U.S. patent application number 13/058304 was filed with the patent office on 2011-07-28 for minigene.
Invention is credited to Luigi Aurisicchio, Ansuman Bagchi, Gennaro Ciliberto, Arthur Fridman, Nicola La Monica, Elisa Scarselli.
Application Number | 20110182926 13/058304 |
Document ID | / |
Family ID | 41319610 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110182926 |
Kind Code |
A1 |
La Monica; Nicola ; et
al. |
July 28, 2011 |
MINIGENE
Abstract
The present invention provides minigenes suitable as a
prophylactic or therapeutic vaccine against conditions such as
cancer, infectious diseases or autoimmune diseases, and
pharmaceutical compositions comprising the minigene. The minigenes
of the present invention comprise (a) a human tissue plasminogen
signal peptide; (b) at least one T-cell epitope; and (c) an E. coli
heat labile enterotoxin B subunit; wherein the at least one T-cell
epitope is linked to the rest of the minigene, and to any other
epitopes, by furin sensitive linkers. In some embodiments of the
invention, the minigene comprises T-cell epitopes from one or more
of CEA, her-2/neu and hTERT. Also provided herein are immunogenic
peptide epitopes of CEA, her-2/neu and hTERT, as well as
immunogenic peptide analogs, and pharmaceutical compositions and
vaccines comprising one or more of said peptides and analogs for
prophylaxis and/or treatment of cancer or other disorder. Methods
of inducing an immune response in a patient, in addition to methods
of treatment using the minigenes, immunogenic peptides, and peptide
analogs disclosed herein are also provided.
Inventors: |
La Monica; Nicola; (Natick,
MA) ; Scarselli; Elisa; (Rome, IT) ;
Ciliberto; Gennaro; (Rome, IT) ; Aurisicchio;
Luigi; (Rome, IT) ; Fridman; Arthur;
(Secaucus, NJ) ; Bagchi; Ansuman; (Plainsboro,
NJ) |
Family ID: |
41319610 |
Appl. No.: |
13/058304 |
Filed: |
August 7, 2009 |
PCT Filed: |
August 7, 2009 |
PCT NO: |
PCT/EP09/60282 |
371 Date: |
March 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61188762 |
Aug 12, 2008 |
|
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|
Current U.S.
Class: |
424/192.1 ;
530/350 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2319/02 20130101; A61P 31/00 20180101; C07K 2319/55 20130101;
C12N 9/6459 20130101; A61P 37/04 20180101; C07K 14/70503 20130101;
C07K 2319/50 20130101 |
Class at
Publication: |
424/192.1 ;
530/350 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 19/00 20060101 C07K019/00; A61P 37/04 20060101
A61P037/04; A61P 35/00 20060101 A61P035/00; A61P 31/00 20060101
A61P031/00 |
Claims
1. A minigene comprising: (a) a human tissue plasminogen signal
peptide; (b) at least one T-cell epitope; and (c) an E. coli heat
labile enterotoxin B subunit; wherein (d) the at least one T-cell
epitope is linked to the rest of the minigene, and to any other
epitopes, by furin sensitive linkers having the sequence RxKR or
RxRR where x is any amino acid.
2. A minigene according to claim 1 wherein the furin sensitive
linkers have the sequence REKR.
3. A minigene according to claim 1 wherein the T-cell epitope is an
immunogenic analogue of a naturally occurring epitope.
4. A minigene according to claim 1, wherein the T-cell epitope is
from a microbial or tumour associated antigen or from the variable
domain of an autoantibody.
5. A minigene according to claim 1 wherein the T-cell epitope is
restricted by an HLA allele.
6. A minigene according to claim 5 comprising from two to twenty
epitopes.
7. A minigene according to claim 4 wherein the at least one T-cell
epitope is from one or more of CEA, her-2/neu and hTERT.
8. A minigene according to claim 5 having more than one T-cell
epitope restricted by more than one HLA allele.
9. A minigene according to claim 6 wherein at least one of the
T-cell epitopes is repeated.
10. A minigene according to claim 1 comprising furin-sensitive
linkers having at least two different sequences.
11. A minigene according claim 1 further comprising a T-helper
epitope.
12. A minigene according to claim 12 wherein the T-helper epitope
is tetanus toxin-derived universal helper peptide p2 or p30.
13. A pharmaceutical composition comprising a minigene according to
claim 1 and a pharmaceutically acceptable excipient adjustment.
14-15. (canceled)
16. A method of treatment of a subject suffering from or prone to
cancer, an infectious disease or an autoimmune disease which
comprises administering to that subject a therapeutically or
prophylactically effective amount of a minigene of claim 1.
17. The pharmaceutical composition of claim 13, wherein the at
least one T-cell epitope is from one or more of CEA, her-2/neu and
hTERT.
18. The minigene according to claim 3, wherein the furin sensitive
linkers have the sequence REKR.
19. The minigene according to claim 18, wherein the T-cell epitope
is restricted by an HLA allele.
20. The minigene according to claim 19, wherein the at least one
T-cell epitope is from one or more of CEA, her-2/neu and hTERT.
21. The pharmaceutical composition of claim 13, further comprising
an adjuvant.
22. The minigene according to claim 6, wherein the epitopes are
from more than one antigen.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a minigene suitable as a
prophylactic or therapeutic vaccine against conditions such as
cancer, infectious diseases or autoimmune diseases, pharmaceutical
compositions comprising the minigene, and the use of the minigene
in therapy.
BACKGROUND OF THE INVENTION
[0002] It is recognised in the art (Ishioka et al, J. Immunol.,
162, 3915-3925, 1999) that immunizations with minigenes containing
T-cell epitopes may have several advantages compared to full-length
proteins. Proteins may have unknown and potentially toxic
biological activity while minigenes deliver only immunologically
relevant genetic information. Immunization with a protein usually
leads to an immune response that is narrowly focused on a few
epitopes (Yewdell and Bennink, Annu. Rev. Immunol., 17, 51-88,
1999), while minigenes can induce significant immune response to
multiple (up to 10 or more) epitopes simultaneously (Thomson et al,
J. Immunol., 160, 1717-1723, 1998). Minigenes are short and
compatible with commonly used delivery vectors including plasmid
DNA and adenoviruses.
[0003] Minigenes are known in the art comprising HLA allele
restricted T-cell epitopes, rather than the full sequence of the
protein against which protection is required. Thus Thomson et al,
supra discloses a minigene comprising multiple contiguous minimal
murine CTL epitopes. Ishioka et al, supra describes a minigene
encoding nine contiguous dominant HLA-A2.1- and A11-restricted
epitopes from the polymerase, envelope and core proteins of
hepatitis B virus and HIV, together with the PADRE (pan-DR epitope)
universal T-cell epitope and an endoplasmic reticulum-translocating
signal sequence.
[0004] The prior art also describes minigenes having spacers
between the epitopes and other components. Thus Wang et al, Scand.
J. Immunol., 60, 219-225, 2004 discloses a minigene having CTL
epitopes of MPT64 and 38 kDa proteins of Mycobacterium
tuberculosis. Minigenes have been prepared with spacers having the
sequence Ala-Ala-Tyr and/or ubiquitin functioning as a
protein-targeting sequence. Pitcovski et al, Vaccine, 24, 636-643,
2006 describes a melanoma multiepitope polypeptide having three
repeats of four modified melanoma antigens linked by five spacer
elements that encode a signal for proteasomal cleavage, which
polypeptide is fused to Escherichia coli heat labile enterotoxin
(LTB). Zhan et al, J. Clin. Invest., 113, 1792-1798, 2004 discusses
an oral DNA minigene vaccine containing the HIV tat translocation
peptide, a spacer (AAA) and then an HLA-A2-restricted CEA T-cell
epitope. The minigene was inserted into a pCMV vector comprising an
ER signal peptide. Lu et al, J. Immunol., 172, 4575-4582, 2004,
describes minigenes having multiple CTL epitopes joined via
furin-sensitive linkers and also containing HIV-1 tat.
[0005] Despite extensive studies with the above minigenes, and the
use of other strategies, the provision of an optimal minigene that
maximizes epitope-specific immune responses remains elusive. To
that end, the minigene provided by the present invention has been
designed to maximise the expression and immunogenicity of each
epitope in the minigene.
DESCRIPTION OF DRAWINGS
[0006] FIG. 1, panel A, shows the amino acid sequence of the human
CEA protein (SEQ ID NO:50), as set forth in NCBI Genbank Accession
No. M17303. Panel B shows an exemplary CEA protein sequence which
is deleted of its C-terminal anchoring domain (SEQ ID NO:51). Panel
C shows an exemplary variant CEA protein sequence (SEQ ID NO:52)
which comprises analogs SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and
SEQ ID NO:9. Modifications to the wt CEA sequence are shown in
bold.
[0007] FIG. 2 summarizes results obtained when comparing the
top-scoring CEA-proteins to the human proteome. Of the 150
top-scoring CEA peptides, 45 had no matches in other human
proteins, 64 matched a fragment of another CEA-like cell adhesion
molecule, 26 matched a fragment of a pregnancy-specific
glycoprotein, and 15 were similar to a fragment of a protein
outside the CEA family. Altogether, 105/150 peptides were rejected
or flagged.
[0008] FIG. 3 shows the binding affinity of CEA epitope candidates.
The stability of the peptide-MHC complex was evaluated by
fluorescent activated cell sorting (FACS). The mean fluorescence
intensity MFI resulting from the FACS analysis is shown for each
epitope.
[0009] FIG. 4 shows the relative binding stability of exemplary CEA
epitopes described herein. The stability of peptide-MHC complex was
evaluated by FACS analysis over time. MFI=mean fluorescence
intensity.
[0010] FIG. 5 shows EI Suite-selected peptides and analogs are
immunogenic in HLA-A2.1 mice. HHD mice were immunized by
subcutaneous injection of peptides. Two weeks later, cell mediated
immune response was measured by ICS on pooled PBMCs.
[0011] FIG. 6 shows analogs of immunogenic CEA peptides identified
by EI Suite strongly increase immune reactivity against the
corresponding wild type epitopes. HHD mice were immunized by SC
injection of peptides. Two weeks later, cell mediated immune
response against natural peptides was measured by ICS on individual
mouse splenocytes.
[0012] FIG. 7 shows the immunogenicity of additional EI
Suite-selected peptides (EXAMPLE 9). Groups of 6 HHD mice were
vaccinated subcutaneously with 100 .mu.g of peptide in combination
with HBVcore128 peptide (140 .mu.g), IFA (1:1) and CpG (50 .mu.g).
Mice received two injections two weeks apart and the immune
response was measured by intracellular staining for IFN.gamma.
three weeks later. Each triangle represents the CMI of a single
mouse; the geometric mean of each group is indicated by a round
dot. Student's t-test between CEA310 and CEA310L2-vaccinated groups
is statistically significant (p=0.007). The vaccination protocol
has been repeated with similar results.
[0013] FIG. 8 shows the binding affinity of hTERT candidate
epitopes and analogues. The level of binding is expressed as a
percent of positive control peptide binding. Wild type/analogue
pairs have the same shading.
[0014] FIG. 9 shows the relative binding stability of hTERT
candidate epitopes and analogues. The y-axis shows the off-rate in
hours.
[0015] FIG. 10 shows that epitopes selected by EI Suite, and
analogues, are immunogenic and that the analogues generate a
stronger immune response than the wild type peptides. The responses
of individual mice are shown as dots and the geometric mean is
shown as a triangle.
[0016] FIG. 11 shows the binding affinity of her-2/neu candidate
epitopes and analogues. The level of binding is expressed as a
percentage of positive control peptide binding. Wild type/analogue
pairs have the same shading.
[0017] FIG. 12 shows the relative binding stability of her-2/neu
candidate epitopes and analogues. The y-axis shows the off-rate in
hours.
[0018] FIG. 13 shows CMI response against wild type peptide in HHD
mice immunized with her-2/neu peptides. The responses of individual
mice are shown as dots and the geometric mean is shown as a
triangle.
[0019] FIG. 14 shows the general form of a minigene scaffold.
[0020] FIG. 15 shows a minigene exemplifying the scaffold defined
in this invention. TPA is human tissue plasminogen activator signal
peptide; 411V10, 691, 589V10 and 682V10 are epitopes and
immunogenic analogues from CEA; triangles are furin sensitive
linker REKR; LTB is E. coli heat labile enterotoxin B subunit.
