constructed by using convenient
restriction endonuclease sites adjacent to the desired deletion. Subsequent to
restriction, overhangs may be filled in and the DNA re-ligated. Alternatively,
random mutagenesis techniques, such as alanine scanning mutagenesis, error
prone polymerase chain reaction mutagenesis, and oligonucleotide-directed
mutagenesis may be used to prepare immunogen polypeptide variants (see,
e.g., Sambrook et al., supra). Species (or variants) of a particular CCNA1-
derived immunogen (or polypeptide fragment thereof) may include a
polypeptide immunogen that has at least 85%, 90%, 95%, or 99% amino acid
sequence identity to any of the exemplary amino acid sequences disclosed
herein (e.g., SEQ ID NOS:1-8, or polypeptides of no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10 or 9 amino acids in which at least one of SEQ ID
NOS:1-8 may be present).
These CCNA1-derived peptide immunogen variants retain one or
more biological activities or functions of the respective CCNA1-derived peptide
that is immunogenic for T-cells as described herein (e.g., SEQ ID NOS:1-8). In
particular, such immunogens that are variants of a herein described CCNA1-
derived peptide retain, in a statistically, clinically, or biologically significant
manner, the capability to induce a T-cell response (including a cytotoxic T
lymphocyte response). Given the many molecular biology, protein expression,
and protein isolation techniques and methods routinely practiced in the art for
introducing mutations in a polypeptide, preparing polypeptide fragments,
isolating the fragments and variants, and analyzing such products,
immunogenic CCNA1 polypeptide variants and fragments thereof having the
desired biological activities can be made readily and without undue
experimentation based on the disclosure herein.
A variety of criteria known to persons skilled in the art indicate
whether an amino acid that is substituted at a particular position in a peptide or
polypeptide is conservative (or similar). For example, a similar amino acid or a
conservative amino acid substitution is one in which an amino acid residue is
replaced with an amino acid residue having a similar side chain. Similar amino
acids may be included in the following categories: amino acids with basic side
chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains
(e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino
acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and
amino acids with aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan). Proline, which is considered more difficult to classify, shares
properties with amino acids that have aliphatic side chains (e.g., leucine, valine,
isoleucine, and alanine). In certain circumstances, substitution of glutamine for
glutamic acid or asparagine for aspartic acid may be considered a similar
substitution in that glutamine and asparagine are amide derivatives of glutamic
acid and aspartic acid, respectively. As understood in the art “similarity”
between two polypeptides is determined by comparing the amino acid
sequence and conserved amino acid substitutes thereto of the polypeptide to
the sequence of a second polypeptide (e.g., using GENEWORKS, Align, the
BLAST algorithm, or other algorithms described herein and practiced in the art).
As described herein for immunogenic peptide fragments of
CCNA1, assays for assessing whether a respective variant folds into a
conformation comparable to the non-variant polypeptide or fragment include, for
example, the ability of the protein to react with mono- or polyclonal antibodies
that are specific for native or unfolded epitopes, the retention of ligand-binding
functions, and the sensitivity or resistance of the mutant protein to digestion
with proteases (see Sambrook et al., supra). Such variants can be identified,
characterized, and/or made according to methods described herein or other
methods known in the art, which are routinely practiced by persons skilled in
the art.
Isolated/recombinant immunogens included in the immunogenic
compositions described herein may be produced and prepared according to
various methods and techniques routinely practiced in the molecular biology
and/or polypeptide purification arts. Construction of an expression vector that is
used for recombinantly producing an immunogen of interest can be
accomplished using any of numerous suitable molecular biology engineering
techniques known in the art, including, without limitation, the standard
techniques of restriction endonuclease digestion, ligation, transformation,
plasmid purification, and DNA sequencing, for example as described in
Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current
Protocols in Molecular Biology (2003)). To obtain efficient transcription and
translation, the polynucleotide sequence in each recombinant expression
construct includes at least one appropriate expression control sequence (also
called a regulatory sequence), such as a leader sequence and particularly a
promoter operably (i.e., operatively) linked to the nucleotide sequence encoding
the immunogen.
Methods that may be used for isolating and purifying a
recombinantly produced immunogenic peptide, by way of example, may include
obtaining supernatants from suitable host cell/vector systems that secrete the
recombinant immunogen into culture media and then concentrating the media
using a commercially available filter. Following concentration, the concentrate
may be applied to a single suitable purification matrix or to a series of suitable
matrices, such as an affinity matrix or an ion exchange resin. One or more
reverse phase HPLC steps may be employed to further purify a recombinant
polypeptide. These purification methods may also be employed when isolating
an immunogen from its natural environment. Methods for large scale
production of one or more of the isolated/recombinant immunogens described
herein include batch cell culture, which is monitored and controlled to maintain
appropriate culture conditions. Purification of the immunogen may be
performed according to methods described herein and known in the art and that
comport with laws and guidelines of domestic and foreign regulatory agencies.
The presence of a malignant condition in a subject refers to the
presence of dysplastic, cancerous and/or transformed cells in the subject,
including, for example neoplastic, tumor, non-contact inhibited or oncogenically
transformed cells, or the like (e.g., hematologic cancers including lymphomas
and leukemias, such as acute myeloid leukemia, chronic myeloid leukemia,
etc.) which are known to the art and for which criteria for diagnosis and
classification are established (e.g., Hanahan and Weinberg, 2011 Cell 144:646;
Hanahan and Weinberg 2000 Cell 100:57; Cavallo et al., 2011 Canc. Immunol.
Immunother. 60:319; Kyrigideis et al., 2010 J. Carcinog. 9:3). In preferred
embodiments contemplated by the present invention, for example, such cancer
cells may be cells of acute myeloid leukemia, B-cell lymphoblastic leukemia, T-
cell lymphoblastic leukemia, or myeloma, including cancer stem cells that are
capable of initiating and serially transplanting any of these types of cancer (see,
e.g., see Park et al. 2009 Molec. Therap. 17:219).
The CCNA1-derived polypeptides that contain epitopes
recognized by and immunogenic for T-cells, as described herein (e.g., SEQ ID
NOS:1-8, and variants thereof), may be functionally characterized according to
any of a large number of art accepted methodologies for assaying T-cell
activity, including determination of T-cell activation or induction and also
including determination of T-cell responses that are antigen-specific. Examples
include determination of T-cell proliferation, T-cell cytokine release, antigen-
specific T-cell stimulation, MHC-restricted T-cell stimulation, CTL activity (e.g.,
by detecting Cr release from pre-loaded target cells and/or by caspase-3
assay (e.g., Jerome et al. 2003 Apoptosis 8:563; He et al., 2005 J. Imm. Meth.
304:43), changes in T-cell phenotypic marker expression, and other measures
of T-cell functions. Procedures for performing these and similar assays are
may be found, for example, in Lefkovits (Immunology Methods Manual: The
Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols
in Immunology; Weir, Handbook of Experimental Immunology, Blackwell
Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in
Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green
and Reed, Science 281:1309 (1998) and references cited therein).
Levels of cytokines may be determined according to methods
described herein and practiced in the art, including for example, ELISA,
ELISPOT, intracellular cytokine staining, and flow cytometry and combinations
thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell
proliferation and clonal expansion resulting from an antigen-specific elicitation
or stimulation of an immune response may be determined by isolating
lymphocytes, such as circulating lymphocytes in samples of peripheral blood
cells or cells from lymph nodes, stimulating the cells with antigen, and
measuring cytokine production, cell proliferation and/or cell viability, such as by
incorporation of tritiated thymidine or non-radioactive assays, such as MTT
assays and the like. The effect of an immunogen described herein on the
balance between a Th1 immune response and a Th2 immune response may be
examined, for example, by determining levels of Th1 cytokines, such as IFN- ,
IL-12, IL-2, and TNF-β, and Type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10,
and IL-13.
The level of a CTL immune response thus may be determined by
any one of numerous immunological methods described herein and routinely
practiced in the art. The level of a CTL immune response may be determined
prior to and following administration of any one of the herein described CCNA1-
derived polypeptides that contain epitopes recognized by, and immunogenic
for, T-cells (or administration of a composition comprising a polynucleotide
encoding such a polypeptide). Cytotoxicity assays for determining CTL activity
may be performed using any one of several techniques and methods routinely
practiced in the art (see, e.g., Henkart et al., “Cytotoxic T-Lymphocytes” in
Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins,
Philadelphia, PA), pages 1127-50, and references cited therein).
A binding partner or an antibody is said to be “immunospecific,”
“specific for” or to “specifically bind” an immunogen of interest if the antibody
reacts at a detectable level with the immunogen or immunogenic fragment
thereof, preferably with an affinity constant, K of greater than or equal to
4 -1 5 -1
about 10 M , or greater than or equal to about 10 M , greater than or equal
6 -1 7 -1
to about 10 M , greater than or equal to about 10 M , or greater than or
equal to 10 M . Affinity of an antibody for its cognate antigen is also
commonly expressed as a dissociation constant K , and an antibody
specifically binds to the immunogen of interest if it binds with a K of less than
or equal to 10 M, less than or equal to about 10 M, less than or equal to
- - -
6 7 8
about 10 M, less than or equal to 10 M, or less than or equal to 10 M.
Affinities of binding partners or antibodies can be readily
determined using conventional techniques, for example, those described by
Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)) and by surface
plasmon resonance (SPR; BIAcore , Biosensor, Piscataway, NJ). For surface
plasmon resonance, target molecules are immobilized on a solid phase and
exposed to a binding partner (or ligand) in a mobile phase running along a flow
cell. If ligand binding to the immobilized target occurs, the local refractive index
changes, leading to a change in SPR angle, which can be monitored in real
time by detecting changes in the intensity of the reflected light. The rates of
change of the SPR signal can be analyzed to yield apparent rate constants for
the association and dissociation phases of the binding reaction. The ratio of
these values gives the apparent equilibrium constant (affinity) (see, e.g., Wolff
et al., Cancer Res. 53:2560-2565 (1993)).
