U.S. patent application number 13/484829 was filed with the patent office on 2012-10-25 for polypeptides.
This patent application is currently assigned to GEMVAX AS. Invention is credited to Jon Amund ERIKSEN, Gustav GAUDERNACK, Mona MOLLER, Stein S BOE-LARSSEN.
Application Number | 20120269858 13/484829 |
Document ID | / |
Family ID | 9905720 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120269858 |
Kind Code |
A1 |
GAUDERNACK; Gustav ; et
al. |
October 25, 2012 |
POLYPEPTIDES
Abstract
The present invention relates to polypeptides, and nucleic acids
DNA encoding these polypeptides, capable of eliciting an immune
reaction against cancer, methods for generating T lymphocytes
capable of recognising and destroying tumour cells, and
pharmaceutical compositions for the treatment, prophylaxis or
diagnosis of cancer.
Inventors: |
GAUDERNACK; Gustav;
(Sandvika, NO) ; S BOE-LARSSEN; Stein; (Oslo,
NO) ; MOLLER; Mona; (Porsgrunn, NO) ; ERIKSEN;
Jon Amund; (Porsgrunn, NO) |
Assignee: |
GEMVAX AS
Oslo
NO
|
Family ID: |
9905720 |
Appl. No.: |
13/484829 |
Filed: |
May 31, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12042837 |
Mar 5, 2008 |
8193326 |
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13484829 |
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11391497 |
Mar 29, 2006 |
7375117 |
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12042837 |
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10451050 |
Jun 19, 2003 |
7078416 |
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PCT/NO01/00498 |
Dec 18, 2001 |
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11391497 |
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Current U.S.
Class: |
424/277.1 ;
514/19.3; 514/44R; 536/23.1 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 9/1241 20130101; A61P 35/00 20180101; A61P 43/00 20180101;
A61P 37/04 20180101 |
Class at
Publication: |
424/277.1 ;
536/23.1; 514/19.3; 514/44.R |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61K 38/02 20060101 A61K038/02; A61P 35/00 20060101
A61P035/00; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2000 |
GB |
0031430.2 |
Claims
1-18. (canceled)
19. A pharmaceutical composition comprising a nucleic acid molecule
that comprises a) a strand that encodes a polypeptide; b) a strand
that is complementary to the strand described in a) above; or c) a
strand that hybridizes under stringent conditions to the strand
described in a) or b) above, wherein the polypeptide comprises a
sequence selected from the group consisting of SEQ ID NOs:3, 4, 5,
6, and 11, and wherein the polypeptide is capable of inducing a T
cell response.
20. The pharmaceutical composition according to claim 19, further
comprising a polypeptide capable of inducing a T cell response
directed against a polypeptide produced by an oncogene or against a
mutant tumor suppressor protein.
21. The pharmaceutical composition according to claim 20, wherein
the oncogene or mutant tumor suppressor protein is p21-ras, Rb,
p53, abl, gip, gsp, ret, or trk.
22. The pharmaceutical composition according to claim 19, further
comprising a pharmaceutically acceptable carrier, diluent,
additive, stabilizer, and/or adjuvant, or a combination thereof,
the composition optionally further including one or more of a
cytokine or a growth factor.
23. The pharmaceutical composition according to claim 19, wherein
the composition is an immunogenic composition.
24. The pharmaceutical composition according to claim 23, wherein
the immunogenic composition is a vaccine.
25. The pharmaceutical composition according to claim 19, further
comprising a nucleic acid encoding a polypeptide capable of
inducing a T cell response directed against a polypeptide produced
by an oncogene or against a mutant tumor suppressor protein.
26. The pharmaceutical composition according to claim 25, wherein
the oncogene or mutant tumor suppressor protein is p21-ras, Rb,
p53, abl, gip, gsp, ret, or trk.
27. A method of treating cancer comprising administering a
therapeutically effective amount of the pharmaceutical composition
according to claim 19 to a patient in need thereof.
28. The method of treating cancer according to claim 27, wherein
the cancer is mammalian cancer.
29. The method of treating cancer according to claim 28, wherein
the cancer is human cancer.
30. The method of treating cancer according to claim 27, wherein
the cancer is breast cancer, prostate cancer, pancreatic cancer,
colorectal cancer, lung cancer, malignant melanoma, leukemia,
lymphoma, ovarian cancer, cervical cancer, or a biliary tract
carcinoma.
31. The method of treating cancer according to claim 27, wherein
the pharmaceutical composition is an immunogenic composition.
32. The method of treating cancer according to claim 31, wherein
the immunogenic composition is a vaccine.
33. A diagnostic for diagnosing cancer comprising a nucleic acid
molecule that comprises a) a strand that encodes a polypeptide; b)
a strand that is complementary to the strand described in a) above;
or c) a strand that hybridizes under stringent conditions to the
strand described in a) or b) above, wherein the polypeptide
comprises a sequence selected from the group consisting of SEQ ID
NOs:3, 4, 5, 6, and 11, and wherein the polypeptide is capable of
inducing a T cell response.
34. The diagnostic for diagnosing cancer according to claim 33,
wherein the cancer is mammalian cancer.
35. The diagnostic for diagnosing cancer according to claim 34,
wherein the cancer is human cancer.
36. The diagnostic for diagnosing cancer according to claim 33,
wherein the cancer is breast cancer, prostate cancer, pancreatic
cancer, colorectal cancer, lung cancer, malignant melanoma,
leukemia, lymphoma, ovarian cancer, cervical cancer, or a biliary
tract carcinoma.
37. The diagnostic for diagnosing cancer according to claim 33,
wherein the diagnostic is provided in a kit.
Description
[0001] The present invention relates to polypeptides, and nucleic
acids DNA encoding these polypeptides, capable of eliciting an
immune reaction against cancer, methods for generating T
lymphocytes capable of recognising and destroying tumour cells, and
pharmaceutical compositions for the treatment, prophylaxis or
diagnosis of cancer.
[0002] Cancer develops through a multistep process involving
several mutational events. These mutations result in altered
expression/function of genes belonging to two categories: oncogenes
and tumour suppressor genes. Oncogenes arise in nature from
proto-oncogenes through point mutations or translocations, thereby
resulting in a transformed state of the cell harbouring the
mutation. Oncogenes code for and is function through a protein.
Proto-oncogenes are normal genes of the cell which have the
potential of becoming oncogenes. In the majority of cases,
proto-oncogenes have been shown to be components of signal
transduction pathways. Oncogenes act in a dominant fashion.
Tumour-suppressor genes on the other hand, act in a recessive
fashion, i.e. through loss of function, and contribute to
oncogenesis when both alleles encoding the functional protein have
been altered to produce non-functional gene products.
[0003] In the field of human cancer immunology, the last two
decades have seen intensive efforts to characterise genuine cancer
specific antigens. In particular, effort has been devoted to the
analysis of antibodies to human tumour antigens. The prior art
suggests that such antibodies can be used for diagnostic and
therapeutic purposes, for instance in connection with an
anti-cancer agent. However, antibodies can only bind to tumour
antigens that are exposed on the surface of tumour cells. For this
reason, the effort to produce a cancer treatment based on the
immune system of the body has been less successful than
anticipated.
[0004] A fundamental feature of the immune system is that it can
distinguish self from nonself molecules and that it does not
normally react against self molecules. It has been shown that
rejection of tissues or organs grafted from other individuals is an
immune response to the foreign antigens on the surface of the
grafted cells. The immune response comprises a humeral response,
mediated by antibodies, and a cellular response. Antibodies are
produced and secreted by B lymphocytes, and typically recognise
free antigen in native conformation. They can therefore potentially
recognise almost any site exposed on the antigen surface. In
contrast to antibodies, T cells, which mediate the cellular arm of
the immune response, recognise antigens only in the context of
major histocompatability complex (MHC) molecules, and only after
appropriate antigen processing. This antigen processing usually
consists of proteolytic fragmentation of the protein, resulting in
polypeptides that fit into the groove of the MHC molecules. This
enables T cells to also recognise polypeptides derived from
intracellular protein fragments/antigens.
[0005] T cells can recognise aberrant polypeptides derived from
anywhere in the tumour cell, in the context of MHC molecules on the
surface of the tumour cell. The T cells can is subsequently be
activated to eliminate the tumour cell harbouring the aberrant
polypeptide. In experimental models involving murine tumours it has
been shown that point mutations in intracellular "self" proteins
may give rise to tumour rejection antigens, consisting of
polypeptides differing in a single amino acid from the normal
polypeptide. The T cells recognising these polypeptides in the
context of MHC molecules on the surface of the tumour cells are
capable of killing the tumour cells and thus rejecting the tumour
from the host (Boon et al., 1989, Cell 58: 293-303).