[0021] FIG. 16 shows the modular structure of a minigene scaffold
containing epitopes from one or more antigens restricted by
multiple MHC alleles.
[0022] FIG. 17 shows an alternative minigene scaffold containing
epitopes from one or more antigens restricted by multiple MHC
alleles.
[0023] FIGS. 18 and 19 show the CMI response against wild type
peptides in HHD/CEA mice immunized with the minigene of Example 16.
The responses of individual mice are shown in black; the group
geometric mean is in grey.
[0024] FIG. 20 shows a minigene containing universal helper peptide
p30.
[0025] FIGS. 21 and 22 show the CMI response against wild type
peptides in HHD/CEA mice immunized with the minigene construct of
Example 20.
[0026] FIG. 23 shows a minigene scaffold designed to evaluate
dependence of immune response on epitope position.
[0027] FIG. 24 shows the immune responses produced by the minigene
shown in FIG. 23 against target epitopes in HHD/CEA mice.
[0028] FIG. 25 shows a ubiquitinized minigene construct containing
one of the three spacer sequences AAY, LRA and RLRA.
[0029] FIG. 26 shows the CMI responses produced by the minigene of
FIG. 25 in HHD/CEA mice in which the linker is (a) AAY, (b) LRA or
(c) RLRA.
[0030] FIG. 27 shows a minigene containing the HIV tat
membrane-translocating sequence.
[0031] FIG. 28 shows the CMI responses produced by the minigene of
FIG. 27 in HHD/CEA mice.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Thus the present invention provides a minigene comprising:
(a) a human tissue plasminogen signal peptide; (b) at least one
T-cell epitope; and (c) an E. coli heat labile enterotoxin B
subunit; wherein (d) the at least one T-cell epitope is linked to
the rest of the minigene, and to any other epitopes, by furin
sensitive linkers having the sequence RxKR or RxRR where x is any
amino acid.
[0033] Without being bound by theory, it is believed that the
tissue plasminogen activator (TPA) signal peptide directs the
minigene product to the endoplasmic reticulum (ER) where
endoprotease furin cleaves the furin sensitive linkers to produce
individual T-cell epitopes. Epitopes are then loaded onto empty
major histocompatibility complex (MHC) molecules for export to the
cell surface. Thus the TPA signal peptide enhances secretion of the
translated product. TPA has the sequence MKRGLCCVLLLCGAVFVSPS (SEQ
ID NO: 46).
[0034] Again, without being bound by theory, it is believed that
the E-coli heat labile enterotoxin B (LTB) subunit of the secreted
polypeptide binds to a receptor or on a professional antigen
presenting cell (APC) resulting in polypeptide internalisation and
APC maturation and activation. As a result an efficient T-cell
response is induced. The LTB may be as described in WO-A-05077977
(IRBM). Thus it may be a truncated in its signal sequence. LTB has
the amino acid sequence
SRAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQHIDS
QKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMEN (SEQ ID NO: 47) where
SR at the N-terminus is an artificial linker.
[0035] In one embodiment, the furin sensitive linkers have the
sequence REKR. In another embodiment, the furin-sensitive linkers
may have two or more different sequences.
[0036] The minigene may further comprise a T-helper epitope, such
as tetanus toxin-derived universal helper peptide p2 or p30, see
Valmori et al, J. Immunol., 149, 717-721, 1992.
[0037] In another embodiment the T-cell epitope is an immunogenic
analogue of a naturally occurring epitope. Without being bound by
theory, it is expected that analogues will form more stable
complexes with MHC molecules compared to the corresponding
wild-type epitopes (Lipford et al, Immunology, 84, 298-303, 1995).
The longer half-life of analogue-MHC complexes on cell surfaces
allows more efficient priming of T-cells which then recognise
cognate wild-type epitopes on the surface of target cells (Keogh et
al, J. Immunol., 167, 787-796, 2001). The minigenes of the
invention may comprise solely wild-type epitopes, or solely
analogues thereof, or a mixture of the two.
[0038] In an embodiment, the T-cell epitope is from a microbial or
tumour associated antigen or from the variable domain of an
autoantibody. The T-cell epitopes may be restricted by an HLA
allele or the epitopes in the minigene may be restricted by more
than one HLA allele. The T-cell epitopes may be restricted by
HLA-A*0201. The T-cell epitope may be repeated one or more times in
the minigene. Where the minigene contains epitopes restricted by
more than one allele, the epitopes may be grouped by allelic
restriction. Alternatively the epitopes, restricted by more than
one allele, may be randomly distributed.
[0039] The minigene may comprise from two to twenty epitopes. It
may comprise from 2 to 15, 2 to 10, 2 to 6, 4 to 20, 4 to 15, 4 to
10, 5 to 20, 5 to 15 or 5 to 20 epitopes. The minigene may comprise
more than 20 epitopes.
[0040] The minigene may comprise T-cell epitopes from one or more
of CEA, her-2/neu and hTERT.
[0041] The development of a minigene as a cancer vaccine capable of
eliciting a clinically-relevant immune response is partly dependent
on the choice of a target antigen that is preferentially expressed
on tumor cells compared to normal cells.
[0042] Human CEA is one human associated antigen TAA that has been
implicated in the pathogenesis of cancer. CEA is normally expressed
during fetal development and in adult colonic mucosa. Aberrant CEA
expression has long been correlated with many types of cancers,
with the first report describing CEA overexpression in human colon
tumors published over thirty years ago (Gold and Freedman, J. Exp.
Med. 121:439-462 (1965)). Overexpression of CEA has since been
detected in nearly all colorectal tumors, as well as in a high
percentage of adenocarcinomas of the pancreas, liver, breast,
ovary, cervix, and lung. Moreover, it was demonstrated in
transgenic mice immunized with a recombinant vaccinia vector
expressing CEA that anti-CEA immune responses could be elicited
without inducing autoimmunity, making CEA a particularly attractive
target for active and passive cancer immunotherapy (Kass et al.
Cancer Res. 59: 676-83 (1999)).
[0043] Therapeutic strategies targeting CEA have included the use
of CEA-based DNA and protein vaccines, and dendritic cell-based
vaccines (for review, see Berinstein, supra; and Sarobe et al.
Current Cancer Drug Targets 4: 443-54 (2004)). Antigenic peptide or
epitope-based vaccines have also been investigated as a means of
promoting the destruction of cancerous cells overexpressing CEA by
an individual's immune system.
[0044] The ribonucleoprotein telomerase is expressed by more than
85% of human cancers, and therefore functions as a nearly universal
TAA. Telomerase maintains the telomeric ends of linear chromosomes,
protecting them from degradation and end-to-end fusion. Most human
cells do not express telomerase and lose telomeric DNA with each
cell division. In contrast, the great majority of human tumours
exhibit strong telomerase activity, express human telomerase
reverse transcriptase (hTERT) and maintain the length of their
telomerases suggesting that the activation of telomerase plays an
important role in the development of human cancers. The telomorase
catalytic subunit hTERT is the rate-limiting component of the
complex, and its expression correlates best with telomerase
activity (Vonderheide et al, Immunity, 10, 673-679, 1999).
Therapeutic strategies targeting hTERT have included hTERT-based
protein vaccines, and dendritic cell-based vaccines (Minev et al,
PNAS, 97, 4796-4801, 2000; Vonderheide et al, Clin. Can. Res., 10,
828-839, 2004).
[0045] HER-2/neu is a transmembrane glycoprotein with tyrosine
kinase activity whose structure is similar to epidermal growth
factor. Amplification of her-2/neu gene and/or overexpression of
the associated protein have been reported in many human
adenocarcinomas of the breast, ovary, uterus, prostate, stomach,
oesophagus, pancreas, kidney and lung.
[0046] Therapeutic strategies targeting her-2/neu have included
CTLs from healthy donor PBMCs stimulated with HLA-A*0201-restricted
epitopes and dendritic cell-based vaccines and an immunogenic
protein with granulocyte-macrophage colony-stimulating factor
(Scardino et al, J. Immunol., 168, 5900-5906, 2002; Brossart et al,
Blood, 96, 3102-3108, 2000; Peoples et al, J. Clin. Oncol., 23,
7536-7545, 2005).
[0047] Immunogenic epitopes of hTERT and her-2/neu are described in
Minev et al, supra, Keogh et al, J. Immunol., 167, 787-796, 2001
and Kono et al, Int. J. Cancer, 78, 202-208, 1998.
[0048] The T-lymphocyte cellular-mediated immune response forms a
critical component of the immune response and plays a crucial role
in the eradication of tumor cells by the mammalian immune system. T
cell-mediated immune responses require the activation of cytotoxic
(CD8+) and helper (CD4+) T lymphocytes. Cytotoxic T lymphocytes
(CTL) and their T-cell receptors (TCR) recognize small peptides
presented by major histocompatibility complex (MHC) class I
molecules on the cell surface (Bjorkman P J., Cell 89:167-170
(1997); Garcia et al., Science 274:209-219 (1996)). The peptides
are derived from intracellular antigens via the endogenous antigen
processing and presentation pathway (Germain R N., Cell 76:287-299
(1994); Pamer et al., Annu Rev Immunol 16:323-358 (1998)). Peptides
for human CD8+ epitopes range from 7 to 14 amino acids, and
typically are 9-10 amino acids in length. TCR recognition of the
peptide-MHC class I molecule complexes on the cell surface triggers
the cytolytic activity of CTL, resulting in the death of cells
presenting the peptide-MHC class I complexes (Kagi et al., Science
265: 528-530 (1994)). MHC class I restricted epitope vaccines have
been shown to confer protection in some animal models. The
development of epitope vaccines encoding human HLA-restricted CTL
epitopes capable of conferring broad, effective, and non-ethnically
biased population coverage is highly desirable.
[0049] Epitope-based vaccines offer a number of advantageous
features compared to vaccines based on full-length TAAs, including
ease and low cost of peptide synthesis. In addition, peptide
vaccines can induce immune responses to subdominant epitopes when
there is tolerance to a dominant epitope, and anchor-modified or
heteroclitic peptide analogs can be constructed that can break
tolerance and/or further increase immunogenicity relative to native
peptides (for review, see Lazoura and Apostolopoulos, Current
Medicinal Chemistry 12: 1481-94 (2005)). The use of peptides as
immunogens also minimizes safety risks associated with the use of
intact proteins.
[0050] The identification of novel CEA epitopes and epitope analogs
that generate effective anti-tumor immune responses without causing
autoimmunity will allow the development of epitope-based cancer
vaccines that elicit a clinically relevant prophylactic and/or
therapeutic immune response against CEA.
[0051] The present invention thus includes the use of immunogenic
peptides ("epitopes") of CEA, hTERT and her-2/neu and analogs
thereof; which are selected based on their binding affinity for a
Class I MHC allele, specifically, HLA-A*0201. The peptides and
analogs described herein were selected based on their ability to
elicit a maximum tumor-specific immune response in a tolerized
setting, as well as for their minimal potential for eliciting
off-target autoimmune activity. More specifically, the CEA, hTERT
and her-2/neu proteins were scanned using a proprietary software
package called EI Suite that ranks protein fragments based on
binding affinity for HLA-A*0201, similarity to fragments of other
human proteins, and amenability to immunogenic enhancement.
[0052] Wild-type CEA epitopes of use in the present invention
include the sequence of amino acids shown in SEQ ID NO:1 or SEQ ID
NO:7. Wild-type hTERT epitopes of use in the present invention may
be selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:24.
Wild-type her-2/neu epitopes of use in the present invention may be
selected from SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:44. One favoured
epitope for her-2/neu is SEQ ID NO:36.
[0053] In addition to wild-type CEA, hTERT and her-2/neu epitopes,
in some embodiments of the invention described herein,
modifications were introduced at specific amino acid positions
within the naturally occurring sequence of the corresponding
wild-type immunogenic CEA, hTERT and her-2/neu peptides to form
anchor-modified analogs. Said analogs may provide an increased
benefit as a vaccine component if they induce an immune response
cross-reactive against CEA which is superior in quality to that
induced by the corresponding wild-type epitope. The immunogenic
peptide analogs of the present invention comprise a sequence of
amino acids selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
and SEQ ID NO:12 for CEA; SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:20
and SEQ ID NO:22 for hTERT; and SEQ ID NO:26, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ
ID NO:42 and SEQ ID NO:45 for her-2/neu.