Antigen-specific T-cell responses are typically determined by
comparisons of observed T-cell responses according to any of the herein
described T-cell functional parameters (e.g., proliferation, cytokine release, CTL
activity, altered cell surface marker phenotype, etc.) that may be made between
T-cells that are exposed to a cognate antigen in an appropriate context (e.g.,
the antigen used to prime or activate the T-cells, when presented by
immunocompatible antigen-presenting cells) and T-cells from the same source
population that are exposed instead to a structurally distinct or irrelevant control
antigen. A response to the cognate antigen that is greater, with statistical
significance, than the response to the control antigen signifies antigen-
specificity.
A biological sample may be obtained from a subject for
determining the presence and level of an immune response to an immunogenic
CCNA1-derived polypeptide as described herein that contains an epitope
recognized by, and immunogenic for, T-cells. A “biological sample” as used
herein may be a blood sample (from which serum or plasma may be prepared),
biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings,
synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any
other tissue or cell preparation from the subject or a biological source.
Biological samples may also be obtained from the subject prior to receiving any
immunogenic composition, which biological sample is useful as a control for
establishing baseline (i.e., pre-immunization) data.
With respect to all immunoassays and methods described herein
for determining an immune response, a person skilled in the art will also readily
appreciate and understand experimental control conditions that are
appropriately included when practicing these methods. Concentrations of
reaction components, types of buffers, temperature, and time periods sufficient
to permit interaction of the reaction components can be determined and/or
adjusted according to methods described herein and with which persons skilled
in the art are familiar.
As generally referred to in the art, and as used herein, sequence
identity and sequence homology may be used interchangeably and generally
refer to the percentage of nucleotides or amino acid residues in a candidate
sequence that are identical with, respectively, the nucleotides or amino acid
residues in a native polynucleotide or polypeptide sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and not considering any conservative substitutions as part of
the sequence identity. Preferably, an isolated peptide capable of eliciting an
antigen-specific T-cell response to human cyclin A1 (CCNA1) as described
herein, or an encoding polynucleotide therefore, according to the embodiments
disclosed herein shares at least about 75%, at least about 80%, at least about
85%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% of the
amino acid residues (or of the nucleotides in a polynucleotide encoding such a
CCNA1-derived polypeptide) with the immunogenic peptides disclosed herein
as SEQ ID NOS:1-8. Such sequence identity may be determined according to
well known sequence analysis algorithms, including those available from the
University of Wisonsin Genetics Computer Group (Madison, WI), such as
FASTA, Gap, Bestfit, BLAST, or others.
It has also been determined according to certain embodiments of
the present invention that N-terminus extensions of the CCNA1 T-cell epitope-
containing peptides described herein can alter the affinity of the peptide binding
to a class I major histocompatibility complex (MHC) antigen in association with
which the peptide may be displayed on the surface of an antigen-presenting cell
(APC), and/or binding of the peptide to the T-cell receptor of a CCNA1-specific
T-cell, while C-terminus extensions may also enhance binding and/or activity of
the CCNA1-derived peptides. Accordingly, certain embodiments contemplate
the use of one or more of the peptides having amino acid sequences set forth in
SEQ ID NOS:1-8, and/or variants of such peptides as described herein, and
certain embodiments may additionally or alternatively include the use of
polypeptides of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9
amino acids that include in their sequences any of these peptides. Hence,
certain contemplated embodiments relate to CCNA1-derived T-cell
immunogenic peptides having amino-terminal and/or carboxy-terminal peptide
extensions in addition to the amino acid sequences set forth in SEQ ID NOS:1-
8, or variants thereof which, as described herein, may be selected for their
ability to elicit an antigen-specific T-cell response to CCNA1, such as a CTL
response.
As understood by a person skilled in the medical art, the terms,
“treat” and “treatment,” refer to medical management of a disease, disorder, or
condition of a subject (i.e., patient, host, who may be a human or non-human
animal) (see, e.g., Stedman’s Medical Dictionary). In general, an appropriate
dose and treatment regimen provide one or more of the herein described
CCNA1-derived peptide immunogens (e.g., SEQ ID NOS:1-8 and variants
thereof), and optionally an adjuvant, in an amount sufficient to provide
therapeutic and/or prophylactic benefit. Therapeutic and/or prophylactic benefit
resulting from therapeutic treatment and/or prophylactic or preventative
methods include, for example an improved clinical outcome, wherein the object
is to prevent or retard or otherwise reduce (e.g., decrease in a statistically
significant manner relative to an untreated control) an undesired physiological
change or disorder, or to prevent or or retard or retard or otherwise reduce the
expansion or severity of such disease or disorder. Beneficial or desired clinical
results from treating a subject include, but are not limited to, abatement,
lessening, or alleviation of symptoms that result from or are associated the
disease or disorder to be treated; decreased occurrence of symptoms;
improved quality of life; longer disease-free status (i.e., decreasing the
likelihood or the propensity that a subject will present symptoms on the basis of
which a diagnosis of a disease is made); diminishment of extent of disease;
stabilized (i.e., not worsening) state of disease; delay or slowing of disease
progression; amelioration or palliation of the disease state; and remission
(whether partial or total), whether detectable or undetectable; and/or overall
survival.
“Treatment” can also mean prolonging survival when compared to
expected survival if a subject were not receiving treatment. Subjects in need of
the methods and compositions described herein include those who already
have the disease or disorder as well as subjects prone to have or at risk of
developing the disease or disorder. Subjects in need of prophylactic treatment
include subjects in whom the disease, condition, or disorder is to be prevented
(i.e., decreasing the likelihood of occurrence or recurrence of the disease or
disorder). The clinical benefit provided by the compositions (and preparations
comprising the compositions) and methods described herein can be evaluated
by design and execution of in vitro assays, preclinical studies, and clinical
studies in subjects to whom administration of the compositions is intended to
benefit. The design and execution of the appropriate preclinical studies and
clinical studies can be readily performed by persons skilled in the relevant
art(s).
The isolated CCNA1-derived peptide immunogens (including
synthetically or recombinantly produced peptides), or recombinant expression
vectors encoding such peptide(s) may be administered to a subject in a
pharmaceutically or physiologically acceptable or suitable excipient or carrier.
Pharmaceutically acceptable excipients are biologically compatible vehicles,
e.g., physiological saline, which are described in greater detail herein, that are
suitable for administration to a human or other non-human subject including a
non-human mammalian subject.
With respect to administration of a recombinant expression vector,
a therapeutically effective amount provides an amount of the polynucleotide
which is capable of producing a clincally desirable result (i.e., a sufficient
amount of the CCNA1-derived peptide immunogen is expressed to induce or
enhance the immune response by T-cells specific for CCNA1 (e.g., cell-
mediated response, including a cytotoxic T cell response) in a statistically
significant manner) in a treated human or non-human animal. As is well known
in the medical arts, the dosage for any one patient depends upon many factors,
including the patient's size, weight, body surface area, age, the particular
compound to be administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. Doses will vary, but a
preferred dose for administration of an immunogenic composition comprising a
6 12
recombinant expression vector is sufficient to provide approximately 10 to 10
copies of the vector polynucleotide molecule.
Pharmaceutical compositions, including the CCNA1-specific T-cell
immunogenic compositions described herein, may be administered in a manner
appropriate to the disease or condition to be treated (or prevented) as
determined by persons skilled in the medical art. An appropriate dose and a
suitable duration and frequency of administration of the compositions will be
determined by such factors as the health condition of the patient, size of the
patient (i.e., weight, mass, or body area), the type and severity of the patient's
disease, the particular form of the active ingredient, and the method of
administration. In general, an appropriate dose and treatment regimen provide
the composition(s) in an amount sufficient to provide therapeutic and/or
prophylactic benefit (such as described herein, including an improved clinical
outcome, such as more frequent complete or partial remissions, or longer
disease-free and/or overall survival, or a lessening of symptom severity). For
prophylactic use, a dose should be sufficient to prevent, delay the onset of, or
diminish the severity of a disease associated with disease or disorder.
Prophylactic benefit of the immunogenic compositions administered according
to the methods described herein can be determined by performing pre-clinical
(including in vitro and in vivo animal studies) and clinical studies and analyzing
data obtained therefrom by appropriate statistical, biological, and clinical
methods and techniques, all of which can readily be practiced by a person
skilled in the art.
In general, the amount of an immunogen, including fusion
polypeptides as described herein, present in a dose, or produced in situ by an
encoding polynucleotide present in a dose, ranges from about 0.01 μg to about
1000 μg per kg of host. The use of the minimum dosage that is sufficient to
provide effective therapy is usually preferred. Patients may generally be
monitored for therapeutic or prophylactic effectiveness using assays suitable for
the condition being treated or prevented, which assays will be familiar to those
having ordinary skill in the art and which are described herein. When
administered in a liquid form, suitable dose sizes will vary with the size of the
patient, but will typically range from about 1 ml to about 500 ml (comprising
from about 0.01 μg to about 1000 μg per kg) for a 10-60 kg subject. Optimal
doses may generally be determined using experimental models and/or clinical
trials. The optimal dose may depend upon the body mass, body area, weight,
or blood volume of the subject. As described herein, the appropriate dose may
also depend upon the patient's (e.g., human) condition, that is, stage of the
disease, general health status, as well as age, gender, and weight, and other
factors familiar to a person skilled in the medical art.
For pharmaceutical compositions comprising a nucleic acid
molecule such as the recombinant expression vectors described herein, the
nucleic acid molecule may be present within any of a variety of delivery
systems known to those of ordinary skill in the art, including nucleic acid, and
bacterial, viral and mammalian expression systems such as, for example,
vector particles and recombinant expression constructs as provided herein.
Techniques for incorporating a polynucleotide (e.g., DNA) into such expression
systems are well known to those of ordinary skill in the art. In other certain
embodiments, the recombinant expression vector, which is typically DNA, may
also be “naked,” as described, for example, in Ulmer et al., Science 259:1745-
49 (1993) and reviewed by Cohen, Science 259:1691-92 (1993). The uptake of
naked DNA may be increased by coating the DNA onto biodegradable beads,
which are efficiently transported into the cells.
