U.S. patent application number 14/196775 was filed with the patent office on 2015-01-08 for p53 peptide vaccine.
The applicant listed for this patent is Academisch Ziekenhuis Leiden h.o.d.n. LUMC. Invention is credited to Cornelis Johannes M. MELIEF, Rienk OFFRINGA, Sjoerd Henricus van der BURG.
Application Number | 20150010586 14/196775 |
Document ID | / |
Family ID | 39722615 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150010586 |
Kind Code |
A1 |
van der BURG; Sjoerd Henricus ;
et al. |
January 8, 2015 |
p53 Peptide Vaccine
Abstract
The invention relates to a peptide derived from p53 that could
be used as a vaccine against cancer.
Inventors: |
van der BURG; Sjoerd Henricus;
(Leiden, NL) ; MELIEF; Cornelis Johannes M.;
(Haarlem, NL) ; OFFRINGA; Rienk; (Leiden,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academisch Ziekenhuis Leiden h.o.d.n. LUMC |
Leiden |
|
NL |
|
|
Family ID: |
39722615 |
Appl. No.: |
14/196775 |
Filed: |
March 4, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12592815 |
Dec 3, 2009 |
8663646 |
|
|
14196775 |
|
|
|
|
PCT/NL2008/050319 |
May 27, 2008 |
|
|
|
12592815 |
|
|
|
|
60941070 |
May 31, 2007 |
|
|
|
60942483 |
Jun 7, 2007 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
506/9 |
Current CPC
Class: |
C07K 14/4746 20130101;
A61P 35/00 20180101; A61K 39/001151 20180801; A61K 45/06 20130101;
C07K 14/00 20130101; A61K 39/0011 20130101 |
Class at
Publication: |
424/185.1 ;
506/9 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06; C07K 14/00 20060101
C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2007 |
EP |
07109287.8 |
Jun 7, 2007 |
EP |
07109802.4 |
Claims
1. A method for designing a peptide derived from a ubiquitously
expressed self-antigen known to be associated with cancer, said
peptide exhibiting a low to intermediate capacity to form stable
cell surface expressed class I-MHC complexes and/or being
inefficiently processed by a proteasome and/or exhibiting a low to
intermediate MHC binding affinity.
2. The method according to claim 1, wherein the peptide exhibits a
low to intermediate capacity to form stable cell surface expressed
class I-MHC complexes and/or is inefficiently processed by a
proteasome.
3. A method for designing a peptide derived from a protein that is
a ubiquitously expressed self-antigen associated with cancer, said
method comprising: (a) selecting a set of peptides comprising an
MHC class I binding residue; (b) determining the stability of
binding of each peptide of the set to an MHC class I molecule;
and/or (c) analyzing the digestion of each peptide of the set by an
immunoproteasome or proteasome, wherein the affinity of the peptide
for an MHC molecule is given by the concentration of an epitope of
the peptide at which 50% (IC.sub.50) of the binding of a reference
epitope is inhibited; and wherein the IC.sub.50 is from about 5
.mu.M to about 50 .mu.M; and/or wherein the IC.sub.50 of the
peptide class I-MHC complex at 20.degree. C. is more than two times
greater than the IC.sub.50 at 4.degree. C., and the IC.sub.50 at
20.degree. C. is <15 .mu.M; and/or after a one hour digestion by
a proteasome, less than 1% of the peptide has been digested.
4. The method of claim 3, wherein step (a) comprises selecting a
set of peptides having a binding residue for HLA-A*0101,
HLA-A*0301, HLA-A*1101 and HLA-A*2401.
5. A method for treating a cell proliferative disorder, comprising
administering to a subject a peptide derived from a protein that is
a ubiquitously expressed self-antigen associated with cancer, said
peptide exhibiting a low to intermediate capacity to form stable
cell surface expressed class I-MHC complexes, and/or being
inefficiently processed by a proteasome and/or exhibiting a low to
intermediate MHC binding affinity, thereby treating said cell
proliferative disorder.
6. The method according to claim 5, wherein the peptide exhibits a
low to intermediate capacity to form stable cell surface expressed
class I-MHC complexes and/or is inefficiently processed by a
proteasome.
7. A method for treating a cell proliferative disorder, comprising
administering to a subject a peptide derived from a protein that is
a ubiquitously expressed self-antigen associated with cancer,
wherein: the affinity of the peptide for an MHC molecule is given
by the concentration of an epitope of the peptide at which 50%
(IC.sub.50) of the binding of a reference epitope is inhibited; and
wherein the IC.sub.50 is from about 5 .mu.M to about 50 .mu.M;
and/or wherein the IC.sub.50 at 20.degree. C. is more than two
times greater than the IC.sub.50 at 4.degree. C., and the IC.sub.50
at 20.degree. C. is <15 .mu.M; and/or after a one hour digestion
by a proteasome, less than 1% of the peptide has been digested;
thereby treating the cell proliferative disorder.
8. The method of claim 3 or 7, wherein said protein is selected
from the group consisting of: p53, MDM-2, HDM2, survivin,
telomerase, cytochrome p450 isoform 1B1, Her-2/neu and CD19.
9. The method of claim 7, wherein detection of IFN-.gamma.
secreting T cells specific for the administered peptide in
peripheral blood mononuclear cells (PBMC) derived from the subject
following administration of the peptide indicates treatment.
10. The method of claim 7, wherein treatment is indicated when
following administration of the peptide to the subject, T-cells
derived from a subject proliferate after in vitro stimulation with
the peptide.
11. The method of claim 7, wherein treatment is indicated when
following administration of the peptide to the subject, there is
detectable antigen spreading following in vitro stimulation of T
cells derived from the subject with the peptide.
12. The method of claim 7, wherein treatment is determined by a
skin biopsy and/or a T cell proliferation assay.
13. The method of claim 7, wherein treatment is indicated by
survival of the subject.
14. The method of claim 3 or 7, wherein the cell proliferative
disorder is selected from the group consisting of: lung, colon,
esophagus, ovary, pancreas, skin, gastric, head and neck, bladder,
sarcoma, prostate, hepatocellular, brain, adrenal, breast,
endometrium, mesothelioma, renal, thyroid, hematologic, carcinoid,
melanoma, parathyroid, cervix, neuroblastoma, Wilms, testes,
pituitary and pheochromocytoma cancer.
15. The method of claim 3 or 7 wherein said peptide is a peptide
derived from p53.
16. The method of claim 15, wherein the peptide comprises or
consists of a peptide selected from the group consisting of: SEQ ID
NOS: 2-10 and 16.
17. The method of claim 16, wherein said peptide comprises or
consists of a peptide selected from the group consisting of: SEQ ID
NOS: 4, 5, 6 or 7.
18. The method of claim 16, wherein an additional peptide selected
from the group consisting of: SEQ ID NOS: 14, 15, 20 or 21 is
present.
19. The method of claim 7, wherein said peptide is administered as
a composition comprising a pharmaceutical excipient and/or an
immune modulator and/or an immune stimulant.
20. The method of claim 7, wherein said peptide is administered in
combination with a dendritic cell (DC) activating agent.
21. The method of claim 7, where said peptide is administered
intradermally and/or subcutaneously in the presence or absence of
an immune modulator and/or an immune stimulant.
22. The method of claim 19, wherein the immune modulator is an
adjuvant.
23. The method of claim 22, wherein said peptide is administered
intradermally within less than 5, 2, 1, 0.5, 0.2 or 0.1 cm from the
site of the lesion.
24. The method of claim 19, wherein the amount of peptide in the
composition is between 1 and 1000 .mu.g.
Description
RELATED APPLICATIONS
[0001] This is a divisional application of U.S. application Ser.
No. 12/592,815, filed Dec. 3, 2009, now U.S. Pat. No. 8,663,646,
which is a continuation of International Application No.
PCT/NL2008/050319, filed May 27, 2008, which claims the benefit of
U.S. Provisional Application No. 60/942,483, filed Jun. 7, 2007,
and European Application No. 07109802.4, filed Jun. 7, 2007, and
U.S. Provisional Application No. 60/941,070, filed May 31, 2007,
and European Application No. 07109287.8, filed May 31, 2007. All of
which are herein incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Feb. 19,
2010, is named 85119306.txt, and is 33,841 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of medicine and
immunology. In particular it relates to an improved p53 peptide
vaccine.
BACKGROUND OF THE INVENTION
[0004] In this application p53, is taken as a representative
example of an ubiquitously expressed self-antigen known to be
associated with cancer. The strategy used to design a vaccine
against p53 could be applied to design a vaccine against any other
ubiquitously expressed self-antigen known to be associated with
cancer.
[0005] The nuclear phosphoprotein p53 is a tumor suppressor protein
that is ubiquitously expressed at low levels in normal tissues,
including thymus, spleen and lymphohematopoetic cells (Rogerl A et
al, Milner J et al, Terada N et al). The normal half-life of
wild-type p53 is less than 30 minutes. Following ubiquitination,
the wild-type (WT) p53 protein is rapidly degraded by proteasomes
(Honda R et al, Momand J et al, Shkedy D et al).
Proteasome-mediated digestion of p53 may lead to the generation of
peptides that are presented by class I MHC molecules. Recognition
of these class I MHC bound wild-type p53 derived peptides at the
surface of thymic APC by immature thymic T-cells with high avidity
for the class I MHC-peptide complex will result in negative
selection (Allen P M et al, Ashton-Rickardt P G et al, Kappler J W
et al). As a consequence, the peripheral T-cell repertoire will not
contain functional p53-specific class I MHC-restricted T-cells.
Theobald et al. elegantly showed that CTL specific for the
naturally processed peptide p53.sub.187-197 were deleted from the
repertoire in WTp53 mice but not in p53-/- mice (Theobald M et al,
1997), demonstrating that negative selection of high avidity
p53-specific CTL can occur in the thymus. Paradoxically, class I
MHC-restricted CTL able to recognize endogenously processed WTp53
at the surface of tumor cells, have been detected in both mice and
man (Theobald M et al 1997, Macagno A, et al, Mayordomo J I et al,
Barfoed A M et al, Chikamatsu K et al, Eura M et al, Houbiers J G
et al, Ropke M et al) suggesting that functional p53-specific class
I MHC-restricted CTL can escape from tolerance induction.
[0006] Several p53 vaccines have already been developed. For
example, WO 00/75336 discloses polyepitopic peptides derived from
p53 having the capacity to be degraded by the proteasome and to
associate with high affinity to class I MHC molecules. Such
properties are supposed to be essential for inducing an immune
response against p53. More likely, T-cells responding to this type
of peptides have either been deleted in the thymus, are tolerized
in the periphery or are of low T-cell receptor affinity to mediate
an effective anti-tumor response (Theobald M & Offring a R.
2003, and Morgan et al.) Thus, it is to be expected that such
peptides derived from an ubiquitously expressed self-antigen, such
as p53, will not be able to trigger a strong and effective immune
response in vivo.
[0007] Therefore, there is still a need for new and improved p53
vaccines, which does not have all the drawbacks of existing p53
vaccines.
DESCRIPTION OF THE INVENTION
[0008] The present invention is based on the surprising finding
that in order to induce an efficient anti p53 response, a peptide
derived from p53 should be inefficiently processed by the
proteasome and/or exhibit low to intermediate capacity to stably
form cell surface class I MHC-peptide complexes and/or a peptide
exhibits a low to intermediate MHC binding affinity. Preferably, a
peptide derived from p53 should be inefficiently processed by the
proteasome and/or exhibit low to intermediate capacity to stably
form cell surface class I MHC-peptide complexes. We have found that
escape of self-specific T-cells from negative selection in the
thymus may occur through low-avidity interactions between the TCR
and MHC. Indeed, WTp53-specific CTL that recognizes their cognate
peptide have been detected in WTp53-mice but with a 10-fold lower
avidity than CTL obtained from p53-/- mice (Theobald M et al, 1997
and Hernandez J et al). Furthermore, a disparity between so-called
household proteasomes and immunoproteasomes in the generation of
certain peptide epitopes may allow positive selection by thymic
epithelium but failure to delete these cells by a lack of
presentation of these MHC-peptide complexes by thymic APC.
Dendritic cells constitutively express immunoproteasomes (Kloetzel
P M & Ossendorp 2004) and high levels of immunoproteasomes have
been detected in the thymus (Zanelli E et al, and Stohwasser R et
al). Morel et al. demonstrated that human CTL recognizing the
melanoma antigen Melan-A as well as CTL that recognized a novel
ubiquitously expressed protein did not recognize APC carrying
immunoproteasomes whereas they were capable of recognizing cells
expressing household proteasomes. Failure to present enough class I
MHC-molecules presenting the same peptide may also allow T-cells to
survive thymic selection (Sebzda E et al). A modest surface
expression of certain class I MHC-restricted peptides can be
achieved by several, not mutually exclusive, mechanisms: 1) to low
expression or to low turn-over of proteins in the cell resulting in
the generation of insufficient numbers of epitopes that allow
recognition by CTL (Vierboom M P et al), 2) peptides with only a
weak binding affinity for MHC may lose the competition with
peptides with better MHC-binding properties and as such are
scarcely expressed at the cell surface, 3) peptides with only a
weak capacity to stably bind to class I MHC may form class I
MHC-peptide complexes at the cell surface which quickly
disintegrate and as such are not stimulatory to T-cells anymore
(van der Burg S H et al 1996) and 4) proteosomal generation of CTL
epitopes may be insufficient to generate effective numbers of
MHC-peptide complexes.
Peptide
[0009] Therefore, in a first aspect, there is provided a peptide
derived from a protein that is ubiquitously expressed self-antigen
and known to be associated with cancer, said peptide comprising an
epitope exhibiting a low to intermediate capacity to form stable
class I MHC-peptide complexes at the cell surface and/or being
inefficiently processed by a proteasome and/or exhibiting a low to
intermediate MHC binding affinity. Preferably, a peptide comprises
an epitope exhibiting a low to intermediate capacity to form stable
class I MHC-peptide complexes at the cell surface and/or being
inefficiently processed by a proteasome.
[0010] In the context of the invention, "exhibiting a low to
intermediate MHC binding affinity" preferably means that the
relative binding affinity of an epitope contained in a peptide is
comprised between 5 and 50 .mu.M. More preferably, the relative
binding affinity is comprised between 10 and 50 .mu.M, even more
preferably between 15 and 50 .mu.M. The relative binding affinity
is preferably assessed by a competition based cellular binding
assay as previously described (van der Burg S H 1995) (see also
example 1). The affinity of a given epitope present within a
peptide is expressed as the epitope concentration to inhibit 50%
(IC50) of the binding of a reference epitope. The length of the
epitope is generally comprised between 8 and 12 amino acids in
length and is typically selected based on typical anchor residues
for HLA-A*0101, A*0301, A*1101 and A*2401 (Rammensee H G et al).
Preferred peptides are the ones as described in example 1.
[0011] In the context of the invention, "exhibiting a low to
intermediate MHC binding affinity" is preferably measured by
measuring the stability for binding to MHC as described in (van der
Burg S H et al 1996).
[0012] Stability of other peptide-HLA complexes was preferably
determined as follows. Peptide binding was performed at 4.degree.
C. and 20.degree. C. and IC.sub.50 were determined. Peptides of
>50% of the initial complexes was lost within 2 hours were
considered unstable. Stable peptides displayed IC.sub.50 at
20.degree. C. that deviated <2 times of the IC.sub.50 at
4.degree. C. Peptides that displayed IC.sub.50 at 20.degree. C. of
more than twice the IC.sub.50 at 4.degree. C. but IC.sub.50<15
.mu.M were considered to bind with intermediate stability. The rest
was designated as unstable peptide binding.
[0013] In the context of the invention, "being inefficiently
processed by a proteasome" preferably means that within the first
hour of digestion by a proteasome less than 1% of total digested
peptide is found. The processing by a proteasome is preferably
assessed by incubating a purified proteasome, more preferably a
human proteasome with a peptide comprising the potential CTL
epitope (30 amino acid length approximately) in a proteasome
digestion buffer during at least one hour at 37.degree. C. The
reaction is subsequently stopped by adding trifluoroacetic acid.
Analysis of the digested peptides is performed with electrospray
ionization mass spectrometry (see example 1). Even more preferably,
the human proteasome is an immunoproteasome from B-LCL JY cells
(Kessler J H et al. 2001).
[0014] The sequence of a peptide used in the present invention is
not critical as long as it is derived from a protein ubiquitously
expressed self-antigen and known to be associated with cancer and
as long as the peptide comprises an epitope exhibiting a low
capacity to form stable class I MHC at the cell surface and/or
which is inefficiently processed by a proteasome and/or exhibiting
a low to intermediate MHC binding affinity. Preferably, a peptide
comprises an epitope exhibiting a low capacity to form stable class
I MHC at the cell surface and/or which is inefficiently processed
by a proteasome
[0015] Accordingly, a peptide is preferably used, which comprises a
contiguous amino acid sequence derived from the amino acid sequence
of a protein ubiquitously expressed self-antigen and know to be
associated with cancer.
[0016] In the context of the invention, a protein is ubiquitously
expressed. Preferably, a protein is ubiquitously expressed when it
is broadly expressed. Broadly preferably means that its expression
is detectable by means of arrays or Northern in at least 5 distinct
types of tissues including the thymus, more preferably at least 7,
including the thymus and even more preferably at least 10,
including the thymus.
[0017] A protein is preferably said to be associated with cancer in
the following illustrating and non-limitative cases: a protein is
over-expressed and/or is mutated and/or is aberrantly expressed in
a given tissue of cancer patients by comparison with the
corresponding tissue of a subject not having cancer. An aberrantly
expressed protein may be de novo expressed in a tissue wherein it
is normally not expressed. A mutated protein may be a splice
variant. A mutated protein may further be produced as an aberrant
fusion protein as a result of a translocation.
[0018] Examples of proteins that are ubiquitously expressed
self-antigens known to be associated with cancer are p53, MDM-2,
HDM2 and other proteins playing a role in p53 pathway, molecules
such as survivin, telomerase, cytochrome P450 isoform 1B1,
Her-2/neu, and CD19 and all so-called house hold proteins.
[0019] In a preferred embodiment, the protein is p53, more
preferably human p53.
[0020] The amino acid sequence of human p53 is depicted in SEQ ID
No.1 Preferably, the length of the contiguous amino acid sequence
derived from the protein, preferably p53 is no more than 45 amino
acids and comprises at least 19 contiguous amino acids derived from
the amino acid sequence of a protein, preferably p53. The length of
the contiguous amino acid sequence derived from a protein,
preferably p53 comprised within the peptide, preferably is
comprised between 19-45, 22-45, 22-40, 22-35, 24-43, 26-41, 28-39,
30-40, 30-37, 30-35, 32-35 33-35, 31-34 amino acids. In another
preferred embodiment, a peptide comprises 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
or 45 or more than 45 contiguous amino acid residues of a protein,
preferably p53. The skilled person will therefore understand that a
peptide of the invention is distinct from a p53 protein, preferably
from the human p53. In another preferred embodiment, a peptide of
the invention consists of any of the contiguous amino acid
sequences from a protein, preferably p53 as defined herein. A
peptide of such length used in the invention may be easily
synthesized.
[0021] In a preferred embodiment, an antigen present in a peptide
derives from a protein, preferably p53 or an immunogenic part,
derivative and/or analogue thereof. This peptide should meet the
activities as earlier defined herein (a peptide comprises an
epitope displaying a low to intermediate capacity to form stable
cell-surface expressed class I MHC-peptide complexes and/or being
inefficiently processed by a proteasome and/or exhibiting a low to
intermediate MHC binding affinity. Preferably, a peptide comprises
an epitope displaying a low to intermediate capacity to form stable
cell-surface expressed class I MHC-peptide complexes and/or being
inefficiently processed by a proteasome). An immunogenic part,
derivative and/or analogue of a protein, preferably p53 comprises
the same immunogenic capacity in kind not necessarily in amount as
said protein itself. A derivative of such a protein can be obtained
by preferably conservative amino acid substitution.