[0006] MHC molecules in humans are normally referred to as HLA
(human leukocyte antigen) molecules. There are two principal
classes of HLA molecules: class I and class II. HLA class I
molecules are encoded by HLA A, B and C subloci and primarily
activate CD8+ cytotoxic T cells. HLA class II molecules, on the
other hand, primarily activate CD4+ (cytotoxic or helper) T cells,
and are encoded by the HLA DR, DP and DQ subloci. Every individual
normally has six different HLA class I molecules, usually two
alleles from each of the three subgroups A, B and C, although in
some cases the number of different HLA class I molecules is reduced
due to the occurrence of the same HLA allele twice. For a general
review, see Roitt, I. M. et al. (1998) Immunology, 5.sup.th
Edition, Mosby, London.
[0007] The HLA gene products are highly polymorphic. Different
individuals express distinct HLA molecules that differ from those
found in other individuals. This explains the difficulty of finding
HLA matched organ donors in transplantations. The significance of
the genetic variation of the HLA molecules in immunobiology lies in
their role as immune-response genes. Through their polypeptide
binding capacity, the presence or absence of certain HLA molecules
governs the capacity of an individual to respond to specific
polypeptide epitopes. As a consequence, HLA molecules influence
resistance or susceptibility to disease.
[0008] T cells may inhibit the development and growth of cancer by
a variety of mechanisms. Cytotoxic T cells, both HLA class I
restricted CD8+ and HLA class II restricted CD4+, may directly kill
tumour cells presenting the appropriate tumour antigens. Normally,
CD4+ helper T cells are needed for cytotoxic CD8+ T cell responses,
but if the polypeptide antigen is presented by an appropriate APC,
cytotoxic CD8+ T cells can be activated directly, which results in
a quicker, stronger and more efficient response.
[0009] In International Application PCT/NO92/00032 (published as
WO92/14756), synthetic polypeptides and fragments of oncogene
protein products which have a point of mutation or translocations
as compared to their proto-oncogene or tumour suppressor gene
protein are described. These polypeptides correspond to, completely
cover or are fragments of the processed oncogene protein fragment
or tumour suppressor gene fragment as presented by cancer cells or
other antigen presenting cells, and are presented as a
HLA-polypeptide complex by at least one allele in every individual.
The polypeptides were shown to induce specific T cell responses to
the actual oncogene protein fragment produced by the cell by
processing and presented in the HLA molecule. In particular, it is
described in WO92/14756 that polypeptides derived from the p21-ras
protein which had point mutations at particular amino acid
positions, namely positions 12, 13 and 61. These polypeptides have
been shown to be effective in regulating the growth of cancer cells
in vitro. Furthermore, the polypeptides were shown to elicit CD4+ T
cell immunity against cancer cells harbouring the mutated p21-ras
oncogene protein through the administration of such polypeptides in
vaccination or cancer therapy schemes. It has subsequently been
shown that these polypeptides also elicit CD8+ T cell immunity
against cancer cells harbouring the mutated p21 ras oncogene
protein through the administration mentioned above (Gjertsen, M. K.
et al., 1997, Int. J. Cancer 72: 784-790).
[0010] International Application PCT/NO99/00143 (published as
WO99/58552) describes synthetic polypeptides and fragments of
mutant protein products arising from frameshift mutations occurring
in genes in cancer cells. These polypeptides correspond to,
completely cover or are fragments of the processed frameshift,
mutant protein fragment as presented by cancer cells or other
antigen presenting cells, and are presented as a HLA-polypeptide
complex by at least one allele in every individual. In particular
polypeptides resulting from frameshift mutations in the BAX and
hTGF.quadrature.-RII genes are disclosed. These polypeptides were
shown to be effective in stimulating CD4+ and CD8+ T cells in a
specific manner.
[0011] However, the polypeptides described above will be useful
only in certain numbers of cancers involving oncogenes with point
mutations, frameshift mutations or translocation in a
proto-oncogene or tumour suppressor gene. There is a strong need
for an anticancer treatment or vaccine that will be effective
against a generic range of cancers.
[0012] The concerted action of a combination of altered oncogenes
and tumour-suppressor genes results in cellular transformation and
development of a malignant phenotype. Such cells are however prone
to senescence and have a limited life-span. In most cancers,
immortalisation of the tumour cells requires the turning on of an
enzyme complex called telomerase. In somatic cells, the catalytic
subunit of the telomerase holoenzyme, hTERT (human telomerase
reverse transcriptase), is not normally expressed. Additional
events, such as the action of proteins encoded by a tumour virus or
demethylation of silenced (methylated) promoter sites, can result
in expression of the genes encoding the components of the
functional telomerase complex in tumour cells.
[0013] Due to the presence of telomerase in most types of cancer
cells, the enzyme has been disclosed as a general cancer vaccine
candidate (International Patent Application No. PCT/NO99/00220,
published as WO00/02581). WO00/02581 describes a method for
preventing or treating cancer by generating a T cell response
against telomerase-expressing cells in a mammal suffering (or
likely to suffer from) cancer. It is demonstrated in WO00/02581
that both CD4+ and CD8+ T cells can be stimulated by administration
of polypeptides having sequences derived from such a telomerase
protein.
[0014] Alternative splice variants of the telomerase pre-mRNA have
been reported in the literature (Kilian, A. et al., 1997, Hum. Mol.
Genet. 6: 2011-2019). Kilian et al. (1997, supra) indicated that it
was noteworthy that several splice variants were located with the
critical RT (reverse transcriptase) domain of hTERT. They stated,
to however, that a full understanding of the significance of the
hTERT splice variants was not obtained and that further functional
characterisation was required.
[0015] Analysis of the complete genomic sequence of the hTERT gene,
has verified that the different mRNA splice variants arise from the
usage of alternative splice sites in the is hTERT pre-mRNA (Wick,
M. et al., 1999, Gene 232: 97-106). Compared with the full-length
hTERT mRNA, at least five additional splice variants have been
detected. A schematic drawing of these variants are provided in
FIG. 1, and FIG. 2 shows an alignment of the proteins encoded. Two
of the splice variants, named .alpha.-del (or DEL1) and .beta.-del
(or DEL2), represent deletions of specific coding sequences. The
.alpha.-del variant has deleted the first 36 nucleotides of exon 6
and encodes a protein which lacks a stretch of 12 internal amino
acids. In the .beta.-del variant 182 nucleotides representing the
entire exons 7 and 8 are missing, leading to a shift in the open
reading frame and a truncated protein with a 44-amino acid long
carboxyl terminus not present in the full-length hTERT protein. The
remaining splice variants result from the use of alternative splice
sites located inside intron regions, resulting in the insertion of
intron sequences within the open reading frame and premature
termination of translation. The .sigma.-insert (or INS1) variant
results from an insertion of the first 38 nucleotides of intron 4.
The .sigma.-insert does not contain a stop codon, but instead, the
open reading frame extends 22 nucleotides into the normal sequence
using an alternative reading frame. The .gamma.-insert (or INS3)
variant is caused by insertion of the last 159 nucleotides from
intron 14. Ins-4 contains the first 600 nucleotides from intron 14
while at the same time having deleted exon 15 and most of exon 16.
The truncated proteins resulting from translation of these splice
variants are shown in FIG. 2.
[0016] Several recent studies have addressed the regulation of
telomerase activity, and some correlation between hTERT mRNA
transcription and telomerase activity has been reported for several
cell lines and tissues (Nakamura, T. M. et al., 1997, Science 277:
955-959; Meyerson, M. et al., 1997, Int. J. Cancer 85: 330-335;
Nakayama, J. et al., 1998, Nature Genet. 18: 65-68; Liu, K. et al.,
1999, Proc. Natl. Acad. Sci. USA 96: 5147-5152). Others studies
have shown that telomerase activity is up-regulated through
phosphorylation of the hTERT protein by protein kinase C.alpha.,
and conversely, down-regulated by the presence of protein kinase C
inhibitors and phosphatase 2A to (Li, H. et al., 1997, J. Biol.
Chem. 272: 16729-16732; Li, H. et al., 1998, J. Biol. Chem. 273:
33436-33442; Bodnar, A. G. et al., 1996, Exp. Cell Res. 228: 58-64;
Ku, W. C. et al., 1997, Biochem. Biophys. Res. Comm. 241: 730-736).