Nucleic acid molecules may be delivered into a cell according to
any one of several methods described in the art (see, e.g., Akhtar et al., Trends
Cell Bio. 2:139 (1992); Delivery Strategies for Antisense Oligonucleotide
Therapeutics, ed. Akhtar, 1995, Maurer et al., Mol. Membr. Biol. 16:129-40
(1999); Hofland and Huang, Handb. Exp. Pharmacol. 137:165-92 (1999); Lee et
al., ACS Symp. Ser. 752:184-92 (2000); U.S. Patent No. 6,395,713;
International Patent Application Publication No. WO 94/02595); Selbo et al., Int.
J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol. 23:103-12 (2002); U.S.
Patent Application Publication Nos. 2001/0007666, and 2003/077829). Such
delivery methods known to persons having skill in the art, include, but are not
restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation
into other vehicles, such as biodegradable polymers; hydrogels; cyclodextrins
(see, e.g., Gonzalez et al., Bioconjug. Chem. 10:1068-74 (1999); Wang et al.,
International Application Publication Nos. WO 03/47518 and WO 03/46185);
poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (also useful for
delivery of peptides and polypeptides and other substances) (see, e.g., U.S.
Patent No. 6,447,796; U.S. Patent Application Publication No. 2002/130430);
biodegradable nanocapsules; and bioadhesive microspheres, or by
proteinaceous vectors (International Application Publication No. WO 00/53722).
In another embodiment, the nucleic acid molecules can also be formulated or
complexed with polyethyleneimine and derivatives thereof, such as
polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)
or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-
triGAL) derivatives (see also, e.g., U.S. Patent Application Publication No.
2003/0077829).
Certain of the presently disclosed invention embodiments include
preventative treatment of a subject or cells, tissues or organs of a subject, that
is suspected of having or of being susceptible to a condition associated with
CCNA1 overexpression. The preventative treatment may be the same as or
different from the regimen (dosing and schedule, as well as choice of
immunogenic CCNA1-derived peptide and/or other therapeutic agents such as
antigen-presenting cells or adoptively transferred T-cells) employed to treat a
subject or cells, tissues or organs of a subject that has been confirmed to have
a condition associated with CCNA1 overexpression. Prevention and/or
treatment may also include the use of vaccines comprising compositions
disclosed herein, for example by way of illustration and not limitation, one or
more CCNA1-derived peptides that are immunogenic for antigen-specific T-
cells.
Particular contemplated embodiments relate to
immunotherapeutic regimens using the herein described compositions and
methods to elicit T-cell immune responses that are directed against leukemic
stem cells (LSC). More particularly, and according to non-limiting theory, as
disclosed herein it is believed that by certain of the present embodiments,
cytotoxic T lymphocyte (CTL) responses are elicited that are specifically
targeted against LSC. These and related embodiments are thus believed to
provide a highly specific approach to eliminate or substantially reduce LSC
populations in a subject, thereby providing benefits for the treatment of
leukemias that are characterized by CCNA1 overexpression, such as acute
myeloid leukemia (AML). These approaches also offer unprecedented
advantages pertaining to the specificity afforded by the restricted pattern of
CCNA1 overexpression, and to the avoidance of undesirable generalized toxic
effects that accompany many less target-specific immunotherapeutic
approaches.
A condition associated with CCNA1 overexpression includes any
disorder or condition in which underactivity, overactivity or improper activity of a
CCNA1 cellular or molecular event is present, and typically results from
unusually high (with statistical significance) levels of CCNA1 expression on
afflicted cells (e.g., leukemic cells such as AML cells or leukemic stem cells),
relative to normal cells. A subject having such a disorder or condition would
benefit from treatment with a composition or method of the presently described
embodiments. Some conditions associated with CCNA1 overexpression thus
may include acute as well as chronic disorders and diseases, such as those
pathological conditions that predispose the subject to a particular disorder.
Some non-limiting examples of conditions associated with CCNA1
overexpression include hyperproliferative disorders, which refer to states of
activated and/or proliferating cells (which may also be transcriptionally
overactive) in a subject including tumors, neoplasms, cancer, malignancy, etc.
In addition to activated and/or proliferating cells, the hyperproliferative disorder
may also include an aberration or dysregulation of cell death processes,
whether by necrosis or apoptosis. Such aberration of cell death processes may
be associated with a variety of conditions, including cancer (including primary,
secondary malignancies as well as metastasis) and other conditions.
According to certain embodiments, virtually any type of cancer
that is characterized by CCNA1 overexpression may be treated through the use
of compositions and methods disclosed herein, including but not limited to
hematological cancers (e.g., leukemia including acute myeloid leukemia (AML),
T or B cell lymphomas, myeloma, and others) are considered. Furthermore,
“cancer” may refer to any accelerated proliferation of cells, including solid
tumors, ascites tumors, blood or lymph or other malignancies; connective tissue
malignancies; metastatic disease; minimal residual disease following
transplantation of organs or stem cells; multi-drug resistant cancers, primary or
secondary malignancies, angiogenesis related to malignancy, or other forms of
cancer. Also contemplated within the presently disclosed embodiments are
specific embodiments wherein only one of the above types of disease is
included, or where specific conditions may be excluded regardless of whether
or not they are characterized by CCNA1 overexpression.
Certain methods of treatment or prevention contemplated herein
include administering a composition that comprises a desired nucleic acid
molecule such that it stably integrates into the chromosome of certain desired
cells. For example, such compositions may be integrated into immune system
cells (e.g., antigen-presenting cells and/or T-cells) in order to promote a
desired, CCNA1-targeted T-cell response.
As used herein, administration of a composition or therapy refers
to delivering the same to a subject, regardless of the route or mode of delivery.
Administration may be effected continuously or intermittently, systemically or
locally. Administration may be for treating a subject already confirmed of
having a recognized condition, disease or disease state, or for subjects
susceptible to or at risk of developing such a condition, disease or disease
state. Co-administration may include simultaneous and/or sequential delivery
of multiple agents in any order and on any dosing schedule.
An effective amount of a therapeutic or pharmaceutical
composition refers to an amount sufficient, at dosages and for periods of time
needed, to achieve the desired clinical results or beneficial treatment, as
described herein. An effective amount may be delivered in one or more
administrations. If the administration is to a subject already known or confirmed
to have a disease or disease-state, the term “therapeutic amount” may be used
in reference to treatment, whereas “prophylactically effective amount” may be
used to describe administrating an effective amount to a subject that is
susceptible or at risk of developing a disease or disease-state as a preventative
course.
Pharmaceutical Compositions
In certain embodiments of the disclosed invention, a
pharmaceutical composition comprising at least one herein disclosed
composition (e.g., an isolated peptide capable of eliciting an antigen-specific T-
cell response to human cyclin A1, or a recombinant expression vector encoding
the same), is administered to a subject. As used herein, a pharmaceutical
composition generally refers to the combination of an active pharmaceutical
drug or other therapeutic agent and an excipient or carrier, whether inert or
active, wherein the pharmaceutical composition comprises at least one isolated
peptide capable of eliciting an antigen-specific T-cell response to CCNA1 (or a
recombinant expression vector encoding the same) that is suitable for
therapeutic use, including prophylactic use, in vivo, in vitro, or ex vivo.
In certain embodiments, the present invention relates to
formulations of one or more compositions disclosed herein in pharmaceutically-
acceptable excipients or carriers for administration to a cell or a subject either
alone, or in combination, with one or more other modalities of therapy. It is
understood that, if desired, a composition as disclosed herein may be
administered in combination with other agents as well, including therapeutic
agents. Such compositions may be synthesized de novo or purified from host
cells or other biological sources.
It will be apparent that any of the pharmaceutical compositions
described herein can contain pharmaceutically acceptable excipients or other
carriers, and may contain acceptable salts. Such salts can be prepared, for
example, from pharmaceutically acceptable non-toxic bases, including organic
bases (e.g. salts of primary, secondary and tertiary amines and basic amino
acids) and inorganic bases (e.g. sodium, potassium, lithium, ammonium,
calcium and magnesium salts).
While any suitable carrier known to those of ordinary skill in the
art may be employed in the pharmaceutical compositions as described herein
(e.g., pharmaceutical compositions that comprise the presently disclosed
isolated peptide capable of eliciting a CCNA1-specific T-cell response, or a
recombinant expression vector encoding the same) the type of carrier will
typically vary depending on the mode of administration. Compositions of the
present invention may in certain embodiments be formulated for any
appropriate manner of administration, including for example, topical, oral, nasal,
mucosal, intravenous, intratumor, rectal, parenteral, intraperitoneal,
subcutaneous and intramuscular administration.
Carriers for use with such pharmaceutical compositions are
biocompatible, and may also be biodegradable. In certain embodiments, the
formulation preferably provides a relatively constant level of active component
release. In other embodiments, however, a more rapid rate of release
immediately upon administration may be desired. The formulation of such
compositions is well within the level of ordinary skill in the art using known
techniques. Illustrative carriers useful in this regard include microparticles of
poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the
like. Other illustrative delayed-release carriers include supramolecular
biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked
polysaccharide or oligosaccharide) and, optionally, an external layer comprising
an amphiphilic compound, such as a phospholipid (see e.g., U.S. Patent No.
,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO
96/06638). The amount of active compound contained within a sustained
release formulation depends upon the route of administration, the rate and
expected duration of release and the nature of the condition to be treated or
prevented.
Certain embodiments of the invention may utilize an alkalinizing
agent, which is typically soluble in aqueous phase under physiological pH
conditions. Such alkalinizing agents are well known to those in the art and may
include alkali or alkaline-earth metal hydroxides, carbonates, bicarbonates,
phosphates, sodium borate, as well as basic salts (as discussed herein).