[0022] In a preferred embodiment, when the protein is p53, several
epitopes displaying a low to intermediate capacity to form stable
cell-surface expressed class I MHC-peptide complexes have already
been identified and are presented in table 5. In this preferred
embodiment, a peptide of the invention comprises any of these HLA
A1, A2, A3, A11 and/or A24 type epitopes:
A1: 229-236 and/or A2 149-157 and/or A3: 101-110, 112-120, 113-120,
117-126, 154-163, 156-163, 360-370, 363-372, 373-381, 376-386
and/or A11: 101-110, 112-120, 283-291, 311-319, 311-320, 312-319,
363-370, 374-382 and/or
A24: 340-348.
[0023] Alternatively or in combination with the previous preferred
embodiment, in another preferred embodiment, when the protein is
p53, epitopes which are inefficiently processed by a proteasome
have already been identified and are presented in table 5. In this
preferred embodiment, a peptide of the invention comprises any of
these HLA A1, A2, A3 and/or A11 type epitopes:
A1: 117-126, 196-205, 229-236 and/or A2: 264-272 and/or A3:
101-110, 154-163, 154-164, 156-163, 156-164, 172-181, 376-386
and/or
A11: 101-110, 156-164, 311-319, 311-320, 312-319, 374-382.
[0024] Alternatively or in combination with one or two of the
previous preferred embodiments, in a preferred embodiment, when the
protein is p53, several epitopes exhibiting a low to intermediate
MHC binding affinity have already been identified and are presented
in table 5. In this preferred embodiment, a peptide of the
invention comprises any of these HLA A1, A2, A3, and/or A11 type
epitopes:
A1: 117-126, 196-205, 205-214, 229-236, 229-236, and/or A2:
113-122, 149-157, 264-272, 322-330, and/or A3: 112-120, 113-120,
117-126, 154-163, 156-163, 172-181, 360-370, 363-372, 373-381
and/or
A11: 112-120, 283-291, 363-370, 374-382.
[0025] In an even more preferred embodiment, first two preferred
embodiments are combined to define several epitopes, when the
protein is p53, said epitopes displaying a low to intermediate
capacity to form stable cell-surface expressed class I MHC-peptide
and being inefficiently processed by a proteasome. In this even
more preferred embodiment, a peptide of the invention comprises any
of these HLA A1, A3, and/or A11 type epitopes:
A1: 229-236 and/or A3: 101-110, 154-163, 156-163, 376-386
and/or
A11: 101-110, 311-319, 311-320, 312-319, 374-382.
[0026] Within this even more preferred embodiment, epitopes A1:
229-236, A3: 154-163, 156-163 and/or A11: 374-382 are most
preferred since each of these also exhibits a low to intermediate
MHC binding affinity.
[0027] In one embodiment, a p53 peptide does not consist of or
comprise an HLA-A2.1 type epitope. In this embodiment, preferably a
p53 peptide does not consist of or comprise an epitope exhibiting a
low to intermediate MHC binding affinity.
[0028] In a more preferred embodiment, when the protein is p53, the
peptide is selected from the following peptides, each peptide
comprises or consists of or overlaps with any of the following
sequences:
p53 86-115, p53 102-131, p53 142-171, p53 157-186, p53 190-219, p53
224-248, p53 225-254, p53 257-286, p53 273-302, p53 305-334, p53
353-382 and p53 369-393. Even more preferably, when the protein is
p53, the peptide is selected from the following peptides, each
peptide comprises or consists of or overlaps with any of the
following sequences: p53 142-171, p53 157-186, p53 190-219, p53
224-248, p53 225-254, p53 241-270, p53 257-286 and p53 273-302.
[0029] In the context of the invention, overlapping means that the
sequence of the peptide partially or completely overlaps with a
given sequence. Preferably, overlapping means partially
overlapping. Partially preferably means that the overlap is of one
or more amino acids at the N-terminus and/or at the C-terminus of
the peptide sequence, more preferably of two or more amino acids at
the N-terminus and/or at the C-terminus, or more. It is also
preferred that the overlap is of one or more amino acids at the
N-terminus and/or two or more amino acids at the C-terminus of the
peptide sequence or vice versa. The skilled person will understand
that all kinds of overlaps are encompassed by the present invention
as long as the obtained peptide exhibits the desired activity as
earlier defined herein.
[0030] Even more preferably, the peptide does not consist of p53
102-137, p53 106-137, p53 149-169, p53 129-156, p53 187-212, p53
187-220, p53 187-205, p53 187-234, p53 226-243 or p53 226-264. Each
of these p53 peptides is known from the prior art to exhibit a high
MHC binding affinity and/or is efficiently processed by a
proteasome
Composition
[0031] In a second aspect of the invention, there is provided a
composition comprising one or more of the peptides as defined
herein above. Preferably the composition comprises at least two or
at least three or at least four, or at least five, or at least six
or more of such peptides.
[0032] Preferred compositions include at least two of, or at least
three of or the following peptides: p53 142-171, p53 157-186, p53
190-219, p53 224-248, p53 225-254, p53 241-270, p53 257-286 and p53
273-302, p53 305-334, p53 353-382 and p53 369-393. More preferred
compositions further include p53 86-115 and/or p53 102-131.
[0033] In yet other preferred embodiments, the composition
comprises at least one of the following pools of peptides, wherein
each peptide comprises or consists of or overlaps with the
following sequences:
pool 1: p53 190-219, p53 206-235, p53 224-248, pool 2: p53 142-171,
p53 157-186, 174-203, pool 3: p53 225-254, p53 241-270, p53
257-286, p53 273-302, p53 289-318, p53 305-334, p53 321-350, p53
337-366, p53 353-382 and p53 369-393, pool 4: p53 102-131, p53
126-155, pool 5: p53 70-99, p53 86-115.
[0034] The art currently knows many ways of generating a peptide.
The invention is not limited to any form of generated peptide as
long as the generated peptide comprises, consists or overlaps with
any of the given sequences and had the required activity as earlier
defined herein. By way of example, a peptide present in the
composition may be obtained from a protein, preferably p53
synthesized in vitro or by a cell, for instance through an encoding
nucleic acid. A peptide may be present as a single peptide or
incorporated into a fusion protein. A peptide may further be
modified by deletion or substitution of one or more amino acids, by
extension at the N- and/or C-terminus with additional amino acids
or functional groups, which may improve bio-availability, targeting
to T-cells, or comprise or release immune modulating substances
that provide adjuvant or (co)stimulatory functions. The optional
additional amino acids at the N- and/or C-terminus are preferably
not present in the corresponding positions in the amino acid
sequence of the protein it derives from, preferably p53 amino acid
sequence.
[0035] Accordingly, in a further aspect a peptide of the invention
and a composition of the invention as herein defined are for use as
a medicament.
[0036] In a further preferred embodiment, a peptide or a peptide
composition further comprises a pharmaceutical excipient and/or an
immune modulator. Any known inert pharmaceutically acceptable
carrier and/or excipient may be added to the composition.
Formulation of medicaments, and the use of pharmaceutically
acceptable excipients are known and customary in the art and for
instance described in Remington; The Science and Practice of
Pharmacy, 21.sup.nd Edition 2005, University of Sciences in
Philadelphia.
[0037] A peptides of the invention is preferably soluble in
physiologically acceptable watery solutions (e.g. PBS) comprising
no more than 35 decreasing to 0%; 35, 20, 10, 5 or 0% DMSO. In such
a solution, a peptide is preferably soluble at a concentration of
at least 0.5, 1, 2, 4, or 8 mg peptide per ml. More preferably, a
mixture of more than one different peptides of the invention is
soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg
peptide per ml in such solutions.
[0038] Any known immune modulator, may be added to the composition.
Preferably, the immune modulator is an adjuvant. More preferably,
the composition comprises a peptide as earlier defined herein and
at least one adjuvant. Preferably, the adjuvant is an oil-in-water
emulsion such as incomplete Freunds Adjuvants, MONTANIDE.TM. ISA51
(Seppic, France), MONTANIDE.TM. 720 (Seppic, France). This type of
medicament may be administered as a single administration.
Alternatively, the administration of a peptide as earlier herein
defined and/or an adjuvant may be repeated if needed and/or
distinct peptides and/or distinct adjuvants may be sequentially
administered.
[0039] Particularly preferred adjuvants are those that are known to
act via the Toll-like receptors. Adjuvants that are capable of
activation of the innate immune system, can be activated
particularly well via Toll like receptors (TLR's), including TLR's
1-10 and/or via a RIG-1 (Retinoic acid-inducible gene-1) protein
and/or via an endothelin receptor. Compounds capable of activating
TLR receptors and modifications and derivatives thereof are well
documented in the art. TLR1 may be activated by bacterial
lipoproteins and acetylated forms thereof, TLR2 may in addition be
activated by Gram positive bacterial glycolipids, LPS, LPA, LTA,
fimbriae, outer membrane proteins, heatshock proteins from bacteria
or from the host, and Mycobacterial lipoarabinomannans. TLR3 may be
activated by dsRNA, in particular of viral origin, or by the
chemical compound poly(I:C). TLR4 may be activated by Gram negative
LPS, LTA, Heat shock proteins from the host or from bacterial
origin, viral coat or envelope proteins, taxol or derivatives
thereof, hyaluronan containing oligosaccharides and fibronectins.
TLR5 may be activated with bacterial flagellae or flagellin. TLR6
may be activated by mycobacterial lipoproteins and group B
Streptococcus heat labile soluble factor (GBS-F) or Staphylococcus
modulins. TLR7 may be activated by imidazoquinolines and
derivatives. TLR9 may be activated by unmethylated CpG DNA or
chromatin--IgG complexes. In particular TLR3, TLR4, TLR7 and TLR9
play an important role in mediating an innate immune response
against viral infections, and compounds capable of activating these
receptors are particularly preferred for use in the invention.
Particularly preferred adjuvants comprise, but are not limited to,
synthetically produced compounds comprising dsRNA, poly(I:C),
unmethylated CpG DNA which trigger TLR3 and TLR9 receptors, IC31, a
TLR9 agonist, IMSAVAC, a TLR4 agonist. In another preferred
embodiment, the adjuvants are physically linked to a peptide as
earlied defined herein. Physical linkage of adjuvants and
costimulatory compounds or functional groups, to the HLA class I
and HLA class II epitope comprising peptides provides an enhanced
immune response by simultaneous stimulation of antigen presenting
cells, in particular dendritic cells, that internalize, metabolize
and display antigen. Another preferred immune modifying compound is
a T cell adhesion inhibitor, more preferably an inhibitor of an
endothelin receptor such as BQ-788 (Buckanovich R J et al, Ishikawa
K, PNAS (1994) 91:4892). BQ-788 is
N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D
-1-methoxycarbonyltryptophanyl-D-norleucine. However any derivative
of BQ-788 or modified BQ-788 compound is also encompassed within
the scope of this invention.
[0040] Furthermore, the use of APC (co)stimulatory molecules, as
set out in WO99/61065 and in WO03/084999, in combination with a
peptide present in the medicament used in the invention is
preferred. In particular the use of 4-1-BB and/or CD40 ligands,
agonistic antibodies or functional fragments and derivates thereof,
as well as synthetic compounds with similar agonistic activity are
preferably administered separately or combined with a peptide
present in the medicament to subjects to be treated in order to
further stimulate the mounting an optimal immune response in the
subject.
[0041] In a preferred embodiment, the adjuvant comprises an
exosome, a dendritic cell, monophosphoryl lipid A and/or CpG
nucleic acid.
[0042] Therefore in a preferred embodiment, a medicament comprises
a peptide or a composition as earlier defined herein and an
adjuvant selected from the group consisting of: oil-in water
emulsions (MONTANIDE.TM. ISA51, MONTANIDE.TM. ISA 720), an adjuvant
known to act via a Toll-like receptor, an APC-costimulatory
molecule, an exosome, a dendritic cell, monophosphoryl lipid A and
a CpG nucleic acid.
[0043] In another preferred embodiment, to promote the presentation
of a peptide by a professional antigen presenting cell or dendritic
cells, the medicament comprising a peptide further comprises a
DC-activating agent.
[0044] Ways of administration are known and customary in the art
are for instance described in Remington; The Science and Practice
of Pharmacy, 21.sup.st Edition 2005, University of Sciences in
Philadelphia. Peptide, peptide compositions and pharmaceutical
compositions and medicaments of the invention are preferably
formulated to be suitable for intravenous or subcutaneous, or
intramuscular administration, although other administration routes
can be envisaged, such as mucosal administration or intradermal
and/or intracutaneous administration, e.g. by injection.
Intradermal administration is preferred herein. Advantages and/or
preferred embodiments that are specifically associated with
intradermal administration are later on defined in a separate
section entitled "intradermal administration".
[0045] It is furthermore encompassed by the present invention that
the administration of at least one peptide and/or at least one
composition of the invention may be carried out as a single
administration. Alternatively, the administration of at least one
peptide and/or at least one composition may be repeated if needed
and/or distinct peptides and/or compositions of the invention may
be sequentially administered.
[0046] Any way of administration of the composition or medicament
of the invention may be used. The composition or medicament of the
invention may be formulated to be suitable for intravenous or
subcutaneous, or intramuscular administration, although other
administration routes may be envisaged, such as mucosal or
intradermal and/or intracutaneous administrations, e.g. by
injection.
[0047] In addition a preferred embodiment comprises delivery of a
peptide, with or without additional immune stimulants such as TLR
ligands and/or anti CD40/anti-4-1 BB antibodies in a slow release
vehicle such as mineral oil (e.g. MONTANIDE.TM. ISA 51) or PLGA.
Alternatively, a peptide of the invention may be delivered by
intradermally, e.g. by injection, with or without immune stimulants
(adjuvants). Preferably for intradermal delivery a peptide of the
invention is administered in a composition consisting of the
peptides and one or more immunologically inert pharmaceutically
acceptable carriers, e.g. buffered aqueous solutions at
physiological ionic strength and/or osmolarity (such as e.g.
PBS).
Use of a Peptide
[0048] In a further aspect of the invention, there is provided a
use of a peptide as earlier defined herein derived from a
ubiquitously expressed self-antigen known to be associated with
cancer, said peptide exhibiting a low to intermediate capacity to
form stable cell surface expressed class I-MHC complexes and/or
being inefficiently processed by a proteasome and/or exhibiting a
low to intermediate MHC binding affinity for the manufacture of a
medicament for the treatment or prevention of cancer. Preferably,
the protein is p53. Preferably, a peptide exhibits a low to
intermediate capacity to form stable cell surface expressed class
I-MHC complexes and/or is inefficiently processed by a
proteasome.
[0049] Preferred peptides for use in the treatment or prevention of
cancer are as already defined herein above.
[0050] All preferred features of the medicament manufactured for
this use have already been defined earlier herein. In a preferred
embodiment, the medicament which is used further comprises an inert
pharmaceutically acceptable carrier and/or an adjuvant. In a
preferred embodiment, the medicament, which is a vaccine, is
administered to a human or animal. In a more preferred embodiment,
the human or animal is suffering from or at risk of suffering from
a cancer, wherein the protein the peptide derives from is
associated with. More preferably, the protein is p53, even more
preferably human p53. Even more preferably, cancer associated with
p53 are selected among the following list: lung, colon, esophagus,
ovary, pancreas, skin, gastric, head and neck, bladder, sarcoma,
prostate, hepatocellular, brain, adenal, breast, endometrium,
mesothelioma, renal, thyroid, hematologic, carcinoid, melanoma,
parathyroid, cervix, neuroblastoma, Wilms, testes, pituitary and
pheochromocytoma cancers.
[0051] Other preferred proteins have been already cited herein.
[0052] Preferably, said disease such as cancer is at least in part
treatable or preventable by inducing and/or enhancing said immune
response using a peptide of the invention.
[0053] A method of the invention is therefore very suited for
providing a subject with immunity against any ubiquitously
expressed self protein known to be associated with cancer and/or
for enhancing said immunity. Methods of the invention are suitable
for any purpose that other immunization strategies are used for. Of
old immunizations are used for vaccination purposes, i.e. for the
prevention of cancer. However, methods of the invention are not
only suitable for preventing cancer. Methods can also be used to
treat existing cancer, of course with the limitations that the
cancer is treatable by inducing and/or enhancing antigen specific T
cell immunity.
Method
[0054] In a further aspect, the invention provides a method for
designing a peptide derived from a protein ubiquitously expressed
self-antigen associated with cancer, said peptide comprising an
epitope exhibiting a low to intermediate capacity to form stable
cell surface expressed class I-MHC complexes and/or being
inefficiently processed by a proteasome and/or exhibiting a low to
intermediate MHC binding affinity and said peptide being suitable
for the manufacture of a medicament for the treatment or prevention
of cancer. Preferred peptide comprises an epitope exhibiting a low
to intermediate capacity to form stable cell surface expressed
class I-MHC complexes and/or being inefficiently processed by a
proteasome.
[0055] All features of this method have already been explained
herein.
[0056] To identify such a peptide the skilled person could follow
the strategy as illustrated in the examples: an epitope exhibiting
a low to intermediate MHC binding affinity may be identified by
measuring the relative binding affinity as earlier defined herein.
The capacity of an epitope to form a low/intermediate stable cell
surface expressed class I-MHC complexes may be measured as earlier
defined herein. Finally, the inefficiently processing of a peptide
by a proteasome may be assessed as earlier defined herein.
Intradermal Administration
[0057] In a preferred embodiment, a peptide or a composition
comprising a peptide or a medicament used in the invention all as
defined herein are formulated to be suitable for intradermal
administration or application. Intradermal is known to the skilled
person. In the context of the invention, intradermal is synonymous
with intracutaneous and is distinct from subcutaneous. A most
superficial application of a substance is epicutaenous (on the
skin), then would come an intradermal application (in or into the
skin), then a subcutaneous application (in the tissues just under
the skin), then an intramuscular application (into the body of the
muscle). An intradermal application is usually given by injection.
An intradermal injection of a substance is usually done to test a
possible reaction, allergy and/or cellular immunity to it. A
subcutaneous application is usually also given by injection: a
needle is injected in the tissues under the skin.
[0058] In another further preferred embodiment, a medicament or
composition or peptide used in the invention does not comprise any
adjuvant such as MONTANIDE.TM. ISA-51, it means the formulation of
the medicament (or composition or peptide) is more simple: an
oil-water based emulsion is preferably not present in a medicament
(or composition or peptide) used. Accordingly, a medicament (or
composition or peptide) used in the invention does not comprise an
adjuvant such as MONTANIDE.TM. ISA-51 and/or does not comprise an
oil-in-water based emulsion. Therefore, in a preferred embodiment,
a medicament (or composition or peptide) used in the invention is a
buffered aqueous solutions at physiological ionic strength and/or
osmolarity, such as e.g. PBS (Phosphate Buffer Saline) comprising
or consisting of one or more peptide as defined earlier herein. The
skilled person knows how to prepare such a solution.
[0059] A medicament (or composition or peptide) as used in the
invention has another advantage, which is that by intradermally
administering low amounts of a peptide as earlier herein defined,
an immunogenic effect may still be achieved. The amount of each
peptide used is preferably ranged between 1 and 1000 .mu.g, more
preferably between 5 and 500 .mu.g, even more preferably between 10
and 100 .mu.g.
[0060] In another preferred embodiment, a medicament (or
composition) comprises a peptide as earlier defined herein and at
least one adjuvant, said adjuvant being not formulated in an oil-in
water based emulsion and/or not being of an oil-in-water emulsion
type as earlier defined herein. This type of medicament may be
administered as a single administration. Alternatively, the
administration of a peptide as earlier herein defined and/or an
adjuvant may be repeated if needed and/or distinct peptides and/or
distinct adjuvants may be sequentially administered. It is further
encompassed by the present invention that a peptide of the
invention is administered intradermally whereas an adjuvant as
defined herein is sequentially administered. An adjuvant may be
intradermally administered. However any other way of administration
may be used for an adjuvant.