Alternative splicing of the hTERT pre-mRNA represents an additional
mechanism for regulating telomerase activity, and has been shown to
mediate down-regulation during fetal kidney development and in
adult ovarian and uterine tissues (Ulaner, G. A. et al., 1998,
Cancer Res. 58: 4168-4172; Ulaner, G. A. et al., 2000, Int. J.
Cancer 85: 330-335). The focus of the abovementioned studies has
been on the .alpha. and .beta. splice variants, presumably because
they delete sequences which are believed to encode critical reverse
transcriptase motifs (Lingner, J. et al., 1997, Science 276:
561-567).
[0017] The present invention provides peptides and nucleic acids
encoding said peptides based on the TERT .gamma. and .sigma. splice
variants, and the novel use of these peptides and nucleic acids in
medicine.
[0018] Thus according to the present invention there is provided a
polypeptide for use in medicine; wherein the polypeptide:
a) comprises a sequence given in SEQ ID NO: 1, 2, 3, 4, 5, 6 or 11;
b) comprises 8 contiguous amino acids from SEQ ID NO: 1, 2, 3, 4,
5, 6 or 11, with the proviso that at least one of said 8 contiguous
amino acids is from SEQ ID NO: 1, 3, 5 or 11; or c) comprises 8
contiguous amino acids that have only one, two or three amino acid
changes (eg. substitutions) relative to the 8 contiguous amino
acids as described in b) above, with the proviso that that at least
one of the 8 contiguous amino acids present is from SEQ ID NO: 1,
3, 5 or 11; wherein the polypeptide is capable of inducing a T cell
response.
[0019] The term "comprises" used herein includes "consists". The
polypeptide (or nucleic acid) of the present invention may be
flanked by one or more amino acid (or nucleic acid) residues unless
otherwise specified. For example, the polypeptide may be part of a
fusion protein which has one or more flanking domain at the N- or
C-terminus to allow for purification of the fusion protein.
[0020] Amino acid changes or modifications (eg. substitutions) in
the polypeptide may in particular be made to the anchor residues
which fit into HLA or MHC molecules for presentation to T cells.
Enhanced binding and immunogenic properties of the polypeptide to
HLA or MHC molecules may thus be achieved (see Bristol, J. A. et
al., 1998, J. Immunol. 160(5): 2433-2441; Clay, T. M. et al., 1999,
J. Immunol. 162(3): 1749-1755).
[0021] The polypeptide described above optionally may:
a) have at least 55% sequence identity with a molecule comprising
the sequence of SEQ ID NO: 1, as determined by an NCBI BLASTP
Version 2.1.2 search with default parameters; b) have at least 55%
sequence identity with a molecule comprising the sequence of SEQ ID
NO: 2, as determined by an NCBI BLASTP Version 2.1.2 search with
default parameters; c) have at least 40% sequence identity with a
molecule comprising the sequence of SEQ ID NO: 3, as determined by
an NCBI BLASTP Version 2.1.2 search with an Expect value of 1000
and other parameters as default; d) have at least 40% sequence
identity with a molecule comprising the sequence of SEQ ID NO: 4,
as determined by an NCBI BLASTP Version 2.1.2 search with an Expect
value of 1000 and other parameters as default; e) have at least 70%
sequence identity with a molecule comprising the sequence of SEQ ID
NO: 5, as determined by an NCBI BLASTP Version 2.1.2 search with an
Expect value of 100000 and other parameters as default; f) have at
least 50% sequence identity with a molecule comprising the sequence
of SEQ ID NO: 6, as determined by an NCBI BLASTP Version 2.1.2
search with an Expect value of 10000 and other parameters as
default; or g) have at least 40% and preferably 60% sequence
identity with a molecule comprising the sequence of SEQ ID NO: 11,
as determined by an NCBI BLASTP Version 2.1.2 search with an Expect
value of 1000 and other parameters as default;
[0022] The NCBI BLASTP program can be found at
http://www.ncbi.nlm.nih.gov/blast/, and default parameters changed
using the Advanced Search. Higher than default "Expect" values may
be required when searching with small query sequences for matches
to be displayed. The term "sequence identity" used herein refers to
amino acid residues in optimally aligned sequences which match
exactly at corresponding relative positions. For example, the NCBI
BLASTP program provides a percentage value of identities between
query and subject ("hit") sequences.
[0023] The polypeptide described above may comprise a sequence as
given in SEQ ID NO: 1, 2, 3, 4, 5, 6 or 11 or may be a fragment of
a sequence as shown in SEQ ID NO: 1, 3, 5, 6 or 11.
[0024] While the polypeptides that are presented by HLA class II
molecules are of varying length (12-25 amino acids), the
polypeptides presented by HLA class I molecules must normally be
nine amino acid residues long in order to fit into the class I HLA
binding groove. A longer polypeptide will not bind if it cannot be
processed internally by an APC or target cell, such as a cancer
cell, before presenting in the class I restricted HLA groove. Only
a limited number of deviations from this requirement of nine amino
acids have been reported, and in those cases the length of the
presented polypeptide has been either eight or ten amino acid
residues long. For reviews on polypeptide binding to MHC molecules
see Rammensee, H.-G. et al. (1995) Immunogenetics 41: 178-228 and
Barinaga (1992), Science 257: 880-881. Male, D. K. et al. (1996,
Advanced Immunology, Mosby, London) provide background information
on the field of immunology.
[0025] The T cell response generated by the polypeptide described
above may be generated after intracellular cleavage of the
polypeptide to provide a fragment that fits into an MHC or HLA
binding groove. Alternatively, the polypeptide described above may
not need intracellular cleavage to fit into an MHC or HLA class I
binding groove. In this case, the polypeptide may be from 8 to 10
amino acids long. Also provided is a polypeptide described above
which does not need intracellular cleavage to fit into an MHC or
HLA class II binding groove. In this case, the polypeptide may be
from 12 to 25 amino acids long.
[0026] The T cell response according to the present invention may
increase the number and/or activity of T helper and/or T cytotoxic
cells.
[0027] Also provided is a polypeptide which does not stimulate a
substantial cytotoxic T cell is response in a patient against one
or more of the following: bone marrow stem cells, epithelial cells
in colonic crypts or lymphocytes.
[0028] Further provided according to the present invention is a
nucleic acid molecule for use in medicine; wherein the nucleic acid
molecule:
a) has a strand that encodes a polypeptide described above, as
described above; b) has a strand that is complementary with a
strand as described in a) above; or c) has a strand that hybridises
with a molecule as described in a) or b) above (eg. under stringent
conditions).
[0029] Stringent hybridisation conditions are discussed in detail
at pp 1.101-1.110 and 11.45-11.61 of Sambrook, J. et al. (1989,
Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor). One example of hybridisation conditions
that can be used involves using a pre-washing solution of
5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and attempting
hybridisation overnight at 55.degree. C. using 5.times.SSC.
Hybridising nucleic acid sequences within the scope of the present
invention include probes, primers or DNA fragments. The term primer
includes a single stranded oligonucleotide which acts as a point of
initiation of template-directed DNA synthesis under appropriate
conditions (eg. in the presence of four different nucleoside
triphosphates and an agent for polymerisation, such as DNA or RNA
polymerase or reverse transcriptase) in an appropriate buffer and
at a suitable temperature.
[0030] Also provided is a vector or cell for use in medicine
comprising a nucleic acid molecule according to the present
invention.
[0031] Further provided is a binding agent for use in medicine;
wherein the binding agent binds to a polypeptide described above as
described above. Said binding agent may be to specific for a
polypeptide as described above. Said binding agent may be an
antibody or a fragment thereof. Said binding agent may be
lectin.
[0032] The term antibody in its various grammatical forms is used
herein to refer to immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antibody combining site or paratope. Such molecules are
also referred to as "antigen binding fragments" of immunoglobulin
molecules. Illustrative antibody molecules are intact
immunoglobulin molecules, substantially intact immunoglobulin
molecules and those portions of an immunoglobulin molecule that
contain the paratope, including those portions known in the art as
Fab, Fab', F(ab')2 and F(v). Antibodies of the present invention
may be monoclonal or polyclonal. The term antibody is also intended
to encompass single chain antibodies, chimeric, humanised or
primatised (CDR-grafted) antibodies and the like, as well as
chimeric or CDR-grafted single chain antibodies, comprising
portions from two different species. For preparation of antibodies
see Harlow, E. and Lane, D. (1988, Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor) and
Harlow, E. and Lane, D. (1999, Using Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
Immunological adjuvants for vaccines comprising lecithin may be
used to stimulate antibody production (see for example U.S. Pat.