In another illustrative embodiment, biodegradable microspheres
(e.g., polylactate polyglycolate) are employed as carriers for the compositions
that are herein disclosed. Suitable biodegradable microspheres are disclosed,
for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128;
,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Another illustrative
carrier/delivery system employs a carrier comprising particulate-protein
complexes, such as those described in U.S. Patent No. 5,928,647, which are
capable of inducing a class I-restricted cytotoxic T lymphocyte response in a
host.
In another illustrative embodiment, calcium phosphate core
particles are employed as carriers, adjuvants, or as controlled release matrices
for the compositions of this invention. In certain embodiments, an adjuvant may
be necessary in order to increase the immune response of the subject.
Adjuvants are well known in the art and may include cytokines, dead viruses or
bacteria or fragments thereof, antibodies, or any other agent that heightens an
immune response.
The pharmaceutical compositions as provided herein will often
further comprise one or more 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,
bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g.,
aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or
weakly hypertonic with the blood of a recipient, suspending agents, thickening
agents and/or preservatives. Alternatively, compositions described herein may
be formulated as lyophilizates.
The pharmaceutical compositions described herein may be
presented in unit-dose or multi-dose containers, such as sealed ampoules or
vials. Such containers are typically sealed in such a way to preserve the
sterility and stability of the formulation until use. In general, formulations may
be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
Alternatively, a pharmaceutical composition may be stored in a freeze-dried
condition requiring only the addition of a sterile liquid carrier immediately prior
to use.
The development of suitable dosing and treatment regimens for
using the particular compositions described herein in a variety of treatment
regimens, including e.g., oral, parenteral, intravenous, intranasal, and
intramuscular administration and formulation, is well known in the art, some of
which are briefly discussed below for general purposes of illustration.
In certain applications, the pharmaceutical compositions disclosed
herein may be delivered via oral administration to an animal. As such, these
compositions may be formulated with an inert diluent or with an assimilable
edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or
they may be compressed into tablets, or they may be incorporated directly with
the food of the diet. The pharmaceutical composition may take the form of
tablets, lozenges, pills, troches, capsules, elixirs, powders, granules,
suspensions, emulsions, syrups, or tinctures. Slow-release or delayed-release
forms may also be prepared (for example, in the form of coated particles, multi-
layer tablets or microgranules).
The compositions may also contain any of a variety of additional
components, for example, pharmaceutically acceptable binders, such as gum
tragacanth, gum acacia, sodium alginate, carboxymethylcellulose, polyethylene
glycol, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, methylcellulose,
polyvinylpyrrolidone, xanthan gum, bentonite, agar, alginic acid and the like; a
lubricant, such as magnesium stearate; a sweetening agent, such as sucrose,
lactose, glucose, aspartame or saccharin may be added; a diluent, such as
lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,
calcium silicate or dicalcium phosphate; a flavoring agent, such as peppermint,
oil of wintergreen, orange, raspberry, bubblegum, or cherry flavoring, coating
agents, such as polymers or copolymers of acrylic acid and/or methacrylic acid
and/or their esters, waxes, fatty alcohols, zein, shellac or gluten; preservatives,
such as sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl
paraben, propyl paraben, or sodium bisulphate; lubricants, such as magnesium
stearate, stearic acid, sodium oleate, sodium chloride or talc; and/or time delay
agents, such as glyceryl monostearate or glyceryl distearate.
In certain embodiments, a tablet or pill may be in the form of a
compression coating or alternatively in the form of a spray coating. A
compression coating may include a small tablet or pill utilized as part of the
compression of a second tablet and wherein the small tablet is located nearly in
the center of the rest of the powder compressed outside. A spray coating may
include an overcoating of a tablet with the coating preparation containing an
active substance.
In certain embodiments, the pharmaceutical compositions of the
present invention include “slow-release” or “immediate release” forms. As used
herein, “slow-release” generally refers to a release of 20% to 60% in 1 hour and
greater than 70% in 6 hours or 40% to 80% in 2 hours, and greater than 70% in
6 hours in 500 ml of water (HCl 0.1N) in USP apparatus 1 (37 C, 100 RPM).
Whereas, “immediate release” generally refers to a release of more than 70% in
minutes, in 500 ml of water (HCl 0.1N) in USP apparatus 1 (37 C, 100
RPM).
In certain embodiments, the tablet or pill weighs in the range of
100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, and any
value therebetween or greater. The oral dosage formulations of certain
embodiments of the present invention may be manufactured according to
known methods in the art, and may be packaged as known, including in a
moisture and/or oxygen and/or light protective packaging material.
In addition, liquid forms of the pharmaceutical compositions may
include a liquid carrier, such as water, oils (olive oil, peanut oil, sesame oil,
sunflower oil, safflower oil, arachis oil, coconut oil), liquid paraffin, ethylene
glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,
glycerol, fatty alcohols, triglycerides, or mixtures thereof.
If the subject composition is administered parenterally, the
composition may also include sterile aqueous or oleaginous solution or
suspension. Suitable non-toxic parenterally acceptable diluents or solvents
include water, Ringer’s solution, isotonic salt solution, 1,3-butanediol, ethanol,
propylene glycol or polythethylene glycols in mixtures with water. Aqueous
solutions or suspensions may further comprise one or more buffering agents,
such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of
course, any material used in preparing any dosage unit formulation should be
pharmaceutically pure and substantially non-toxic in the amounts employed. In
addition, the active compounds may be incorporated into sustained-release
preparation and formulations. Dosage unit form, as used herein, refers to
physically discrete units suited as unitary dosages for the subject to be treated;
each unit may contain a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms are largely
dictated by and directly dependent on the unique characteristics of the active
compound and the particular therapeutic or prophylactic effect to be achieved,
as well as the limitations inherent in the art of compounding such an active
compound for treatment in subjects. Exemplary and non-limiting dosage
ranges may be from 0.1-10 mg/kg, 1.0-20 mg/kg, 5.0-50 mg/kg, 10-100 mg/kg,
or any values therebetween.
Typically, these formulations will contain at least about 0.01% of
the active compound or more by weight of the active substance. However, the
percentage of the active ingredient(s) may be varied and may conveniently be
between about 1-99%, including about 60% or 70% or more of the weight or
volume of the total formulation. Naturally, the amount of active compound(s) in
each therapeutically useful composition may be prepared is such a way that a
suitable dosage will be obtained in any given unit dose of the compound.
Factors such as solubility, bioavailability, biological half-life, route of
administration, product shelf life, as well as other pharmacological
considerations will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and treatment
regimens may be desirable.
In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein topically, orally, subcutaneously,
parenterally, intravenously, intramuscularly, or even intraperitoneally. Such
approaches are well known to the skilled artisan, some of which are further
described, for example, in U.S. Patent No. 5,543,158; U.S. Patent No.
,641,515 and U.S. Patent No. 5,399,363. In certain embodiments, solutions of
the active compounds as free base or pharmacologically acceptable salts may
be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid
polyethylene glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations generally will contain a
preservative to prevent the growth of microorganisms.
In one embodiment, for parenteral administration in an aqueous
solution, the solution should be suitably buffered if necessary and the liquid
diluent first rendered isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal administration. In this connection, a sterile
aqueous medium that can be employed will be known to those of skill in the art
in light of the present disclosure. For example, one dosage may be dissolved in
1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis
fluid or injected at the proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).
Some variation in dosage will necessarily occur depending on the condition of
the subject being treated. Moreover, for human administration, preparations
preferably meet sterility, pyrogenicity, and the general safety and purity
standards as required by FDA Office of Biologics standards.
In another embodiment of the invention, the compositions
disclosed herein may be formulated in a neutral or salt form. Illustrative
pharmaceutically-acceptable salts include the acid addition salts (formed with
the free amino groups of the protein) which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such organic acids
as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as, for
example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and
such organic bases as isopropylamine, trimethylamine, histidine, procaine and
the like. Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective.
The carriers can further comprise any and all solvents, dispersion
media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic
and absorption delaying agents, buffers, carrier solutions, suspensions,
colloids, and the like. The use of such media and agents for pharmaceutical
active substances is well known in the art. Except insofar as any conventional
media or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active ingredients
can also be incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and compositions
that do not produce an allergic or similar untoward reaction when administered
to a human.
In certain embodiments, the pharmaceutical compositions may be
delivered by intranasal sprays, inhalation, and/or other aerosol delivery
vehicles. Methods for delivering genes, nucleic acids, and peptide
compositions directly to the lungs via nasal aerosol sprays have been
described, e.g., in U.S. Patent No. 5,756,353 and U.S. Patent No. 5,804,212.
Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga
et al., J Controlled Release 1998 Mar 2; 52(1-2):81-7) and lysophosphatidyl-
glycerol compounds (U.S. Patent No. 5,725,871) are also well-known in the
pharmaceutical arts. An illustrative example of transmucosal drug delivery in
the form of a polytetrafluoroetheylene support matrix is described in U.S. Patent
No. 5,780,045.
In certain embodiments, liposomes, nanocapsules, microparticles,
microencapsulation, lipid particles, vesicles, and the like, are used for the
introduction of the presently disclosed compositions into suitable host
cells/organisms. In particular, such compositions may be formulated for
delivery either encapsulated in a lipid particle, a liposome, a vesicle, a
nanosphere, microsphere or microparticle, a nanoparticle or the like.
Alternatively, compositions disclosed herein can be bound, either covalently or
non-covalently, to the surface of such carrier vehicles.
The formation and use of liposome and liposome-like preparations
as potential drug carriers is generally known to those of skill in the art.
Liposomes have been used successfully with a number of cell types that are
normally difficult to transfect by other procedures, including T-cell suspensions,
primary hepatocyte cultures and PC 12 cells (Renneisen et al., J Biol Chem.
1990 Sep 25;265(27):16337-42; Muller et al., DNA Cell Biol. 1990 Apr;9(3):221-
9). In addition, liposomes are free of the DNA length constraints that are typical
of viral-based delivery systems. Liposomes have been used effectively to
introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses,
transcription factors, allosteric effectors and the like, into a variety of cultured
cell lines and animals. Furthermore, the use of liposomes does not appear to
be associated with autoimmune responses or unacceptable toxicity after
systemic delivery.