[0061] The intradermal administration of a peptide is very
attractive since the injection of the vaccine is realized at or as
close by as possible to the site of the disease resulting in the
local activation of the disease draining lymph node, resulting in a
stronger local activation of the immune system. In a preferred
embodiment, the intradermal administration is carried out directly
at the site of the lesion or disease. At the site of the lesion is
herein understood to be within less than 5, 2, 1, 0.5, 0.2 or 0.1
cm from the site of the lesion.
[0062] Upon intradermally administering a medicament as defined
herein, not only Th2 but also Th1 responses are triggered. This is
surprising since it was already found that cutaneous antigen
priming via gene gun lead to a selective Th2 immune response
(Alvarez D. et al, 2005). Furthermore, the immune response observed
is not only restricted to the skin as could be expected based on
(Alvarez D. et al, 2005). We demonstrate that specific T cells
secreting IFN.gamma. circulate through the secondary lymph system
as they are detected in the post challenged peripheral blood.
[0063] Another crucial advantage of a medicament (or composition or
peptide) of the invention is that relatively low amounts of a
peptide may be used, in one single shot, in a simple formulation
and without any adjuvant known to give undesired side-effects as
MONTANIDE.TM. ISA-51. Without wishing to be bound by any theory, we
believe that the intradermal peptide(s) used in the invention
specifically and directly targets the epidermal Langerhans cells
(LC) present in the epithelium. Langerhans cells are a specific
subtype of DC which exhibit outstanding capacity to initiate
primary immune responses (Romani N. et al 1992). These LC may be
seen as natural adjuvants recruited by the medicament used in the
invention.
[0064] In another preferred embodiment, the invention relates to
the use of a peptide as defined herein for the manufacture of a
medicament for the treatment or prevention of a disease as defined
herein, wherein the medicament is for intradermal administration as
earlier defined and wherein in addition a same and/or distinct
peptide as defined herein is further used for the manufacture of a
medicament for the treatment or prevention of the same disease,
wherein the medicament is for subcutaneous administration.
[0065] A medicament for intradermal administration has already been
defined herein. A peptide used for subcutaneous administration may
be the same as the one used for intradermal administration and has
already been defined herein. The skilled person knows how to
formulate a medicament suited for subcutaneous administration.
Preferably, a medicament suited for subcutaneous administration
comprises a peptide as already herein defined in combination with
an adjuvant. Preferred adjuvants have already been mentioned
herein. Other preferred adjuvants are of the type of an oil-in
water emulsions such as incomplete Freund's adjuvant or IFA,
MONTANIDE.TM. ISA-51 or MONTANIDE.TM. ISA 720 (Seppic France). In a
further preferred embodiment, a medicament suited for subcutaneous
administration comprises one or more peptides, an adjuvant both as
earlier defined herein and an inert pharmaceutically acceptable
carrier and/or excipients all as earlier defined herein.
Formulation of medicaments, and the use of pharmaceutically
acceptable excipients are known and customary in the art and for
instance described in Remington; The Science and Practice of
Pharmacy, 21.sup.nd Edition 2005, University of Sciences in
Philadelphia. The second medicament used in the invention is
formulated to be suitable for subcutaneous administration.
[0066] In this preferred embodiment, a medicament suited for
intradermal administration may be simultaneously administered with
a medicament suited for subcutaneous administration. Alternatively,
both medicaments may be sequentially intradermally and subsequently
subcutaneously administered or vice versa (first subcutaneous
administration followed by intradermal administration). In this
preferred embodiment as in earlier preferred embodiment dedicated
to the intradermal administration, the intradermal and/or
subcutaneous administration of a peptide as earlier herein defined
and/or of an adjuvant may be repeated if needed and/or of distinct
peptides and/or of distinct adjuvants may be sequentially
intradermally and/or subcutaneously administered. It is further
encompassed by the present invention that a peptide of the
invention is administered intradermally and/or subcutaneously
whereas an adjuvant as defined herein is sequentially administered.
The adjuvant may be intradermally and/or subcutaneously
administered. However any other way of administration may be used
for the adjuvant.
[0067] We expect the combination of an intradermal and a
subcutaneous administration of a medicament (or a composition or a
peptide) according to the invention is advantageous. DC in the
epidermis are clearly different from DC in the dermis and in the
subcutis. The intracutaneous (intradermal) immunization will cause
antigen processing and activation of epidermal DC
(Langerin-positive langerhans cells) that through their dendritic
network are in close contact with the keratinocytes. This will also
optimally activate inflammatory pathways in the interactions
between Langerhans cell and keratinocytes, followed by trafficking
of antigen loaded and activated Langerhans cell to the
skin-draining lymph nodes.
[0068] The subcutaneous administration will activate other DC
subsets, that will also become loaded with antigen and travel
independently to the skin-draining lymph nodes. Conceivably, the
use of a medicament which may be administered both intradermally
and subcutaneously may lead to a synergistic stimulation of T-cells
in these draining nodes by the different DC subsets.
[0069] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition the verb "to consist" may
be replaced by "to consist essentially of" meaning that a peptide
or a composition as defined herein may comprise additional
component(s) than the ones specifically identified, said additional
component(s) not altering the unique characteristic of the
invention. In addition, reference to an element by the indefinite
article "a" or "an" does not exclude the possibility that more than
one of the element is present, unless the context clearly requires
that there be one and only one of the elements. The indefinite
article "a" or "an" thus usually means "at least one".
[0070] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety. The following examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIG. 1. Proteasomal cleavage products. FIG. 1 discloses SEQ
ID NOS 91-93, respectively, in order of appearance.
[0072] FIG. 2. Explanation of the mechanism of formation of the
proteasomal cleavage products.
[0073] FIG. 3. Flow chart of phase I and II of the clinical trial
in ovarian cancer patient as presented in example 2 and in colon
cancer patient as presented in example 3. For ovarian cancer
patient, fours shots of vaccine are used, whereas for colon cancer
patient, two shots of vaccine are used. For colon cancer patient,
the skin biopsy was carried out at the second vaccine site.
[0074] FIGS. 4A-G. P53-peptide specific responses in clinical trial
of ovarian cancer patients as measured by proliferation assay
(n=8). FIGS. 4A-F. Vaccine induced response (*) if post-vaccination
sample .gtoreq.1000 cpm & proliferation index .gtoreq.3 and if
post-vaccination cpm value .gtoreq.2.times. pre-vaccination cpm
value. FIG. 4G. Responses to memory recall mix (MRM). Light grey
bars: pre-vaccination, black bars: post-vaccination.
[0075] FIGS. 5A-D. P53-peptide specific responses in clinical trial
of colon cancer patients as measured by proliferation assay and
cytokine production. The p53-specific proliferative capacity (FIGS.
5A and 5C) and cytokine production (FIGS. 5B and 5D) of 2 male
patients with colorectal cancer is shown before and after
vaccination with p53-long peptide vaccine. Patients were vaccinated
twice with overlapping p53 peptides covering the amino acid
sequence 70-248 (indicated by the pools of 2 peptide: V70-115,
V102-155, V142-203, V190-248). Patients PBMC were also tested
against the N-terminal region of p53 (aa 1-78) and C-terminal
region of p53 (aa 241-393).
[0076] FIGS. 6A-F. P53-peptide specific responses as measured by
IFN-.gamma. ELISPOT (n=7) in ovarian cancer patients.
[0077] Vaccine induced response (*) if {mean number of spots-(mean
number of spots in medium+2SD)>10 spots} AND post-vaccination
value.gtoreq.2.times. pre-vaccination value. Light grey bars:
pre-vaccination, black bars: post-vaccination.
[0078] FIGS. 7A-D. P53-peptide specific responses in skin biopsies
of vaccination sites obtained from ovarian cancer patients as
measured by proliferation assay (n=7).
[0079] Biopsies are taken three weeks after the last vaccination
from the last injection site. Vaccine induced response (*) if
counts per minute .gtoreq.1000 and proliferation index
.gtoreq.3.
[0080] FIG. 8. Vaccine-induced p53-specific T-cells can migrate
into areas where p53 antigen is present and recognize naturally
processed and presented p53 protein.
[0081] A biopsy of the second vaccine site of a patient with
colorectal cancer was taken and skin-infiltrating T-cells were
expanded. The skin-infiltrating T-cells were tested against several
pools of p53 peptides (indicated by the number of the first and
last amino acid of the amino acid sequence of the p53 protein that
is covered by the pool of peptides used) as well as p53 protein and
control protein. The bars indicate the mean and standard deviation
of triplicate wells.
[0082] FIG. 9. Vaccination with p53-SLP vaccine induces T-cell
memory responses and antigen-spreading in patients with colorectal
cancer.
[0083] PBMC isolated pre-vaccination, after 2 vaccinations and at 6
months (#1) or 9 months (#2) after vaccination were tested for the
presence of p53-specific T-cells in a proliferation assay by
stimulating the PBMC for 6 days with several pools of p53 peptides
(indicated by the number of the first and last amino acid of the
amino acid sequence of the p53 protein that is covered by the pool
of peptides used). The bars indicate the mean and standard
deviation of 8-wells.
[0084] FIG. 10. Responses in ovarian cancer patients to individual
p53 synthetic long peptides as measured by proliferation assay
after four immunisations. Positive response (*) if .gtoreq.1000 cpm
& proliferation index .gtoreq.2.
[0085] FIG. 11. P53-peptide specific responses in skin biopsies of
vaccination sites obtained from ovarian cancer patients as measured
by proliferation assay.
[0086] Biopsies from the last injection site were obtained from 17
ovarian cancer patients three weeks after the last vaccination.
Insufficient numbers of lymphocytes for proliferation assay could
be cultured from two biopsies (015 & 020). Positive response
(*) if counts per minute .gtoreq.1000 and proliferation index
.gtoreq.3.
[0087] FIG. 12. P53-specific T-cell responses as measured by
proliferation assay before the first and after the last vaccination
as well as after subsequent chemotherapy in ovarian cancer
patients. Post-chemotherapy samples were obtained 12 months (009)
resp. 9 months (019) after the last vaccination and at least one
month after the last chemotherapy. Vaccine-induced response (*) if
cpm .gtoreq.1000 & S.I. .gtoreq.3 and if the cpm after
vaccination/chemotherapy was .gtoreq.2 the pre-vaccination
value.
[0088] FIGS. 13A and B. Serum CA-125 levels in ovarian cancer
patients before, during and after vaccination. Missing values: 04
visit 4/5/FU; 05 visit 3/4/5/FU; 11 visit FU.
[0089] FIG. 14: An overview of the number, day of appearance and
injected antigen that induced a positive skin reactions in the
group of 19 healthy donors (HD). Skin reactions were considered
positive when papules greater then 2 mm in diameter arose no less
then 2 days after injection. The indicated layout is used for the 8
peptide pools, the first and last amino acid in the protein of the
peptide pool used is indicated. The layout printed in bold
indicates at least one positive reaction within this timeframe; a
filled square represents a new developed, positive skin reaction to
the indicated peptide pool.
[0090] FIG. 15. Detection of HPV16 specific T cells by IFN.gamma.
ELIspot in the pre-challenge blood sample of healthy donors is
significantly correlated with the appearance of an early (<13
days) positive skin reaction to the recognized peptide pool
(p=0.0003, two tailed Fisher's Extract test). Specific responses
were calculated by subtracting the mean number of spots+2.times.SD
of the medium control from the mean number of spots in experimental
wells. The number of specific spots per 100.000 PBMC is given.
Responses were considered positive if peptide pool specific T cell
frequencies were .gtoreq.5 in 100.000 PBMCs.
[0091] FIG. 16. Association between the appearance of a positive
skin reaction and the simultaneous detection (IFN.gamma. ELIspot)
of circulating HPV 16 specific T cells in the post-challenge blood
sample of healthy donors (p<0.0001, two tailed Fisher's exact
test). From a total of 88 skin tests, 39 were positive. Twenty-five
of these 39 reactions were associated with a positive reaction in
ELIspot (T cell frequency .gtoreq.5 in 100.000 PBMCs). Of the 49
skin test sites that did not show a skin reaction, 10 were
associated with a positive ELIspot.
[0092] FIGS. 17A-C.
[0093] FIG. 17A. HPV16 specific T cell responses detected by
IFN.gamma. ELIspot in the post-challenge blood sample of healthy
donors displaying a positive skin reaction. The mean number of
spots per 100.000 PBMCs are depicted. Memory response mix (MRM) was
used as a positive control. The filled bar indicates the positive
skin reaction site of which a punch biopsy was taken and put in to
culture.
[0094] FIG. 17B. T lymphocytes exfiltrating from punch biopsies
were, after a 14- to 28 day period of cytokine driven expansion,
tested for their capacity to proliferate upon stimulation with
monocytes pulsed with peptides (10 .mu.g/ml)--as injected in the
skin test--or with protein (20 .mu.g/ml). Phytohemagglutinine (PHA)
served as a positive control. Proliferation was measured by
[.sup.3H]thymidine incorporation and a proliferative response was
defined specific as the stimulation index (SI).gtoreq.3. Healthy
donor 17 (HD17) is an example of a positive skin reaction site
consisting of non specific T cells.
[0095] FIG. 17C. Supernatants of the proliferative responses in B
were analysed for the presence of IFN.gamma., interleukin 4 (IL4),
IL5 and tumor necrosis factor .alpha., IL2, IL10 (not shown) by
cytometric bead array. Cutoff values were based on the standard
curves of the different cytokines (100 .mu.g/ml IFN.gamma. and 20
.mu.g/ml for the remaining cytokines). Antigen-specific cytokine
production was defined as a cytokine concentration above cutoff
level and >2.times. the concentration of the medium control.
Healthy donor 15 (HD15) displays a high background level of IL5,
but is increased >2.times. after antigen stimulation.
[0096] FIG. 18.
[0097] T cell culture of the skin biopsy of pool 4 (E6.sub.41-65,
E6.sub.55-80, E6.sub.71-95) of healthy donor 15 (HD15) consists of
both HPV16 specific CD4+ and CD8+ T cells. The specificity of the
culture was tested in an intracellular cytokine staining (ICS)
against the protein (20 .mu.g/ml) and the peptides (10 .mu.g/ml)
corresponding with the injected skin test. Remarkably, in 3 out of
4 biopsies CD8+ HPV16-specific T cells were detected.
EXAMPLES
Example 1
Relation Between HLA Binding, Proteasomal Digestion and
Tolerance
[0098] A modest surface expression of certain class I
MHC-restricted peptides can be achieved by several, not mutually
exclusive, mechanisms: 1) to low expression or to low turn-over of
proteins in the cell resulting in the generation of insufficient
numbers of epitopes that allow recognition by CTL (Vierboom M P et
al), 2) peptides with only a weak binding capacity for MHC may lose
the competition with peptides with better MHC-binding properties
and as such are scarcely expressed at the cell surface, and 3)
proteosomal generation of CTL epitopes may be insufficient to
generate effective numbers of MHC-peptide complexes.
[0099] As part of normal cell regulation, p53 protein is targeted
for proteasome-mediated degradation and shortage of protein
entering the proteasome is therefore not likely to play an
important role in the escape of negative selection. We have,
therefore, analyzed the binding capacity of p53 derived peptides as
well as the capacity of immunoproteasomes and household proteasomes
to generate these peptides in vitro.
Results and Discussion
[0100] From a large set of peptides that were selected based on the
presence of so-called major anchor residues for HLA-A*0101,
HLA-A*0301, HLA-A*1101 and HLA-A*2401, 43 peptides were found to
bind with intermediate to high affinity (Table 1). These peptides
and 7 HLA-A*0201 binding peptides (Houbiers J G et al, Nijman H W
et al, Theobald M et al 1995) were further in vitro analyzed for
both the stability of binding van der Burd S H et al 1995 and 1996)
as well as for the liberation of the exact C-terminus by
immunoproteasomes and household proteasomes (Kessler J H et al
2001) (Table 1). In general, the results obtained with both types
of proteasomes were comparable. Except for the peptides
p53.sub.110-120, p53.sub.111-120, p53.sub.112-120, and
p53.sub.113-120 binding to both HLA-A*0301 and/or HLA-A*1101 and
carrying the same C-terminal lysine, no disparity in epitope
generation between the household and immunoproteasomes was detected
(Table 1).
[0101] At present, 5 different CTL epitopes in p53 have been
identified (Table 2). Four of these epitopes were restricted by
HLA-A*0201 and one by HLA-A*2401. Peptide binding analysis revealed
that peptide p53.sub.264-272 displayed intermediate binding
affinity and peptide p53.sub.149-157 displayed a weak capacity to
form stable peptide-HLA-A*0201 complexes, the other 4 peptides
displayed a good and stable binding to their restricting HLA
molecules. Interestingly, proteasomal cleavage analysis of 30
residue long precursor peptides demonstrated that only two
(p53.sub.149-157 and p53.sub.187-197) out of these five peptides
were efficiently generated by both household and immunoproteasomes
(FIG. 1). For peptide p53.sub.264-272 a fragment corresponding to
the part after C-terminal cleavage was found after 4 hours of
digestion, but the peptide itself or N-terminally extended
pre-cursors were not detected (FIG. 1). Recently, it was
demonstrated that pre-cursor peptides could be detected after in
vitro digestion by 20S proteasomes, albeit at trace amounts
(Theobald M et al 1998). Taken together, there are two epitopes
p53.sub.65-73 and p53.sub.125-134 which display good binding
capacity but are not/inefficiently processed by 20S proteasomes
whereas a third peptide (p53.sub.149-157) is well processed by both
types of proteasomes but demonstrates a weak capacity to form
stable peptide-HLA complexes. Furthermore, p53.sub.264-272 binds
with intermediate affinity to HLA-A*0201 but is not well processed
by proteasomes (Table 2). Most likely, the number of MHC-peptide
complexes formed by these peptides at the surface of thymic APC is
insufficient to delete the corresponding p53-specific CTL, allowing
these cells to egress into the periphery. The one peptide
(p53.sub.187-197) that displays good binding capacity to
HLA-A*0201, forms stable peptide-HLA complexes and is well
generated by both immunoproteasomes and household proteasomes
(Table 2) induced T-cell tolerance (Theobald M et al 1997), as was
expected. Bearing this in mind, it will be interesting to find out
whether the other inefficiently processed peptides, presented in
table 1, which bind to either HLA-A*0101, HLA-A*0301, HLA-A*1101 or
HLA-A*2401, form genuine CTL epitopes.
[0102] We recently reported on the identification of CTL epitopes
in the cancer associated self-antigen PRAME (Kessler J H et al
2001). Comparison of the binding capacity and liberation of the
C-terminus by proteasomes of p53- and PRAME-derived peptides
reveals that the PRAME peptides are well processed whereas the
p53-derived CTL epitopes are not (Table 2). PRAME is expressed in a
variety of tumors, testis and at low levels in normal endometrium
but is not expressed in the thymus and as a result PRAME-specific
CTL are not deleted in the thymus. Together these data suggest that
the combination of stable peptide binding and proteasomal
liberation of exact C-termini may lead to accurate prediction of
CTL epitopes for which CTL will be available (e.g. viral antigens,
tumor antigens such as PRAME) whereas for ubiquitously expressed
antigens such as p53 it may reveal to which peptides tolerance will
exist.