[0054] Also disclosed are polynucleotides encoding said immunogenic
CEA, hTERT and her-2/neu peptides, peptide analogs, and variant
CEA, hTERT or her-2/neu proteins, as well as recombinant expression
vectors, including but not limited to, adenovirus and plasmid
vectors, comprising said polynucleotides. In some embodiments, the
vector is an adenovirus vector, which, in preferred embodiments, is
selected from the group consisting of: Ad5, Ad6, and Ad24. In
further exemplary embodiments, the polynucleotides comprise a
sequence of nucleotides that is operably linked to a promoter. Also
provided are recombinant host cells comprising the expression
vectors described herein.
[0055] Further disclosed are pharmaceutical compositions comprising
one or more of the immunogenic CEA, hTERT or her-2/neu peptides,
analogs, variant CEA, hTERT or her-2/neu proteins, or nucleic acids
encoding said peptides, analogs, and proteins, together with a
pharmaceutically acceptable carrier. In preferred embodiments, the
pharmaceutical composition comprises a plurality of immunogenic
peptides or analogs thereof. In some embodiments, the
pharmaceutical composition further comprises an adjuvant.
[0056] Also disclosed are vaccine compositions comprising one or
more of the CEA, hTERT or her-2/neu peptide epitopes, analogs,
variant proteins or comprising one or more polynucleotides encoding
said epitopes, analogs, or variant proteins disclosed throughout
the specification. In preferred embodiments, the vaccine
compositions comprise a plurality of isolated polynucleotides,
encoded peptides, or analogs thereof.
[0057] Further disclosed is a method of eliciting an immune
response to CEA, hTERT or her-2/neu in a patient in need thereof,
said method comprising introducing into the patient the
pharmaceutical compositions or vaccines disclosed herein.
[0058] The present invention further provides methods for
inhibiting the development of a cancer in a mammal, or treating or
minimizing an existing cancer, by eliciting an immune response to
CEA, hTERT or her-2/neu, such methods comprising administering a
vaccine or pharmaceutical composition comprising one or more
immunogenic CEA, hTERT or her-2/neu peptide described herein, or
analog thereof, or polynucleotide encoding said peptide or analog,
as described herein. In preferred embodiments of the methods
herein, the immune response is enhanced relative to the response
elicited by a wild-type CEA, hTERT or her-2/neu peptide.
[0059] As used throughout the specification, the following
definitions and abbreviations apply:
[0060] The term "promoter" refers to a recognition site on a DNA
strand to which the RNA polymerase binds. The promoter forms an
initiation complex with RNA polymerase to initiate and drive
transcriptional activity. The complex can be modified by activating
sequences termed "enhancers" or inhibiting sequences termed
"silencers".
[0061] The term "cassette" refers to a nucleotide or gene sequence
that is to be expressed from a vector, for example, a nucleotide or
gene sequence encoding one or more of the CEA peptide epitopes,
analogs, or modified CEA proteins described herein. In general, a
cassette comprises a gene sequence that can be inserted into a
vector, which in some embodiments, provides regulatory sequences
for expressing the nucleotide or gene sequence. In other
embodiments, the nucleotide or gene sequence provides the
regulatory sequences for its expression. In further embodiments,
the vector provides some regulatory sequences and the nucleotide or
gene sequence provides other regulatory sequences. For example, the
vector can provide a promoter for transcribing the nucleotide or
gene sequence and the nucleotide or gene sequence provides a
transcription termination sequence. The regulatory sequences that
can be provided by the vector include, but are not limited to,
enhancers, transcription termination sequences, splice acceptor and
donor sequences, introns, ribosome binding sequences, and poly(A)
addition sequences.
[0062] The term "vector" refers to some means by which DNA
fragments can be introduced into a host organism or host tissue.
There are various types of vectors including plasmid, virus
(including adenovirus), bacteriophages and cosmids.
[0063] The term "first generation," as used in reference to
adenoviral vectors, describes adenoviral vectors that are
replication-defective. First generation adenovirus vectors
typically have a deleted or inactivated E1 gene region, and
preferably have a deleted or inactivated E3 gene region.
[0064] The term "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures. Those in need of
treatment include those already with the disorder as well as those
prone to have the disorder or those in which the disorder is to be
prevented.
[0065] A "disorder" is any condition that would benefit from
treatment with the molecules of the present invention, including
the CEA peptide epitopes, CEA epitope analogs, modified CEA
proteins and nucleic acid molecules encoding said epitopes,
analogs, and modified proteins. Encompassed by the term "disorder"
are chronic and acute disorders or diseases including those
pathological conditions which predispose the mammal to the disorder
in question. The molecules of the present invention are intended
for use as treatments for disorders or conditions characterized by
aberrant cell proliferation, including, but not limited to,
pancreatic cancer, liver cancer, breast cancer, colorectal cancer,
ovarian cancer, cervical cancer, and lung cancer.
[0066] The term "effective amount" means sufficient vaccine
composition is introduced to produce the adequate levels of the
polypeptide, so that a clinically significant immune response
results. One skilled in the art recognizes that this level may
vary.
[0067] The term "nucleic acid" or "nucleic acid molecule" is
intended for ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),
probes, oligonucleotides, fragment or portions thereof, and
primers.
[0068] "Wild-type CEA", "wild-type hTERT", "wild-type her-2/neu" or
"wild-type protein" or "wt protein" refers to a CEA protein
comprising a naturally occurring sequence of amino acids, which
sequence is encoded by the major allele of CEA found in the human
population, free of induced mutations or modifications.
[0069] The term "variant protein", "variant CEA", "variant hTERT"
or "variant her-2/neu" refers to a CEA, hTERT or her-2/neu protein
comprising modifications to at least one specific amino acid
residue of the CEA protein relative to the full-length wild-type
CEA, hTERT or her-2/neu protein.
[0070] The term "mammalian" refers to any mammal, including a human
being.
[0071] The abbreviation "Ag" refers to an antigen. The term
"antigen" refers to any biologic or macromolecular substance that
can be recognized by a T-cell or an antibody molecule.
[0072] The abbreviations "Ab" and "mAb" refer to an antibody and a
monoclonal antibody, respectively.
[0073] The terms "major histocompatibility complex (MHC)" and
"human leukocyte antigen (HLA)" are used interchangeably to refer
to a locus of genes that encode proteins which present a vast
variety of peptides onto the cell surface for specific recognition
by a T-cell receptor. A subclass of MHC genes, called Class I MHC
molecules, present peptides to CD8.sup.+T-cells.
[0074] "Epitope" refers to a peptide which is a portion of an
antigen, wherein the peptide comprises an amino acid sequence that
is capable of stimulating an immune response. The MHC class I
epitopes disclosed herein are useful in pharmaceutical compositions
(e.g., vaccines) for stimulating an immune response directed to
CEA. In preferred embodiments, epitopes according to this
definition represent peptides which are likely to be non-covalently
bound to the binding cleft of a class I MHC molecule on the surface
of antigen presenting cells in a manner which facilitates its
interaction with T-cell receptors (TCR). The term "epitope" is used
interchangeably herein with the term "immunogenic peptide."
[0075] The term "wild type epitope" refers to an epitope comprising
a sequence of nine or ten amino acids that can be found in the
naturally occurring wild-type CEA protein as set forth in SEQ ID
NO:1.
[0076] The terms "9-mer" and "10-mer" refer to a linear sequence of
nine or ten amino acids that occur in a target antigen. It is
generally understood that a collection of sequences that includes
all possible 9-mers and 10-mers present in a parent sequence,
comprise sequences which overlap by eight or nine residues,
respectively.
[0077] The term "anchor residues" refers to the amino acid residues
of an immunogenic peptide fragment that provide a contact point
between the peptide and the MHC molecule. The anchor residues
comprise side chains that fit into the peptide-binding clefts of
said MHC molecules.
[0078] "Binding motif" refers to a specific pattern or combination
of anchor residues within protein sequences which are correlated
with the ability to bind to a specified HLA allele or
serotypes.
[0079] "Immunogen" refers to specific antigens that are capable of
inducing or stimulating an immune response. Not all antigens are
immunogenic.
[0080] "Enhanced immunogenicity" refers an increased ability to
activate the immune system when compared to the immune response
elicited by the wild-type peptide. A variant peptide or analog can
be said to have "increased immunogenicity" if it induces a higher
level of T-cell activation relative to the level of activation
induced by the corresponding wild-type peptide as measured in a
standard in-vitro T-cell activation assay. In a preferred
embodiment, the frequency of vaccination-induced epitope-specific
T-cells will be increased at least 10-fold by the administration of
an immunoenhanced analogs relative to the level of T-cell
activation (i.e., number of epitope-specific CTLs) induced by
immunization with the parent peptide. A 50-fold increase in T-cell
activity is an especially preferred level of immunoenhancement.
[0081] "Immunogenic analog" or "epitope analog" refers to a peptide
epitope with one or more residues of the wild-type amino acid
sequence substituted with an alternative amino acid sequence
identified by the immunoenhancement filter of EI Suite. Coordinated
substitutions are often carried out to regulate or modify (e.g.,
increase) immunogenicity of a natural peptide.
[0082] The terms "prediction" and "predicting" are used herein
refer to the use of the present teachings to estimate properties
(e.g., ability to bind to MHC class I allele, likelihood of being
efficiently processed and presented by APC, uniqueness to target
antigen, immunogenicity) of amino acid sequences representing
putative T-cell epitopes.
[0083] The terms "MHC class I binder" and "MHC peptide" are used to
refer to peptides having a high known or predicted binding affinity
for a mammalian class I major histocompatibility complex (MHC)
molecule.
[0084] "Immunogenic composition" refers to a composition that is
capable of inducing an immune response, a reaction, an effect,
and/or an event. In some embodiments, such responses, reactions,
effects, and/or events can be induced in vitro or in vivo. For
example, the induction, activation, or expansion of cells involved
in cell mediated immunity, such as CTLs represents an immune
response, effect or an event. Representative immunogenic
compositions include an immunoenhanced full-length target antigen
or a minigene vaccine.
[0085] "Vaccine" refers to an immunogenic composition that is
capable of eliciting a clinically relevant prophylactic and/or
therapeutic immune response that prevents, cures, or ameliorates
disease.
[0086] "Epitope vaccine" generally refers to a composition of
several epitopes derived from one or more target proteins of the
same, or different pathogen or tumor cell, specific to one or more
alleles of interest. The list of epitopes used may include those
optimized for natural processing, immunogenicity, uniqueness (e.g.,
lack of similarity to other self-antigens), population coverage,
and predicted disease relevance. For example, an immunogenic
composition comprising more than one putative T-cell epitope
derived from at least one target antigen linked together, with or
without additional amino acids ("spacers") between the epitopes can
be used as an epitope vaccine. Thus, an epitope vaccine can
stimulate immune responses directed to single or multiple epitopes
of one or more antigens.
[0087] "EI Suite" refers to a software package useful for
identifying immunogenic epitopes described in WO-A-06124408.
[0088] The CEA, hTERT and her-2/neu epitopes and analogs provided
herein were selected based on their binding affinity for a class I
MHC allele, specifically, HLA-A*0201. Said peptides and analogs
were additionally selected based on their ability to elicit a
maximum tumor-specific immune response in a tolerized setting, as
well as for their minimal potential for eliciting off-target
autoimmune activity.
[0089] More specifically, the CEA, hTERT and her-2/neu epitopes and
analogs were initially selected by scanning the CEA, hTERT and
her-2/neu protein sequences using a proprietary software package
called EI Suite that ranks protein fragments based on binding
affinity for a Class I MHC allele, in this case HLA-A*0201,
similarity to fragments of other human and murine proteins, and
amenability to immunogenic enhancement (see WO 2006/0257944).
Epitopes and analogs predicted to be MHC class I binders were
analyzed in vitro for binding affinity to T2 cells, which are
HLA-A*0201 positive, MHC class II negative and TAP deficient.
Moreover, the immunogenicity of the selected peptide epitopes and
analogs was determined in HHD transgenic mice. HHD mice are
transgenic for the HHD complex (human .beta.2-microglobulin fused
to HLA-A2.1 .alpha.1 and .alpha.2 domain, H-2D.sup.b .alpha.3
domain) and are devoid of H-2D.sup.b and murine
.beta.-microglobulin. (Pascolo et al. J. Exp. Med. 185(12):2043-51
(1997)). For this reason, the immune response elicited in these
mice is specifically restricted to human HLA-A2.1, making this line
a suitable model for epitope identification and optimization.