No. 4,803,070).
[0033] Further provided according to the present invention is a T
lymphocyte for use in medicine; wherein the T lymphocyte is capable
of killing a cell expressing a polypeptide described above
according to the present invention or of helping in the killing of
such a cell. Said T lymphocyte may be a T cytotoxic cell or a T
helper cell.
[0034] Also provided is a clonal cell line for use in medicine
comprising a plurality of T lymphocytes as described above. Also
provided is a mixture of T lymphocytes for use in medicine
comprising a T helper cell or a clonal cell line of such cells and
a T cytotoxic cell or a clonal cell line of such cells.
[0035] Also provided is a method of generating T lymphocytes
capable of recognising and destroying tumour cells in a mammal,
comprising taking a sample of T lymphocytes from a mammal and
culturing the T lymphocyte sample in the presence of at least one
polypeptide described above in an amount sufficient to generate
hTERT .gamma.-insert protein specific T lymphocytes and/or hTERT
.sigma.-insert protein specific T lymphocytes.
[0036] Also provided is a B lymphocyte which may be useful in
generating antibodies according to the present invention.
Hybridomas which are capable of generating is antibodies according
to the present invention are also included (see for example Koehler
et al., 1975, Nature 256: 495-497; Kosbor et al., 1983, Immunol.
Today 4: 72; Cote et al., 1983, PNAS USA 80: 2026-2030; Cole et
al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss
Inc., New York, pp. 77-96).
[0037] Further provided according to the present invention is the
use of a polypeptide as described above, a nucleic acid as
described above, a vector or cell as described above, a binding
agent as described above, a T lymphocyte as described above, a cell
line as described above, or a mixture of T lymphocytes as described
above, in the preparation of a medicament for treating cancer, or
in the preparation of a diagnostic for diagnosing cancer. The
cancer may be a mammalian cancer. In particular, the cancer may be
human cancer. For example, the cancer may be breast cancer,
prostate cancer, pancreatic cancer, colo-rectal cancer, lung
cancer, malignant melanoma, leukaemia, lymphoma, ovarian cancer,
cervical cancer or a biliary tract carcinoma.
[0038] Said medicament may be a vaccine.
[0039] The polypeptides described here are particularly suited for
use in a vaccine capable of safely eliciting either CD4+ or CD8+ T
cell immunity. As the polypeptides may be synthetically produced,
medicaments including the polypeptides do not include transforming
cancer genes or other sites or materials which might produce
deleterious effects. The polypeptides may be targeted for a
particular type of T cell response without the side effects of
other unwanted responses.
[0040] Said medicament may be an antisense molecule or is capable
of generating an antisense molecule in vivo.
[0041] Said diagnostic may be provided in a kit. The kit may
comprise means for generating a detectable signal (eg. a
fluorescent label, a radioactive label) or a detectable change (eg.
an enzyme-catalysed change). The kit may include instructions for
use in diagnosing cancer.
[0042] Further provided is a pharmaceutical composition comprising
a polypeptide as described above, a nucleic acid as described
above, a vector or cell as described above, a binding agent as
described above, a T lymphocyte as described above, a cell line as
described above, or a mixture of T lymphocytes as described
above.
[0043] Said pharmaceutical composition may comprise a polypeptide
capable of inducing a T cell response directed against a
polypeptide produced by an oncogene or against a mutant tumour
suppressor protein, or a nucleic acid encoding such a polypeptide,
or a binding agent that binds such a polypeptide, or a T cell that
is capable of killing a cell expressing such a polypeptide or of
helping in the killing of such a cell. Example of such oncogenes or
mutant tumour suppressor proteins include p21-ras, Rb, p53, abl,
gip, gsp, ret or trk. The oncogene target may be the p21-ras
polypeptides described in International Application No.
PCT/NO92/00032 (Publication No. WO92/14756).
[0044] Also provided is a combined preparation comprising a
component from the pharmaceutical compositions described above for
simultaneous, separate or sequential use in anticancer therapy.
[0045] Also provided is a pharmaceutical composition or a combined
preparation as described above further comprising a
pharmaceutically acceptable carrier, diluent, additive, stabiliser,
and/or adjuvant; said composition or combined preparation
optionally further including one or more of: a cytokine or growth
factor (eg. IL-2, IL-12, and/or GM-CSF) and another polypeptide
arising from a frameshift mutation (eg. a frameshift mutation in
the BAX or hTGF.beta.-RII gene.)
[0046] The stimulatory effect on CD4+ and CD8+ T cells in a
specific manner by polypeptides resulting from frameshift mutations
in the BAX and hTGF.beta.-RII genes was disclosed in WO99/58552
(see above).
[0047] The pharmaceutical composition or combined preparation
described above may be a vaccine.
[0048] The pharmaceutical composition or combined preparation
described above may comprise or be capable of producing antisense
molecules.
[0049] Also provided is a method for the preparation of a
pharmaceutical composition as is described above, comprising the
steps of combining the above described components with a
pharmaceutically acceptable carrier, diluent, additive, stabiliser
and/or adjuvant.
[0050] A pharmaceutical composition according to the present may
comprise any of the following mixtures:
[0051] a) a mixture of at least one polypeptide described above
together with another polypeptide having a different sequence;
[0052] b) a mixture of at least one polypeptide described above
together with another polypeptide having an overlapping sequence,
so that the polypeptides are suitable to fit different MHC or HLA
alleles;
[0053] c) a mixture of both mixtures a) and b);
[0054] d) a mixture or several mixtures a);
[0055] e) a mixture of several mixtures b); or
[0056] f) a mixture of several mixtures a) and several mixtures
b).
[0057] The polypeptides in the mixture may be covalently linked
with each other to form larger polypeptides or even cyclic
polypeptides. The polypeptides themselves may be in a linear or
cyclic form.
[0058] Also provided according to the present invention is a
diagnostic composition comprising a polypeptide as described above,
a nucleic acid as described above, a vector or cell as described
above, a binding agent as described above, a T lymphocyte as
described above, a cell line as described above, or a mixture of T
lymphocytes above.
[0059] Also provided according to the present invention is a
diagnostic kit as described above.
[0060] Also provided according to the present invention is a method
of treatment or prophylaxis of cancer of the human or animal body
comprising administering a therapeutically effective amount of
pharmaceutical composition described above to a patient or animal
in need of same. The invention includes a method of treatment or
prophylaxis of patient or animal afflicted with cancer, the method
comprising administering to said patient or animal a pharmaceutical
composition described above in an amount sufficient to elicit a
T-cell response against said cancer. The method of treatment may
also include stimulation in vivo or ex vivo with a pharmaceutical
composition described above. Ex vivo therapy may include isolating
dendritic cells or other suitable antigen presenting cells from a
patient or animal, loading said cells with at least one polypeptide
or nucleic acid described above, and infusing these loaded cells
back into the patient or animal. The polypeptides or nucleic acids
described above may also be used in a method of vaccination of a
patient in order to obtain resistance against cancer. Oncogenes are
sometimes associated with viruses. The present invention is also
suitable for the treatment of certain viral disorders.
[0061] The polypeptides according to the present invention may be
administered in an amount in the range of 1 microgram (1 .mu.g) to
1 gram (1 g) to an average human patient or individual to be
vaccinated. It is preferred to use a smaller dose in the range of 1
microgram (1 .mu.g) to 1 milligram (1 mg) for each
administration.
[0062] The exact dosages, ie. pharmaceutically acceptable dosages,
and administration regime of pharmaceutical compositions and
medicaments of the present invention may be readily determined by
one skilled in the art, for example by using for example
dose-response assays.
[0063] The administration may take place one or several times as
suitable to establish and/or maintain the desired T cell immunity.
The polypeptides according to the present invention may be
administered together, either simultaneously or separately, with
compounds such as cytokines and/or growth factors, i.e.,
interleukin-2 (IL-2), interleukin-12 (IL-12), granulocyte
macrophage colony stimulating factor (GM-CSF) or the like in order
to strengthen the immune response as known in the art. The
polypeptides can be used in a vaccine or a therapeutic composition
either alone or in combination with other materials. For example,
the polypeptide or polypeptides may be supplied in the form of a
lipopeptide conjugate which is known to induce a high-affinity
cytotoxic T cell response (Deres, K. et al., 1989, Nature 342:
561-564).