[0103] It has now been well established that CTL epitopes may be
generated via alternative processing pathways (Benham A M et al,
Glas R et al, Geier E et al, Reimann J et al) and that this results
in HLA-class I molecules containing peptides with diverse carboxy
termini and from proteins dispersed throughout the cell (Luckey C J
et al 1998). A wide variety of tumors display enhanced expression
levels of p53, due to mutations in the p53 gene or other genes of
the p53 regulatory pathway, as a result of decreased proteasomal
digestion (Honda R et al). Interestingly, the expression of the
dominant HLA-A*0201-restricted influenza matrix CTL epitope is
enhanced when cells are treated with proteasome inhibitors (Luckey
C J et al 1998). Furthermore, it was demonstrated that 50-60% of
normal expression levels of HLA-A*0201 molecules re-appeared at
surface despite the presence of proteasome inhibitors (Luckey C J
et al 2001). This suggests that proteins that are not/less well
degraded by proteasome are more likely to be processed by other
ways. This may mean that over-expression of p53 results in an
enhanced expression of (amongst others HLA-A*0201-restricted) CTL
epitopes generated via other routes than the proteasome (FIG. 2).
Activation of p53-specific CTL may occur after uptake of
tumor-derived p53 by peripheral APC. This results in the
presentation of p53-peptides in the MHC class II pathway (van der
Brug S H et al 2001 and Tilkin A F et al) and may also lead to
presentation in MHC class I (Reimann J et al) (FIG. 2).
Concluding Remarks
[0104] We recently demonstrated that the efficient liberation of
the exact C-terminus of putative CTL epitopes present in 25-30
amino acid long precursor peptides by 20S proteasomes, accurately
identified natural processed peptides for which CTL are present in
the peripheral blood (Kessler J H et al 2001). With respect to
ubiquitously expressed antigens that as part of their normal
regulation are degraded by the proteasome, some necessary
differentiations should be made. A combination of a high capacity
to form stable MHC peptide complexes and an efficient liberation by
proteasomal digestion renders a peptide to induce tolerance. In
contrast, peptides handicapped by either one of these two may form
natural targets for CTL that made it to the periphery.
Material and Methods
Peptides, Peptide Binding and Stabilization Assays
[0105] Peptides 8-11 residues in length were selected based on
typical anchor residues for HLA-A*0101, A*0301, A*1101 and
HLA-A*2401 (Rammensee H G et al). The capacity of peptides to bind
was tested in a competition based cellular binding assay as
previously described (van der Burg S H et al 1995). FL-labeled
reference peptides were synthesized as Cys-derivative (van der Burg
S H et al 1995). Fluorescence labeled reference peptides used were:
YLEPAC(FL)AK (SEQ ID NO:87) (HLA-A*0101) and KVFPC(FL)ALINK (SEQ ID
NO:88) (HLA-A*1101) (Sette A et al), KVFPC(FL)ALINK (SEQ ID NO:89)
(HLA-A*0301) (van der Burg S H et al 1995) and RYLKC(FL)QQLL (SEQ
ID NO:90) (A*2401) (Dai L C et al). B-cell lines used are: CAA
(A*0101), EKR (A*0301), BVR (A*1101), VIJF (A*2401). The relative
binding capacity of the peptides is expressed as the peptide
concentration to inhibit 50% (IC.sub.50) of the binding of the
reference peptide. Affinity is categorized as follows: good
IC.sub.50<5 .mu.M, intermediate IC.sub.50=5-15 .mu.M, and low
IC.sub.50>15-50 .mu.M. Peptide stabilization for HLA-A*0201 was
performed as previously described (van der Burg S H et al, 1996).
Peptides of >50% of the initial complexes was lost within 2
hours were considered unstable. Stability of other peptide-HLA
complexes was determined as follows. Peptide binding was performed
at 4.degree. C. and 20.degree. C. and IC.sub.50 were determined.
Stable peptides displayed IC.sub.50 at 20.degree. C. that deviated
<2 times of the IC.sub.50 at 4.degree. C. Peptides that
displayed IC.sub.50 at 20.degree. C. of more than twice the
IC.sub.50 at 4.degree. C. but IC.sub.50<15 .mu.M were considered
to bind with intermediate stability. The rest was designated as
unstable peptide binding.
In Vitro Proteasome Mediated Digestions and Mass-Spectrometry
[0106] 20S immuno-proteasomes were purified from a mouse B-cell
line (RMA) and a human B-LCL cell line (JY) and 20S household
proteasomes were purified from human tumor cell line (HeLa) as
described (Kessler J H et al 2001). To assess kinetics, digestions
were performed with different incubation periods as indicated.
Briefly, peptides containing the (potential) CTL epitopes (30 mers,
20 .mu.g) were incubated with 1 .mu.g of purified proteasome at
37.degree. C. for 1 h, 4 h and 24 h in 300 .mu.l proteasome
digestion buffer, trifluoroacetic acid (30 .mu.l) was added to stop
the digestion and samples were stored at -20.degree. C. before mass
spectrometric analysis. Electrospray ionization mass spectrometry
was performed on a hybrid quadrupole time-of-flight mass
spectromter, a Q-TOF (Micromass), equipped with an on-line
nanoelectrospray interface with an approximate flow rate of 250
mL/min as described (Kessler J H et al 2001). The peaks in the mass
spectrum were searched in the digested precursor peptide using the
Biolynx/proteins software (Micromass) supplied with the mass
spectrometer. The intensity of the peaks in the mass spectra was
used to establish the relative amounts of peptides generated after
proteasome digestion. The relative amounts of the peptides are
given as a percentage of the total amount of peptide digested by
the proteasome at the indicated incubation time. Major cleavage
sites are defined as more than >1% of total digested peptide
within the first hour.
Example 2
Vaccination Study with p53 Peptides in Ovarian Cancer
Patients--Immunological Results in 7 Vaccinated Patients
Objectives of the Trial
2.1 General Objectives
[0107] Primary objective: [0108] To evaluate the safety and
tolerability of a p53 specific "long peptide" vaccine in
combination with a defined adjuvant with known mode of action
(MONTANIDE.TM. ISA) (phase I part of the study) [0109] To evaluate
the immunogenicity of a p53 specific "long peptide" vaccine in
combination with a defined adjuvant with known mode of action
(MONTANIDE.TM. ISA) (phase II part of the study)
2.2 End-Points
[0110] Primary endpoint of the phase I study is safety and
tolerability Primary endpoint of the phase II study is
immunogenicity (p53 specific T cell responses)
2.3. Trial Design
[0111] A phase I/II vaccination study was performed in patients
with ovarian cancer, using 10 overlapping p53 peptides in
combination with an adjuvant with a sustained dendritic cell
activating ability (MONTANIDE.TM.-ISA-51). The flow chart phase is
given in FIG. 3.
2.4. Patient Selection Criteria
2.4.1. Inclusion Criteria
[0112] Histological proven epithelial ovarian carcinoma [0113] At
least 4 weeks after termination of first line treatment (debulking
surgery and platinum based chemotherapy) [0114] Rising CA125 serum
levels after first line treatment and no measurable disease
according to the RECIST (Response Evaluation Criteria in Solid
Tumours) criteria (Therasse P et al). [0115] Rising CA125 serum
levels after first line treatment with measurable disease according
to the RECIST (Response Evaluation Criteria in Solid Tumours)
criteria (Therasse P et al) but not willing or otherwise not fit to
receive second line chemotherapy.
[0116] A rising in CA125 is known as a prognostic marker for
ovarian cancer (Ferrandina G et al, Goonewardene et al and Rustin
et al). [0117] Age 18 years or older, an expected life expectancy
of at least 3 months [0118] Absence of any psychological, familial,
sociological or geographical condition potentially hampering
compliance with the study protocol and follow-up schedule; those
conditions should be discussed with the patient before registration
in the trial [0119] Before patient registration/randomization,
written informed consent must be given according to Good Clinical
Practice (GCP), and national/local regulations. [0120] Performance
status 0 to 2 (WHO scale) [0121] Adequate hepatic, renal, and bone
marrow function as defined: [0122] INR <1.5; WBC
>3.0.times.109/L; thrombocytes >100.times.109/L; hemoglobin
>6.0 mmol/1.
2.4.2. Exclusion Criteria
[0122] [0123] Pregnancy and/or breast feeding. [0124] Other
malignancies (previous or current), except basal or squamous cell
carcinoma of the skin. [0125] High dose of immunosuppressive
agents. [0126] Prior therapy with a biological response modifier.
[0127] Uncontrolled hypertension, unstable angina pectoris,
arrhythmias requiring treatment or myocardial infarction within the
preceding 3 months. [0128] Uncontrolled congestive heart failure or
severe cardiomyopathy. [0129] Signs or symptoms of CNS metastases.
[0130] Severe neurological or psychiatric disorders.
2.4.3 Withdrawal Criteria
[0130] [0131] progressive disease necessitating other forms of
anti-tumor therapy [0132] unacceptable toxicity (gr 3-4 toxicity
due to vaccination persisting for more than 2 weeks) [0133] patient
refusal [0134] lost to follow-up
2.5. Therapeutic Regimens, Expected Toxicity.
[0135] Most Th and CTL responses recognize peptides within the
residues 70-251 of the p53 protein. Therefore the vaccine
encompasses this region of the p53 protein. The peptides used are
given in table 3.
[0136] The clinical grade peptides for vaccination are prepared in
the Peptide Laboratory, section IGFL, department of Clinical
Pharmacy and Toxicology, Leiden University Medical Center.
Technical details on production processes or product are described
in the relevant IMPD. The p53 peptides are 9 30-mers, and 1 25-mer
overlapping each other by 15 residues (see table 3). This set of 10
peptides is expected to contain the possible CTL epitopes for all
class I alleles as well as the possible T-helper epitopes for all
class II alleles.
[0137] MONTANIDE.TM.-ISA-51 is used as an adjuvant: MONTANIDE.TM.
ISA 51 or purified incomplete Freund's adjuvant is composed of
10.+-.2% (w/w) mannide oleate (MONTANIDE.TM. 80) in 90.+-.2% (w/w)
Drakeol 6VR, a pharmaceutical-grade mineral oil. MONTANIDE.TM. ISA
51 is marketed as a sterile, pyrogen-free adjuvant for human use by
Seppic (Paris, France). Long term (35 years) monitoring of 18000
patients--that received incomplete Freund's adjuvanted vaccine--and
22000 controls, did not show a significant difference in death rate
due to cancer: 2.18% and 2.43% for vaccines with and without
adjuvant, respectively.
2.6. Dosage and Treatment Overview
[0138] A phase I vaccination study at first in 5 patients with
rising CA125 levels is performed. The dose of peptides consists of
300 .mu.g/peptide. This dose is chosen based on prior clinical
trials of peptide vaccination (Rosenberg S A et al, Hersey P et al)
and results from the phase I/II HPV E6/E7 long peptide trial
performed at LUMC (unpublished data). Vaccination is carried out 4
times with a 3 weeks interval with clinical and immunological
evaluation in between. The vaccine is injected deep subcutaneously,
at four different sites, in a dose of 300 .mu.g per peptide in
DMSO/20 mM phosphate buffer pH 7.5/MONTANIDE.TM. ISA-51 adjuvant.
The first injection is in the right upper arm, the second in the
left upper arm, the third in the left upper leg and the fourth and
last in the right upper leg. Prior to vaccination, mononuclear
blood cells (PBMC; stored in 10% DMSO in liquid nitrogen) and serum
is isolated for evaluation of the baseline immune status towards
p53 prior to vaccination. The effect of vaccination on p53 immunity
is tested in PBMC and serum samples taken 2 weeks after the second-
and final (booster) vaccination. T cell responses against the
individual peptides in the vaccine are measured. In addition, a
skin biopsy is taken from the fourth vaccination site, 2 weeks
after vaccination, to isolate infiltrating T-cells. These T-cells
are tested with respect to their specificity and polarization.
[0139] If no grade 3 or 4 toxicity occurs in any of the 5 patients
entered in the phase I study the phase II vaccination study is
started. If one patient experiences unexpected grade 3 or 4
toxicity, the number of patients in the phase I will be expanded to
10. It should be noted that in previous peptide vaccination studies
no or minor toxicity occurred. Due to the relative good general
condition normally encountered in these patients, this patient
group is ideal to test the immunogenicity of the vaccine in a
classical phase II trial. At first 14 patients are entered in this
phase II study. P53 specific T cell responses before and after
vaccination are compared. If no responders (response arbitrarily
defined as >30% increase p53 specific T cell responses) are
found among the first 19 patients the study is discontinued because
of apparent lack of immunogenicity of the vaccine. In case of
responses the total number of patients in the study depends on the
number of observed responses.
[0140] Vaccination in the phase I/II study is started 3 months
after the last first line chemotherapy course and continued for a
total of 4 vaccinations spaced by 3 weeks. If the phase II
vaccination shows no results in terms of positive T cell responses,
this approach will be discontinued.
2.7. Supportive Care in Case of Toxicity
[0141] In case of skin toxicity, systemic antihistamines or topical
steroids are allowed. Patients are not allowed to receive growth
factors for myelosuppression. Analgetics are allowed.
2.8. Concomitant Therapy
[0142] No other chemotherapy, immunotherapy, hormonal agents
(excluding topical steroids for skin rashes), radiation therapy,
experimental drugs, radiotherapy, and/or surgery are allowed while
patients are on study. Any disease progression necessitating other
forms of anti-tumor therapy is a cause for patient's withdrawal
from the study. Systemic corticosteroids are not permitted during
the study. Patients should receive full supportive care.
2.9 Clinical Evaluation, Laboratory Tests and Follow-Up
2.9.1 Before Treatment Start
[0143] Less than 14 days before registration within the trial the
following parameters are required to recorded: [0144] relevant
medical history including date of first diagnosis, histological
type, concurrent diseases, and any concurrent use of medication.
[0145] Physical examination including WHO performance, height,
weight, vital signs, base-line clinical symptoms. [0146]
Electrocardiogram (ECG) and urine analysis to exclude an
asymptomatic cystitis [0147] Hematology: hemoglobin, thrombocytes,
WBC. [0148] Biochemistry: serum creatinine, INR, bilirubin, ASAT,
ALAT, LDH, INR,
[0149] When the patient is registered within the trial, study
treatment starts within 7 days after inclusion.
2.9.2. During Treatment
[0150] Less than 28 days before start of vaccination (300 ml),
during vaccination (3.times.50 ml) and approximately 14 days after
the last vaccination (300 ml) PBMC are collected to measure p53
specific T cell responses.
[0151] Two weeks after the last vaccination a punch biopsy is taken
from the fourth vaccination site. Immediately before administration
of each vaccination, 10 to 14 days after the last administration of
the vaccine, the following examinations are carried out: (1)
physical examination including neurological evaluation, vital
signs, O.sub.2-saturation and ECG before and three-four hours after
each vaccination. (2) Evaluation of all adverse events (worst grade
of events occurring during this vaccination should be recorded), as
well as weight and WHO performance, (3) Hematology and biochemistry
including hemoglobin, thrombocytes, WBC, serum creatinine, INR,
bilirubin, ASAT, ALAT, LDH and INR. (4) Any concomitant
medication.
2.9.3. After the End of Treatment (Follow-Up)
[0152] If the patient has not progressed after vaccination, the
extent of disease is recorded every 3 months following the same
procedures as during treatment. In case of progression, the
patients are followed for survival every 3 months. Initiation of
any form of other anti-tumor treatment is recorded. Table 4
summarizes the whole vaccination and analysis process.
[0153] Toxicity evaluation is recorded during vaccination.
Hematology and biochemistry including hemoglobin, thrombocytes,
WBC, serum creatinine, INR, bilirubin, ASAT, ALAT, LDH and INR.
2.10. Immuno-Monitoring
[0154] At five different time points during the study PBMC are
collected: before vaccination (300 ml), and after each vaccination
(3.times.50 ml) and after the last vaccination (300 ml). PBMC and
serum is isolated.
[0155] Evaluation of the p53-specific T cell responses in
vaccinated subjects of the phase I/II study is carried out as
follows:
[0156] Positive CD4+ T helper cell responses after vaccination are
defined by:
a) Significantly increased (a vaccination induced reaction is
considered positive when the proliferation index of
post-vaccination samples is at least twice as high as the
proliferation index of pre-vaccination samples and the
proliferation index of post-vaccination samples should at least be
2 and/or b) Significantly increased IFN.gamma. production of PBMC,
that have been stimulated twice with peptide in vitro, against p53
peptides as measured by ELISA following 1 day culture, and/or c) A
significantly increased percentage of CD4+ PBMC that produce
IFN.gamma. upon a 6 hour in vitro stimulation with p53 peptides in
the presence of brefeldin A, utilizing tricolor intracellular FACS
staining with antibodies against CD4, the early activation marker
CD69 and IFN.gamma., and/or d) A significantly increased number of
antigen-specific T cells that produce IFN.gamma., IL-4 or IL-10 as
measured in the ELISPOT assay in which a frequency of .gtoreq.
1/10,000 PBMC is considered positive and a twofold increase in the
number of spots between pre-vaccination and post-vaccination is
chosen as a positive response to the vaccine.
2.11. Statistical Consideration
2.11.2. Analysis
[0157] Patients not satisfying the inclusion criteria are
ineligible. Patients not evaluated because of withdrawal or for
other reasons (e.g. patients refusal, lost to follow-up, protocol
violation) are clearly indicated. A total of 19 full evaluable
patients need to be entered in the study with a maximum of 20%
drop-out, this number might increase to 23 patients).
[0158] The p53 specific immune response of the vaccine measured
before and after the vaccinations will be compared by Sign test
(2-sided, 5% significance level). Response rates and rates of grade
3-4 toxicity encountered during vaccination will be estimated and
the 95% exact confidence limits calculated. Time to progression
will be computed by Kaplan-Meier curves and will be compared to a
historical control group by the logrank test.
2.12. Translational Research
[0159] The following analyses are performed:
[0160] The p53-specific T-cell response is analyzed using freshly
isolated PBMC for p53-specific proliferation. The cytokines
(IFN.gamma., TNF.alpha., IL-10, IL-5, IL-4, IL-2) that are
specifically produced upon this antigenic stimulation are tested by
the cytokine bead array (according to the protocol of the
manufacturer Becton-Dickinson). PBMC freshly isolated before,
during and after the fourth vaccination, are tested. Responses are
classified as Th1 (IFN.gamma., TNF.alpha.), Th2 (IL-4, IL-5,
IL-10), Th0 (IFN.gamma., IL-4, IL-5, IL-10) or impaired with
respect to cytokine production (no production). The presence of
high numbers of p53-specific T-cells in the peripheral blood may
not in all cases lead to the migration of these T-cells to the
vaccine injection site, despite the presence of peptide antigen.
The success or failure of T-cells to migrate into the skin may be a
reflection of what goes on at the site of disease. Therefore, 2
weeks after the last vaccination a biopsy is taken from the fourth
vaccination site. T-cells that have infiltrated the vaccination
site are isolated from the biopsy. The specificity, polarization
and type of the infiltrating T-cells is tested by cytokine bead
array (protocol of manufacturer) and by FACS.
2.13. Results
2.13.1. Proliferation Assay
[0161] FIG. 4 depicts the proliferation assay carried out as
indicated. It demonstrates that the vaccine containing peptides
covering p53.sub.70-248 is able to induce an immune response in
most patients. Clearly, peptides derived from the region of
p53.sub.102-248 most efficiently induce a T cell response after
vaccination as measured by proliferation after in vitro stimulation
with pools of vaccine peptides (FIG. 4b-d). The first pool of
vaccine peptides, covering p53.sub.70-101, hardly induces T cell
proliferation after in vitro stimulation (FIG. 4a). T cells
isolated from vaccinated patients were also stimulated with
peptides that were not present in the vaccine. This concerned
peptides covering the N-terminal and the C-terminal region of the
p53 protein. As shown by FIG. 4f, the N-terminal region is able to
induce a T cell response in some ovarian cancer patients. This
could indicate that the vaccine is very efficient at inducing
killer T cells, eventually resulting in epitope spreading. On the
other hand, the C-terminal region of p53 in these patients does not
induce a measurable T cell response.
2.13.2. IFN.gamma. ELISPOT
[0162] The vaccine was considered to induce a positive response
if:
the mean number of spots-(mean number of spots in medium+2SD)>10
spots [0163] post-vaccination SI>=twice the prevaccination SI.