[0090] The isolated immunogenic CEA, hTERT and her-2/neu peptides
were predicted to be optimal vaccine candidates by EI Suite and
were determined to bind HLA-A*0201 and to be immunogenic in HHD
transgenic mice. CEA, hTERT and her-2/neu epitopes present in the
major wild-type CEA allele and identified in this manner are
disclosed in SEQ ID NOs: 1, 7, 13, 14, 16, 18, 19, 21, 23, 24, 25,
27, 28, 30, 32, 34, 36, 38, 40, 41, 43 and 44.
[0091] Class I MHC molecules are heterodimers of non-covalently
bound MHC-encoded heavy (or alpha) chain, and a non-MHC-encoded
.beta.2-microglobulin light chain. There are four separate regions:
1) the peptide binding region, 2) the immunoglobulin-like region,
3) the transmembrane region and 4) a cytoplasmic region. The
peptide-binding region is a groove which functions to accommodate a
peptide ligand of 8-10 amino acid residues. To this end, the CEA,
hTERT and her-2/neu epitopes and analogs consist of a linear
sequence of nine or ten amino acids. In preferred embodiments, the
epitopes and analogs are nine or ten amino acids in length.
[0092] The binding of peptide ligands to the MHC binding groove is
specific and is stabilized at both ends by contacts between atoms
in the free amino and carboxyl termini of the peptide and invariant
sites that are found at each end of the cleft of all MHC class I
molecules. Because the amino acid side chains at these positions
insert into pockets in the MHC molecule and function to anchor the
peptide to the MHC molecule they are commonly referred to as anchor
residues. The bound peptide lies in an extended conformation along
the groove. Anchor residues can be divided into primary and
secondary. Primary anchor positions exhibit strong preferences for
relatively well-defined sets of amino acid residues. Secondary
positions show weaker and/or less well-defined preferences that can
often be better described in terms of less favored, rather than
more favored residues.
[0093] The anchor residues confer sequence selectivity and binding
specificity to the interaction between the peptide ligand and the
MHC molecule. The main anchor residues of human HLA class I
molecules occur at positions 2 and the C-terminus of the peptides.
Generally speaking, peptide-binding to a particular MHC molecule
requires the peptide to have one or more specific amino acids at a
fixed position, frequently the terminal or penultimate amino acid
of the peptide. Since more stable binding will generally improve
immunogenicity, anchor residues are preferably conserved or
optimized in the design of analogs, regardless of their
position.
[0094] As discussed above, it is known in the art that HLA class I
binders modified at anchor positions (position 2 and the C-terminus
position of the peptide), are often more immunogenic than the
wild-type peptide due to improved binding to the HLA molecule (G.
Lipford et. al., Immunology 84: 298-303 (1995)). At the same time,
T-cells specific for the modified peptide generally also recognize
the wild-type peptide, since the mutations are restricted to
residues that do not make contact with the T-cell receptor. Based
on this knowledge, the immunogenicity enhancement filter of EI
Suite was utilized to identify anchor-modified analogs that
comprise substitutions/mutations that optimize peptide/MHC binding
interactions at the anchor positions. Said immunoenhancement filter
identified substitutions within anchor residues of CEA T-cell
epitope candidates to improve their immunogenicity. Immunoenhanced
peptide analogs that are cross-reactive to the target antigen are
beneficial for use in cancer vaccines targeting tumor-associated
antigens to overcome tolerance and poor immunogenicity.
[0095] To this end, there is provided anchor-modified peptide
analogs which can elicit an immune response against CEA, hTERT or
her-2/neu that is stronger than the immune response elicited by the
corresponding wild-type epitope. The peptide analogs of the present
invention comprise a sequence of amino acids selected from those
shown in Tables 1, 2 and 3. Said peptide analogs consist of a
linear sequence of nine or ten amino acids.
[0096] Also disclosed are nucleotides encoding the immunogenic
epitopes and epitope analogs which are useful either alone or in
combination to construct DNA-based vaccines and minigenes targeting
CEA, hTERT or her-2/neu. Said nucleotides are useful in genetic
vaccines to elicit or enhance immunity to the protein product
expressed by the CEA, hTERT and her-2/neu tumor-associated
antigens.
[0097] Accordingly, the present invention relates to an isolated
nucleic acid molecule or polynucleotide comprising a sequence of
nucleotides encoding an epitope analog, said analog comprising a
sequence of amino acids as set forth in Tables 1, 2 and 3. The
nucleic acid molecules of the present invention are substantially
free from other nucleic acids.
[0098] Also disclosed are recombinant vectors and recombinant host
cells, both prokaryotic and eukaryotic, which contain the nucleic
acid molecules disclosed throughout this specification. The
isolated DNA molecules, associated vectors, and hosts of the
present invention are useful for the development of a cancer
vaccine.
[0099] Vectors may be comprised of DNA or RNA. For most cloning
purposes, DNA vectors are preferred. Typical vectors include
plasmids, modified viruses, baculovirus, bacteriophage, cosmids,
yeast artificial chromosomes, and other forms of episomal or
integrated DNA that can encode a CEA, hTERT or her-2/neu fusion
protein. It is well within the purview of the skilled artisan to
determine an appropriate vector for a particular gene transfer or
other use.
[0100] An expression vector containing a CEA, hTERT or her-2/neu
peptide-encoding nucleic acid molecule may be used for high-level
expression of CEA, hTERT or her-2/neu peptides in a recombinant
host cell. Expression vectors may include, but are not limited to,
cloning vectors, modified cloning vectors, specifically designed
plasmids or viruses. Also, a variety of bacterial expression
vectors may be used to express recombinant CEA, hTERT or her-2/neu
peptide sequences in bacterial cells if desired. In addition, a
variety of fungal cell expression vectors may be used to express
recombinant CEA, hTERT or her-2/neu peptide sequences in fungal
cells. Further, a variety of insect cell expression vectors may be
used to express recombinant peptides in insect cells.
[0101] Also disclosed are host cells transformed or transfected
with vectors comprising the nucleic acid molecules of the present
invention. Recombinant host cells may be prokaryotic or eukaryotic,
including but not limited to, bacteria such as E. coli, fungal
cells such as yeast, mammalian cells including, but not limited to,
cell lines of bovine, porcine, monkey and rodent origin; and insect
cells including but not limited to Drosophila and silkworm derived
cell lines. In one embodiment of the invention, the host cell is a
yeast cell which is selected from the group consisting of:
Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris,
Kluyvermyces fragilis, Kluveromyces lactis, and Schizosaccharomyces
pombe. Such recombinant host cells can be cultured under suitable
conditions to produce a CEA, hTERT or her-2/neu peptide epitope or
epitope analog. In one embodiment of the present invention, the
host cell is human. As defined herein, the term "host cell" is not
intended to include a host cell in the body of a transgenic human
being, human fetus, or human embryo.
[0102] The nucleic acids may be assembled into an expression
cassette which comprises sequences designed to provide for
efficient expression of the peptide, analog, variant protein, or
minigene in a human cell. The cassette preferably contains a
peptide epitope or analog-encoding gene, or minigene, with related
transcriptional and translations control sequences operatively
linked to it, such as a promoter, and termination sequences. The
promoter may be the cytomegalovirus promoter without the intron A
sequence (CMV), although those skilled in the art will recognize
that any of a number of other known promoters, or other eukaryotic
gene promoters may be used. A preferred transcriptional terminator
is the bovine growth hormone terminator, although other known
transcriptional terminators may also be used. The combination of
CMV-BGH terminator is particularly preferred.
[0103] The CEA, hTERT or her-2/neu peptide expression cassette is
inserted into a vector. The vector is preferably an adenoviral or
plasmid vector, although linear DNA linked to a promoter, or other
vectors, such as adeno-associated virus or a modified vaccinia
virus, retroviral or lentiviral vector may also be used.
[0104] In one embodiment of the invention, the vector is an
adenovirus vector (used interchangeably herein with "adenovector").
Adenovectors can be based on different adenovirus serotypes such as
those found in humans or animals. Examples of animal adenoviruses
include bovine, porcine, chimp, murine, canine, and avian (CELO).
Preferred adenovectors are based on human serotypes, more
preferably Group B, C, or D serotypes. Examples of human adenovirus
Group B, C, D, or E serotypes include types 2 ("Ad2"), 4 ("Ad4"), 5
("Ad5"), 6 ("Ad6"), 24 ("Ad24"), 26 ("Ad26"), 34 ("Ad34") and 35
("Ad35"). In particularly preferred embodiments of the present
invention, the expression vector is an adenovirus type 5 or 6 (Ad 5
or Ad6) vector.
[0105] If the vector chosen is an adenovirus, it is preferred that
the vector be a so-called first-generation adenoviral vector. These
adenoviral vectors are characterized by having a non-functional E1
gene region, and preferably a deleted adenoviral E1 gene region.
Adenovectors do not need to have their E1 and E3 regions completely
removed. Rather, a sufficient amount the E1 region is removed to
render the vector replication incompetent in the absence of the E1
proteins being supplied in trans; and the E1 deletion or the
combination of the E1 and E3 deletions are sufficiently large
enough to accommodate a gene expression cassette.
[0106] In some embodiments, the expression cassette is inserted in
the position where the adenoviral E1 gene is normally located. In
addition, these vectors optionally have a non-functional or deleted
E3 region. In some embodiments of the invention, the adenovirus
genome used is deleted of both the E1 and E3 regions
(.DELTA.E1.DELTA.E3). The adenoviruses can be multiplied in known
cell lines which express the viral E1 gene, such as 293 cells, or
PERC.6 cells, or in cell lines derived from 293 or PERC.6 cell
which are transiently or stablily transformed to express an extra
protein. For examples, when using constructs that have a controlled
gene expression, such as a tetracycline regulatable promoter
system, the cell line may express components involved in the
regulatory system. One example of such a cell line is T-Rex-293;
others are known in the art.
[0107] For convenience in manipulating the adenoviral vector, the
adenovirus may be in a shuttle plasmid form. This invention is also
directed to a shuttle plasmid vector which comprises a plasmid
portion and an adenovirus portion, the adenovirus portion
comprising an adenoviral genome which has a deleted E1 and optional
E3 deletion, and has an inserted expression cassette comprising an
epitope, analog, or CEA minigene. In preferred embodiments, there
is a restriction site flanking the adenoviral portion of the
plasmid so that the adenoviral vector can easily be removed. The
shuttle plasmid may be replicated in prokaryotic cells or
eukaryotic cells.
[0108] In one embodiment, the expression cassette is inserted into
an Ad6 (.DELTA.E1.DELTA.E3) adenovirus plasmid (See Emini et al.,
WO2003031588A2, which is hereby incorporated by reference). This
vector comprises an Ad6 adenoviral genome deleted of the E1 and E3
regions. In some embodiments of the invention, the expression
cassette is inserted into the pMRKAd5-HV0 adenovirus plasmid (See
Emini et al., WO 02/22080, which is hereby incorporated by
reference). This plasmid comprises an Ad5 adenoviral genome deleted
of the E1 and E3 regions. The design of the pMRKAd5-HV0 plasmid was
improved over prior adenovectors by extending the 5' cis-acting
packaging region further into the E1 gene to incorporate elements
found to be important in optimizing viral packaging, resulting in
enhanced virus amplification. Advantageously, these enhanced
adenoviral vectors are capable of maintaining genetic stability
following high passage propagation.
[0109] Standard techniques of molecular biology for preparing and
purifying DNA constructs enable the preparation of the
adenoviruses, shuttle plasmids, and DNA immunogens mentioned
herein.
[0110] The vectors described above may be used in immunogenic
compositions and vaccines for preventing or decreasing the
likelihood of the development of adenocarcinomas associated with
aberrant CEA, hTERT or her-2/neu expression and/or for treating
existing cancers. The vectors allow for vaccine development and
commercialization by providing an immunogenic CEA, hTERT or
her-2/neu peptide which can elicit an enhanced immune response,
relative to full-length wild-type CEA, hTERT or her-2/neu when
administered to a mammal such as a human being.