[0064] The polypeptides according to the present invention may be
administered to an individual or animal in the form of DNA
vaccines. The DNA encoding the polypeptide(s) may be in the form of
cloned plasmid DNA or synthetic oligonucleotide. The DNA may be
delivered together with cytokines, such as IL-2, and/or other
co-stimulatory molecules. The cytokines and/or co-stimulatory
molecules may themselves be delivered in the form of plasmid or
oligonucleotide DNA.
[0065] Response to a DNA vaccine has been shown to be increased by
the presence of immunostimulatory DNA sequences (ISS). These can
take the form of hexameric motifs containing methylated CpG,
according to the formula:
5'-purine-purine-CG-pyrimidine-pyrimidine-3'. DNA vaccines
according to the present invention may therefore incorporate these
or other ISS, in the DNA encoding the hTERT .gamma.-insert protein
and/or the hTERT .sigma.-insert protein, in the DNA encoding the
cytokine or other co-stimulatory molecules, or in both. A review of
the advantages of DNA vaccination is provided by Tighe et al.
(1998, Immunology Today, 19(2): 89-97).
[0066] Also provided according to the present invention is the
polypeptide as described above, optionally in isolated form,
wherein the polypeptide is not a polypeptide consisting of the
sequences shown in FIG. 4.
[0067] The polypeptide sequence shown in FIG. 4 represents the
disclosure in FIG. 5C of Kilian et al. (1997, supra) of 46 amino
acid residues at the C-terminal end of the circa 1100 amino acid
residue hTERT .gamma.-insert splice variant, which includes 44
amino acids of SEQ ID NO: 1. The sequence provided in Kilian et al.
(1997, supra) shows the "alternative C-terminus" of the hTERT
.gamma.-insert splice variant protein. (Kilian et al., 1997, supra,
indicate that the corresponding DNA sequence in provided by GenBank
Accession number AF015950.) Kilian et al. (1997, supra) do not
disclose as a separate entity the polypeptide according to SEQ ID
NOs: 1, 2 or 5 at the C-terminal end of the hTERT .gamma.-insert
splice variant protein, and they do not disclose or suggest
medicinal use of the polypeptide according to SEQ ID NO: 1, 2 or
5.
[0068] The polypeptides described herein may be produced by
conventional processes, for example, by the various polypeptide
synthesis methods known in the art. Alternatively, they may be
fragments of a hTERT .gamma.-insert protein and/or a hTERT
.sigma.-insert protein produced by cleavage, for example, using
cyanogen bromide, and subsequent purification. Enzymatic cleavage
may also be used. The hTERT .gamma.-insert protein and the hTERT
.sigma.-insert protein or peptides may also be in the form of
recombinant expressed proteins or polypeptides.
[0069] Also provided is the nucleic acid as described herein,
optionally in isolated form; wherein the nucleic acid is not a
nucleic acid encoding the polypeptide excluded above and is also
not a nucleic acid as shown in FIG. 4 or FIG. 5.
[0070] The nucleic acid sequence at the 3'-end of the circa 3100 bp
hTERT .gamma.-insert splice variant, part of which encodes the
C-terminal end of the corresponding protein that includes SEQ ID
NO: 1, is provided in FIG. 4 of Kilian et al. (1997, supra). The
nucleic acid sequence at the exon-intron borders of the hTERT
splice variants INS1 (equivalent to the .sigma.-insert splice
variant) and INS3 (equivalent to the .gamma.-insert splice
variant), as disclosed in FIG. 2B of Wick et al. (1999, supra), are
shown in FIG. 5. The nucleic acids shown in FIG. 5 as disclosed in
FIG. 2B of Wick et al. (1999, supra) include nucleotides which
encode amino acid residues present in SEQ ID NOs: 1-6 and 11. Wick
at al. (1999, supra) make no specific reference to the existence of
the nucleic acids shown as distinct entities or to their medical
use. Wick et al., 1999, supra, provide reference to the complete
nucleotide sequence of their hTERT gene in GenBank Accession
numbers AF128893 and AF128894.
[0071] Nucleic acids encoding the polypeptides of the present
invention may be made by oligonucleotide synthesis. This may be
done by any of the various methods available in the art. A nucleic
acid encoding telomerase protein may be cloned from a genomic or
cDNA library, using conventional library screening. The probe may
correspond to a portion of any sequence of a known hTERT
.gamma.-insert and/or hTERT .sigma.-insert gene. Alternatively, the
nucleic acid can be obtained by using the Polymerase Chain Reaction
(PCR). The nucleic acid is preferably DNA, and may suitably be
cloned into a vector. Subclones may be generated by using suitable
restriction enzymes. The cloned or subcloned DNA may be propagated
in a suitable host, for example a bacterial host. Alternatively,
the host can be a eukaryotic organism, such as yeast or
baculovirus. The hTERT .gamma.-insert and the hTERT .sigma.-insert
proteins or polypeptides may be produced by expression in a
suitable host. In this case, the DNA is cloned into an expression
vector. A variety of commercial expression kits are available. The
methods described in Sambrook, J. et al. (1989, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor) may be used for these purposes.
[0072] Also provided is the vector or cell as described herein,
optionally in isolated form.
[0073] Further provided is the binding agent as described herein,
optionally in isolated form.
[0074] Yet further provided is the T lymphocyte as described
herein, optionally in isolated form.
[0075] Also provided is the clonal cell line as described herein,
optionally in isolated form.
[0076] Further provided is the mixture of T lymphocytes as
described herein.
[0077] Also provided is a machine readable data carrier (eg. a
disk) comprising the sequence of a polypeptide or of a nucleic acid
as described herein.
[0078] Yet further provided is a method comprising using the
sequence of a polypeptide or a nucleic acid molecule as described
herein to perform sequence identity studies, sequence homology
studies, or hybridisation studies. Said method may include using
said sequence to predict structure and/or function (eg. to predict
anti-cancer activity). Also provided is the use of this method in a
drug development or screening procedure. Further provided is a drug
identified or selected by this procedure.
[0079] Also provided is a computer or database that displays or
stores a sequence of a polypeptide or a nucleic acid molecule as
described herein or that is set up to perform a method as described
above.
[0080] Also provided is the invention as substantially hereinbefore
described with reference to the accompanying figures and
examples.
[0081] The phrases "amino acid residue" and "amino acid" are
broadly defined to include modified and unusual amino acids as
defined in WIPO Standard ST.25, and incorporated herein by
reference.
[0082] The term treatment or therapy used herein includes
prophylactic treatment or therapy where applicable.
[0083] The contents of each of the references discussed herein,
including the references cited therein, are herein incorporated by
reference in their entirety.
[0084] The invention will be further apparent from the following
description, with reference to the several accompanying figures,
which show, by way of example only, various polypeptides and their
use according to the present invention.
[0085] Of the figures:
[0086] FIG. 1 is a schematic drawing of the full-length hTERT mRNA
and splice variants found in cancer cell lines;
[0087] FIG. 2 shows a protein alignment between a portion of the
hTERT protein and proteins resulting from translation of splice
variants;
[0088] FIG. 3 shows the carboxyl termini of the hTERT
.gamma.-insert and .sigma.-insert splice variant proteins;
[0089] FIG. 4 shows prior art sequences relating to the hTERT
.gamma.-insert splice variant as disclosed in FIG. 5C of Kilian et
al. (1997, supra);
[0090] FIG. 5 show prior art sequences relating to the hTERT
.gamma.-insert and .sigma.-insert splice variants as disclosed in
FIG. 2B of Wick et al. (1999, supra);
[0091] FIG. 6 shows results from RT-PCR analysis of the regions
comprising the .gamma.-insert (A) and .sigma.-insert (13) splice
variants of hTERT;
[0092] FIG. 7 shows proliferative T cell responses induced in human
blood samples by a polypeptide having the amino acid sequence of
SEQ ID NO: 11;
[0093] FIG. 8 shows proliferation of T cell clones induced by a
polypeptide having the amino acid sequence of SEQ ID NO:11; and
[0094] FIG. 9 shows proliferation of other T cell clones induced by
a polypeptide having the amino acid sequence of SEQ ID NO:11.
[0095] In FIG. 1, the position of introns present in the hTERT
pre-mRNA is indicated by the letter "i" followed by an appropriate
number. Insertion and deletion variants are shown as square boxes;
shaded fill represents sequences that encode protein sequence not
present in the full-length hTERT protein. Position and orientation
of oligonucleotide primers used to analyse the different splice
variants is indicated by arrows.
[0096] In FIG. 2, amino acid numbering is shown above the sequence.
Amino acids are represented by their standard one letter
abbreviation known in the art.