The results are depicted in FIG. 6. This assay shows similar
results compared to the proliferation assay: The vaccine peptides
derived from the p53.sub.102-248 region have induced T cells
specific for epitopes contained in that area (FIG. 6b-d), whereas
the peptides derived from the p53.sub.70-101 region do not (FIG.
6a). Moreover, this assay further confirms that the principal of
epitope spreading might take place in some patients (FIG. 6f).
Example 3
Vaccination Study with p53 Peptides in Colorectal Cancer
Patients-Immunological Results in 2 Vaccinated Patients
[0164] The principle of the study is similar as in example 2 (see
also FIG. 3). The differences being that in this study, patients
only received two vaccinations (instead of four).
[0165] The p53-specific proliferative capacity (FIGS. 5A and C) and
cytokine production (FIGS. 5B and D) of 2 male patients with
colorectal cancer is shown before and after vaccination with
p53-long peptide vaccine. Patients were vaccinated twice with
overlapping p53 peptides covering the amino acid sequence 70-248
(indicated by the pools of 2 peptide: V70-115, V102-155, V142-203,
V190-248). Patients PBMC were also tested against the N-terminal
region of p53 (aa 1-78) and C-terminal region of p53 (aa 241-393).
Note that in both cases, vaccination results in the activation of
T-cell reactivity against the C-terminal region (outside the
vaccine) too, indicative for epitope-spreading of the immune
response following vaccination. P53-specific proliferation is
associated with the production of IFN.gamma., IL-10 and IL-5, as
measured by Cytokine bead Array (CBA).
Example 4
Vaccination Study with p53 Peptides in Colorectal Cancer
Patients-Immunological Responses in 9 Vaccinated Patients
[0166] Table 6 shows an overview of p53 specific T cell responses
in colorectal cancer patients that have been vaccinated twice with
the p53 SLP vaccine. In conclusion, all patients exhibited a p53
specific T cell response. The peptides that seem to be the most
immunogenic are located in the most N-terminal portion of the p53
protein as present in the vaccine (aa 190-248): 8 out of 9 patients
responded against these peptides. However, also peptides derived
from aa 102-155 and 142-203 are able to induce p53 specific
responses (6 out of 9 patients respond to one or more peptides
derived from these regions). Clearly, the C-terminal portion of p53
seems to be less immunogenic. Importantly, peptides derived from
the N-terminal portion of p53 which was not present in the p53 SLP
vaccine still were able to induce a T cell response in vitro. This
phenomenon is known as antigen spreading and occurs through
stimulation of T-cells by DC that have taken up tumor-derived p53
released by dying tumor cells. As this is observed after
vaccination only it is indicative for an effective vaccine-induced
anti-tumor response.
[0167] To determine whether the p53 SLP vaccine is able to induce
p53 specific T cells that can migrate to areas where the p53
antigen is presented, vaccine sites were isolated and
skin-infiltrating T cells were expanded. FIG. 8 shows the results
from 1 patient with colorectal cancer. T cells have migrated to the
vaccine site and are mostly specific for vaccine peptides 5-10 (aa
142-248). Furthermore, also peptides encoding the N-terminal
portion of the p53 protein (aa 241-393) can be recognized by
infiltrating T cells, while this part of the protein is not present
in the p53-SLP vaccine. Again evidence for antigen spreading after
effective vaccination. Importantly, the results also show that
vaccine induced T cells that have migrated to the area of antigen
presentation can also recognize cells that have processed whole p53
protein. This indicates that the natural processing pathway
processes p53 protein into epitopes that can be recognized by the
vaccine induced T cells. This implies that tumor cells that also
process whole p53 protein in a natural manner can also be
recognized and lysed by these T cells.
[0168] To determine whether the vaccinated colon carcinoma patients
were also able to induce a memory T cell response, PBMC isolated
before and after 2 vaccinations and after 6 or 9 months were tested
for the presence of p53 specific T cells. In FIG. 9, shows that the
p53-specific T-cell responses which were not present before
vaccination and induced after 2 vaccinations were still present in
the circulation of the tested patients at 6-9 months after
vaccination which is indicative for p53-specific memory T-cell
responses. Moreover, both patients display a response to amino
acids 241-393 of the p53-protein after 2 vaccinations and even at 6
months of follow up (#1), indicating that T cells induced as a
result of epitope spreading were also still present.
Example 5
Vaccination Study with p53 Peptides in Ovarian Cancer
Patients--Immunological Results in all (18) Vaccinated Patients
[0169] Table 7 summarizes immunological and clinical responses of
all ovarian cancer patients treated with the p53-SLP vaccine.
Immunological Responses
[0170] In 100% of the ovarian cancer patients receiving all four
immunizations (N=18), vaccine-induced p53-specific responses
against the vaccine peptides were present at two or more time
points (I-IV) after immunization as measured by IFN-.gamma. ELISPOT
(Table 8). Vaccine-induced p53-specific responses were directed
against at least two of the vaccine peptide pools in all ovarian
cancer patients. After four immunizations, circulating IFN-.gamma.
secreting p53-specific T-cells could be detected in 61.1% (11/18)
of patients.
[0171] Vaccine-induced p53-specific proliferative responses against
the vaccine peptides were observed in 82.4% (14/17) of ovarian
cancer patients (Table 9). As depicted in FIG. 12, proliferative
responses against the vaccine peptides could still be demonstrated
9-12 months after the last immunization, even though patients had
since been treated with chemotherapy. Furthermore, also in the
ovarian cancer patients, p53-specific responses against the first
(aa 1-78) and the last (aa 241-393) portion of the p53-protein not
covered by the vaccine peptides, were observed in 11.8% (2/17) and
23.5% (4/17) of the patients respectively after four immunizations
(Table 9). FIG. 10 illustrates the proliferative capacity of PBMC
in response to ex-vivo stimulation with single vaccine peptides as
analyzed in 7 patients with ovarian cancer after four
immunizations. Responses were observed to all peptides, except for
vaccine peptide 1 (aa70-99). To analyze the capacity of
p53-specific T-cells to migrate to sites where p53 antigen is
presented, a proliferation assay was performed with lymphocytes
cultured from skin biopsies taken at the fourth injection site
(n=17). Insufficient numbers of lymphocytes could be cultured from
the skin biopsies of two patients (P15 and P20). P53-specific
responses were observed in lymphocytes cultured from skin biopsies
in 52.9% (9/17) of patients. Most responses were observed against
vaccine peptide p8-p10 (aa 190-248) (FIG. 11). Importantly, all
patients with p53-specific responses in lymphocytes cultured from
skin biopsies also showed vaccine-induced p53-specific responses in
PBMC as analyzed by proliferation assay (Table 7), although
responses were not always directed against the same epitopes.
[0172] P53-autoantibodies were present in 40% (8/20) of the
patients before immunization. After one or more immunizations,
p53-autoantibodies were present in 45% (9/20) of patients. A
vaccine-induced increase in p53-autoantibody titer was detected in
15% (3/20) of the patients (Table 7).
Clinical Responses
[0173] Because of rapidly progressive disease, two patients
received only two immunizations (P04, P12). Clinical responses of
the remaining 18 ovarian cancer patients were evaluated based on
CA-125 levels and evaluation of CT scans according to the RECIST
and GCIG criteria (Table 10). One patient had a partial response as
measured by CA-125 (P05), and six patients had stable CA-125 levels
(P02, P03, P06, P09, P17, P21, P23) (FIG. 13). Two of these
patients (11.1%) also had stable disease on CT-scan (P17, P23). In
both patients, vaccine-induced p53-specific responses were detected
in PBMC. P23 also showed p53-specific responses in lymphocytes
cultured from the skin biopsy. The other patients (16/18; 88.9%)
were classified as having progressive disease. All patients with
progressive disease had developed new lesions since their last
CT-scan.
Example 6
Demonstration of the Advantage of Intradermal Administration of a
Vaccine Peptide
[0174] In this example, peptides were derived from a HPV protein
are used in an intradermal vaccine. The advantages of an
intradermal vaccine as demonstrated herein are generalisable to any
other peptides, among other derived from a protein that is
ubiquitously expressed self-antigen and known to be associated with
cancer, such as p53.
Materials and Methods
Study Design
[0175] A cross-sectional pilot study to analyse HPV16 E2-, E6-, and
E7-specific T-cell responses as measured by intradermal injection
of pools of clinical grade HPV16 peptides in the upper arm was
performed in patients with HPV-related disorders of the cervix and
in healthy individuals. Since a delayed type hypersensitivity
reaction represents a memory T-cell response, there was no
prerequisite for HPV16-positivity at the time of analysis.
Subjects
[0176] A group of nineteen healthy individuals (HD) participated in
this study after providing informed consent. The group of healthy
individuals displayed a median age of 31 years old (range, 20-51
years) and was comprised of 80% women and 20% males. Peripheral
blood mononuclear cells (PBMCs) were obtained from all subjects
immediately before administration of the skin test. The late
appearance of positive skin tests in healthy individuals resulted
in the isolation of a second blood sample from 11 of 19 healthy
volunteers. The study design was approved by the Medical Ethical
Committee of the Leiden University Medical Centre.
DTH Skin Test
[0177] Skin tests, based on Delayed Type Hypersensitivity reactions
(DTH), can be used as a sensitive and simple method for in vivo
measurement of HPV-specific cellular immune responses (Hopfl R et
al, 2000; Hopfl R et al, 1991). The skin test preparations
consisted of 8 pools of long clinical-grade synthetic peptides
spanning the whole HPV 16 E6 and E7 protein and the most
immunogenic regions of HPV 16 E2 protein (de Jong A et al, 2004).
These clinical grade peptides were produced in the interdivisional
GMP-Facility of the LUMC. Each pool of the skin test consisted of 2
or 3 synthetic peptides, indicated by the first and last amino acid
of the region in the protein covered by the peptides. Pool 1:
E2.sub.31-60, E2.sub.46-75, Pool 2: E2.sub.301-330, E2.sub.316-345,
Pool 3: E6.sub.1-31, E6.sub.19-50, Pool 4: E6.sub.41-65,
E6.sub.55-80, E6.sub.71-95, Pool 5: E6.sub.85-109, E6.sub.91-122,
Pool 6: E6.sub.109-140, E6.sub.127-158, Pool 7: E7.sub.1-35,
E7.sub.22-56, Pool 8: E7.sub.43-77, E7.sub.64-98. The sequence of
E2, E6, and E7 of HPV16 is respectively represented by SEQ ID
NO:22, 23, and 24. Per peptide pool 0.05 ml of 0.2 mg/ml peptides
in 16% DMSO in 20 mM isotonic phosphate buffer (10 .mu.g/peptide)
was injected intracutaneously. The pools of peptides and a negative
control (dissolvent only) were injected separately at individual
skin test sites of the upper arm Skin test sites were inspected at
least three times, at 72 hours and 7 days after injection (Hopfl R
et al 2000, 2001) of the peptides and at 3 weeks following the
first report of a very late skin reaction in one of the first
healthy subjects. Reactions were considered positive when papules
greater than 2 mm in diameter arose no less than 2 days after
injection. From positive skin reaction sites punch biopsies (4 mm)
were obtained, cut in small pieces and cultured in IMDM containing
10% human AB serum, 10% TCGF and 5 ng/ml IL7 and IL15 to allow the
emigration of lymphocytes out of the skin tissue. After 2 to 4
weeks of culture the expanded T cells were harvested and tested for
their HPV-specific reactivity.
Antigen for In Vitro Immune Assays
[0178] A set of peptides, similar to the peptides used in the skin
test, were used for T-cell stimulation assays and
IFN.gamma.-ELISPOT assays. The four HPV 16 E2 peptides consisted of
30-mer peptides overlapping 15 residues, HPV 16 E6 consisted of
32-mers and HPV 16 E7 of 35-mers, both overlapping 14 residues. The
peptides were synthesized and dissolved as previously described
(van der Burg S H et al, 1999). Notably, in the IFN.gamma. ELISPOT
assays peptide pool 4 and 5 slightly differed from the peptide
pools used in the skin test, pool 4 contained peptides
E6.sub.37-68, E6.sub.55-86, E6.sub.73-104 and pool 5 comprised
peptides E6.sub.73-104, E6.sub.91-122.
[0179] Memory response mix (MRM 50.times.), consisting of a mixture
of tetanus toxoid (0.75 Limus flocculentius/ml; National Institute
of Public Health and Environment, Bilthoven, The Netherlands),
Mycobacterium tuberculosis sonicate (5 .mu.g/ml; generously donated
by Dr. P. Klatser, Royal Tropical Institute, Amsterdam, The
Netherlands), and Candida albicans (0.15 mg/ml, HAL Allergenen
Lab., Haarlem, The Netherlands) was used as a positive control.
Recombinant HPV 16 E2, E6 and E7 proteins were produced in
recombinant Escherichia coli as described previously (van der Burg
S H et al, 2001).
Analysis of Antigen-Specific Th Cells by IFN.gamma. ELISPOT
[0180] The presence of HPV 16-specific Th Cells was analyzed by
ELISPOT as described previously (van der Burg S H et al, 2001)
Briefly, fresh PBMCs were seeded at a density of 2.times.10.sup.6
cells/well of a 24-well plate (Costar, Cambridge, Mass.) in 1 ml of
IMDM (Bio Whittaker, Verviers, Belgium) enriched with 10% human AB
serum, in the presence or absence of the indicated HPV 16 E2, E6
and E7 peptide pools. Peptides were used at a concentration of 5
m/ml/peptide. After 4 days of incubation at 37.degree. C., PBMCs
were harvested, washed, and seeded in four replicate wells at a
density of 10.sup.5 cells per well in 100 .mu.l IMDM enriched with
10% FCS in a Multiscreen 96-well plate (Millipore, Etten-Leur, The
Netherlands) coated with an IFN.gamma. catching antibody (Mabtech
AB, Nacha, Sweden). Further antibody incubations and development of
the ELISPOT was performed according to the manufacturer's
instructions (Mabtech). Spots were counted with a fully automated
computer-assisted-video-imaging analysis system (Bio Sys). Specific
spots were calculated by subtracting the mean number of
spots+2.times.SD of the medium control from the mean number of
spots in experimental wells (van der Burg S H et al, 2001).
T Cell Proliferation Assay
[0181] T-cell cultures of the skin biopsies were tested for
recognition of the specific peptides and protein in a 3-day
proliferation assay (van der Burg S H et al, 2001). Briefly,
autologous monocytes were isolated from PBMCs by adherence to a
flat-bottom 96-well plate during 2 h in X-vivo 15 medium (Cambrex)
at 37.degree. C. The monocytes were used as APCs, loaded overnight
with 10 .mu.g/ml peptide and 20 .mu.g/ml protein Skin
test-infiltrating-lymfocytes were seeded at a density of
2-5.times.10.sup.4 cells/well in IMDM supplemented with 10% AB
serum. Medium alone was taken along as a negative control,
phytohemagglutinine (0.5 .mu.g/ml) served as a positive control.
Proliferation was measured by [.sup.3H]thymidine (5 .mu.Ci/mmol)
incorporation. A proliferative response was defined specific as the
stimulation index (SI).gtoreq.3. Supernatants of the proliferation
assays were harvested 48 hours after incubation for the analysis of
antigen-specific cytokine production.
Analysis of Cytokines Associated with HPV16-Specific Proliferative
Responses
[0182] The simultaneous detection of six different Th1 and Th2
cytokines: IFN.gamma., tumor necrosis factor .alpha., interleukin 2
(IL2), IL4, IL5 and IL10 was performed using the cytometric bead
array (Becton Dickinson) according to the manufacturer's
instructions. Cut-off values were based on the standard curves of
the different cytokines (100 pg/ml IFN.gamma. and 20 pg/ml for the
remaining cytokines). Antigen-specific cytokine production was
defined as a cytokine concentration above cutoff level and
>2.times. the concentration of the medium control (de Jong A et
al, 2004).
Intracellular Cytokine Staining (ICS)
[0183] The specificity and character of the T cell cultures derived
from positive skin reaction sites was tested by ICS as reported
previously (de Jong A et al, 2005). Briefly, skin test infiltrating
lymphocytes were harvested, washed and suspended in IMDM+10% AB
serum and 2-5.times.10.sup.4 cells were added to autologous
monocytes that were pulsed overnight with 50 .mu.l peptide (10
.mu.g/ml) or protein (20 .mu.g/ml) in X vivo medium. Medium alone
was taken along as a negative control, phytohemagglutinine (0.5
.mu.g/ml) served as a positive control. Samples were simultaneously
stained with FITC-labelled mouse-antihuman IFN.gamma. (0.5 g/ml, BD
PharMingen), PE-labelled mouse-antihuman IL5 (0.2 mg/ml, BD
PharMingen), APC-labelled anti-CD4 (BD Bioscience) and
PerCP-labelled anti-CD8 (BD Bioscience). After incubation at
4.degree. C., the cells were washed, fixed with 1% paraformaldehyde
and analyzed by flow cytrometry (FACSscan, BD Biosciences)
Statistical Analysis
[0184] Fisher's Exact test (2-tailed) was used to analyze the
relationship between the detection of IFN.gamma.-producing
HPV-specific T-cells in PBMC, the presence of a skin test reaction
or the presence of HPV-specific T-cells in skin biopsies, as well
as differences between patients and healthy controls with respect
to the size or the number of the skin reactions within these
groups. Statistical analyzes were performed using Graphpad Instat
Software (version 3.0) and Graphpad Prism 4.
Results
[0185] Skin Reactions to Intracutaneous Injection with HPV 16 E2,
E6- and E7 Peptides
[0186] We studied skin reactions in healthy subjects after
intracutaneous injection with HPV 16 E2, -E6 and -E7 peptides.
Positive skin reactions appeared as flat reddish papules of 2 to 20
mm of diameter, arising within 2 to 25 days after injection. A
positive skin reaction was detected in 46 of the 152 skin tests in
the healthy volunteers. Over all, each peptide-pool in the skin
test could give rise to a positive skin reaction. Most frequently
reactions against E2.sub.31-75 (10 out of 19 subjects),
E6.sub.37-104 (9/16) and E7.sub.43-98 (7/19) were observed in the
control group. This reaction pattern resembles that of what we
previously observed in PBMC (de Jong A et al, 2002; Welters et al,
2003) (FIG. 14). These skin reactions corresponded with the
presence of a peptide specific T cell response as detected in the
PBMC of these individuals (data not shown).
Skin Reactions in Healthy Donors are Associated with Higher
Frequencies of HPV 16-Specific T-Cells in the Peripheral Blood.
[0187] In order to compare the results of the skin test with the
presence of circulating HPV 16-specific type 1 T cells, an
IFN.gamma. ELIspot assay was performed with PBMC's collected before
the intradermal peptide-challenge was given. In 5 out of 19 healthy
volunteers we were able to detect a HPV 16-specific immune response
by IFN.gamma.-ELIspot. The detection of .gtoreq.5 circulating
HPV16-specific T-cells per 100.000 PBMC in the pre-challenge blood
sample of healthy individuals was associated with an early
(.ltoreq.13 days) positive skin reaction to the same peptide
sequence (p=0.0003, two tailed Fisher's exact test; FIG. 15). No
HPV 16-specific circulating T-cells were detected in the
pre-challenge blood sample healthy donors to peptides that induced
a late positive skin reaction (14 to 25 days). This suggests that
the frequency of circulating antigen-specific cells determine the
delay time for skin reactions to appear.
[0188] In order to assess the frequency of HPV-specific T-cells at
the time that a late skin reaction appeared additional blood
samples from 11 healthy volunteers were collected. In these
individuals 39 out of 88 skin tests were positive. In 25 of the 39
positive skin reactions and in 10 of 49 negative skin reactions
.gtoreq.5 HPV 16-specific T-cells were detected per 100.000 PBMC.