[0111] In accordance with the method described above, the vaccine
vector may be administered for the treatment or prevention of a
cancer in any mammal, including but not limited to: lung cancer,
breast cancer, and colorectal cancer. In a preferred embodiment of
the invention, the mammal is a human. The cancer may be of the
ovary, uterus, stomach, oesophagus, prostate or kidney.
[0112] Further, one of skill in the art may choose any type of
vector for use in the treatment and prevention method described.
Preferably, the vector is an adenovirus vector or a plasmid vector.
In a preferred embodiment of the invention, the vector is an
adenoviral vector comprising an adenoviral genome with a deletion
in the adenovirus E1 region, and an insert in the adenovirus E1
region, wherein the insert comprises an expression cassette
comprising: a polynucleotide comprising a sequence of nucleotides
that encodes at least one immunogenic CEA, hTERT or her-2/neu
analog as described herein and as set forth in SEQ ID NOs:3-7 and
9-13, and a promoter operably linked to the polynucleotide. In a
preferred embodiment the adenovirus vector is an Ad 5 vector, an
Ad6 vector, or an Ad 24 vector.
[0113] Also disclosed is a vaccine plasmid comprising a plasmid
portion and an expression cassette portion, the expression cassette
portion comprising: (a) a sequence of nucleotides that encodes an
immunogenic T-cell peptide epitope analog of CEA as set forth in
SEQ ID NOs:3-7 and 9-13; and (b) a promoter operably linked to the
polynucleotide.
[0114] The amount of expressible DNA or transcribed RNA to be
introduced into a vaccine recipient will depend partially on the
strength of the promoters used and on the immunogenicity of the
expressed gene product. In general, an immunologically or
prophylactically effective dose of about 1 ng to 100 mg, and
preferably about 0.25-5 mg of a plasmid vaccine vector is
administered directly into muscle tissue. An effective dose for
recombinant adenovirus is approximately 10.sup.6-10.sup.12
particles and preferably about 10.sup.7-10.sup.11 particles.
[0115] It may be desirable for the vaccine vectors to be in a
physiologically acceptable solution, such as, but not limited to,
sterile saline or sterile buffered saline. Alternatively, it may be
advantageous to administer an agent which assists in the cellular
uptake of DNA, such as, but not limited to calcium ion. These
agents are generally referred to as transfection facilitating
reagents and pharmaceutically acceptable carriers. Those of skill
in the art will be able to determine the particular reagent or
pharmaceutically acceptable carrier as well as the appropriate time
and mode of administration.
[0116] It is a common goal of vaccine development to augment the
immune response to the desired antigen to induce long lasting
protective and therapeutic immunity. Co-administration of vaccines
with compounds that can enhance the immune response against the
antigen of interest, known as adjuvants, has been extensively
studied. In addition to increasing the immune response against the
antigen of interest, some adjuvants may be used to decrease the
amount of antigen necessary to provoke the desired immune response
or decrease the number of injections needed in a clinical regimen
to induce a durable immune response and to provide protection from
disease and/or induce regression of disease.
[0117] Therefore, the vaccines and immunogenic compositions
described herein may be formulated with an adjuvant in order to
primarily increase the immune response. Adjuvants which may be used
include, but are not limited to, adjuvants containing CpG
oligonucleotides, (see A. M. Krieg, Biochimica et Biophysica Acta,
1489, 107-116, 1999) or other molecules acting on toll-like
receptors such as TLR9 (for review, see, Daubenberger, C. A., Curr.
Opin. Mol. Ther. 9(1):45-52 (2007)), T-helper epitopes, lipid-A and
derivatives or variants thereof, liposomes, cytokines, (e.g.
granulocyte macrophage-colony stimulating factor (GMCSF)), CD40,
CD28, CD70, IL-2, heat-shock protein (HSP) 90, CD134 (OX40), CD137,
non ionic block copolymers, incomplete Freund's adjuvant, and
chemokines. Additional adjuvants for use with the compositions
described herein are adjuvants containing saponins (e.g. QS21),
either alone or combined with cholesterol and phospholipid in the
characteristic form of an ISCOM ("immune stimulating complex," for
review, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25
(1996); and Skene and Sutton, Methods 40: 53-59 (2006)).
Additionally, aluminum-based compounds, such as aluminum hydroxide
(Al(OH).sub.3), aluminum hydroxyphosphate (AlPO.sub.4), amorphous
aluminum hydroxyphosphate sulfate (AAHS) or so-called "alum"
(KAl(SO.sub.4).12H.sub.2O), many of which have been approved for
administration into humans by regulatory agencies worldwide, may be
combined with the compositions provided herein.
[0118] As stated above, the epitopes and analogs and minigenes of
the present invention can be included in an immunogenic composition
or vaccine, which can then be administered to a human patient in
need thereof to induce an immune response. Moreover, the
administration of immunogenic compositions and vaccines comprising
analogs to a patient in need thereof can effectively elicit an
enhanced cellular immune response that is cross-reactive to human
CEA, hTERT or her-2/neu protein relative to native epitopes.
[0119] Immunogenic compositions may be used alone at appropriate
dosages which allow for optimal induction of a cellular immune
response against CEA, hTERT or her-2/neu with minimal potential
toxicity. In addition, co-administration or sequential
administration of other agents may be desirable.
[0120] The compositions may be administered to a patient by
intramuscular injection, subcutaneous injection, intradermal
introduction, or impression though the skin. Other modes of
administration such as intraperitoneal, intravenous, or inhalation
delivery are also contemplated. In preferred embodiments of the
invention, the vaccines and pharmaceutical compositions are
administered by intramuscular administration.
[0121] The minigenes of the present invention may be administered
with an adjuvant, such as those described above. In particular, the
adjuvant may be a natural or synthetic immunomodulatory
oligodeoxynucleotide generally containing a CpG dinucleotide,
particularly when this dinucleotide occurs in certain base contexts
(CpG-S motifs). Alternatively the adjuvant may be an anti-CTLA4
antibody such as that described in Hodi et al, PNAS, 100,
4712-4717, 2003.
[0122] The minigenes of the present invention may also be
administered simultaneously, sequentially or subsequently with a
peptide containing promiscuous T-cell epitopes, such as tetanus
toxoid peptide p2 or p30, see Valmori et al, supra.
[0123] Electroporation may be used to improve uptake of the
minigenes according to the present invention into the subject to be
vaccinated, for example using the method described in Babiuk et al,
Vaccine, 20, 3399-3408, 2002.
[0124] The present invention thus also relates to a pharmaceutical
composition comprising a minigene of the invention and a
pharmaceutically acceptable excipient. The composition may be for
the treatment or prophylaxis of cancer or an autoimmune disease or
condition.
[0125] The present invention also relates to minigenes of the
present invention for use in a method of treatment of the human
body by therapy, such as the treatment or prophylaxis of cancer or
an autoimmune disease or condition.
[0126] Thus, there is also disclosed a method of treating a subject
suffering from or prone to cancer or an autoimmune disease or
condition by therapy or prophylaxis which comprises administering
to that subject a therapeutically or prophylactically effective
amount of a minigene according to the present invention.
[0127] The present invention also provides the use of a minigene of
the invention for the manufacture of a medicament for the treatment
or prophylaxis of cancer or an autoimmune disease or condition.
[0128] In general the minigene is administered to humans in an
amount of 0.1-10 mg, particularly 1 to 5 mg, especially about 2.5
mg. The administration is generally intramuscular and is preferably
followed by electroporation.
[0129] The following examples illustrate, but do not limit the
invention.
Example 1
Epitope Identification by EI Suite
[0130] In order to identify epitope candidates that may elicit
maximum tumor-specific immune response in a tolerized setting,
while minimizing potential for off-target autoimmune activity, the
entire CEA protein was scanned with several independent filters,
using a proprietary software package called EI Suite (as described
in WO 2006/124408). EI Suite ranks protein fragments based on
binding affinity for a Class I MHC allele (in this case,
HLA-A*0201), similarity to fragments of other human and murine
proteins, and amenability to immunogenic enhancement.
[0131] A total of 12 HLA-A*0201-restricted peptides and peptide
analogs were identified by EI Suite as having the highest potential
for use in an epitope-based vaccine or for in vitro monitoring of
vaccine-induced CEA-specific CTL responses. The selected CEA
peptide epitopes are shown in Table 1
TABLE-US-00001 SEQ ID Position Peptide NO. Type CEA.691 IMIGVLVGV 1
Wild type CEA.411V10 VLYGPDDPTV 2 Anchor-modified analog CEA.690L2
GLMIGVLVGV 3 Anchor-modified analog CEA.589V10 VLYGPDTPIV 4
Anchor-modified analog CEA.682V10 GLSAGATVGV 5 Anchor-modified
analog CEA.307V10 GLNRTTVTTV 6 Anchor-modified analog CEA.687
ATVGIMIGV 7 Wild type CEA.100V10 IIYPNASLLV 8 Anchor-modified
analog CEA310L2 RLTVTTITV 9 Anchor-modified analog CEA605V9
YLSGANLNV 10 Anchor-modified analog CEA687L2 ALVGIMIGV 11
Anchor-modified analog CEA691L2 ILIGVLVGV 12 Anchor-modified
analog
and include 2 wild-type CEA peptides and 10 peptide analogs.
Examples 2-4 provide the details of peptide selection.
Example 2
The A*0201 Binding Affinity Filter
[0132] HLA binding affinity has been shown to correlate with T-cell
recognition for peptides derived from viral antigens (Sette et al.
J. Immunol. 153: 5586-92 (1994)). More recently, this relationship
has also been observed for tumor epitopes (Keogh et al. J. Immunol.
167: 787-796 (2001)). Selecting potential epitopes on the basis of
HLA binding affinity is, therefore, an efficient alternative to
large-scale epitope mapping or other T cell-dependent
strategies.
[0133] Therefore, using EI Suite, the sequence of the CEA protein
was scanned for peptides that were predicted to be strong binders
to the HLA class I allele A*0201 (referred to as A2.1 below). A
total of 1267 peptides representing all possible 9- and 10-mer
frames of the 702 amino acid protein were evaluated. Each peptide
was scored based on the degree of adherence to a statistical motif
inferred from several publicly available epitope databases,
including: SYFPEITHI (Rammensee et al. Immunogenetics 50: 213-19
(1999)), MHCBN (Bhasin et al. Bioinformatics 19(5): 665-66 (2003),
and FIMM (Schonbach et al. Nucleic Acids Res. 28(1): 222-24
(2000)). 150 top-scoring fragments were retained for further
analysis.
Example 3
Comparison of CEA Peptides with Fragments of Other Human
Proteins
[0134] A vaccine containing only CEA-specific epitopes may prevent
the off-target T-cell response that sometimes occurs when immune
tolerance is broken to a shared antigen. The CTL-mediated
destruction of melanocytes (van Elsas et al. J. Exp. Med. 190:
355-366 (1999)) and pancreatic islet .beta.-cells (Ludewig et al.
J. Exp. Med. 2000, 191: 795-804 (2000)) following immunotherapy are
two examples of such off-target immune response. Moreover, CEA
peptides that are also found in other proteins that are expressed
in multiple normal tissues are more likely to have been presented
to T-cells in a tolerizing setting Immune tolerance to such
peptides may therefore be more difficult to overcome. For this
reason, EI Suite was used to select, from the 150 top-scoring CEA
peptides, those unique to the CEA protein.
[0135] It is known that T-cells can often recognize cognate
epitopes comprising modifications at the HLA contact positions
(positions 2 and 9/10 for HLA-A2.1) (Keogh (2001), supra). In
contrast, mismatches within the TCR contact region (positions 1 and
3-8/9, for HLA-A2.1) are generally expected to lead to a loss of
recognition. Therefore, to minimize the likelihood of
vaccine-induced autoimmunity, CEA peptides whose TCR contact region
was identical to a fragment of another human protein were rejected
as epitope candidates even if they were predicted to be strong HLA
binders and potentially immunogenic. Of 150 top-scoring CEA
peptides, 49 were rejected because their TCR contact residues were
identical to a fragment of another human protein.
[0136] However, a certain degree of degeneracy in peptide
recognition exists with respect to changes in the TCR contact
region (Sparbier et al. Curr. Opin. Imm. 11: 214-21 (1999)). In
fact, depending on the peptide and the T-cell clone, changes in the
TCR contact region may result in an enhanced recognition, loss of
recognition, or an intermediate response (Scardino et al. Eur. J.