In certain embodiments, liposomes are formed from phospholipids
that are dispersed in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
Alternatively, in other embodiments, the invention provides for
pharmaceutically-acceptable nanocapsule formulations of the compositions of
the present invention. Nanocapsules can generally entrap compounds in a
stable and reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1 m) may be
designed using polymers able to be degraded in vivo. Such particles can be
made as described, for example, by Couvreur et al., Crit Rev Ther Drug Carrier
Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998
Mar;45(2):149-55; Zambaux et al. J Controlled Release 1998 Jan 2;50(1-3):31-
40; and U.S. Patent No. 5,145,684.
Dosing Schedules
Routes and frequency of administration of the therapeutic
compositions described herein, as well as dosage, will vary from individual to
individual, and may be readily established using standard techniques. In
general, the pharmaceutical compositions may be administered by injection
(e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally
(e.g., by aspiration) or orally. Suitable dosage formulations and methods of
administering the agents are readily determined by those of skill in the art. In
certain instances, the isolated peptide capable of eliciting a CCNA1-specific T-
cell response to human cyclin A1 (or a recombinant expression vector encoding
the same) may be administered at about 1 mg/kg, 5 mg/kg, 10mg/kg, 20mg/kg,
25mg/kg, 30mg/kg, 35mg/kg 40mg/kg, 45mg/kg, 50mg/kg, 60mg/kg, 70mg/kg,
75mg/kg, 80mg/kg, 90mg/kg, 100mg/kg, 125mg/kg, 150mg/kg, 175mg/kg,
200mg/kg or any value therebetween or greater. In certain instances, doses
(and optionally, at least one other therapeutic agent dose) may be provided
between 1 day and 14 days over a 30 day period. In certain instances, doses
(and optionally, at least one other therapeutic agent dose) may be provided 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days over a 60 day period. Alternate
protocols may be appropriate for individual subjects. A suitable dose is an
amount of a compound that, when administered as described above, is capable
of altering or ameliorating symptoms, or is at least 10-50% above the basal
(i.e., untreated) level, which can be monitored by measuring specific levels of
blood components, for example, detectable levels of circulating leukemic cells.
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 remissions, complete or
partial, or longer disease-free survival) in treated subjects as compared to non-
treated subjects. Increases in preexisting immune responses to a tumor protein
generally correlate with an improved clinical outcome. Such immune responses
may generally be evaluated using standard proliferation, cytotoxicity or cytokine
assays, which are routine in the art and may be performed using samples
obtained from a subject before and after treatment.
Standard techniques may be used for recombinant DNA, peptide
and oligonucleotide synthesis, immunoassays and tissue culture and
transformation (e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to manufacturer's
specifications or as commonly accomplished in the art or as described herein.
These and related techniques and procedures may be generally performed
according to conventional methods well known in the art and as described in
various general and more specific references in microbiology, molecular
biology, biochemistry, molecular genetics, cell biology, virology and
immunology techniques that are cited and discussed throughout the present
specification. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.; Current Protocols in Molecular Biology (John Wiley and Sons, updated
July 2008); Short Protocols in Molecular Biology: A Compendium of Methods
from Current Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL
Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology
(Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.
Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR:
Current Technology and Applications, Edited by Julie Logan, Kirstin Edwards
and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK;
Anand, Techniques for the Analysis of Complex Genomes, (Academic Press,
New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular
Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait,
Ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, Eds., 1985);
Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell
Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular
Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-
VCH); PCR Protocols (Methods in Molecular Biology) (Park, Ed., 3 Edition,
2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the
treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold
Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods
In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,
London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir andCC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition,
(Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells:
Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed.,
2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization
(Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem
Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology)
(Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods
in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells:
Methods and Protocols (Methods in Molecular Biology) (Darwin J. Prockop,
Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell
Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T.
Jordan Eds., 2001); Hematopoietic Stem Cell Protocols (Methods in Molecular
Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and
Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008).
Unless specific definitions are provided, the nomenclature utilized
in connection with, and the laboratory procedures and techniques of, molecular
biology, analytical chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and
commonly used in the art. Standard techniques may be used for recombinant
technology, molecular biological, microbiological, chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, and delivery, and treatment
of patients.
Each embodiment described in this specification is to be applied
mutatis mutandis to every other embodiment unless expressly stated otherwise.
Unless the context requires otherwise, throughout the present
specification and claims, the word “comprise” and variations thereof, such as,
“comprises” and “comprising” are to be construed in an open, inclusive sense,
that is, as “including, but not limited to”. By “consisting of” is meant including,
and typically limited to, whatever follows the phrase “consisting of.” By
“consisting essentially of” is meant including any elements listed after the
phrase, and limited to other elements that do not interfere with or contribute to
the activity or action specified in the disclosure for the listed elements. Thus,
the phrase “consisting essentially of” indicates that the listed elements are
required or mandatory, but that no other elements are required and may or may
not be present depending upon whether or not they affect the activity or action
of the listed elements.
In this specification and the appended claims, the singular forms
“a,” “an” and “the” include plural references unless the content clearly dictates
otherwise. As used herein, in particular embodiments, the terms “about” or
“approximately” when preceding a numerical value indicates the value plus or
minus a range of 5%, 6%, 7%, 8% or 9%. In other embodiments, the terms
“about” or “approximately” when preceding a numerical value indicates the
value plus or minus a range of 10%, 11%, 12%, 13% or 14%. In yet other
embodiments, the terms “about” or “approximately” when preceding a numerical
value indicates the value plus or minus a range of 15%, 16%, 17%, 18%, 19%
or 20%.
Reference throughout this specification to “one embodiment” or
“an embodiment” or “an aspect” means that a particular feature, structure or
characteristic described in connection with the embodiment is included in at
least one embodiment of the present invention. Thus, the appearances of the
phrases “in one embodiment” or “in an embodiment” in various places
throughout this specification are not necessarily all referring to the same
embodiment. Furthermore, the particular features, structures, or characteristics
may be combined in any suitable manner in one or more embodiments. When
steps of a method are described or claimed, and the steps are described as
occurring in a particular order, the description of a first step occurring (or being
performed) “prior to” (i.e., before) a second step has the same meaning if
rewritten to state that the second step occurs (or is performed) “subsequent” to
the first step.
The following Examples are presented by way of illustration and
not limitation.
EXAMPLES
EXAMPLE 1
IDENTIFICATION OF HUMAN CYCLIN A1 (CCNA1) AS AN
IMMUNOTHERAPEUTIC TARGET FOR LEUKEMIA AND
CHARACTERIZATION OF CCNA1 T-CELL IMMUNOGENIC EPITOPES
In this Example, analyses of differential gene expression were
performed to identify cyclin A1 (CCNA1), an intracellular protein, as a candidate
new T ‐cell target protein. By way of brief background, CCNA1 was reported to
regulate the progression of male germ cells through meiosis I and was
therefore selectively expressed in testis (Yang et al., 1997 Cancer Res 57
(5):913 ‐920; Wolgemuth et al., 2004 Int J Androl 27 (4):192 ‐199. doi:10.1111/
j.1365 ‐ 2605.2004. 00480.x IJA480 [pii]). Published reports have shown that
CCNA1 ‐/ ‐ mice were viable, and were phenotypically normal with the exception
of male infertility (Krug et al., 2009 Int J Oncol 34 (1):129 ‐136; Nickerson et al.,
2007 Dev Biol 306 (2):725 ‐735. doi:S0012 ‐1606 (07) 00783 ‐X [pii] 10.1016/
j.ydbio. 2007.04.009). CCNA1 was also shown to be aberrantly expressed in
AML as well as other malignancies (Yang et al., 1997 Cancer Res 57
(5):913 ‐920; Stirewalt et al., 2008 Genes Chromosomes Cancer 47 (1):8 ‐20.
doi:10.1002/gcc.20500). In AML, CCNA1 sustained the malignant phenotype
through pro ‐proliferative and anti ‐apoptotic activities (Chan et al., 2009
Oncogene 28 (43):3825 ‐3836. doi: onc 2009236 [pii] 10.1038/ onc.2009.236;
Jang et al., 2008 Cancer Res 68 (12):4559 ‐4570. doi: 68/12/ 4559 [pii] 10.1158/
0008 ‐5472.CAN ‐ 08 ‐0021; Ji et al., 2007 Int J Cancer 121 (4):706 ‐713.
doi:10.1002 /ijc. 22634), and over ‐expression of CCNA1 in mice caused
dysplastic myelopoiesis and transplantable myeloid leukemias in 15% of the
mice (Liao et al., 2001 Proc Natl Acad Sci USA 98 (12):6853 ‐6858.
doi:10.1073/ pnas.12154 0098 12154 0098 [pii]).
Here, CCNA1 is shown to act as a testis ‐leukemia ‐antigen
harboring a multitude of potentially immunogenic MHC class I epitopes that can
be used to generate CD8+ T-cells from healthy donors. T-cells so generated
were able to recognize and lyse leukemic cells.
Material and Methods
Human samples. For quantitative realtime PCR (qRT PCR),
mononuclear cells of AML patients from peripheral blood and bone marrow
(BM), BM mononuclear cells from chronic myeloid leukemia (CML) patients and
patients with myelodysplastic syndrome (MDS), and GM ‐CSF ‐mobilized CD34+
cells were isolated by leukopheresis or Ficoll ‐Hypaque (Biochrom, Cambrige,
UK). All AML samples contained more than 60% malignant cells (average
84%). Cells were collected at Fred Hutchinson Cancer Research Center
(FHCRC), Seattle, WA, USA, and at Charité Campus Benjamin Franklin, Berlin,
Germany. For the generation of T-cell lines, leukopheresis products were
obtained from two healthy donors at the FHCRC, Seattle. All samples were
collected after written informed consent and with approval of the institutional
review boards of the respective institutions.