[0097] In FIG. 3, SEQ ID NO: 1 reflects the truncated tail of the
hTERT .gamma.-insert protein and SEQ ID NO: 2 reflects the same
polypeptide with an extension at the amino terminus with the nine
amino acids normally found in these positions in the naturally
occurring hTERT .gamma.-insert expression product (underlined). SEQ
ID NO: 3 reflects the truncated tail of the hTERT .sigma.-insert
protein. SEQ ID NO: 4 reflects SEQ ID NO: 3 with an extension at
the amino terminus with the nine amino acids normally found in
these positions in the naturally occurring hTERT .sigma.-insert
expression product (underlined).
[0098] In FIG. 4, the exon/intron junctions of insert splice
variant 3 (equivalent to the hTERT .gamma.-insert splice variant)
is shown as provided in FIG. 5C of Kilian et al. (1997, supra). The
following information is provided by Kilian et al. (1997, supra):
the to nucleic acid sequence is shown above a protein translation
sequence, with the putative unspliced intron given in bold type;
putative exon/intron junctions are marked with |; the nucleic acid
sequence numbering corresponds as follows: nucleotide 1 corresponds
to nucleotide 139 of the sequence in GenBank Accession number
AF015950; and amino acids corresponding to the putative c-AbI/SH3
binding site are is underlined. The amino acid sequence shown by
Kilian et al. (1997; supra) represents the C-terminal end of a
circa 1100 amino acid residue hTERT .gamma.-insert splice variant
protein.
[0099] In FIG. 5, nucleotides of the exon-intron borders of the
hTERT splice variants INS1 (equivalent to the .sigma.-insert splice
variant) and INS3 (equivalent to the .gamma.-insert splice
variant), as disclosed in FIG. 2B of Wick et al. (1999, supra), are
represented. Intronic and exonic sequences are shown in lower-case
and upper-case letters, respectively. Wick et al., (1999, supra)
indicate that the nucleotide sequence of their hTERT gene has been
deposited as GenBank Accession numbers AF128893 and AF128894.
[0100] It has been established that the hTERT .gamma.-insert and
.sigma.-insert splice variants are expressed in cancer cell lines
and tumours but are undetectable, or present at very low levels, in
normal cells. The present application therefore discloses general
cancer vaccine candidates with improved specificity in comparison
with vaccines based on the functional variant of the telomerase
(hTERT) protein. Both .gamma. and .sigma. inserts results in
formation of an early stop codon and the expression of a protein
that is truncated at the carboxyl terminus. The truncated hTERT
.gamma.- and .sigma.-insert proteins have no telomerase activity
themselves. In the case of a .gamma.-insert the truncated tail of
the protein is a sequence of 44 amino acids (SEQ ID NO: 1). A
.sigma.-insert results in a protein in which the truncated tail is
a sequence of 20 amino acids (SEQ ID NO: 3). They are predominantly
expressed in cancer cell lines but are undetectable, or present at
very low levels, in normal cells and are therefore targets for
specific immunotherapy. According to the present invention,
polypeptides corresponding to the carboxyl end (truncated tail) of
proteins expressed by hTERT .gamma.-insert and/or .sigma.-insert
splice variants, are useful as anticancer agents or vaccines with
the function to trigger the cellular arm of the immune system
(T-cells) in humans against cancer cells. In a preferred embodiment
of the invention, the polypeptide comprises the sequence according
to SEQ ID NO: 5. In another preferred embodiment of the invention,
the polypeptide comprises the sequence according to SEQ ID NO: 6.
It yet another preferred embodiment, the polypeptide comprises the
sequence according to SEQ ID NO: 11.
Experimental
[0101] The experiments outlined herein describe the
characterisation of hTERT splice variants in various cancer cell
lines compared with normal cells. Synthesis of polypeptides
according to the present invention, and experiments for testing the
efficacy of the polypeptides for use in cancer therapy are
detailed. An experiment showing induction and proliferation of
human T cells by the peptide having an amino acid sequence
according to SEQ ID NO: 11 is described.
RT-PCR Analysis of the .gamma.-Insert and .sigma.-Insert Splice
Variants of hTERT
RNA Analysis:
[0102] Poly(A).sup.+ mRNA from completely lysed cells was isolated
directly from crude lysates is using magnetic oligo(dT) beads
(Dynal A S; Jakobsen, K. S. et. al., 1990, Nucleic Acids Res. 18:
3669). Cytosolic mRNA fractions were prepared by incubating cells
in 1% IGEPAL (Sigma) at 0.degree. C. for one minute, followed by
centrifugation [10000 g; 1 min.; 4.degree. C.] to remove nuclei.
Poly(A).sup.+ mRNA was then isolated from the supernatant using
oligo(dT) beads as described above.
cDNA Synthesis and PCR:
[0103] First strand cDNA synthesis was carried out by standard
procedures using M-MLV RNaseH/ reverse transcriptase (Promega
Corp.), and the PCR reactions were performed by using HotStar Taq
DNA polymerase (Qiagen) and run for 35 cycles on a PTC-200 thermal
cycler (MJ Research). To obtain detectable products from PBM and
CD34+ cells, 10% of the reaction was used as template in a second
PCR reaction and amplified by 15 additional cycles.
[0104] For analysis of the .gamma.-insert splice variant the
plus-strand primer variant the plus-strand primer hTERT-p3195
(5-GCC TCC CTC TGC TAC TCC ATC CT--SEQ ID NO: 7) and minus-strand
primer hTERT-m3652 (5-CGT CTA GAG CCG GAC ACT CAG CCT TCA--SEQ ID
NO: 8) were used. Applied on the full-length hTERT cDNA and the
.gamma.-insert variant, these primers produce fragments of 465 and
624 nucleotides, respectively. The analysis of the .sigma.-insert
variant was performed by using primers hTERT-P6 (5-GCC AAG TTC CTG
CAC TGG CTG A--SEQ ID NO: 9) and hTERT-m2044 (5-GCT CTA GAA CAG TGC
CTT CAC CCT CG--SEQ ID NO: 10). The amplification product resulting
from using these primers with full-length hTERT cDNA and the
.sigma.-insert variant comprises 369 and 407 nucleotides,
respectively. To verify that these PCR products represent genuine
splice variants, the fragments were isolated from the gel and
analysed by direct sequencing using an ABI prism 310 automated
sequencer (PE Corp.).
Results:
[0105] Telomerase activity is subject to complex regulation at the
post-transcriptional level, and methods used to detect the presence
or absence of telomerase proteins should involve direct
measurements of the protein itself, or alternatively, mRNA
variants. Furthermore, the abundance of the different hTERT splice
variants found in cells is not necessarily correlated with the
levels found in the cytosolic fraction of the same cells (see FIG.
6). Such deviations may be explained by differences in the
efficiency with which mRNA variants are transported from the
nucleus to the cytosolic compartment, and/or by differential
stability of the specific splice variants in the cytosol. It is
well known in the art that such mechanisms are part of the concept
of gene regulation. Nevertheless, the studies conducted to explain
hTERT regulation, including those cited above, have used total RNA
or mRNA isolated from completely lysed cells for their analysis.
Kits and reagents required to perform this kind of RNA isolation
are widely available in the commercial market. To obtain a correct
picture of gene expression, studies on mRNA abundance should
include analysis of mRNA specific to the cytosolic compartment.
[0106] FIG. 6 shows results from RT-PCR analysis of the regions
comprising the .gamma.- (A) and .sigma.-insert variants (B) of
hTERT. HL60, K562, and Jurkat denote the cancer cell lines
analysed. HL60 is a promyelocytic leukemia cell line (Sokoloski, J.
A. et al., 1993, Blood 82: 625-632), K562 an erythroid leukemia
cell line (Lozzio, C. B. et al., 1975, Blood 45: 321-324), while
Jurkat is derived from acute T-lymphocyte leukemia cells (Gillis,
S. et al., 1980, J. Exp. Med. 152: 1709-1719). The HL60, K562, and
Jurkat cancer cell lines are commercially available (for example,
from ATCC, Oslo). PBM1, PBM2, PBM3 and PBM4 represent peripheral
blood mononuclear (PBM) cell populations isolated from four
different healthy donors. CD34 denotes CD34-positive stem cells
isolated from a healthy donor, and CC1/CC2 denotes colon cancer
biopsies obtained from two cancer patients at the Norwegian Radium
Hospital, Oslo, with CC2a and CC2b being two tissue samples
dissected from the same tumour. RT-PCR reactions performed with
mRNA isolated by complete lysis of cells and with mRNA isolated
from cytosolic fractions are marked with the letters "T" and "C",
respectively. "M" indicates lane with molecular weight marker.