Imm. 31: 3261-3270 (2001)). In light of this fact, an expanded
search of the human proteome was performed, allowing a single
mismatch in the TCR binding region, in addition to a mismatch, if
any, at positions 2 or 9/10. Of 150 top-scoring CEA peptides, 105
had sufficiently close matches with other proteins by this
definition. These peptides were flagged to indicate that, if
selected as vaccine candidates, they must undergo additional
screening to rule out cross-recognition of proteins other than the
target protein CEA. Interestingly, 15/150 (10%) top-scoring CEA
peptides had matches outside the CEA family.
[0137] The output of the self-similarity filter for the 150
top-scoring CEA peptides is summarized in FIG. 2. Most of the
matches were within proteins in the CEACAM branch (CEA-like cell
adhesion molecule) of the CEA family (Nomenclature Announcement,
Exp. Cell Res. 252: 243-249 (1999)). In fact, 44/150 (29%)
top-scoring CEA peptides were identical to a fragment of another
CEACAM, and a few occurred in a majority of expressed human
CEACAMs, including biliary glycoprotein (BGP, CEACAM1) and
non-specific cross-reacting antigen (NCA, CEACAM6). BGP and NCA are
both known to be expressed broadly in normal epithelia, including
pancreas, lung, liver, kidney, and cervix (Hammarstrom et al. Semin
Cancer Biol. 9: 67-81 (1999)). NCA is also known to be
overexpressed in colorectal carcinoma (Koops et al. Eur J Biochem
253(3): 778-786 (1998)).
[0138] 17% (26/150) top-scoring CEA peptides matched a
pregnancy-specific glycoprotein (PSG) (Hammarstrom et al. 1999,
supra). CEA-like cell adhesion molecules (CEACAM) and
pregnancy-specific glycoproteins (PSG) form two branches of the CEA
family, with CEA being in the CEACAM subset. PSGs have a unique
expression profile compared to CEACAM members, with ubiquitous
expression noted in the placenta during fetal development, and a
more restrictive expression pattern seen in the normal adult
tissues pancreas, salivary glands, and brain (Hammarstrom et al.
1999, supra).
Example 4
Anchor-Modified Peptide Analogs
[0139] It is known that the use of peptides modified at one or more
HLA contact positions, called anchor-modified peptide analogs, may
improve HLA binding affinity (See, e.g. Lipford et al. Immunology
84: 298-303 (1998)). As a result of improved HLA binding, the
modified peptide is often more immunogenic than the original
wild-type (wt) peptide. Furthermore, analog-specific T-cells
generally recognize the wt peptide because of the identical amino
acid residues in the TCR contact region.
[0140] Therefore, anchor-modified immunogenic analogs were
identified for 11 of 14 CEA peptides that were not rejected by the
self-similarity filter and were predicted to be moderate or better
A2.1 binders (the score of 2 or higher). Most of the analogs
identified comprise an Ito V mutation at the C-terminus Peptides
with this mutation had a greater affinity for A2.1 and elicited
T-cells that recognized the wt peptide more consistently than any
other alteration in the HLA contact region (Keogh et al. J.
Immunol. 167: 787-796 (2001)). Analog CEA.690L2 was not modified at
the C-terminus because the wt peptide CEA.690 already comprises a
valine in this position. Instead, a substitution to a leucine, a
preferred amino acid at position 2 in the A*0201 binding motif, was
made to improve binding affinity.
[0141] Anchor modifications for weaker HLA binders (binding
score<2) were not investigated. These peptides are thought to be
present at low density on the cell surface in complex with the HLA
molecule, having been out-competed by stronger binding peptides
(Pardoll, D. M. Nat Rev 1 mm 2: 227-238 (2002)).
Example 5
Binding Affinity of T-cell Epitopes to T2 Cells
[0142] To determine the binding ability of EI Suite-selected wild
type peptides and analogs, an in vitro cellular binding assay was
performed using the TAP-deficient cell line T2. T2 cells are
HLA-A*0201 positive, MHC class II negative and TAP deficient,
meaning that they lack a functional transporter associated with
antigen presentation, so that they accumulate empty unstable class
I molecules.
[0143] T2 cells were incubated with 50 .mu.M peptide in serum-free
RPMI 1640 supplemented with 5 .mu.g/mL human .beta.2m (Fluka) for
18 hours at 37.degree. C. HLA-A*0201 expression was then measured
by flow cytometry using the anti-HLA-A2.1 monoclonal antibody (mAb)
BB7.2 followed by incubation with fluorescein isothiocyanate
(FITC)-conjugated F(ab')2 goat antimouse Ig (Biosource). The
results are expressed as fluorescence index (FI) defined as a ratio
(median channel of fluorescence) between the sample and a control
without any peptide. An increase over the control of at least 65%
(FI>2) was arbitrarily chosen as the cutoff point.
[0144] The binding assay consisted of the exogenous addition of
peptides and .beta.2 microglobulin protein: peptide binding
up-regulates surface HLA expression and HLA-A2 molecules on the
surface are measured using FACS by means of an antibody capable of
recognizing the peptide-MHC complex (Kuzushima et al. Blood;
98:1872-81 (2001); Passoni et al. Blood 99:2100-06 (2002)).
[0145] Results of the binding assay are shown in FIG. 3. Most of
the modified epitopes (411V10, 690L2, 589V10, 682V10, 307V10)
showed a marked increase of binding to HLA-A*0201 compared to their
wild-type counterparts.
Example 6 Measurement of HLA-A*0201/peptide complex stability
[0146] To assess the HLA-A*0201/peptide complex stability, T2 cells
(10.sup.6/mL) were incubated overnight with 50 .mu.M of each
peptide in serum-free RPMI 1640 supplemented with 100 ng/mL human
.beta.2m at 37.degree. C. Cells were then washed 4 times to remove
free peptides, incubated for 1 hour with 10 ng/mL Brefeldin A
(Sigma-Aldrich) to block cell surface expression of newly
synthesized HLA-A*0201 molecules, washed, and incubated at
37.degree. C. for 0, 2, 4, 6, or 8 hours. Subsequently, cells were
stained with anti-HLA-A2.1 mAb BB7.2. For each time point,
peptide-induced HLA-A*0201 expression was calculated as mean
fluorescence value of peptide incubated T2 cells/mean fluorescence
value T2 cells in the absence of the peptide. Dissociation complex
50 (DC50) was defined as the time required for the loss of 50% of
the HLA-A*0201/peptide complexes stabilized at t=0.
[0147] Binding stability of the peptide-MHC complex was also
evaluated over time. The CAP-1 peptide was used as positive
control. Again, results demonstrate an improved stability of
peptide analogs, in particular for 411V10, 589V10 and 682V10,
compared to their wild-type counterparts (FIG. 4).
Example 7
Immunogenicity of T-Cell Epitopes in HHD Transgenic Mice
[0148] HLA-A2.1 (HHD) transgenic mice were bred at Charles River
Laboratories (Lecco, Italy). These mice are transgenic for the HHD
complex (human .beta.2-microglobulinfused to HLA-A2.1 .alpha.1 and
.alpha.2 domain, H-2D.sup.b .alpha.3 domain) and are devoid of
H-2D.sup.b and murine .beta.-microglobulin. (Pascolo et al. J. Exp.
Med. 185(12):2043-51 (1997)). For this reason, the immune response
elicited in these mice is specifically restricted to human
HLA-A2.1, making this line a suitable model for epitope
identification and optimization.
[0149] To determine the in vivo immunogenicity of CEA analogs and
wild-type peptides, HHD transgenic mice were co-immunized with 100
.mu.g of the CEA peptide and 140 g of the HBV core 128 helper T
cell peptide (I-A.sup.b-restricted, sequence TPPAYRPPNAPIL (SEQ ID
NO:16). Both immunogens were emulsified in incomplete Freund's
adjuvant (IFA) together with 50 .mu.g of CpG TLR9 agonist and the
emulsion was injected subcutaneously (s.c.) at the tail base of
each animal.
[0150] Groups of 4 HHD mice were injected subcutaneously with the
following components: 1) wild type peptide or analog (100 mcg); 2)
HBV core peptide (140 mcg); 3) CpG oligonucleotide (50 mcg); 4)
incomplete Freund adjuvant. Mice were given a second injection 15
days later with the same component.
[0151] Two weeks after the last injection, mice were bled and
peripheral blood lympho-monocytes (PBMC) and/or splenocytes were
recovered for immunological assays. PBMC or splenocytes were
analyzed by intracellular staining (ICS) for interferon gamma
release upon stimulation with wild type or analog peptides (see
EXAMPLE 8). Results demonstrate that the peptide analogs were more
immunogenic than their wild type counterparts (FIGS. 5 and 6). The
enhancement in immune response for the peptide analogs relative to
the corresponding wt peptide ranged from 11.8- to 310-fold
depending on the epitope. Importantly, the response elicited by
analogs was fully cross reactive with corresponding natural
peptides.
Example 8
Intracellular Staining (ICS) for IFN.gamma.
[0152] One to two millions mouse splenocytes or PBMC in 0.6 ml RPMI
10% FCS were incubated with peptides (5 .mu.g/ml final
concentration of each peptide) and brefeldin A (1 .mu.g/ml; BD
PharMingen, Franklin Lakes, N.J.) at 37.degree. C. and 5% CO.sub.2
for 12-16 hours. Cells were then washed with FACS buffer (PBS1%
FBS, 0.01% NaN.sub.3) and incubated with purified anti-mouse
CD16/CD32 Fc block (BD PharMingen) for 15 min at 4.degree. C. Cells
were then washed and stained with surface antibodies: CD4-PE
conjugated anti-mouse (BD PharMingen), PercP CD8 conjugated anti
mouse (BD PharMingen) and APC-conjugated anti-mouse CD3e (BD
PharMingen) for 30 minutes at room temperature in the dark. After
the washing, cells were fixed and permeabilized with
Cytofix-Cytoperm Solution (BD PharMingen) for 20 min at 4.degree.
C. in the dark. After washing with PermWash Solution (BD
PharMingen) cells were incubated with the IFN.gamma.-FITC
antibodies (BD PharMingen). Cells were then washed, fixed with
formaldehyde 1% in PBS and analyzed on a FACS-Calibur flow
cytometer, using CellQuest software (Becton Dickinson, Franklin
Lakes, N.J.).
Example 9
Identification of Additional Immunogenic CEA Epitopes
[0153] Additional HLA-A2.1 restricted peptides from CEA were
identified by EI Suite as potential immunogenic epitopes for
peptide and/or minigene vaccine, using the procedure described in
EXAMPLES 1-3. These peptides were modified at specific positions as
described in Example 4, to increase their binding affinity to MHC-I
and consequently enhance their immunogenic potency.
[0154] In order to assess the immunogenicity of these peptides and
correlate the immunogenic outcome with biochemical binding
properties, groups of 6 HHD mice were immunized subcutaneously with
100 .mu.g of each peptide admixed with HBVcore128 helper epitope
and a TLR9 agonist (Coley's CpG) in incomplete Freund adjuvant. Two
weeks later, mice received a second injection with the same peptide
mixture. After three weeks the immune response against the natural
target epitope was analyzed by intracellular staining for
IFN.gamma. as described in EXAMPLE 8. Results show that CEA691 and
CEA605 were immunogenic, but little or no improvement was conferred
by the corresponding analogs (FIG. 7). On the other hand, CEA310
epitope was poorly immunogenic while CEA310L2 analog was extremely
powerful in eliciting a cross reactive immune response. In fact,
this analog was 123 fold more immunogenic than the natural peptide.
Similarly, CEA687 was found to be significantly immunogenic in
mice, resulting in 4 out of 6 mice responding to the vaccination.
However, the analog CEA687L2 was about 3.4 fold more immunogenic
and 100% of the mice responded to the treatment. Importantly, a
very good correlation between in vitro binding affinity (i-Topia
assay) and in vivo immunogenicity data was found for these epitopes
(data not shown).
[0155] These data demonstrate that CEA691L2, CEA605V9, CEA310L2 and
CEA687L2 would be useful for the development of a peptide-based
vaccine targeting CEA, or for the construction of an epitope
modified minigene (EMM) vaccine, or any other modality that
incorporates these analogs, such as a genetic vaccine encoding CEA,
in which positions 691-699, 310-318, etc, are replaced with the
corresponding analog sequences, as described throughout the
specification.