Cell lines. Epstein ‐Barr virus (EBV) transformed lymphoblastoid
cell lines (LCLs) were generated as described (Riddell et al., 1991 J Immunol
146 (8):2795 ‐2804). TM ‐LCL were used as feeder cells in the Rapid Expansion
Protocol (REP) (Ho et al., 2006 J Immunol Methods 310 (1 ‐2):40 ‐52. doi:
S0022 ‐1759 (05) 00429 ‐1 [pii] 10.1016/ j.jim.2005. 11.023). The T-cell/B ‐cell
hybrid cell line T2 used for presentation of epitopes expressed only HLA
A*0201, but was TAP deficient. LCL 721.221 expressed no HLA class I due to
a radiation induced deletion of the relevant alleles, and was stably transduced
with the retroviral vector pLBPC containing HLA A*0201 (Akatsuka et al., 2002
Tissue Antigens 59 (6):502 ‐511. doi: tan 590607 [pii]). LCLs and T2 cells were
maintained as described (Ho et al., 2006 J Immunol Methods 310 (1 ‐2):40 ‐52.
doi: S0022 ‐1759 (05) 00429 ‐1 [pii] 10.1016/ j.jim.2005. 11.023). Cell lines
K562 (CML), THP ‐1, HL60, KG1 (AML) and U937 (monocytic cell line) were
maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 g/ml
streptomycin (Invitrogen, Carlsbad, CA), and 10% fetal bovine serum (FBS),
and for THP ‐1, 50 M ‐mercaptoethanol (Sigma, St. Louis, MO) was also
added. CTLs and dendritic cells (DCs) were maintained as described (Ho et
al., 2006 J Immunol Methods 310 (1 ‐2):40 ‐52).
Microarray data analysis. Two panels of microarray data sets
(Affymetrix, Santa Clara, CA) were used in this study: (1) nine AML LSC
samples (Lineage–, CD34+, CD38 ‐, CD90, (Majeti et al., 2009 Proc Natl Acad
Sci USA 106 (9):3396 ‐3401), seven corresponding leukemic blast samples
(Lineage, CD34), four HSC samples (Lineage, CD34+, CD38–, CD90+ (Majeti
et al., 2009 Proc Natl Acad Sci USA 106 (9):3396 ‐3401)) and data sets of
peripheral blood mononuclear cells (PBMCs), CD34+ BM mononuclear cells
and tissues (NCBI GEO server GSM279585 ‐279588, 414970, 414972, 414975,
419165 ‐419174, 457175 ‐457177, 483480 ‐483496, 80576, 80582, 80602,
80615, 80619, 80653, 80689, 80712, 80734, 80738, 80739, 80759, 80792,
80824, 80826, 80867, 80869, HG U133 plus 2.0 format); and (2) 30 samples of
AML cells (>75% malignant cells, Stirewalt et al., 2008 Genes Chromosomes
Cancer 47 (1):8 ‐20; and unpublished) and 58 tissue samples (NCBI GEO
server GSM18873, 18874, 18881, 18882, 18899 ‐18906, 18909, 18910, 18917,
18918, 18921, 18922, 18943 ‐18962, 18965, 18966, 18969 ‐18974, 18977,
18978, 18981, 18982, 18995 ‐18998, 19001, 19002, 19013, 19014,
44671 ‐44693, 44699 ‐44706, HG U133A format). Samples were normalized
using the invariant set method (dChip 2.0 software, Li et al., 2001 Proc Natl
Acad Sci USA 98 (1):31 ‐36). Before analyzing the panels at a single ‐probe ‐set
level, unsupervised hierarchical clustering was performed to rule out clustering
in accordance to the origin of the samples rather than of the biological
background of the data sets. Expression values of probe set 205899_at in
LSCs were compared with other cell types using a two ‐tailed Mann ‐Whitney
test. CCNA1 expression values of seven paired samples of LSC and the
corresponding leukemic blasts (Lineage–, CD34–) were compared using a
two ‐tailed Wilcoxon Signed Rank test.
Quantitative realtime PCR. Total RNA was extracted using Trizol
reagent (Invitrogen) or RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse
transcription was performed using Superscript III (Invitrogen) or Omniscript
(Qiagen). A panel of cDNAs from pooled healthy tissues was purchased from
Clontech (Mountain View, CA), and five samples of healthy BM were purchased
from Cambrex (Rutherford, NJ). Quantitative two ‐step RT PCR was performed
on an ABI 7500 machine (Applied Biosystems, Carlsbad, CA) with TA=60°C
using the following primers and probes:
Glyceraldehyde 3 ‐phosphate dehydrogenase (GAPDH)_fwd:
GAG TCA ACG GAT TTG GTC GT; [SEQ ID NO:11]
GAPDH_probe, labeled with 6FAM (6-carboxyfluorescein) at the
5’ end and TAMRA (carboxytetramethylrhodamine) at the 3’ end:
GAT ATT GTT GCC ATC AAT GAC CCC T [SEQ ID NO:12];
GAPDH_rev: GAC AAG CTT CCC GTT CTC AG [SEQ ID NO:13];
CCNA1_fwd: CAT GAA GAA GCA GCC AGA CA [SEQ ID
NO:14];
CCNA1_probe, labeled with 6FAM (6-carboxyfluorescein) at the 5’
end and TAMRA (carboxytetramethylrhodamine) at the 3’ end:
TTC GAG CAG AGA CCC TGT ATC TGG [SEQ ID NO:15];
CCNA1_rev: TTC GAA GCC AAA AGC ATA GC [SEQ ID NO:16].
Crossing points were plotted against standard curves of
pCR4 ‐TOPO plasmids (Invitrogen) containing the respective PCR product as
described (Keilholz et al., 2004 Clin Cancer Res 10 (5):1605 ‐1612 ). All
reactions were performed in duplicate. CCNA1 expression is given as copies
per copies of GAPDH.
Cytokines and peptides. Recombinant human IL ‐1, IL ‐4, IL ‐7,
IL ‐15, and TNFα were obtained from R&D Systems (Minneapolis, MN), IL ‐2 and
GM ‐CSF from Chiron (Emeryville, CA), PGE2 from MP Biomedicals (Irvine,
CA), and IL ‐21 from Peprotech (Rocky Hill, NJ). A peptide library of a total of
103 15 ‐mers with an overlap of 11 amino acids (AA) spanning CCNA1 (isoform
c, NM_001111046) was purchased from Sigma (St. Louis, MO). Shorter
peptides were purchased from Sigma or from JPT (Berlin, Germany).
Generation of CCNA1specific T cell clones. T ‐cell lines were
generated as described with minor modifications (Ho et al., 2006 J Immunol
Methods 310 (1 ‐2):40 ‐52) . Briefly, DCs were derived from the plastic adherent
fraction of PBMCs by culture over two days (days ‐2 to 0) in DC media
(CellGenix, Freiburg, Germany) supplemented with GM ‐CSF (800 U/ml) and
IL ‐4 (1000 U/ml). On day ‐1, maturation cytokines TNFα (1100 U/ml), IL ‐1
(2000 U/ml), IL ‐6 (1000 U/ml) and PGE2 (1 g/ml) were added. On day 0, DCs
were harvested, washed and pulsed with peptide (single peptides at 10 g/ml or
peptide pools at 2 g/ml) over 2 to 4 h in serum ‐free DC media. CD8 T cells
were isolated from PBMCs using anti ‐CD8 microbeads (Miltenyi, Auburn, CA)
and stimulated with DCs at an effector target (E:T) ratio of 1:5 to 1:10 in the
presence of IL ‐21 (30 ng/ml). On day 3, IL ‐2 (12.5 U/ml), IL ‐7 (5 ng/ml) and
IL ‐15 (5 ng/ml) were added. Cells were restimulated between days 10 and 14
using the plastic adherent faction of irradiated autologous PBMCs as antigen
presenting cells (APCs) after being peptide ‐pulsed for two hours and in the
presence of IL ‐21. After restimulation, cells were supplemented from day 1 on
with IL ‐2 (25 U/ml), IL ‐7 (5 ng/ml) and IL ‐15 (5 ng/ml). T ‐cell clones were
generated by plating cells at limiting dilution and expanding with TM ‐LCLs
coated with OKT3 (OrthoBiotech, Bridgewater, NJ) and allogeneic PBMCs as
feeders (REP protocol) as described (Ho et al., 2006 J Immunol Methods 310
(1 ‐2):40 ‐52).
IFNγ intracellular staining (ICS). APCs were pulsed with 10 g/ml
peptide overnight and washed once. Effector cells were coincubated with
APCs for 6 h in RPMI containing 10% FBS in the presence of monensin. Cells
were then stained with anti ‐CD8 ‐FITC, permeabilized using the BD
Cytofix/Cytoperm kit, and stained with anti ‐IFNγ ‐allophycocyanin (all from BD
Bioscience, Franklin Lakes, NJ).
HLA stabilization assay. T2 cells were pulsed with 100 g/ml
peptide in serum ‐free RPMI containing 1 g/ml ‐2 ‐microglobulin (Sigma) for 16
h. Cells were then washed and incubated for 4 h in the presence of 5 g/ml
brefeldin A (Sigma), and then stained with FITC ‐labeled anti ‐HLA A, B, C (clone
W6/32, BD Bioscience).
Caspase-3 assay. Target cells were membrane ‐labeled with
PKH26 (Sigma) according to the manufacturer’s instructions. T ‐cell clones
were used at the end of the Rapid Expansion Protocol (REP, Riddell and
Greenberg, 1990 J. Imm. Meth. 128:189; Ho et al., 2006 J. Imm. Meth. 310:40)
cycle (day 12 or later). Targets and T-cells were incubated at an E:T ratio of
3:1 in 96 ‐well round ‐bottom plates at 37°C for 4 h. As a negative control,
targets were incubated without effectors, and, as a positive control, targets
were incubated in the presence of 4 M camptothecin (Sigma) or 1 M
staurosporine (Sigma). Cells were then fixed and permeabilized using the BD
Cytofix/Cytoperm™ kit according to the supplier’s instructions, and stained with
anti ‐active caspase ‐3 antibody conjugated either to FITC or Alexa-Fluor-647
(C92-625, BD Bioscience).