Position of PCR fragments representing the .gamma.- and
.sigma.-insert splice variants and the respective full-length hTERT
products (+) is indicated on the right side of the panels.
[0107] The RT-PCR analysis showed that both .gamma.- and
.sigma.-insert splice variants were readily detectable in all
cancer cell lines and in one of the tumour samples analysed (CC2b),
and with the .sigma.-insert variant appearing as the most abundant
in cytosolic fractions. In contrast, we were not able to detect
these variants in cytosolic mRNA populations isolated from PBM
cells despite the extensive PCR amplification performed with these
samples. The identity of the weak 395-bp fragment produced with the
.sigma.-insert primers on PBM and CD34-positive cells is at present
unknown.
Polypeptide Synthesis and Analysis for Applications Relating to
Cancer
Polypeptide Synthesis:
[0108] The polypeptides were synthesised by using continuous flow
solid phase peptide synthesis. N-a-Fmoc-amino acids with
appropriate side chain protection were used. The Fmoc-amino acids
were activated for coupling as pentafluorophenyl esters or by using
either TBTU or diisopropyl carbodiimide activation prior to
coupling. 20% piperidine in DMF was used for selective removal of
Fmoc after each coupling. Cleavage from the resin and final removal
of side chain protection was performed by 95% TFA containing
appropriate scavengers. The polypeptides were purified and analysed
by reversed phase HPLC. The identity of the polypeptides was
confirmed by using electro-spray mass spectroscopy.
Polypeptide Testing and Cancer Therapy:
[0109] In order for a cancer vaccine according to the present
invention, and methods for specific cancer therapy based on T cell
immunity to be effective, two conditions must be met:
(a) the polypeptide is at least 8 amino acids long and is a
fragment of the hTERT .gamma.-insert protein or the hTERT
.sigma.-insert protein and (b) the polypeptide is capable of
inducing, either in its full length or after processing by antigen
presenting cell, T cell responses.
[0110] The following experimental methods may be used to determine
if these two conditions are met for a particular polypeptide.
First, it should be determined if the particular polypeptide gives
rise to T cell immune responses in vitro. It will also need to be
established if the synthetic polypeptides correspond to, or are
capable after processing to yield, polypeptide fragments
corresponding to polypeptide fragments occurring in cancer cells
harbouring the hTERT .gamma.-insert protein and/or the hTERT
.alpha.-insert protein or antigen presenting cells that have
processed naturally occurring hTERT .gamma.-insert protein and/or
hTERT .sigma.-insert protein. The specificity of T cells induced in
vivo by hTERT .gamma.-insert and/or hTERT .sigma.-insert
polypeptide vaccination may also be determined.
In Vitro T Cell Response Analysis:
[0111] It is necessary to determine if hTERT .gamma.-insert and/or
hTERT .sigma.-insert expressing tumour cell lines can be killed by
T cell clones obtained from peripheral blood from carcinoma
patients after hTERT .gamma.-insert and/or hTERT .sigma.-insert
polypeptide vaccination. T cell clones are obtained after cloning
of T-cell blasts present in peripheral blood mononuclear cells
(PBMC) from a carcinoma patient after hTERT .gamma.-insert and/or
hTERT .sigma.-insert polypeptide vaccination. The polypeptide
vaccination protocol includes several in vivo injections of
polypeptides intracutaneously with GM-CSF or another commonly used
adjuvant. Cloning of T cells is performed by plating responding T
cell blasts at 5 blasts per well onto Terasaki plates. Each well
contains 2.times.10.sup.4 autologous, irradiated (30 Gy) PBMC as
feeder cells. The cells are propagated with the candidate hTERT
.gamma.-insert and/or hTERT .sigma.-insert polypeptide at 25 .mu.M
and 5 U/ml recombinant interleukin-2 (rIL-2) (Amersham, Aylesbury,
UK) in a total volume of 20 ml. After 9 days T cell clones are
transferred onto flat-bottomed 96-well plates (Costar, Cambridge,
Mass.) with 1 mg/ml phytohemagglutinin (PHA, Wellcome, Dartford,
UK), 5 U/ml rIL-2 and allogenic irradiated (30 Gy) PBMC
(2.times.10.sup.5) per well as feeder cells. Growing clones are
further expanded in 24-well plates with PHA/rIL-2 and
1.times.10.sup.6 allogenic, irradiated PBMC as feeder cells and
screened for polypeptide specificity after 4 to 7 days.
[0112] T cell clones are selected for further characterisation. The
cell-surface phenotype of the T cell clone is determined to
ascertain if the T cell clone is CD4+ or CD8+. T cell clone is
incubated with autologous tumour cell targets at different effector
to target ratios to determine if lysis of tumour cells occurs.
Lysis indicates that the T cell has reactivity directed against a
tumour derived antigen, for example, hTERT .gamma.-insert and/or
hTERT .sigma.-insert proteins.
Correlation Between Polypeptides and In Vivo hTERT Insert
Fragments:
[0113] In order to verify that the antigen recognised is associated
with hTERT .gamma.-insert protein is or hTERT .sigma.-insert
protein, and to identify the HLA class I or class II molecule
presenting the putative hTERT .gamma.-insert or hTERT
.sigma.-insert polypeptide to the T cell clone, different hTERT
.gamma.-insert and/or hTERT .sigma.-insert expressing tumour cell
lines carrying one or more HLA class I or II molecules in common
with those of the patient, are used as target cells in cytotoxicity
assays. Target cells are labelled with .sup.51Cr or
.sup.3H-thymidine (9.25.times.10.sup.4 Bq/mL) overnight, washed
once and plated at 5000 cells per well in 96 well plates. T cells
are added at different effector to target ratios and the plates are
incubated for 4 hours at 37.degree. C. and then harvested before
counting in a liquid scintillation counter (Packard Topcount). For
example, the bladder carcinoma cell line T24 (12Val.sup.+,
HLA-A1.sup.+, B35.sup.+), the melanoma cell line FMEX (12Val.sup.+,
HLA-A2.sup.+, B35.sup.+) and the colon carcinoma cell line SW 480
(12Val.sup.+, HLA-A2.sup.+, B8.sup.+) or any other telomerase
positive tumour cell line may be used as target cells. A suitable
cell line which does not express hTERT .gamma.-insert and/or hTERT
.sigma.-insert proteins may be used as a control, and should not be
lysed. Lysis of a particular cell line indicates that the T cell
clone being tested recognises an endogenously-processed hTERT
.gamma.-insert and/or hTERT .sigma.-insert epitope in the context
of the HLA class I or class II subtype expressed by that cell
line.
Characterisation of T Cell Clones:
[0114] The HLA class I or class II restriction of a T cell clone
may be determined by blocking experiments. Monoclonal antibodies
against HLA class I antigens, for example the panreactive HLA class
I monoclonal antibody W6/32, or against class II antigens, for
example, monoclonals directed against HLA class II DR, DQ and DP
antigens (B8/11, SPV-L3 and B7/21), may be used. The T cell clone
activity against the autologous tumour cell line is evaluated using
monoclonal antibodies directed against HLA class I and class II
molecules at a final concentration of 10 .mu.g/ml. Assays are set
up as described above in triplicate in 96 well plates and the
target cells are preincubated for 30 minutes at 37.degree. C.
before addition of T cells.
[0115] The fine specificity of a T cell clone may be determined
using polypeptide pulsing experiments. To identify the hTERT
.gamma.-insert and/or hTERT .sigma.-insert polypeptide actually
being recognised by a T cell clone, a panel of nonamer polypeptides
is tested. .sup.51Cr or .sup.3H-thymidine labelled, mild acid
eluted autologous fibroblasts are plated at 2500 cells per well in
96 well plates and pulsed with the polypeptides at a concentration
of 1 .mu.M together with b2-microglobulin (2.5 .mu.g/mL) in a 5%
CO.sub.2 incubator at 37.degree. C. before addition of the T cells.
Assays are set up in triplicate in 96 well plates and incubated for
4 hours with an effector to target ratio of 5 to 1. Controls can
include T cell clone cultured alone, with APC in the absence of
polypeptides or with an irrelevant melanoma associated polypeptide
MART-1/Melan-A polypeptide.
[0116] An alternative protocol to determine the fine specificity of
a T cell clone may also be used. In this alternative protocol, the
TAP deficient T2 cell line is used as antigen presenting cells.