Example 10
T-cell Epitope List for hTERT
[0156] 2247 peptides representing all possible 9- and 10-mer frames
of hTERT protein (SwissProt accession number O14746) were ranked
using EI Suite starting from the full hTERT sequence as described
above for CEA, and a total of 12 peptides were selected, including
eight wild type epitopes and four analogs (Table 2).
TABLE-US-00002 TABLE 2 hTERT epitope candidates and analogs
selected by EI Suite. Modified amino acid residues are highlighted.
Position SEQ in Wild type ID hTERT Length Type Sequence sequence
NO: 30 9 w.t. RLGPQGWRL 13 540 9 w.t. ILAKFLHWL 14 540 9 V9
ILAKFLHW ILAKFLHWL 15 540 10 w.t. ILAKFLHWLM 16 540 10 V10
ILAKFLHWL ILAKFLHWLM 17 544 10 w.t. FLHWLMSVYV 18 688 10 w.t.
RAWRTFVLRV 19 688 10 L2 R WRTFVLRV RAWRTFVLRV 20 865 9 w.t.
RLVDDFLLV 21 865 9 F1 LVDDFLLV RLVDDFLLV 22 934 9 w.t. LLDTRTLEV 23
986 9 w.t. FLDLQVNSL 24
Example 11
In-Vitro Binding of EI Suite-Selected hTERT Epitopes and Analogs to
HLA-A*0201
[0157] Candidate epitopes and analogs are analyzed for the ability
to bind to HLA-A*0201in-vitro using iTopia.TM. Epitope Discovery
System (http://www.immunomics.com). This assay provides a method to
determine the binding affinity and stability of a test peptide to
any of several Class I HLA molecules, using fluorescent-labeled
antibody that specifically recognizes HLA only when peptide is
bound in the MHC-I groove.
[0158] The binding affinity to HLA-A*0201 of EI Suite-selected
epitopes and analogs are summarized in FIG. 8. Eight out of 8 wild
type peptides show strong or medium binding to HLA-A*0201, as
evidenced by a binding affinity of 30% of the positive control or
greater (the 30% threshold is suggested in the iTopia.TM. technical
manual). In addition, four out of 4 analogs (540.V9, 540.V10,
688.L2, and 865.F1) show an increase in binding affinity to
HLA-A*0201 compared to the corresponding wild type peptide.
[0159] Similarly, the stability of the peptide-HLA complex is
evaluated over time (FIG. 9). The data shows that 10/11 peptides
tested have an off-rate of 4 hr or greater (off-rate data is
unavailable for peptide hTERT.934). Furthermore, three out of 4
analogs show an improved stability compared to the corresponding
wild type peptide.
Example 12
Immunogenicity of T-cell Epitopes in HHD Mice
[0160] HHD transgenic mice carry the HLA-A*0201/Db (or HHD) fusion
protein and are knock-out for murine MHC class I molecule. For this
reason, the immune response elicited in these mice is specifically
restricted to the human HLA-A*0201 allele, making this model
suitable for epitope identification and optimization.
[0161] Groups of five or six HHD mice are injected subcutaneously
with the following components: 1) wild type peptide or analog (50
mcg); 2) HBV core peptide (140 mcg); 3) CpG oligonucleotide (50
mcg); 4) Incomplete Freund Adjuvant. A second injection is
performed 15 days later.
[0162] Two weeks after the last injection, mice are sacrificed,
their splenocytes collected, and cell mediated immune response
against wild type epitopes is measured for each mouse separately by
ICS (FIG. 10).
[0163] Nine out of 12 EI Suite-suggested peptides elicit a
significant cell-mediated response, defined as the mean response of
0.1% CD8+IFNg+ or greater. Furthermore, four out of 4 analogs show
an enhanced immunogenicity against the wild type peptide, compared
to when the wild type peptide is used as an immunogen.
Example 13
List of T-cell Epitopes and Analogs for HER2
[0164] 2493 peptides representing all possible 9- and 10-mer frames
of HER2 protein (SwissProt accession number P04626) were ranked
using EI Suite from the sequence of her-2/neu as described above
for CEA, and a total of 21 peptides were selected, including twelve
wild type epitopes and nine analogs (Table 3).
TABLE-US-00003 TABLE 3 HER2 epitope candidates and analogs selected
by EI Suite. Modified amino acid residues are highlighted. Position
Wild SEQ in Type ID HER2 Length Type Sequence Sequence NO: 5 9 w.t.
ALCRWGLLL 25 5 9 V9 ALCRWGLL ALCRWGLLL 26 106 9 w.t. QLFEDNYAL 27
369 9 w.t. KIFGSLAFL 28 369 9 L2 K FGSLAFL KIFGSLAFL 29 435 9 w.t.
ILHNGAYSL 30 435 9 V9 ILHNGAYS ILHNGAYSL 31 444 10 w.t. TLQGLGISWL
32 444 10 V10 TLQGLGISW TLQGLGISWL 33 484 10 w.t. QLFRNPHQAL 34 484
10 V10 QLFRNPHQA QLFRNPHQAL 35 657 9 w.t. AVVGILLVV 36 657 9 L2 A
VGILLVV AVVGILLVV 37 657 10 w.t. AVVGILLVVV 38 657 10 L2 A VGILLVVV
AVVGILLVVV 39 661 10 w.t. ILLVVVLGVV 40 665 9 w.t. VVLGVVFGI 41 665
9 V9 VVLGVVFG VVLGVVFGI 42 689 9 w.t. RLLQETELV 43 1023 10 w.t.
YLVPQQGFFC 44 1023 10 V10 YLVPQQGFF YLVPQQGFFC 45
Example 14
In-Vitro Binding of EI Suite-Selected HER2 Epitopes and Analogs to
HLA-A*0201
[0165] Candidate epitopes and analogs are analyzed for the ability
to bind to HLA-A*0201 in-vitro using iTopia.TM. Epitope Discovery
System (http://www.immunomics.com). This assay provides a method to
determine the binding affinity and stability of a test peptide to
any of several Class I HLA molecules, using fluorescent-labeled
antibody that specifically recognizes HLA only when peptide is
bound in the MHC-I groove.
[0166] The binding affinity to HLA-A*0201 of EI Suite-selected
epitopes and analogs are summarized in FIG. 11. Nine out of twelve
wild type peptides show strong or medium binding to HLA-A*0201, as
evidenced by a binding affinity of 30% of the positive control or
greater (the 30% threshold is suggested in the iTopia.TM. technical
manual). In addition, eight out of nine analogs show an increase in
binding affinity to HLA-A*0201 compared to the corresponding wild
type peptide.
[0167] Similarly, the stability of the peptide-HLA complex is
evaluated over time (FIG. 12). The data shows that 17/21 peptides
tested have an off-rate of 4 hr or greater. Furthermore, seven out
of 9 analogs show an improved stability compared to the
corresponding wild type peptide.
Example 15
Immunogenicity of T-cell Epitopes in HHD Mice
[0168] HHD transgenic mice carry the HLA-A*0201/Db (or HHD) fusion
protein and are knock-out for murine MHC class I molecule. For this
reason, the immune response elicited in these mice is specifically
restricted to the human HLA-A*0201 allele, making this model
suitable for epitope identification and optimization.
[0169] Groups consisting of 4 HHD mice are injected subcutaneously
with the following components: 1) wild type peptide or analogs (100
mcg); 2) HBV core peptide (140 mcg); 3) CpG oligonucleotide (50
mcg); 4) Incomplete Freund Adjuvant. A second injection was
performed 14 days later.
[0170] Two weeks after the last injection mice are sacrificed,
their splenocytes are collected, and cell mediated immune response
against wild type epitopes is measured for each single mouse by
ICS. Thirteen out of twenty one peptides elicit a significant
cell-mediated response, defined as the mean response of 0.1%
CD8+IFNg+ or greater (FIG. 13). Furthermore, seven out of nine
analogs show an enhanced immunogenicity against the wild type
peptide, compared to when the wild type peptide is used as an
immunogen.
Example 16
Example of a Minigene Scaffold
[0171] A minigene exemplifying the scaffold defined in this
invention is shown in FIG. 15. The minigene contains four epitopes
and immunogenic analogs from the carcinoembryonic antigen (CEA)
previously shown to be immunogenic individually in HHD/CEA double
transgenic mice and to elicit responses cross-reactive with the
wild-type epitope. The epitopes and analogs are as follows:
TABLE-US-00004 1. CEA.411V10 (VLYGPDDPTV); (SEQ ID NO: 2) 2.
CEA.691 (IMIGVLVGV), (SEQ ID NO: 12) 3. CEA.589V10 (VLYGPDTPIV),
(SEQ ID NO: 4) and 4. 682V10 (GLSAGATVGV). (SEQ ID NO: 5)
[0172] The epitopes/analogs are linked by furin-sensitive linker
REKR.
Example 17
The Modular Structure of Minigenes Containing Epitopes from
Multiple Antigens and Restricted by Multiple HLA Alleles
[0173] Any one or more confirmed or predicted epitopes or analogs
from any tumor-associated or microbial antigen, or multiple
antigens, could be used in place of, or in addition to, the four
sequences used in Example 16. For example, one or more of the
HLA-A*0201-restricted epitopes and analogs identified from CEA,
her-2/neu or hTERT identified above. Predicted or confirmed
epitopes or analogs restricted by MHC alleles other than
HLA-A*0201, or epitopes restricted by a combination of MHC alleles,
may also be used (see FIG. 16). Furthermore, some or all of the
epitopes/analogs may be repeated two or more times. Finally, any
other furin-sensitive linker, or a combination of furin-sensitive
linkers, may be used.
[0174] An alternate design of such a minigene can also be
considered where neighboring epitopes (from one or more antigens)
are restricted by different MHC alleles (FIG. 17).
Example 18
The Minigene of Example 16 Generates Significant Immune Response
In-Vivo Against Individual Epitopes
[0175] HHD/CEA double transgenic mice were generated by crossing
HHD (Pascolo et al. J. Exp. Med. 1997; 185: 2043-2051) with CEA
transgenic mice (Clarke et al., Cancer Res 1998; 58:1469-779). This
mouse model allows to assess and measure the immunogenicity of a
CEA cancer vaccine in the presence of immunologic tolerance, thus
providing an useful and more predictive tool. These mice were used
to evaluate the immunogenicity of the scaffold shown in FIG. 15.
Five HHD/CEA mice were injected intramuscularly with 50 .mu.g of
the minigene followed by electroporation (EP). On days 7, 14, and
21, mice were boosted with additional injections. On day 40, mice
were sacrificed and splenocytes were stimulated with the wild type
epitopes. Results are shown in FIG. 18. It is evident that
vaccination with this minigene generated significant immune
responses against CEA691 and the wild-type epitopes corresponding
to two of the three analogs (589V10, and 682V10) included in the
minigene. However, 411 epitope show poor reactivity. The possible
reasons for this observation are: 1) low immunogenicity of 411V10
analog; 2) position-dependent lack of epitope processing within the
folded polypeptide; 3) epitope competition within the same
antigen-presenting cell (APC).
Example 19
Further Evidence that the Minigene of Example 16 Generates
Significant Immune Response In-Vivo
[0176] The minigene of Example 16 was tested in an independent
experiment in four additional HHD/CEA mice. The results, shown in
FIG. 19, confirm that the scaffold of Example 16 reproducibly
elicits robust immune responses against the epitopes in the
minigene.
Example 20
Addition of a Helper Epitope (p30) to the Minigene Scaffold
[0177] The modular structure of the minigene scaffold described in
this invention allows for the inclusion of a separate source of
T-helper epitopes, which have been shown to increase CD8.sup.+cell
mediated immune response. For example, minigene shown in FIG. 20
contains a tetanus toxin-derived universal helper peptide p30.
Example 21
A Separate Helper Epitope is not Necessary for the Construct in
Example 16
[0178] To evaluate whether addition of p30 could further increase
the immunogenicity of the construct in Example 16, five HHD/CEA
mice were immunized with the p30-containing minigene described in
Example 20. The immunization protocol was the same as described in
Example 3. The immune response against the wild type peptides,
shown in FIG. 21, was of similar magnitude compared to that
elicited by the minigene of Example 16, which did not contain p30
(compare FIG. 21 with FIG. 18 in Example 18).