51 51
Chromium release assay. A standard Cr release assay was
performed as described (Ho et al., 2006 J Immunol Methods 310 (1 ‐2):40 ‐52)
using 5000 target cells and T ‐cells at the end of the REP cycle (day 12 or later)
at E:T ratios of 10 ‐1.25:1 per well in triplicate. Spontaneous release was
assessed by incubating targets in the absence of effectors. Percentage of
specific lysis was calculated using the formula 100 x (experimental
release ‐spontaneous release)/(maximum release ‐spontaneous release).
Results
Selective expression of CCNA1 in AML LSC, leukemic blasts and
testis. To systematically screen for candidate target genes for T ‐cell mediated
therapy that were selectively expressed in the AML LSC compartment with or
without expression in other leukemic cell populations, nine LSC microarray data
sets were analyzed along with samples of different hematopoietic cell subsets
and non ‐hematopoietic tissues. Suitable candidate genes were identified by
mathematical filtering and manual vetting of the model ‐based expression
values. Based on the microarray data and published data on oncogenicity and
cellular location of the respective genes, eight candidates including WT1 were
identified, but only probe set 205899_at representing CCNA1 revealed selective
expression in LSCs and AML blasts in the two independent microarray data
sets and a third set of samples analyzed using qRT PCR.
In the first microarray set, CCNA1 was overexpressed in six out of
nine analyzed LSC samples and testis. Expression values of CCNA1 were
significantly higher in LSCs than in HSCs/CD34+ BM mononuclear cells,
PBMCs, and a panel of tissue samples including testis (Figure 1A). Statistical
testing was performed on the array set actually used to select the target. Since
no significant difference in CCNA1 expression was observed between the LSCs
and leukemic blasts derived from the same patients (Figure 1B), the expression
pattern of CCNA1 was confirmed in additional sample sets not selected for
LSCs. Parts of the second microarray data panel, in which CCNA1 was
previously identified as being expressed at significantly higher levels in AML
cells compared to BM CD34+ mononuclear cells, PBMCs, mobilized CD34+
cells and BM, have already been published (Stirewalt et al., 2008 Genes
Chromosomes Cancer 47 (1):8 ‐20). These AML data sets were now analyzed
along with data sets of non ‐hematopoietic tissues and lymphatic organs
including two specimens of testis tissue. Again, expression of CCNA1 was only
detected in AML and testis, and its expression was significantly higher in AML
than in the tissue samples from testis (p=0.0017).
To confirm the in silico findings and the validity of probe set
205899_at, CCNA1 was quantified in AML samples, in other hematopoietic cell
subsets, and in a panel of non ‐hematopoietic tissues using qRT ‐PCR. Of 33
AML samples analyzed, 17 samples revealed expression of CCNA1 at levels at
least twice as high as in every physiological sample measured except testis.
No difference was observed between the CCNA1 expression levels in BM and
G ‐CSF mobilized CD34+ mononuclear cells, which in both cases were very low.
Lowest expression levels were found in maximally proliferating T ‐cells after
OKT3 stimulation (Figure 2 A). Analyzing different French ‐American ‐British
(FAB) AML subtypes and BM samples from patients with CML and MDS,
variable percentages of aberrant CCNA1 expression were observed in AML,
with the highest expression levels in acute promyelocytic leukemia (APL) and
no overexpression in patients with secondary AML, MDS or CML (Figure 2B).
The median expression of CCNA1 copy numbers per GAPDH in the AML
samples was approximately one order of magnitude higher than the expression
of WT1 in the same sample set. In physiological tissues, CCNA1 was only
expressed at detectable levels in testis (Figure 2C).
Mapping of multiple immunogenic oligopeptides on CCNA1. For
the identification of MHC class I ‐restricted T ‐cell epitopes, a reverse
immunology approach was used. Four different CCNA1 mRNA variants have
been described that code for three different isoforms, with isoform c
distinguishable by having a shorter N ‐terminus. As no functional domains have
been identified on the longer N ‐termini of isoforms a and b, and the respective
transcripts for these isoforms could not be amplified by nested PCR, neither
from testis nor AML samples, a peptide library representing only the shorter
CCNA1 isoform c was used, so that immune escape due to targeting of
epitopes in the N ‐termini could not occur as a consequence of cells switching
CCNA1 isoforms and expressing only this shorter isoform.
After four stimulations of CD8 T-cells originating from HLA
A*0201 ‐positive donors 2196 and 2264 with the peptide library, more than 60%
of cells in both T ‐cell lines appeared specific. Following (a) stimulation of T-
cells with autologous LCL as APC pulsed with peptide pools, single 15 ‐mers
and subsequent shorter peptides, and (b) analysis of T-cell responses by
intracellular staining for IFNγ, eight immunogenic oligopeptides were mapped.
Donor 2196 CD8 T-cells identified immunogenic CCNA1 peptides that are
identified here by the amino acid residue positions in the CCNA1 isoform c
sequence:
CCNA1120 ‐131 VDTGTLKSDLHF [SEQ ID NO:1],
CCNA1218 ‐226 AETLYLAVN [SEQ ID NO:2],
CCNA1227 ‐235 FLDRFLSCM [SEQ ID NO:3], and
CCNA1253 ‐261 ASKYEEIYP [SEQ ID NO:4].
Donor 2264 CD8 T-cells identified the following immunogenic
peptides from the CCNA1 isoform c polypeptide:
CCNA1118 ‐127 YEVDTGTLKS [SEQ ID NO:5],
CCNA1167 ‐175 YAEEIYQYL [SEQ ID NO:6],
CCNA1330 ‐339 LEADPFLKYL [SEQ ID NO:7],
CCNA1341 ‐351 SLIAAAAFCLA [SEQ ID NO:8].
Using the two normal donors to assess potential T-cell responses
to CCNA1 isoform c sequences, the eight immunogenic peptides stimulated T-
cells in a manner characterized by MHC restriction against at least three
different HLA class I molecules. CCNA1 was expressed strictly intracellularly
and epitopes from it were processed and presented in the context of Class I
MHC molecules. CCNA1 thus represented a suitable target for T ‐cell based
therapy approaches.
T-cell clones were generated against the epitopes defined by the
CCNA1 isoform c peptides 118 ‐127 [SEQ ID NO:5], 227 ‐235 [SEQ ID NO:3],
167 ‐175 [SEQ ID NO:6], and 341 ‐351 [SEQ ID NO:8]. Using 721.221 cells with
and without stable transfected HLA A*0201 as APCs for T ‐cell lines in an IFNγ
intracellular staining (ICS) assay, epitopes 218 ‐226 [SEQ ID NO:2], 227 ‐235
[SEQ ID NO:3], and 341 ‐351 [SEQ ID NO:8] were found to be presented in a
HLA A*0201 ‐restricted manner. T ‐cell lines antigen-specifically directed against
all three epitopes could be generated using cells from both donors.
Characterization of two HLA A*0201 restricted epitopes. The
9 ‐mer FLDRFLSCM (CCNA1227 ‐235, [SEQ ID NO:3]) was identified as the
minimal immunogenic amino acid (AA) sequence from the library peptides
56/57 that stimulated a responses as revealed from analysis of a T ‐cell line
from donor 2196 (Figure 3A). The 9 ‐mer enhanced the stabilization of HLA
A*0201 on the T2 surface when compared to the 15 ‐mer and an irrelevant
‐mer (Figure 3 B). The IFNγ production of T ‐cell clones against this epitope
was HLA A*0201 restricted (Figure 3 C). The 11 ‐mer SLIAAAAFCLA
(CCNA1341 ‐351, [SEQ ID NO:8]) was identified surprisingly as the minimal
immunogenic AA sequence from library peptides 85/86, which stimulated a
response in a T ‐cell line from donor 2264 (Figure 3D). Even though the 10 ‐mer
SLIAAAAFCL (10 ‐mer 1) MHC complex was more stable than the complex with
the 11 ‐mer, only the latter was able to activate the analyzed T ‐cell clones
(Figure 3D, 3E). The IFNγ production of T ‐cell clones against this epitope was
dependent on peptide 341 ‐351 [SEQ ID NO:8] and HLA A*0201 expression
(Figure 3F).
Cytotoxic Activity of T-cell clones specific for CCNA1 227-235 and
CCNA1 341-351 against the leukemic cell line THP1. To assess a suitable
CCNA1 expressing leukemic cell line as a target cell, CCNA1 was quantified in
five myeloid leukemia cell lines (Figure 4A). THP ‐1, an HLA A*0201 ‐positive
FAB M5b AML line, was found to express the highest levels of CCNA1. Clones
2196.D9, 2196.D11 and 2196.E1, which were all specific for CCNA1 227 ‐235
[SEQ ID NO:3], were tested for reactivity against THP ‐1. All three clones
produced IFNγ after co ‐incubation with autologous LCLs that had been pulsed
with the peptide epitope (Figure 3C, 3F and data not shown), but only clones
D9 and D11 displayed significant IFNγ production in response to THP ‐1. These
responses were enhanced by incubating the THP ‐1 target cells with 1000 U/ml
IFNγ for 16 h before coincubation with the effectors (Figure 4B). Clone D11
showed significant lytic activity against THP ‐1 in a standard 6 h Cr release
assay, while the low avidity clone E1 did not (Figure 4C). Specific caspase ‐3
cleavage was observed not only for clones 2196.D9 and 2196.D11 but also for
clone 2264.E30, which was directed against CCNA1341 ‐351 [SEQ ID NO:8],
indicating proper processing and presentation of both described HLA A*0201
epitopes (Figure 4C, 4D).