At this point a significant correlation was found between the
detection of circulating HPV-specific IFN.gamma.-producing T-cells
in the post-challenged blood sample and the presence of a skin
reaction (p<0.0001, Fisher's exact test; FIG. 16). This shows
that the frequency of HPV16-specific T cells in the blood of
healthy volunteers is significantly higher following an intradermal
challenge with HPV 16 peptide and indicates that intracutaneous
injection of peptide antigens enhances the number of HPV
16-specific T cells in the blood of healthy volunteers.
Biopsies of Positive Skin Reaction Sites Consist of Both
Th1/Th2-CD4+ and CD8+ HPV16-Specific T Cells.
[0189] Approximately 25% of the positive skin reactions of healthy
volunteers were not associated with the detection of HPV
16-specific IFN.gamma.-producing T-cells in the blood, suggesting
that other, non IFN.gamma.-producing types of T-cells may
infiltrate the skin after intradermal injection of HPV16
peptides.
[0190] In order to characterize the cells in a positive skin
reaction site punch biopsies were taken. In total, 8 biopsies were
taken from different positive skin reaction sites of 7 healthy
controls and cultured with a cocktail of cytokines that allowed the
outgrowth of T-cells in vitro without antigenic stimulans. In 7 of
8 cases, T-cells ex-filtrated the tissue and expanded within 3-4
weeks. The expanded T-cells were tested for their specificity in a
short term proliferation assay. FIG. 17 shows examples of T-cell
cultures that specifically proliferated upon stimulation with
autologous monocytes pulsed with the pool of peptides, also
injected in this site during the skin test (HD2, HD10, HD15) as
well as to monocytes pulsed with HPV16 E6 protein (FIG. 17AB). This
indicates that these T-cells were capable of recognizing their
cognate HLA-peptide complexes after the antigen was naturally
processed and presented. Analysis of the supernatants of these
proliferative T-cell cultures revealed a mixed Th1/Th2 cytokine
profile in that the HPV 16-specific T-cells produced IFN.gamma.,
IL-4 and IL-5 (FIG. 17C).
[0191] In each case that HPV-specific T-cells were detected in the
biopsy culture (4 out of 8) this coincided with the detection of
circulating HPV 16-specific IFN.gamma.-producing T-cells in the
post-challenge blood sample by ELIspot (compare FIGS. 17A and B).
In 3 of the other 4 positive skin reaction biopsies (HD2, HD17,
HD18) the T-cells did not respond to HPV16 peptides (FIG. 17; HD17)
and in one case no T-cells ex-filtrated the tissue at all (HD13).
In these 4 cases we were not able to detect circulating HPV
16-specific IFN.gamma.-producing T-cells in the post-challenge
blood sample.
[0192] Co-staining of the biopsy-T cells by CD4 and CD8 cell
surface markers showed that not only HPV 16-specific CD4.sup.+ but
also HPV 16-specific CD8.sup.+ T cells infiltrated the skin site
upon intradermal challenge with HPV16 peptide (FIG. 18). Overall,
in 3 out of 4 biopsies infiltrated by HPV16-specific T-cells, we
were able to detect HPV16-specific CD8.sup.+ T cells. The CD8.sup.+
T cells isolated from the biopsy (pool 6) of HD2 responded to both
overlapping peptides of the injected skin test: HPV16
E6.sub.109-140 and E6.sub.127-158 (data not shown), while the
CD8.sup.+ T cells of both subjects HD15 and HD16 responded to HPV16
E6.sub.37-68 (see example for HD15, FIG. 18). Taken together, the
population of immune cells migrating into the skin upon an
intradermal challenge with HPV16 peptides comprises HPV 16-specific
CD4.sup.+ Th1-, Th2- and CD8.sup.+ cytotoxic T cells. This
infiltration is paralleled by the appearance of circulating
HPV16-specific IFN.gamma.-producing T-cells in the blood.
Discussion
[0193] Skin tests are commonly used as a simple assay for in vivo
measurement of cell mediated immunity. We have validated the use of
the skin test assay for the measurement of HPV 16 specific cellular
immune response against the early antigens E2, E6 and E7 in vivo by
comparing the results with that of parallel measurements of T cell
reactivity by in vitro assays.
[0194] In the group of healthy volunteers early skin reactions
appeared between 4 to 12 days after intradermal antigen challenge.
In these individuals, known to display HPV16 specific type 1 T cell
responses in vitro (de Jong A et al, 2002; Welters et al, 2003),
the appearance of an early skin reaction (within 13 days) was
significantly associated with the detection of IFN.gamma.-producing
HPV16-specific T cells at a frequency of at least 1 per 20.000 PBMC
(FIG. 15, p<0.001). The same cut-off criteria for a positive
reaction in the IFN.gamma. ELIspot assay are recommended by
Jeffries et al (Jeffries D J et al, 2006), who used mathematical
tools to define the appropriate cut-off of the ELISPOT in relation
to Mantoux-tests. The low number of circulating memory T cells
(FIG. 15) may explain why the skin reactions appear somewhat
delayed compared to classical DTH tests. The T cells need to be
boosted or reactivated and start to divide before enough cells are
produced to cause a local inflammatory reaction: the positive skin
test. Indeed, at the time a positive skin reaction appears, a
higher frequency of HPV 16-specific Th1 responses can be detected
in the peripheral blood (FIG. 16).
[0195] Historically it has been postulated that the Th1 cell induce
DTH responses, however, several studies have now shown that also
Th2 cells infiltrating the skin test sites (Wang S et al, 1999;
Woodfolk J A et al, 2001). Similarly, this study shows that the
positive skin test sites of healthy volunteers contain both Th1 and
Th2 type HPV16-specific T cells (FIGS. 17 and 18).
[0196] In addition, positive skin reactions may also be the result
of the influx of non-specific T cells as became evident from two in
depth studies of positive skin test sites used to assay the
specific immune response following vaccination of patients with
renal cell cancer or melanoma (Bleumer I et al, 2007). Also this
study showed that a number of positive skin test sites from healthy
subjects were infiltrated with T-cells that did not respond to the
injected HPV16 antigens. So far, the reason for a-specific positive
skin reactions remains unclear. Unexpectedly, we observed the
majority of skin reactions in healthy individuals to appear 2 to 3
weeks after intradermal injection of the antigen. While, these late
positive skin reactions were not correlated with detection of
circulating HPV-specific CD4.sup.+ memory T cells in pre-challenge
blood (FIG. 15) the immunological constitution of these skin test
sites are similar to that of classic DTH tests (Platt J L et al,
1983; Poulter L W et al, 1982) and comprised of HPV16-specific
CD4.sup.+ Th1- and Th2-cells as well as HPV16-specific CD8.sup.+ T
cells (FIGS. 17 and 18). We hypothesize that these reactions might
be the result of T cell priming. This has also been noted in 29% of
patients whom underwent a 2-step tuberculin skin testing protocol
and whom were only positive at the second test round (Akcay A et
al, 2003). In general, vaccine-induced T cell responses peak at 10
to 14 days after vaccination and not at three weeks. However, one
should bear in mind that in such protocol a higher antigen dose as
well as strong adjuvants are injected. It is therefore reasonable
to assume that the T cell responses induced by intradermal
challenge develop more slowly and peak at a later period. Since the
intradermal peptide challenge in healthy volunteers results in the
induction of both HPV16-specific CD4.sup.+ and CD8.sup.+ T cells
it, therefore, should be considered as a single, low dose
vaccination.
[0197] The main objective of this pilot study was to validate the
use of the HPV16 specific skin test to detect type 1 immune
responses in vivo. In healthy volunteers, a positive skin reaction
within 13 days is indeed correlated with the presence of
circulating IFN.gamma.-producing memory T cells as detected by the
IFN.gamma. ELIspot in vitro. Importantly, we also observed
discrepancies between the outcomes obtained by skin test and
ELIspot. In a number of cases HPV16-specific circulating
IFN.gamma.-producing T cells were detected in the post-challenge
blood samples but without a concomitant skin reaction and vice
versa (FIG. 16), and this may be considered as a false negative or
false positive result. In order to fully understand the impact of
this on the interpretation of the detection of type 1 immunity
against HPV, we have begun a field trial in a large group of HPV
positive patients and healthy volunteers in Indonesia.
TABLE-US-00001 TABLE 1 HLA binding and C-terminal cleavage by
proteasomes of protential CTL epitopes. Table 1 discloses SEQ ID
NOS 25-77, respectively, in order of appearance. Proteasomal
Cleavage Peptide Binding.sup.1 of C-terminus.sup.2 Affinity
Stability IP (RMA) IP (JY) HH (HeLa) HLA-A*0101 117-126 GTAKSVTCTY
intermediate + - - - 196-205 RVEGNLRVEY intermediate + - - -
205-214 YLDDRNTFRH intermediate + 226-234 GSDCTTIHY high + + +
229-236 CTTIHYNY intermediate - - - HLA-A*0201.sup.3 24-32
KLLPENNVL intermediate NT + + + 65-73 RMPEAAPPV high 6 h - - -
113-122 FLHSGTAKSV low 6 h + + + 149-157 STPPPGTRV low <2 h + +
+ 187-197 GLAPPQHLIRV high 6 h + + + 264-272 LLGRNSFEV intermediate
6 h + + + 322-330 PLDGEYFTL intermediate ? + + + HLA-A*0301 101-110
KTYQGSYGFR high +/- - - - 110-120 RLGFLHSGTAK high + + + - 111-120
LGFLHSGTAK high +/- + + - 112-120 GFLHSGTAK intermediate - + + -
113-120 FLHSGTAK intermediate +/- + + - 117-126 GTAKSVTCTY
intermediate - +/- + +/- 122-132 VTCTYSPALNK intermediate + - - -
124-132 CTYSPALNK high + - - - 129-139 ALNKMFCQLAK high + + + +
132-139 KMFCQLAK high + + + + 154-163 GTRVRAMAIY intermediate - - -
- 154-164 GTRVRAMAIYK high + - - - 156-163 RVRAMAIY intermediate -
- - - 156-164 RVRAMAIYK high + - - - 172-181 VVRRCPHHER
intermediate + - - - 360-370 GGSRAHSSHLK intermediate - - - -
361-370 GSRAHSSHLK high + 363-370 RAHSSHLK high + 363-372
RAHSSHLKSK intermediate +/- 363-373 RAHSSHLKSKK high + 373-381
KGQSTSRHK intermediate +/- + + + 376-386 STSRHKKLMFK high +/- - - -
HLA-A*1101 101-110 KTYQGSYGFR high +/- - - - 111-120 LGFLHSGTAK
high + + + - 112-120 GFLHSGTAK intermediate - + + - 124-132
CTYSPALNK high + - - - 132-139 KMFCQLAK high + + + + 156-164
RVRAMAIYK high + - - - 311-319 NTSSSPQPK high +/- - - - 311-320
NTSSSPQPKK high +/- - - - 312-319 TSSSPQPK high +/- - - - 283-291
RTEEENLRK intermediate +/- - - - 363-370 RAHSSHLK intermediate +/-
- - - 374-382 GQSTSRHKK intermediate +/- - - - HLA-A*2401 18-26
TFSDLWKLL high + + + + 102-111 TYQGSYGFRL high + + + + 106-113
SYGFRLGF high + + + + 106-114 SYGFRLGFL high +/- + + + 125-134
TYSPALNKMF high + - - - 204-212 EYLDDRNTF high + 340-348 MFRELNEAL
high +/- - - - .sup.1Affinity of peptide binding is categorized as
follows: good IC.sub.50 < 5 .mu.M, intermediate IC.sub.50 = 15
.mu.M, and low IC.sub.50 > 15-50 .mu.M. To determine the
stability of the peptide-MHC complex, peptide binding was performed
at 4.degree. C. and 20.degree. C. and IC.sub.50 were determined
Stable peptides displayed IC.sub.50 at 20.degree. C. that deviated
<2 times of the IC.sub.50 at 4.degree. C. Peptides that
displayed IC.sub.50 at 20.degree. C. of more than twice the
IC.sub.50 at 4.degree. C. but IC.sub.50 < 15 .mu.M were
considered to bind with intermediate stability. The rest was
designated as unstable peptide binding. .sup.2Proteasome cleavage
of C-terminus. 30 residue long peptides were digested by both mouse
(RMA-cells) and human (B-LCL JY) derived immunoproteasomes (IP) and
human (HeLa cells) derived household (HH) proteasomes.
.sup.3HLA-A*0201 binding peptides. Peptide binding capacity was
previously determined by (16, 24, 25). Peptide stability
TABLE-US-00002 TABLE 2 Relation between peptide binding,
proteasomal digestion and tolerance. Table 2 discloses SEQ ID NOS
78-86, respectively, in order of appearance. Sequence and HLA-
Restriction Proteasomal of Naturally processed Cleavage CTL
epitopes Tolerance of C-terminus HLA- WTp53 p53-/- Peptide Binding
IP IP HH Position Sequence restriction Human mice mice Affinity
Stability (RMA) (JY) (HeLa) P53 65-73 RMPEAAPPV HLA-A*0201 NO high
6 h - - - P53 149-157 STPPPGTRV HLA-A*0201 NO low <2 h + + + P53
187-197 GLAPPQHLIRV HLA-A*0201 YES NO high 6 h + + + P53 125-134
TYSPALNKMF HLA-A*2401 NO high + - - - P53 264-272 LLGRNSFEV
HLA-A*0201 NO inter- 6 h - - - mediate PRA 100-108 VLDGLDVLL
HLA-A*0201 NO inter- 2.5 h + mediate PRA 142-151 SLYSFPEPEA
HLA-A*0201 NO high 3 h + PRA 300-309 ALYVDSLFFL HLA-A*0201 NO high
>4 h + PRA 425-433 SLLQHLIGL HLA-A*0201 NO high >4 h +
TABLE-US-00003 TABLE 3 10 p53 peptides used in the phase I/ II
vaccination study. Table 3 discloses SEQ ID NOS 21, 2, 3, 20, 4, 5,
15, 6, 14 and 7, respectively, in order of appearance. Amino acid
Sequence Number 70-99 APPVAPAPAAPTPAAPAPAPSWPLSSSVPS 1 86-115
APAPSWPLSSSVPSQKTYQGSYGFRLGFLH 2 102-131
TYQGSYGFRLGFLHSGTAKSVTCTYSPALN 3 126-155
YSPALNKMFCQLAKTCPVQLWVDSTPPPGT 4 142-171
PVQLWVDSTPPPGTRVRAMAIYKQSQHMTE 5 157-186
VRAMAIYKQSQHMTEVVRRCPHHERCSDSD 6 174-203
RRCPHHERCSDSDGLAPPQHLIRVEGNLRV 7 190-219
PPQHLIRVEGNLRVEYLDDRNTFRHSVVVP 8 206-235
LDDRNTFRHSVVVPYEPPEVGSDCTTIHYN 9 224-248 EVGSDCTTIHYNYMCNSSCMGGMNR
10
TABLE-US-00004 TABLE 4 whole vaccination and analysis process in
ovarian cancer patients ##STR00001##
TABLE-US-00005 TABLE 5 Analysis of the affinity and stability of
the epitopes for HLA binding as well as proteasome processing of
some preferred peptides Predicted epitopes HLA ISA peptide HLA type
binding Prot. vaccine (p53 aa) Affinity Stability Cleavage
Tolerance p53 86-115 A3 (101-110) high +/- - A11 (101-110) high +/-
- p53 102-131 A1 (117-126) int + - A2 (113-122) low 6 h + A3
(111-120) high +/- + A3 (112-120) int - + A3 (113-120) int - +/- A3
(117-126) int - +/- A11 (112-120) int - + A24 (106-114) high +/- +
p53 142-171 A2 (149-157) low - + no A3 (154-163) int - - A3
(154-164) high + - A3 (156-163) int - - A3 (156-164) high + - A11
(156-164) high + - p53 157-186 A3 (172-181) int + - p53 190-219 A1
(196-205) int + - A1 (205-214) int + ? A24 (204-212) high + ? p53
224-248 A1 (226-234) high + + A1 (229-236) low - - p53 225-254 A1
(229-236) low - - p53 257-286 A2(264-272) int 6 h - p53 273-302 A11
(283-291) int +/- ? p53 305-334 A11 (311-319) high +/- - A11
(311-320) high +/- - A11 (312-319) high +/- - p53 337-366 A24
(340-348) high +/- - p53 353-382 A3 (360-370) int - ? A3 (361-370)
high + ? A3 (363-370) high + ? A3 (363-372) int - ? A3 (363-373)
high + ? A3 (373-381) int - + A11 (363-370) int +/- ? p53 369-393
A3 (376-386) high - - A11 (374-382) int +/- -
TABLE-US-00006 TABLE 6 Summary of p53-specific T-cell responses of
patients with colorectal cancer vaccinated twice with the p53-SLP
vaccine. Patient 1-78.sup.1 70-115 102-155 142-203 190-248 241-393
1 ELISPOT - - - +.sup.2 + - LST - - + + + + 2 ELISPOT - - - + + -
LST - - - + + + 3 ELISPOT - + - - - - LST - - - - + - 4 ELISPOT + +
+ + + + LST - - - + - - 5 ELISPOT - - - - - - LST - - - - - - 7
ELISPOT - - - - - + LST - - + - + - 8 ELISPOT - - + + + - LST - - -
- - - 9 ELISPOT - + + + + - LST - - - - + - 10 ELISPOT - - - - - -
LST - - + + + - Total positive pts 1 3 6 6 8 4 .sup.1The number of
the first and last amino acid of the amino acid sequence of the p53
protein that is covered by the pool of peptides used is depicted.
The columns with the first and last amino acid in bold depict the
parts of the p53 protein tat are used in the vaccine. .sup.2A
plus-sign indicates that this patient displayed a vaccine-induced
p53-specific T-cell response to this pool of p53 peptides.
TABLE-US-00007 TABLE 7 Cellular and humoural vaccine-induced T-cell
responses and clinical responses to p53-SLP vaccine in ovarian
cancer patients p53-specific T-cell Vaccine-induced T-cell
Vaccine-induced T-cell responses in p53-specific responses in PBMC
responses in PBMC lymphocytes from antibodies after Clinical
Response Patient (IFN-.gamma. ELISPOT).sup.1 (proliferation
assay).sup.2 skin biopsies.sup.3 immunisation.sup.4 CA-125.sup.6
CT.sup.7 P01 + + + - PD PD P02 + + + - SD PD P03 + + - +(4.8).sup.5
SD PD P04 na na na - na.sup.8 na.sup.8 P05 - + + + PR PD P06 - + -
+ SD PD P08 + + + - PD PD P09 - - - + SD PD P11 + nt na - PD PD P12
na na na - na.sup.8 na.sup.8 P13 + + + +(2.9) PD PD P14 - - - - PD
PD P15 - - nt + PD PD P17 - + - - SD SD P18 + + - + PD PD P19 + + +
+(2.5) PD PD P20 - + nt - PD PD P21 + + + - SD PD P22 + + + - PD PD
P23 + + + + SD SD .sup.1Vaccine-induced T cell responses after 4
immunisations as measured by IFN-.gamma. ELISPOT. - no
vaccine-induced response, + a vaccine-induced response.
.sup.2Vaccine-induced T-cell responses after 4 immunisations as
measured by proliferation assay. - no vaccine-induced response, + a
vaccine-induced response. .sup.3P53-specific responses in
lymphocytes cultured from skin biopsies as measured by
proliferation assay. - no p53-specific T-cell reactivity, +
p53-specific T-cell reactivity. .sup.4Serum p53 IgG titers after
immunisation as measured by quantitative ELISA. - no p53-specific
antibodies, + p53-specific antibodies. .sup.5The fold of
vaccine-induced increase in p53-specific antibody titer.
.sup.6CA-125 levels evaluated according to GCIG criteria. .sup.7CT
scan evaluated according to RECIST criteria. .sup.8Patient no
evaluated by CA-125 level or CT scan due to clinically evident
rapidly progressive disease. na = not available. nt = not
terminated.