This cell line expresses only small amounts of HLA-A2 antigen, but
increased levels of HLA class I antigens at the cell surface can be
induced by addition of b2-microglobulin. .sup.3H-labelled target
cells are incubated with the different test polypeptides and
control polypeptides at a concentration of 1 .mu.M together with
b2-microglobulin (2.5 .mu.g/mL) for one hour at 37.degree. C. After
polypeptide pulsing, the target cells are washed extensively,
counted and plated at 2500 cells per well in 96 well plates before
addition of the T cells. The plates are incubated for 4 hours at
37.degree. C. in 5% CO.sub.2 before harvesting. Controls include T
cell clone cultured alone or with target cells in the absence of
polypeptides. Assays were set up in triplicate in 96 well plates
with an effector to target ratio of 20 to 1.
[0117] The sensitivity of a T cell clone to a particular
polypeptide identified above may also be determined using a
dose-response experiment. Polypeptide sensitised fibroblasts can be
used as target cells. The target cells are pulsed with the
particular peptide as described above for fine specificity
determination, with the exception that the peptides are added at
different concentrations before the addition of T cells. Controls
include target cells alone and target cells pulsed with the
irrelevant melanoma associated peptide Melan-A/Mart-1.
Induction and Proliferation of Human T Cell Response to the hTERT
.sigma.-Insert Peptide
[0118] In this experiment, peripheral blood mononuclear cells
(PBMC) from four healthy humans (donors "14328", "14313", "23244"
and "23255") and were isolated and primed for seven days with
dendritic cells pulsed with the SEQ ID NO: 11 peptide derived from
the hTERT .sigma.-insert polypeptide, followed by two cycles
consisting of is seven days re-stimulation with peptide-pulsed
autologous PBMC. The dendritic cells were derived from monocytes
from peripheral blood. T cells from the resulting bulk culture were
tested in triplicate with or without peptide-pulsed antigen
presenting cells (APC) before harvesting after 3 days. To measure
the proliferative capacity of the cultures, .sup.3H-thymidine
(3.7.times.10.sup.4 Bq/well) was added to the culture overnight
before harvesting. Cultures with non-pulsed APC or without APC
served as controls. The results showing the proliferative capacity
of the cultures are shown in FIG. 7. Further details of the
protocol used are set out below.
[0119] T cell clones were obtained from the resulting bulk cultures
from non-vaccinated donors 14313 and 23255. The clones were
obtained from T cell blasts preset in PRMCs as described in the
above section "In vitro T cell response analysis". The results of
proliferation of the T cell clones with peptide-pulsed and
non-peptide pulsed anitgen presenting cells are shown in FIG. 8
(donor 14313) and FIG. 9 (donor 23255).
[0120] Results in FIGS. 7, 8 and 9 are given as mean counts per
minute (cpm) of triplicate measurements. The data demonstrates that
blood from humans contain circulating T cells specific for a
peptide (SEQ ID NO: 11) derived from the peptide derived from the
hTERT .sigma.-insert polypeptide, and furthermore that such T cells
can be expanded in vitro following stimulation with the relevant
peptide.
[0121] Thus, the experiments of FIGS. 7, 8 and 9 show that the
hTERT .sigma.-insert polypeptide is immunogenic in man. In vitro
(or in vivo) stimulation can this give rise to hTERT .sigma.-insert
protein-specific T cell responses with the potential to recognise
the same antigen when overexpressed by a tumour growing in a cancer
patient. This particular experiment demonstrates that in principle
the peptide of SEQ ID NO: 11 could be developed as a cancer vaccine
in humans.
Protocol for Induction of MHC Class II Restricted T Cell
Response
Day 0:
[0122] PBMCs were separated out from 50 ml of blood (from buffy
coat). The cells were counted and re-suspended in complete
RPMI-1640/15% pool serum.
[0123] Bulk cultures were set up with 1-2 wells on a 24-well plate
of PBMCs at 2.times.10.sup.6 cells/ml in 1-1.5 ml. 25 .mu.M of SEQ
ID NO:11 peptide derived from the hTERT .sigma.-insert polypeptide
were added.
Day 9-10:
[0124] Bulk cultures were harvested and stimulated with irradiated
PBMCs and peptide. If there was high cell death, cultures were
lymphoprep separated, otherwise they were counted and resuspended
in RPMI/15% pool serum. (Lymphoprep centrifugation of bulk cultures
is carried out in 15 ml Falcon tubes by 1; adding 8 ml of cell
suspension, and 2; underlay with 2 ml of lymphoprep. Spin at 1500
rpm for 30 min, and wash twice with salt water.) One vial of
autologous PBMCs was defrosted, washed, counted and resuspended in
RPMI/15% FCS. The PBMCs were irradiated (25 GY, 5 min 58 sec).
Cells were plated out in 24-well plates. 0.5-2.0.times.10.sup.6 T
cells from bulk cultures were stimulated with 1.times.10.sup.6
irradiated feeder cells (PBMCs) and 25 SEQ ID NO:11 peptide. The
final volume was 1 ml.
Day 12:
[0125] IL-2 (10 U/ml) was added. Medium was also added if necessary
by replacing half the volume. Cultures were split if necessary.
Day 17:
[0126] The T cells in bulk culture were re-stimulated as on day 10,
with autologous, irradiated PBMC's and SEQ ID NO: 11 peptide.
Day 19:
[0127] IL-2 (10 U/ml) was added to day 17 re-stimulated bulk
cultures.
Day 24:
[0128] A proliferation assay for testing T cells for peptide
specificity was set up in 96-well plates, each condition in
triplicate:
TABLE-US-00001 Triplicates 1-3 4-6 7-9 10-12 Controls PBMC.sup.1
Tc.sup.2 PBMC.sup.1 + PBMC.sup.1 + Tc.sup.2 Tc.sup.2 + IL-2.sup.3
Test PBMC.sup.1 + PBMC.sup.1 + Tc.sup.2 + samples Tc.sup.2 +
Pep.sup.4 Pep.sup.4 + IL-2.sup.3 .sup.150 000 irradiated PBMCs,
.sup.250 000 T cells from bulk culture, .sup.31 U/ml, .sup.425
.mu.g/ml SEQ ID NO: 11 peptide
[0129] On day 2-3 of proliferation assay, .sup.3H-Thymidine (20
.mu.l) was added, and incubated at 37 C overnight before
harvesting.
[0130] In a variant of the protocol (as used in the above example)
the PBMCs were, on Day 0, primed with dendritic cells pulsed with
SEQ ID NO: 11.
Sequence CWU 1
1
11144PRTHomo sapiens 1Ala Glu Glu Asn Ile Ser Val Val Thr Pro Ala
Val Leu Gly Ser Gly1 5 10 15Gln Pro Glu Met Glu Pro Pro Arg Arg Pro
Ser Gly Val Gly Ser Phe 20 25 30Pro Val Ser Pro Gly Arg Gly Ala Gly
Leu Gly Leu 35 40253PRTHomo sapiens 2Tyr Ser Ile Leu Lys Ala Lys
Asn Ala Ala Glu Glu Asn Ile Ser Val1 5 10 15Val Thr Pro Ala Val Leu
Gly Ser Gly Gln Pro Glu Met Glu Pro Pro 20 25 30Arg Arg Pro Ser Gly
Val Gly Ser Phe Pro Val Ser Pro Gly Arg Gly 35 40 45Ala Gly Leu Gly
Leu 50320PRTHomo sapiens 3Val Ala Val Leu Trp Phe Asn Phe Leu Phe
Lys Gln Lys Pro Ser Val1 5 10 15Ser Pro Arg Gly 20429PRTHomo
sapiens 4Ala Arg Thr Phe Arg Arg Glu Lys Arg Val Ala Val Leu Trp
Phe Asn1 5 10 15Phe Leu Phe Lys Gln Lys Pro Ser Val Ser Pro Arg Gly
20 2559PRTHomo sapiens 5Phe Leu Phe Lys Gln Lys Pro Ser Val1
5618PRTHomo sapiens 6Arg Val Ala Val Leu Trp Phe Asn Phe Leu Phe
Lys Gln Lys Pro Ser1 5 10 15Val Ser723DNAHomo sapiens 7gcctccctct
gctactccat cct 23827DNAHomo sapiens 8cgtctagagc cggacactca gccttca
27922DNAHomo sapiens 9gccaagttcc tgcactggct ga 221026DNAHomo
sapiens 10gctctagaac agtgccttca ccctcg 261121PRTHomo sapiens 11Arg
Val Ala Val Leu Trp Phe Asn Phe Leu Phe Lys Gln Lys Pro Ser1 5 10
15Val Ser Pro Arg Gly 20
* * * * *
References