Recognition and Lysis of Primary AML Cells by CD8+ T-cell
clones specific for CCNA1 227-235. To determine if CTLs specific for a cyclin-
A1 epitope recognized primary AML cells, cyclin-A1 expressing blasts from two
A*0201-positive and two A*0201-negative patients were tested with clone
2196.D11 , (“D11 ”) which recognized epitope 227-235. 2196. D11 was first
b b b
tested for induction of apoptosis in a four-hour caspase-3 assay. For maximal
apoptosis of these targets, staurosporine was used. As the different AML
samples showed different rates of spontaneous apoptosis, the data were
normalized by calculating specific caspase-3 cleavage as 100 x (experimental-
spontaneous)/(staurosporine-spontaneous). Using an E:T ratio of 5:1,
2196.D11 induced significant apoptosis of the A*0201-positive AML
specimens, but not A*0201-negative ones (Figure 6A). To determine if the
observed caspase-3 cleavage reflected classical lytic activity, a standard four-
hour Cr release assay was performed over a range of E:T ratios. Significant
lysis of the A*0201-positive specimens was observed at an E:T as low as
1.25:1, while no specific lysis was detectable in the A*0201-negative targets.
Thus, primary AML cells were killed in an HLA-restricted fashion (Figure 6B).
H001 and 1690-59 were HLA A*0201 positive, and R10009 and R50400 were
HLA A*0201-negative.
According to the criteria of the National Cancer Institute’s list of
weighted “ideal” cancer antigen criteria/characteristics (Cheever et al., 2009
Clin Cancer Res 15 (17):5323 ‐ 5337), from the present disclosure CCNA1
appeared to be a highly suitable antigen for targeting AML because it was
highly expressed in AML cells including the stem cell compartment of
approximately 50% of patients, and the tissue distribution of CCNA1 expression
was highly restricted. Both WT1 and CCNA1 were expressed at significantly
higher levels in leukemia stem cells (LSCs) than in hematopoietic stem cells
(HSCs) (Majeti et al., 2009 Proc Natl Acad Sci USA 106 (9): 3396 ‐3401 ).
However, unlike WT1, which was expressed in normal spleen, ovary and kidney
at levels higher than in leukemic blasts, significant CCNA1 expression was
found only in testis, which is generally considered an immune privileged site
(Fijak et al., 2006 Immunol Rev 213:66 ‐81. doi: IMR438 [pii] 10.1111/
j.1600 ‐065X .2006.00438.x). Consequently, cytotoxic side effects of targeting
CCNA1 appear unlikely. CCNA1 has been reported to be oncogenic in mice,
with overexpression resulting in the development of AML; CCNA1 expression
sustained the malignant phenotype in AML (Chan et al., 2009 Oncogene 28
(43):3825 ‐3836. doi: onc 2009236 [pii] 10.1038/ onc.2009.236; Jang et al.,
2008 Cancer Res 68 (12):4559 ‐4570. doi: 68/12/ 4559 [pii] 10.1158/
0008 ‐5472.CAN ‐ 08 ‐0021; Ji et al., 2007 Int J Cancer 121 (4):706 ‐713.
doi:10.1002 /ijc. 22634).
Demonstration of specific in vitro cytotoxicity of T ‐cell clones
generated against endogenously expressed and presented
malignancy ‐associated self ‐antigens has been a subject of previous reports
(Wilde et al., 2009 Blood 114 (10):2131 ‐2139. doi: blood ‐2009 ‐ 03 ‐209387 [pii]
.1182/ blood ‐2009 ‐03 ‐ 209387; Doubrovina et al., 2004 Clin Cancer Res 10
(21):7207 ‐7219. doi: 10/21/ 7207 [pii] 10.1158/ 1078 ‐0432. CCR ‐04 ‐ 1040;
Chaise et al., 2008 Blood 112 (7):2956 ‐2964. doi: blood ‐2008 ‐ 02 ‐137695 [pii]
.1182/ blood ‐2008 ‐02 ‐ 137695). Efficient cytotoxic activity of CCNA1 ‐specific
T ‐cell clones was observed against leukemia cells, as described herein. The
level of CCNA1 expression appeared to be about one order of magnitude
higher than that reported by others for WT1, the expression levels of which may
oscillate as a function of the cell cycle. The CCNA1 protein is known to be
regulated by the ubiquitin proteasome ‐mediated pathway (Ekberg et al., 2009
Mol Cell Biochem 320 (1 ‐2):115 ‐ 124. doi: 10.1007/ s11010 ‐008 ‐9913 ‐3),
consistent with optimal presentation of its epitopes on the surface of malignant
cells.
Due to its role in gametogenesis, and because it appears
analogous to the published classification for cancer ‐testis ‐antigens, based on
the present disclosure CCNA1 is hereby classified as a leukemia ‐testis ‐antigen
of the non ‐X type (Simpson et al., 2005 Nat Rev Cancer 5 (8):615 ‐625. doi:
nrc1669 [pii] 10.1038/ nrc1669). CCNA1 was expressed in LSCs and as
disclosed herein appears to be the first described non ‐X
leukemia ‐testis ‐antigen. The tissue-selective expression pattern of CCNA1, its
high expression levels in AML, its function in oncogenesis, and the multitude of
CCNA1 immunogenic T ‐cell epitopes described herein make CCNA1 an
optimal target for T ‐cell based therapeutic approaches including those
described herein, and also including vaccination and/or adoptive T ‐cell transfer.
Additional References: Yang et al., 2010 Cell Oncol 32
(1 ‐2):131 ‐143. doi: G7V87116 LNJ27 053 [pii] 10.3233/ CLO ‐2009 ‐0510; Brait
et al., 2008 Cancer Epidemiol Biomarkers Prev 17 (10): 2786 ‐2794. doi:17/
/2786 [pii] 10.1158/ 1055 ‐9965. EPI ‐08 ‐0192; Spisak et al., 2010 Dis Markers
28 (1):1 ‐14. doi: K32K0082 1215536H [pii] 10.3233/ DMA ‐2010 ‐ 0677;
Farhadieh et al., 2009 ANZ J Surg 79 (1 ‐2):48 ‐54. doi: ANS4799 [pii] 10.1111/
j.1445 ‐2197. 2008. 04799.x; Wegiel et al., 2008 J Natl Cancer Inst 100
(14):1022 ‐1036. doi: djn214 [pii] 10.1093/ jnci/djn214; Coletta et al., 2008
Cancer Res 68 (7):2204 ‐2213. doi: 68/7/ 2204 [pii] 10.1158/ 0008 ‐5472.
CAN ‐07 ‐3141; Cho et al., 2006 Cancer Sci 97 (10):1082 ‐1092. doi: CAS292
[pii] 10.1111/ j.1349 ‐7006. 2006. 00292.x; Fijak et al., 2006 Immunol Rev
213:66 ‐81. doi: IMR438 [pii] 10.1111/ j.1600 ‐065X .2006.00438.x; Rammensee
et al., 1999 Immunogenetics 50 (3 ‐4):213 ‐219. doi:90500213.251 [pii];
Lundegaard et al., 2008 Nucleic Acids Res 36 (Web Server issue):W509 ‐512.
doi:gkn202 [pii] 10.1093/nar/gkn202.
The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign patent
applications and non-patent publications referred to in this specification and/or
listed in the Application Data Sheet are incorporated herein by reference, in
their entirety. Aspects of the embodiments can be modified, if necessary to
employ concepts of the various patents, applications and publications to
provide yet further embodiments.
These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the following claims, the
terms used should not be construed to limit the claims to the specific
embodiments disclosed in the specification and the claims, but should be
construed to include all possible embodiments along with the full scope of
equivalents to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
Equivalents
While particular steps, elements, embodiments and applications
of the present invention have been shown and described herein for purposes of
illustration, it will be understood, of course, that the invention is not limited
thereto since modifications may be made by persons skilled in the art,
particularly in light of the foregoing teachings, without deviating from the spirit
and scope of the invention. Accordingly, the invention is not limited except as
by the appended claims.
Claims (5)
1. An isolated human cyclin A1 (CCNA1)-specific T cell comprising at least one recombinant expression vector encoding a T-cell receptor polypeptide that specifically binds in a human class I HLA-restricted manner to a CCNA1 polypeptide epitope of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 amino acids comprising the amino acid sequence set forth in SEQ ID NO:1, 3, 4, 5, 6, or 8.
2. The isolated human CCNA1-specific T cell of claim 1 wherein the human class I HLA is a human class I HLA-A*0201.
3. A composition for use in the ex vivo manufacture of a medicament for treating a condition characterized by CCNA1 overexpression in cells of a subject, comprising the CCNA1-specific T cell of claim 1 in an amount that is therapeutically effective following adoptive transfer to the subject.
4. The isolated human CCNA1-specific T cell of claim 1 wherein the polypeptide epitope is a polypeptide of general formula: N-X-C, [I] wherein: (a) N-X-C is a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 amino acids in which X comprises an amino acid sequence that is selected from: CCNA1(120-131) VDTGTLKSDLHF [SEQ ID NO:1], CCNA1(227-235) FLDRFLSCM [SEQ ID NO:3], CCNA1(253-261) ASKYEEIYP [SEQ ID NO:4], CCNA1(118-127) YEVDTGTLKS [SEQ ID NO:5], CCNA1(167-175) YAEEIYQYL [SEQ ID NO:6], CCNA1(341-351) SLIAAAAFCLA [SEQ ID NO:8], (b) N is the amino terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are independently selected from natural amino acids and non-natural amino acids, and (c) C is the carboxy terminus of the peptide and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids that are independently selected from natural amino acids and non-natural amino acids.
5. An isolated human cyclin A1 (CCNA1)-specific T cell of any one of claims 1, 2 or 4 substantially as herein described and with or without reference to any one or more of the Examples and/or
Applications Claiming Priority (3)
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US201161558953P | 2011-11-11 | 2011-11-11 | |
US61/558,953 | 2011-11-11 | ||
NZ624549A NZ624549B2 (en) | 2011-11-11 | 2012-11-09 | Cyclin a1-targeted t-cell immunotherapy for cancer |
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NZ723130A NZ723130A (en) | 2020-02-28 |
NZ723130B2 true NZ723130B2 (en) | 2020-05-29 |
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