TABLE-US-00008 TABLE 8 Vaccine-induced p53-specific immune
responses in PBMC of ovarian cancer patients immunised with the
p53-SLP vaccine as analysed by IFN-.gamma. ELISPOT After one
vaccination (I) After two vaccinations (II) After three
vaccinations (III) After four vaccinations (IV) vac vac vac vac vac
vac vac vac vac vac vac vac vac vac vac vac Patient.sup.1
p1-p2.sup.2 p3-p4 p5-p7 p8-p10 p1-p2 p3-p4 p5-p7 p8-p10 p1-p2 p3-p4
p5-p7 p8-p10 p1-p2 p3-p4 p5-p7 p8-p10 P01 - .sup. 62.sup.3 17 106 -
14 - 33 - - - - - 20 - 56 P02 - - - - - - - 28 - - - 21 - - 66 27
P03 - 52 37 166 - 29 15 81 - 43 58 132 - 15 20 57 P04 - - - - - - -
- na na na na na na na na P05 - 19 19 11 - - - - - - 29 18 - - - -
P06 - 12 47 22 - - - 21 - - - - - - - - P08 110 131 241 220 76 130
200 303 42 113 44 219 - 59 32 156 P09 - 31 38 55 - 10 18 20 - 14 12
- - - - - P11 - 71 120 138 - - 78 - - - 35 - - - 28 - P12 26 - 67
73 na na na na na na na na na na na na P13 - 65 65 81 37 83 133 144
16 - 125 93 - - 49 21 P14 - 60 - 22 - - 12 48 - - - - - - - - P15 -
- 18 - - 22 20 - - - 14 - - - - - P17 - 63 46 21 - - 52 20 - - - -
- - - - P18 - 88 106 120 - 41 61 45 - - - 60 - 41 13 - P19 - - 183
94 - - 75 60 - - - - - - - 23 P20 - - - 34 - 11 - 28 - 10 - 53 - -
- - P21 22 119 100 178 - 88 81 32 - 33 38 32 - 12 16 - P22 14 80
257 367 21 57 201 295 - 77 217 345 - - - 64 P23 - 50 106 21 - - 35
- - 20 55 22 - 13 - - .sup.1Patients analysed for p53-specific
responses before and after every immunisation (time points I-IV) by
IFN-.gamma. ELISPOT. .sup.2The pool of p53 vaccine peptides used to
stimulate patient-derived PBMC in vitro for 4 days. .sup.3Only
vaccine-induced p53-specific responses are shown (see definition in
Material and Methods). Responses are depicted as number of specific
spots per 10.sup.5 PBMC (mean of experimental wells - (mean + 2
.times. SD) of medium control). - = no vaccine-induced p53-specific
response; na = PBMC were not available.
TABLE-US-00009 TABLE 9 Vaccine-induced p53-specific T-cell
responses after four immunisations in freshly isolated PBMC of
ovarian cancer patients immunised with the p53-SLP vaccine as
analysed by proliferation assay. Vaccine peptides vac vac vac
Non-vaccine peptides Patient.sup.1 vac p1-p2.sup.2 p3-p4 p5-p7
p8-p10 p1-p4 p16-p24 P01 - 13.sup.3 6.8 6.4 - - P02 - - - 15.8 - -
P03 - 24 14.5 5.3 - - P05 - 3.9 2.5 3.3 - - P06 - - - 3.4 - - P08 -
- 4.4 7.8 - 4.0 P09 - - - - - - P11 nt nt nt nt nt nt P13 - 2.3 4.9
3 - - P14 - - - - - - P15 - - - - - - P17 - - - 7.1 - - P18 2.3 3.1
- - - - P19 - 16.5 7.4 - - - P20 3.7 5.1 - 2.4 - - P21 6.9 25.4 4.6
8.0 6.4 5.4 P22 - - 67.6 14.0 - 22.3 P23 6.4 3.2 - - 3.6 2.1 Total
4 (23.5%) 8 8 11 2 (11.8%) 4 (23.5%) (47.1%) (47.1%) (64.7%)
.sup.1Patients analysed for p53-specific responses before and after
four immunisations by proliferation assay. .sup.2The pool of p53
peptides (vaccine peptides or non-vaccine peptides) used to
stimulate patient-derived PBMC in vitro for 6 days. Proliferation
was measured by .sup.3H-thymidine incorporation. .sup.3Responses
are depicted as the mean of p53-induced proliferation after four
immunisations divided by the mean of p53-induced proliferation
before immunisation. A response >2 was considered a
vaccine-induced response. Otherwise the response was considered
negative (-). nt = not tested.
TABLE-US-00010 TABLE 10 Clinical Responses to p53-SLP immunotherapy
after four immunisations according to serum CA-125 levels and CT
scan in ovarian cancer patients. Target Non- New Overall Best
Patient lesions.sup.1 target lesions.sup.1 Lesions.sup.1
CA-125.sup.2 Reponse.sup.2 001 PD Yes PD PD 002 PD Yes SD PD 003 PD
PD Yes SD PD 004 ND ND ND ND ND* 005 Yes PR PD 006 PD Yes SD PD 008
Yes PD PD 009 PD No SD PD 011 PD Yes PD PD 012 ND ND ND ND ND* 013
PD PD Yes PD PD 014 PD Yes PD PD 015 Yes PD PD 017 No SD SD 018 PD
Yes PD PD 019 Yes PD PD 020 PD Yes PD PD 021 PD PD Yes SD PD 022 SD
Yes PD PD 023 SD No SD SD .sup.1Evaluated according to RECIST
criteria; .sup.2Evaluated according to GCIG criteria. PD =
progressive disease; SD = stable disease; PR = partial response; ND
= dot done *clinically progressive after 2 immunisations
REFERENCES LIST
[0198] Akcay, A., Erdem, Y., Altun, B., Usalan, C., Agca, E.,
Yasavul, U., Turgan, C., and Caglar, S. The booster phenomenon in
2-step tuberculin skin testing of patients receiving long-term
hemodialysis. Am. J. Infect. Control, 31: 371-374, 2003. [0199]
Allen, P. M. Peptides in positive and negative selection: a
delicate balance, Cell. 76: 593-6, 1994. [0200] Alvarez D. et al,
J. of Immunology, (2005), 174:1664-1674 [0201] Ashton-Rickardt, P.
G., Bandeira, A., Delaney, J. R., Van Kaer, L., Pircher, H. P.,
Zinkernagel, R. M., and Tonegawa, S. Evidence for a differential
avidity model of T cell selection in the thymus, Cell. 76: 651-63,
1994. [0202] Barfoed, A. M., Petersen, T. R., Kirkin, A. F., Thor
Straten, P., Claesson, M. H., and Zeuthen, J. Cytotoxic
T-lymphocyte clones, established by stimulation with the HLA-A2
binding p5365-73 wild type peptide loaded on dendritic cells In
vitro, specifically recognize and lyse HLA-A2 tumour cells
overexpressing the p53 protein, Scand J. Immunol. 51: 128-33, 2000.
[0203] Benham, A. M., Gromme, M., and Neefjes, J. Allelic
differences in the relationship between proteasome activity and MHC
class I peptide loading, J. Immunol. 161: 83-9, 1998. [0204]
Bleumer, I., Tiemessen, D. M., Oosterwijk-Wakka, J. C., Voller, M.
C., De Weijer, K., Mulders, P. F., and Oosterwijk, E. Preliminary
analysis of patients with progressive renal cell carcinoma
vaccinated with CA9-peptide-pulsed mature dendritic cells. J.
Immunother., 30: 116-122, 2007. [0205] Buckanovich R J et al,
(2008), Nature Medicine 14:28-36. [0206] Chikamatsu, K., Nakano,
K., Storkus, W. J., Appella, E., Lotze, M. T., Whiteside, T. L.,
and DeLeo, A. B. Generation of anti-p53 cytotoxic T lymphocytes
from human peripheral blood using autologous dendritic cells [In
Process Citation], Clin Cancer Res. 5: 1281-8, 1999. [0207] Dai, L.
C., West, K., Littaua, R., Takahashi, K., and Ennis, F. A. Mutation
of human immunodeficiency virus type 1 at amino acid 585 on gp41
results in loss of killing by CD8+A24-restricted cytotoxic T
lymphocytes, J. Virol. 66: 3151-4, 1992. [0208] de Jong, A., van
der Burg, S. H., Kwappenberg, K. M., van der Hulst, J. M., Franken,
K. L., Geluk, A., van Meijgaarden, K. E., Drijfhout, J. W., Kenter,
G., Vermeij, P., Melief, C. J., and Offring a, R. Frequent
detection of human papillomavirus 16 E2-specific T-helper immunity
in healthy subjects. Cancer Res., 62: 472-479, 2002. [0209] de
Jong, A., van der Hulst, J. M., Kenter, G. G., Drijfhout, J. W.,
Franken, K. L., Vermeij, P., Offring a, R., van der Burg, S. H.,
and Melief, C. J. Rapid enrichment of human papillomavirus
(HPV)-specific polyclonal T cell populations for adoptive
immunotherapy of cervical cancer. Int. J. Cancer, 114: 274-282,
2005. [0210] de Jong, A., van Poelgeest, M. I., van der Hulst, J.
M., Drijfhout, J. W., Fleuren, G. J., Melief, C. J., Kenter, G.,
Offring a, R., and van der Burg, S. H. Human papillomavirus type
16-positive cervical cancer is associated with impaired CD4+ T-cell
immunity against early antigens E2 and E6. Cancer Res., 64:
5449-5455, 2004. [0211] Eura, M., Chikamatsu, K., Katsura, F.,
Obata, A., Sobao, Y., Takiguchi, M., Song, Y., Appella, E.,
Whiteside, T. L., and DeLeo, A. B. A wild-type sequence p53 peptide
presented by HLA-A24 induces cytotoxic T lymphocytes that recognize
squamous cell carcinomas of the head and neck, Clin Cancer Res. 6:
979-86, 2000. [0212] Ferdinanda Gabriela, Manuela Ludovisi, Giacomo
Corrado, Vito Carone, Marco Petrillo and Giovanni Scambia,
Prognostic role of Ca125 response criteria and RECIST criteria:
Analysis of results from the MITO-3 phase III trial of gemcitabine
versus pegylated doxorubicin in recurrent ovarian cancer, (2008),
Gynecologic Oncology, 109: 187-193. [0213] Geier, E., Pfeifer, G.,
Wilm, M., Lucchiari-Hartz, M., Baumeister, W., Eichmann, K., and
Niedermann, G. A giant protease with potential to substitute for
some functions of the proteasome, Science. 283: 978-81, 1999.
[0214] Glas, R., Bogyo, M., McMaster, J. S., Gaczynska, M., and
Ploegh, H. L. A proteolytic system that compensates for loss of
proteasome function, Nature. 392: 618-22, 1998. [0215] Goonewardene
T I, Hall M R, Rustin G J., Management of asymptomatic patients on
follow-up for ovarian cancer with rising CA-125 concentrations.
Lancet Oncol. (2007) 8(9):813-21. [0216] Hernandez, J., Lee, P. P.,
Davis, M. M., and Sherman, L. A. The use of HLA A2.1/p53 peptide
tetramers to visualize the impact of self tolerance on the TCR
repertoire [In Process Citation], J. Immunol. 164: 596-602, 2000.
[0217] Hersey P, Menzies S W, Coventry B, et al. Phase I/II study
of immunotherapy with T-cell peptide epitopes in patients with
stage IV melanoma. Cancer Immunol. Immunother. 2005; 54(3):208-18.
[0218] Honda, R., Tanaka, H., and Yasuda, H. Oncoprotein MDM2 is a
ubiquitin ligase E3 for tumor suppressor p53, FEBS Lett. 420: 25-7,
1997. [0219] Hopfl, R., Heim, K., Christensen, N., Zumbach, K.,
Wieland, U., Volgger, B., Widschwendter, A., Haimbuchner, S.,
Muller-Holzner, E., Pawlita, M., Pfister, H., and Fritsch, P.
Spontaneous regression of CIN and delayed-type hypersensitivity to
HPV-16 oncoprotein E7. Lancet, 356: 1985-1986, 2000. [0220] Hopfl,
R., Sandbichler, M., Sepp, N., Heim, K., Muller-Holzner, E.,
Wartusch, B., Dapunt, O., Jochmus-Kudielka, I., ter Meulen, J.,
Gissmann, L., and Skin test for HPV type 16 proteins in cervical
intraepithelial neoplasia. Lancet, 337: 373-374, 1991. [0221]
Houbiers, J. G., Nijman, H. W., van der Burg, S. H., Drijfhout, J.
W., Kenemans, P., van de Velde, C. J., Brand, A., Momburg, F.,
Kast, W. M., and Melief, C. J. In vitro induction of human
cytotoxic T lymphocyte responses against peptides of mutant and
wild-type p53, Eur J. Immunol. 23: 2072-7, 1993. [0222] Jeffries,
D. J., Hill, P. C., Fox, A., Lugos, M., Jackson-Sillah, D. J.,
Adegbola, R. A., and Brookes, R. H. Identifying ELISPOT and skin
test cut-offs for diagnosis of Mycobacterium tuberculosis infection
in The Gambia. Int. J. Tuberc. Lung Dis., 10: 192-198, 2006. [0223]
Kappler, J. W., Roehm, N., and Marrack, P. T cell tolerance by
clonal elimination in the thymus, Cell. 49: 273-80, 1987. [0224]
Kessler, J. H., Beekman, N. J., Bres-Vloemans, S. A., Verdijk, P.,
van Veelen, P. A., Kloosterman-Joosten, A. M., Vissers, D. C., ten
Bosch, G. J., Kester, M. G., Sijts, A., Wouter Drijfhout, J.,
Ossendorp, F., Offring a, R., and Melief, C. J. Efficient
identification of novel HLA-A(*)0201-presented cytotoxic T
lymphocyte epitopes in the widely expressed tumor antigen PRAME by
proteasome-mediated digestion analysis, J Exp Med. 193: 73-88,
2001. [0225] Kloetzel P M & Ossendorp F Curr Opin Immunol. 2004
February; 16(1):76-81. [0226] Luckey, C. J., Marto, J. A.,
Partridge, M., Hall, E., White, F. M., Lippolis, J. D.,
Shabanowitz, J., Hunt, D. F., and Engelhard, V. H. Differences in
the expression of human class I MHC alleles and their associated
peptides in the presence of proteasome inhibitors, J. Immunol. 167:
1212-21, 2001. [0227] Luckey, C. J., King, G. M., Marto, J. A.,
Venketeswaran, S., Maier, B. F., Crotzer, V. L., Colella, T. A.,
Shabanowitz, J., Hunt, D. F., and Engelhard, V. H. Proteasomes can
either generate or destroy MHC class I epitopes: evidence for
nonproteasomal epitope generation in the cytosol, Journal of
Immunology J1-JI. 161: 112-121, 1998. [0228] Macagno, A., Gilliet,
M., Sallusto, F., Lanzavecchia, A., Nestle, F. O., and Groettrup,
M. Dendritic cells up-regulate immunoproteasomes and the proteasome
regulator PA28 during maturation, Eur J. Immunol. 29: 4037-42,
1999. [0229] Mayordomo, J. I., Loftus, D. J., Sakamoto, H., De
Cesare, C. M., Appasamy, P. M., Lotze, M. T., Storkus, W. J.,
Appella, E., and DeLeo, A. B. Therapy of murine tumors with p53
wild-type and mutant sequence peptide-based vaccines, J Exp Med.
183: 1357-65, 1996. [0230] Milner, J. Different forms of p53
detected by monoclonal antibodies in non-dividing and dividing
lymphocytes, Nature. 310: 143-5, 1984. [0231] Morel, S., Levy, F.,
Burlet-Schiltz, O., Brasseur, F., Probst-Kepper, M., Peitrequin, A.
L., Monsarrat, B., Van Velthoven, R., Cerottini, J. C., Boon, T.,
Gairin, J. E., and Van den Eynde, B. J. Processing of some antigens
by the standard proteasome but not by the immunoproteasome results
in poor presentation by dendritic cells, Immunity. 12: 107-17,
2000. [0232] Momand, J., Zambetti, G. P., Olson, D. C., George, D.,
and Levine, A. J. The mdm-2 oncogene product forms a complex with
the p53 protein and inhibits p53-mediated transactivation, Cell.
69: 1237-45, 1992. [0233] Morgan et al. Science. 2006 Oct. 6;
314(5796):126-9. [0234] Nijman, H. W., Houbiers, J. G., van der
Burg, S. H., Vierboom, M. P., Kenemans, P., Kast, W. M., and
Melief, C. J. Characterization of cytotoxic T lymphocyte epitopes
of a self-protein, p53, and a non-self-protein, influenza matrix:
relationship between major histocompatibility complex peptide
binding affinity and immune responsiveness to peptides, J.
Immunother. 14: 121-6, 1993. [0235] Platt, J. L., Grant, B. W.,
Eddy, A. A., and Michael, A. F. Immune cell populations in
cutaneous delayed-type hypersensitivity. J. Exp. Med., 158:
1227-1242, 1983. [0236] Poulter, L. W., Seymour, G. J., Duke, O.,
Janossy, G., and Panayi, G. Immunohistological analysis of
delayed-type hypersensitivity in man. Cell Immunol., 74: 358-369,
1982. [0237] Rammensee, H. G., Friede, T., and Stevanoviic, S. MHC
ligands and peptide motifs: first listing, Immunogenetics. 41:
178-228, 1995. [0238] Reimann, J. and Schirmbeck, R. Alternative
pathways for processing exogenous and endogenous antigens that can
generate peptides for MHC class I-restricted presentation, Immunol
Rev. 172: 131-52, 1999. [0239] Rogel, A., Popliker, M., Webb, C.
G., and Oren, M. p53 cellular tumor antigen: analysis of mRNA
levels in normal adult tissues, embryos, and tumors, Mol Cell Biol.
5: 2851-5, 1985. [0240] Romani N. et al, Springer Semin
Immunopathol., (1992), 13:265-279. [0241] Ropke, M., Hald, J.,
Guldberg, P., Zeuthen, J., Norgaard, L., Fugger, L., Svejgaard, A.,
Van der Burg, S., Nijman, H. W., Melief, C. J., and Claesson, M. H.
Spontaneous human squamous cell carcinomas are killed by a human
cytotoxic T lymphocyte clone recognizing a wild-type p53-derived
peptide, Proc Natl Acad Sci USA. 93: 14704-7, 1996. [0242]
Rosenberg S A, Yang J C, Schwartzentruber D J, et al. Immunologic
and therapeutic evaluation of a synthetic peptide vaccine for the
treatment of patients with metastatic melanoma. Nat. Med. 1998;
4(3):321-7. [0243] Rustin G J, Bast R C Jr, Kelloff G J, Barrett J
C, Carter S K, Nisen P D, Sigman C C, Parkinson D R, Ruddon R W,
Use of CA-125 in clinical trial evaluation of new therapeutic drugs
for ovarian cancer. Clin Cancer Res. 2004; 10:3919-26. [0244]
Sebzda, E., Wallace, V. A., Mayer, J., Yeung, R. S., Mak, T. W.,
and Ohashi, P. S. Positive and negative thymocyte selection induced
by different concentrations of a single peptide, Science. 263:
1615-8, 1994. [0245] Sette, A., Sidney, J., del Guercio, M. F.,
Southwood, S., Ruppert, J., Dahlberg, C., Grey, H. M., and Kubo, R.
T. Peptide binding to the most frequent HLA-A class I alleles
measured by quantitative molecular binding assays, Mol. Immunol.
31: 813-22, 1994. [0246] Shkedy, D., Gonen, H., Bercovich, B., and
Ciechanover, A. Complete reconstitution of conjugation and
subsequent degradation of the tumor suppressor protein p53 by
purified components of the ubiquitin proteolytic system, FEBS Lett.
348: 126-30, 1994. [0247] Stohwasser, R., Standera, S., Peters, I.,
Kloetzel, P. M., and Groettrup, M. Molecular cloning of the mouse
proteasome subunits MC14 and MECL-1: reciprocally regulated tissue
expression of interferon-gamma-modulated proteasome subunits, Eur
J. Immunol. 27: 1182-7, 1997. [0248] Terada, N., Lucas, J. J., and
Gelfand, E. W. Differential regulation of the tumor suppressor
molecules, retinoblastoma susceptibility gene product (Rb) and p53,
during cell cycle progression of normal human T cells, J. Immunol.
147: 698-704, 1991. [0249] Therasse P, Arbuck S G, Eisenhauer E A,
et al. New guidelines to evaluate the response to treatment in
solid tumors. European Organization for Research and Treatment of
Cancer, National Cancer Institute of the United States, National
Cancer Institute of Canada. J. Natl. Cancer Inst. 2000;
92(3):205-16. [0250] Theobald M & Offring a R. Expert Rev Mol.
Med. 2003 Mar. 28; 2003:1-13. [0251] Theobald, M., Biggs, J.,
Dittmer, D., Levine, A. J., and Sherman, L. A. Targeting p53 as a
general tumor antigen, Proc Natl Acad Sci USA. 92: 11993-7, 1995.
[0252] Theobald, M., Biggs, J., Hernandez, J., Lustgarten, J.,
Labadie, C., and Sherman, L. A. Tolerance to p53 by A2.1-restricted
cytotoxic T lymphocytes, J Exp Med. 185: 833-41, 1997. [0253]
Theobald, M., Ruppert, T., Kuckelkorn, U., Hernandez, J., Haussler,
A., Ferreira, E. A., Liewer, U., Biggs, J., Levine, A. J., Huber,
C., Koszinowski, U. H., Kloetzel, P. M., and Sherman, L. A. The
sequence alteration associated with a mutational hotspot in p53
protects cells from lysis by cytotoxic T lymphocytes specific for a
flanking peptide epitope, J Exp Med. 188: 1017-28, 1998. [0254]
Tilkin, A. F., Lubin, R., Soussi, T., Lazar, V., Janin, N.,
Mathieu, M. C., Lefrere, I., Carlu, C., Roy, M., Kayibanda, M., and
et al. Primary proliferative T cell response to wild-type p53
protein in patients with breast cancer, Eur J. Immunol. 25: 1765-9,
1995. [0255] van der Burg, S. H., Ras, E., Drijfhout, J. W.,
Benckhuijsen, W. E., Bremers, A. J., Melief, C. J., and Kast, W. M.
An HLA class I peptide-binding assay based on competition for
binding to class 1 molecules on intact human B cells.
Identification of conserved HIV-1 polymerase peptides binding to
HLA-A*0301, Hum Immunol. 44: 189-98, 1995. [0256] van der Burg, S.
H., Visseren, M. J., Brandt, R. M., Kast, W. M., and Melief, C. J.
Immunogenicity of peptides bound to MHC class 1 molecules depends
on the MHC-peptide complex stability, J. Immunol. 156: 3308-14,
1996. [0257] van der Burg, S. H., Kwappenberg, K. M., Geluk, A.,
van der, K. M., Pontesilli, O., Hovenkamp, E., Franken, K. L., van
Meijgaarden, K. E., Drijfhout, J. W., Ottenhoff, T. H., Melief, C.
J., and Offring a, R. Identification of a conserved universal Th
epitope in HIV-1 reverse transcriptase that is processed and
presented to HIV-specific CD4+ T cells by at least four unrelated
HLA-DR molecules. J. Immunol., 162: 152-160, 1999. [0258] van der
Burg, S. H., Ressing, M. E., Kwappenberg, K. M., de Jong, A.,
Straathof, K., de Jong, J., Geluk, A., van Meijgaarden, K. E.,
Franken, K. L., Ottenhoff, T. H., Fleuren, G. J., Kenter, G.,
Melief, C. J., and Offring a, R. Natural T-helper immunity against
human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in
patients with HPV 16-positive cervical lesions: identification of 3
human leukocyte antigen class II-restricted epitopes. Int. J.
Cancer, 91: 612-618, 2001. [0259] van der Burg, S. H., de Cock, K.,
Menon, A. G., Franken, K. L., Palmen, M., Redeker, A., Drijfhout,
J., Kuppen, P. J., van de Velde, C., Erdile, L., Tollenaar, R. A.,
Melief, C. J., and Offring a, R. Long lasting p53-specific T cell
memory responses in the absence of anti-p53 antibodies in patients
with resected primary colorectal cancer, Eur J. Immunol. 31:
146-55, 2001.
[0260] Vierboom, M. P., Zwaveling, S., Bos, G. M. J., Ooms, M.,
Krietemeijer, G. M., Melief, C. J., and Offringa, R. High
steady-state levels of p53 are not a prerequisite for tumor
eradication by wild-type p53-specific cytotoxic T lymphocytes,
Cancer Res. 60: 5508-13, 2000. [0261] Wang, S., Fan, Y., Brunham,
R. C., and Yang, X. IFN-gamma knockout mice show Th2-associated
delayed-type hypersensitivity and the inflammatory cells fail to
localize and control chlamydial infection. Eur. J. Immunol., 29:
3782-3792, 1999. [0262] Welters, M. J., de Jong, A., van den Eeden,
S. J., van der Hulst, J. M., Kwappenberg, K. M., Hassane, S.,
Franken, K. L., Drijfhout, J. W., Fleuren, G. J., Kenter, G.,
Melief, C. J., Offring a, R., and van der Burg, S. H. Frequent
display of human papillomavirus type 16 E6-specific memory t-Helper
cells in the healthy population as witness of previous viral
encounter. Cancer Res, 63: 636-641, 2003. [0263] Woodfolk, J. A.
and Platts-Mills, T. A. Diversity of the human allergen-specific T
cell repertoire associated with distinct skin test reactions:
delayed-type hypersensitivity-associated major epitopes induce Th1-
and Th2-dominated responses. J. Immunol., 167: 5412-5419, 2001.
[0264] Zanelli, E., Zhou, P., Cao, H., Smart, M. K., and David, C.
S. Genomic organization and tissue expression of the mouse
proteasome gene Lmp-7, Immunogenetics. 38: 400-7, 1993.
Sequence CWU 1
1
931393PRTHomo sapiens 1Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu
Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu
Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro Ser Gln Ala
Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu Gln Trp
Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro
Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70 75 80 Thr
Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90
95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly
100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr
Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr
Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr Pro Pro Pro Gly
Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys Gln Ser Gln
His Met Thr Glu Val Val Arg Arg Cys 165 170 175 Pro His His Glu Arg
Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190 His Leu Ile
Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200 205 Arg
Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu 210 215
220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr
Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg
Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro Gly Arg Asp
Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro
His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro
Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro
Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335
Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340
345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser
His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys
Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390
230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser
Val Pro Ser Gln Lys 1 5 10 15 Thr Tyr Gln Gly Ser Tyr Gly Phe Arg
Leu Gly Phe Leu His 20 25 30 330PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 3Thr Tyr Gln Gly Ser
Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly 1 5 10 15 Thr Ala Lys
Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn 20 25 30
430PRTArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro
Gly Thr Arg Val 1 5 10 15 Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln
His Met Thr Glu 20 25 30 530PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 5Val Arg Ala Met Ala Ile
Tyr Lys Gln Ser Gln His Met Thr Glu Val 1 5 10 15 Val Arg Arg Cys
Pro His His Glu Arg Cys Ser Asp Ser Asp 20 25 30 630PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr 1
5 10 15 Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Val Pro 20
25 30 725PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Glu Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn
Tyr Met Cys Asn 1 5 10 15 Ser Ser Cys Met Gly Gly Met Asn Arg 20 25
830PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn
Tyr Met Cys Asn Ser 1 5 10 15 Ser Cys Met Gly Gly Met Asn Arg Arg
Pro Ile Leu Thr Ile 20 25 30 930PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 9Leu Glu Asp Ser Ser
Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val 1 5 10 15 Arg Val Cys
Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu 20 25 30
1030PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg
Thr Glu Glu Glu Asn 1 5 10 15 Leu Arg Lys Lys Gly Glu Pro His His
Glu Leu Pro Pro Gly 20 25 30 1130PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 11Lys Arg Ala Leu Pro
Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 1 5 10 15 Lys Pro Leu
Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly 20 25 30
1230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg
Ala His Ser Ser His 1 5 10 15 Leu Lys Ser Lys Lys Gly Gln Ser Thr
Ser Arg His Lys Lys 20 25 30 1325PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 13Leu Lys Ser Lys Lys Gly
Gln Ser Thr Ser Arg His Lys Lys Leu Met 1 5 10 15 Phe Lys Thr Glu
Gly Pro Asp Ser Asp 20 25 1430PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 14Leu Asp Asp Arg Asn Thr
Phe Arg His Ser Val Val Val Pro Tyr Glu 1 5 10 15 Pro Pro Glu Val
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn 20 25 30 1530PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Arg Arg Cys Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala 1
5 10 15 Pro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg Val 20
25 30 1630PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile
Leu Thr Ile Ile Thr 1 5 10 15 Leu Glu Asp Ser Ser Gly Asn Leu Leu
Gly Arg Asn Ser Phe 20 25 30 1730PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 17Leu Arg Lys Lys Gly
Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 1 5 10 15 Lys Arg Ala
Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro 20 25 30
1830PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 18Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln
Ile Arg Gly Arg Glu 1 5 10 15 Arg Phe Glu Met Phe Arg Glu Leu Asn
Glu Ala Leu Glu Leu 20 25 30 1930PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 19Arg Phe Glu Met Phe
Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 1 5 10 15 Ala Gln Ala
Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser 20 25 30
2030PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln
Leu Ala Lys Thr Cys 1 5 10 15 Pro Val Gln Leu Trp Val Asp Ser Thr
Pro Pro Pro Gly Thr 20 25 30 2130PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 21Ala Pro Pro Val Ala
Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro 1 5 10 15 Ala Pro Ala
Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser 20 25 30
22365PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Met Glu Thr Leu Cys Gln Arg Leu Asn Val Cys
Gln Asp Lys Ile Leu 1 5 10 15 Thr His Tyr Glu Asn Asp Ser Thr Asp
Leu Arg Asp His Ile Asp Tyr 20 25 30 Trp Lys His Met Arg Leu Glu
Cys Ala Ile Tyr Tyr Lys Ala Arg Glu 35 40 45 Met Gly Phe Lys His
Ile Asn His Gln Val Val Pro Thr Leu Ala Val 50 55 60 Ser Lys Asn
Lys Ala Leu Gln Ala Ile Glu Leu Gln Leu Thr Leu Glu 65 70 75 80 Thr
Ile Tyr Asn Ser Gln Tyr Ser Asn Glu Lys Trp Thr Leu Gln Asp 85 90
95 Val Ser Leu Glu Val Tyr Leu Thr Ala Pro Thr Gly Cys Ile Lys Lys
100 105 110 His Gly Tyr Thr Val Glu Val Gln Phe Asp Gly Asp Ile Cys
Asn Thr 115 120 125 Met His Tyr Thr Asn Trp Thr His Ile Tyr Ile Cys
Glu Glu Ala Ser 130 135 140 Val Thr Val Val Glu Gly Gln Val Asp Tyr
Tyr Gly Leu Tyr Tyr Val 145 150 155 160 His Glu Gly Ile Arg Thr Tyr
Phe Val Gln Phe Lys Asp Asp Ala Glu 165 170 175 Lys Tyr Ser Lys Asn
Lys Val Trp Glu Val His Ala Gly Gly Gln Val 180 185 190 Ile Leu Cys
Pro Thr Ser Val Phe Ser Ser Asn Glu Val Ser Ser Pro 195 200 205 Glu
Ile Ile Arg Gln His Leu Ala Asn His Pro Ala Ala Thr His Thr 210 215
220 Lys Ala Val Ala Leu Gly Thr Glu Glu Thr Gln Thr Thr Ile Gln Arg
225 230 235 240 Pro Arg Ser Glu Pro Asp Thr Gly Asn Pro Cys His Thr
Thr Lys Leu 245 250 255 Leu His Arg Asp Ser Val Asp Ser Ala Pro Ile
Leu Thr Ala Phe Asn 260 265 270 Ser Ser His Lys Gly Arg Ile Asn Cys
Asn Ser Asn Thr Thr Pro Ile 275 280 285 Val His Leu Lys Gly Asp Ala
Asn Thr Leu Lys Cys Leu Arg Tyr Arg 290 295 300 Phe Lys Lys His Cys
Thr Leu Tyr Thr Ala Val Ser Ser Thr Trp His 305 310 315 320 Trp Thr
Gly His Asn Val Lys His Lys Ser Ala Ile Val Thr Leu Thr 325 330 335
Tyr Asp Ser Glu Trp Gln Arg Asp Gln Phe Leu Ser Gln Val Lys Ile 340
345 350 Pro Lys Thr Ile Thr Val Ser Thr Gly Phe Met Ser Ile 355 360
365 23158PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 23Met His Gln Lys Arg Thr Ala Met Phe Gln Asp
Pro Gln Glu Arg Pro 1 5 10 15 Arg Lys Leu Pro Gln Leu Cys Thr Glu
Leu Gln Thr Thr Ile His Asp 20 25 30 Ile Ile Leu Glu Cys Val Tyr
Cys Lys Gln Gln Leu Leu Arg Arg Glu 35 40 45 Val Tyr Asp Phe Ala
Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly 50 55 60 Asn Pro Tyr
Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile 65 70 75 80 Ser
Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu 85 90
95 Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn
100 105 110 Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu
Asp Lys 115 120 125 Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr
Gly Arg Cys Met 130 135 140 Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg
Glu Thr Gln Leu 145 150 155 2498PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 24Met His Gly Asp Thr
Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln 1 5 10 15 Pro Glu Thr
Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser 20 25 30 Glu
Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp 35 40
45 Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr
50 55 60 Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr
Leu Glu 65 70 75 80 Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro
Ile Cys Ser Gln 85 90 95 Lys Pro 2510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Gly
Thr Ala Lys Ser Val Thr Cys Thr Tyr 1 5 10 2610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Arg
Val Glu Gly Asn Leu Arg Val Glu Tyr 1 5 10 2710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Tyr
Leu Asp Asp Arg Asn Thr Phe Arg His 1 5 10 289PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Gly
Ser Asp Cys Thr Thr Ile His Tyr 1 5 298PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Cys
Thr Thr Ile His Tyr Asn Tyr 1 5 309PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Lys
Leu Leu Pro Glu Asn Asn Val Leu 1 5 319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Arg
Met Pro Glu Ala Ala Pro Pro Val 1 5 3210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Phe
Leu His Ser Gly Thr Ala Lys Ser Val 1 5 10 339PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Ser
Thr Pro Pro Pro Gly Thr Arg Val 1 5 3411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Gly
Leu Ala Pro Pro Gln His Leu Ile Arg Val 1 5 10 359PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Leu
Leu Gly Arg Asn Ser Phe Glu Val 1 5 369PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Pro
Leu Asp Gly Glu Tyr Phe Thr Leu 1 5 3710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Lys
Thr Tyr Gln Gly Ser Tyr Gly Phe Arg 1 5 10 3811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Arg
Leu Gly Phe Leu His Ser Gly Thr Ala Lys 1 5 10 3910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 39Leu
Gly Phe Leu His Ser Gly Thr Ala Lys 1 5 10 409PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Gly
Phe Leu His Ser Gly Thr Ala Lys 1 5 418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Phe
Leu His Ser Gly Thr Ala Lys 1 5 4210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Gly
Thr Ala Lys Ser Val Thr Cys Thr Tyr 1 5 10 4311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Val
Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys 1 5 10 449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Cys
Thr Tyr Ser Pro Ala Leu Asn Lys 1
5 4511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys 1 5
10 468PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Lys Met Phe Cys Gln Leu Ala Lys 1 5
4710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Gly Thr Arg Val Arg Ala Met Ala Ile Tyr 1 5 10
4811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys 1 5
10 498PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Arg Val Arg Ala Met Ala Ile Tyr 1 5
509PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Arg Val Arg Ala Met Ala Ile Tyr Lys 1 5
5110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Val Val Arg Arg Cys Pro His His Glu Arg 1 5 10
5211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Gly Gly Ser Arg Ala His Ser Ser His Leu Lys 1 5
10 5310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Gly Ser Arg Ala His Ser Ser His Leu Lys 1 5 10
548PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Arg Ala His Ser Ser His Leu Lys 1 5
5510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Arg Ala His Ser Ser His Leu Lys Ser Lys 1 5 10
5611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Arg Ala His Ser Ser His Leu Lys Ser Lys Lys 1 5
10 579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 57Lys Gly Gln Ser Thr Ser Arg His Lys 1 5
5811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys 1 5
10 5910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg 1 5 10
6010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Leu Gly Phe Leu His Ser Gly Thr Ala Lys 1 5 10
619PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Gly Phe Leu His Ser Gly Thr Ala Lys 1 5
629PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Cys Thr Tyr Ser Pro Ala Leu Asn Lys 1 5
638PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Lys Met Phe Cys Gln Leu Ala Lys 1 5
649PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Arg Val Arg Ala Met Ala Ile Tyr Lys 1 5
659PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Asn Thr Ser Ser Ser Pro Gln Pro Lys 1 5
6610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 1 5 10
678PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 67Thr Ser Ser Ser Pro Gln Pro Lys 1 5
689PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Arg Thr Glu Glu Glu Asn Leu Arg Lys 1 5
698PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 69Arg Ala His Ser Ser His Leu Lys 1 5
709PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Gly Gln Ser Thr Ser Arg His Lys Lys 1 5
719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 71Thr Phe Ser Asp Leu Trp Lys Leu Leu 1 5
7210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu 1 5 10
738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Ser Tyr Gly Phe Arg Leu Gly Phe 1 5
749PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 74Ser Tyr Gly Phe Arg Leu Gly Phe Leu 1 5
7510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe 1 5 10
769PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Glu Tyr Leu Asp Asp Arg Asn Thr Phe 1 5
779PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 77Met Phe Arg Glu Leu Asn Glu Ala Leu 1 5
789PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 78Arg Met Pro Glu Ala Ala Pro Pro Val 1 5
799PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Ser Thr Pro Pro Pro Gly Thr Arg Val 1 5
8011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val 1 5
10 8110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 81Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe 1 5 10
829PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 82Leu Leu Gly Arg Asn Ser Phe Glu Val 1 5
839PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Val Leu Asp Gly Leu Asp Val Leu Leu 1 5
8410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Ser Leu Tyr Ser Phe Pro Glu Pro Glu Ala 1 5 10
8510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 85Ala Leu Tyr Val Asp Ser Leu Phe Phe Leu 1 5 10
869PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 86Ser Leu Leu Gln His Leu Ile Gly Leu 1 5
878PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 87Tyr Leu Glu Pro Ala Cys Ala Lys 1 5
8810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 88Lys Val Phe Pro Cys Ala Leu Ile Asn Lys 1 5 10
8910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 89Lys Val Phe Pro Cys Ala Leu Ile Asn Lys 1 5 10
909PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Arg Tyr Leu Lys Cys Gln Gln Leu Leu 1 5
9130PRTHomo sapiens 91Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg
Met Pro Glu Ala Ala 1 5 10 15 Pro Pro Val Ala Pro Ala Pro Ala Ala
Pro Thr Pro Ala Ala 20 25 30 9230PRTHomo sapiens 92His Ser Gly Thr
Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu 1 5 10 15 Asn Lys
Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 20 25 30
9330PRTHomo sapiens 93Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg
Asn Ser Phe Glu Val 1 5 10 15 Arg Val Cys Ala Cys Pro Gly Arg Asp
Arg Arg Thr Glu Glu 20 25 30
* * * * *