[0179] The addition of a universal helper epitope does not improve
the immunogenicity of the scaffold in Example 16.
Example 22
Further Evidence that a Separate Helper Epitope is not Necessary
for the Construct of Example 16
[0180] Four additional HHD/CEA mice were immunized with the
p30-containing minigene of Example 20 using the protocol of Example
19. The immune response, shown in FIG. 22, was again similar to
that generated by the construct of Example 16 which did not contain
p30 (compare FIG. 22 with FIG. 19 in Example 19).
Example 23
The Immune Response is Independent of the Epitope Position in the
Minigene
[0181] The protocol of Example 19 was used to test if the immune
response against the wild-type epitopes is dependent on the
position in the minigene where each epitope or analog appears.
Consider a minigene construct similar to that of Example 22, except
the analogs 411V10 and 682V10 were switched (see FIG. 23).
[0182] The results, shown in FIG. 24, exhibit no significant
differences between immune responses against all four wild-type
epitopes compared to those in Example 22.
Example 24
The Immune Response Generated by the Minigene of Example 16 is
Higher than that Elicited by Proteasome-Targeting Minigenes
[0183] In a variant construct (see FIG. 25), the minigene comprised
of the same four epitope/analogs was fused to a mutant form of
ubiquitin (G76V) (see Stack et al, Nature Biotech., 18, 1298-1302,
2000) via a flexible linker peptide (VGKGGSGG (SEQ ID NO:48)).
Ubiquitin fusion has been shown to improve the induction of
CD8.sup.+T-cell response by targeting the protein for rapid
degradation by the proteasome (see Rodriguez et al, J. Virol, 72,
5174-5181, 1998). The design also included the use of one of three
spacer sequences, AAY (see Wang et al, Sc. J. Immunol., 60,
219-225, 2004), LRA, or RLRA, designed to ensure efficient epitope
processing by the proteasome.
[0184] HHD/CEA mice (n=5 per group) were immunized with a minigene
shown in FIG. 25 containing either AAY or LRA as spacers, using the
immunization protocol of Example 17. A similar test was conducted
for the minigene scaffold using the spacer RLRA and the protocol of
Example 18.
[0185] The results, as shown in FIG. 26, lead to the conclusion
that the minigene of Example 16 performs at least as well as any of
the three ubiquitin-containing constructs, if not better (compare
results in FIG. 26a-b with that of FIG. 18, and the results in FIG.
26c with FIG. 19).
Example 25
The Immune Response Generated by the Minigene of Example 1 is not
Improved by Replacing LTB with a Membrane-Translocating Sequence
(MTS)
[0186] The LTB functional element in the minigenes described in
this invention allows the translated and secreted product to enter
MHC Class I processing pathway. Another method known in the art to
direct exogenous peptides for presentation to CD8+T-cells is to
include a membrane-translocating sequence such as HIVtat peptide
AAARKKRRQRRRR (SEQ ID NO:49) (see Kim et al, J. Immunol, 159,
1666-1668, 1997). We investigated whether replacing LTB with an MTS
could enhance CD8+T-cell response against the individual epitopes.
Four HHD/CEA mice were immunized with a minigene containing HIVtat
MTS, see FIG. 27. The immunization protocol was the same as in
Examples 17 and 20.
[0187] The immune response elicited by the construct is shown in
FIG. 28 and was lower compared to that elicited by an
LTB-containing minigene, especially against 691 and 589 epitope
(compare with FIG. 21 in Example 21).
Sequence CWU 1
1
5719PRThomo sapiens 1Ile Met Ile Gly Val Leu Val Gly Val1
5210PRTartificial sequenceAnchor-modified analog 2Val Leu Tyr Gly
Pro Asp Asp Pro Thr Val1 5 10310PRTartificial
sequenceAnchor-modified analog 3Gly Leu Met Ile Gly Val Leu Val Gly
Val1 5 10410PRTartificial sequenceAnchor-modified analog 4Val Leu
Tyr Gly Pro Asp Thr Pro Ile Val1 5 10510PRTartificial
sequenceAnchor-modified analog 5Gly Leu Ser Ala Gly Ala Thr Val Gly
Val1 5 10610PRTartificial sequenceAnchor-modified analog 6Gly Leu
Asn Arg Thr Thr Val Thr Thr Val1 5 1079PRThomo sapiens 7Ala Thr Val
Gly Ile Met Ile Gly Val1 5810PRTartificial sequenceAnchor-modified
analog 8Ile Ile Tyr Pro Asn Ala Ser Leu Leu Val1 5
1099PRTartificial sequenceAnchor-modified analog 9Arg Leu Thr Val
Thr Thr Ile Thr Val1 5109PRTartificial sequenceAnchor-modified
analog 10Tyr Leu Ser Gly Ala Asn Leu Asn Val1 5119PRTartificial
sequenceAnchor-modified analog 11Ala Leu Val Gly Ile Met Ile Gly
Val1 5129PRTartificial sequenceAnchor-modified analog 12Ile Leu Ile
Gly Val Leu Val Gly Val1 5139PRThomo sapiens 13Arg Leu Gly Pro Gln
Gly Trp Arg Leu1 5149PRThomo sapiens 14Ile Leu Ala Lys Phe Leu His
Trp Leu1 5159PRTartificial sequenceanalog 15Ile Leu Ala Lys Phe Leu
His Trp Val1 51610PRThomo sapiens 16Ile Leu Ala Lys Phe Leu His Trp
Leu Met1 5 101710PRTartificial sequenceanalog 17Ile Leu Ala Lys Phe
Leu His Trp Leu Val1 5 101810PRThomo sapiens 18Phe Leu His Trp Leu
Met Ser Val Tyr Val1 5 101910PRThomo sapiens 19Arg Ala Trp Arg Thr
Phe Val Leu Arg Val1 5 102010PRTartificial sequenceanalog 20Arg Leu
Trp Arg Thr Phe Val Leu Arg Val1 5 10219PRThomo sapiens 21Arg Leu
Val Asp Asp Phe Leu Leu Val1 5229PRTartificial sequenceanalog 22Phe
Leu Val Asp Asp Phe Leu Leu Val1 5239PRThomo sapiens 23Leu Leu Asp
Thr Arg Thr Leu Glu Val1 5249PRThomo sapiens 24Phe Leu Asp Leu Gln
Val Asn Ser Leu1 5259PRThomo sapiens 25Ala Leu Cys Arg Trp Gly Leu
Leu Leu1 5269PRTartificial sequenceanalog 26Ala Leu Cys Arg Trp Gly
Leu Leu Val1 5279PRThomo sapiens 27Gln Leu Phe Glu Asp Asn Tyr Ala
Leu1 5289PRThomo sapiens 28Lys Ile Phe Gly Ser Leu Ala Phe Leu1
5299PRTartificial sequenceanalog 29Lys Leu Phe Gly Ser Leu Ala Phe
Leu1 5309PRThomo sapiens 30Ile Leu His Asn Gly Ala Tyr Ser Leu1
5319PRTartificial sequenceanalog 31Ile Leu His Asn Gly Ala Tyr Ser
Val1 53210PRThomo sapiens 32Thr Leu Gln Gly Leu Gly Ile Ser Trp
Leu1 5 103310PRTartificial sequenceanalog 33Thr Leu Gln Gly Leu Gly
Ile Ser Trp Val1 5 103410PRThomo sapiens 34Gln Leu Phe Arg Asn Pro
His Gln Ala Leu1 5 103510PRTartificial sequenceanalog 35Gln Leu Phe
Arg Asn Pro His Gln Ala Val1 5 10369PRThomo sapiens 36Ala Val Val
Gly Ile Leu Leu Val Val1 5379PRTartificial sequenceanalog 37Ala Leu
Val Gly Ile Leu Leu Val Val1 53810PRThomo sapiens 38Ala Val Val Gly
Ile Leu Leu Val Val Val1 5 103910PRTartificial sequenceanalog 39Ala
Leu Val Gly Ile Leu Leu Val Val Val1 5 104010PRThomo sapiens 40Ile
Leu Leu Val Val Val Leu Gly Val Val1 5 10419PRThomo sapiens 41Val
Val Leu Gly Val Val Phe Gly Ile1 5429PRTartificial sequenceanalog
42Val Val Leu Gly Val Val Phe Gly Val1 5439PRThomo sapiens 43Arg
Leu Leu Gln Glu Thr Glu Leu Val1 54410PRThomo sapiens 44Tyr Leu Val
Pro Gln Gln Gly Phe Phe Cys1 5 104510PRTartificial sequenceanalog
45Tyr Leu Val Pro Gln Gln Gly Phe Phe Val1 5 104620PRThomo sapiens
46Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe1
5 10 15Val Ser Pro Ser 2047105PRTE. coli 47Ser Arg Ala Pro Gln Ser
Ile Thr Glu Leu Cys Ser Glu Tyr Arg Asn1 5 10 15Thr Gln Ile Tyr Thr
Ile Asn Asp Lys Ile Leu Ser Tyr Thr Glu Ser 20 25 30Met Ala Gly Lys
Arg Glu Met Val Ile Ile Thr Phe Lys Ser Gly Ala 35 40 45Thr Phe Gln
Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys 50 55 60Lys Ala
Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr Tyr Leu Thr65 70 75
80Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys Thr Pro Asn
85 90 95Ser Ile Ala Ala Ile Ser Met Glu Asn 100 105488PRTartificial
sequenceflexible linker peptide 48Val Gly Lys Gly Gly Ser Gly Gly1
54913PRTHIV 49Ala Ala Ala Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg1
5 1050702PRThomo sapiens 50Met Glu Ser Pro Ser Ala Pro Pro His Arg
Trp Cys Ile Pro Trp Gln1 5 10 15Arg Leu Leu Leu Thr Ala Ser Leu Leu
Thr Phe Trp Asn Pro Pro Thr 20 25 30Thr Ala Lys Leu Thr Ile Glu Ser
Thr Pro Phe Asn Val Ala Glu Gly 35 40 45Lys Glu Val Leu Leu Leu Val
His Asn Leu Pro Gln His Leu Phe Gly 50 55 60Tyr Ser Trp Tyr Lys Gly
Glu Arg Val Asp Gly Asn Arg Gln Ile Ile65 70 75 80Gly Tyr Val Ile
Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser 85 90 95Gly Arg Glu
Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile 100 105 110Ile
Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp 115 120
125Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu
130 135 140Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu
Asp Lys145 150 155 160Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr
Gln Asp Ala Thr Tyr 165 170 175Leu Trp Trp Val Asn Asn Gln Ser Leu
Pro Val Ser Pro Arg Leu Gln 180 185 190Leu Ser Asn Gly Asn Arg Thr
Leu Thr Leu Phe Asn Val Thr Arg Asn 195 200 205Asp Thr Ala Ser Tyr
Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg 210 215 220Arg Ser Asp
Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro225 230 235
240Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn
245 250 255Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser
Trp Phe 260 265 270Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu
Phe Ile Pro Asn 275 280 285Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr
Cys Gln Ala His Asn Ser 290 295 300Asp Thr Gly Leu Asn Arg Thr Thr
Val Thr Thr Ile Thr Val Tyr Ala305 310 315 320Glu Pro Pro Lys Pro
Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu 325 330 335Asp Glu Asp
Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr 340 345 350Thr
Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg 355 360
365Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr
370 375 380Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Glu
Leu Ser385 390 395 400Val Asp His Ser Asp Pro Val Ile Leu Asn Val
Leu Tyr Gly Pro Asp 405 410 415Asp Pro Thr Ile Ser Pro Ser Tyr Thr
Tyr Tyr Arg Pro Gly Val Asn 420 425 430Leu Ser Leu Ser Cys His Ala
Ala Ser Asn Pro Pro Ala Gln Tyr Ser 435 440 445Trp Leu Ile Asp Gly
Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile 450 455 460Ser Asn Ile
Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn465 470 475
480Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val
485 490 495Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser
Lys Pro 500 505 510Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu
Pro Glu Ala Gln 515 520 525Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly
Gln Ser Leu Pro Val Ser 530 535 540Pro Arg Leu Gln Leu Ser Asn Gly
Asn Arg Thr Leu Thr Leu Phe Asn545 550 555 560Val Thr Arg Asn Asp
Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser 565 570 575Val Ser Ala
Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly 580 585 590Pro
Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly 595 600
605Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln
610 615 620Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln
Val Leu625 630 635 640Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly
Thr Tyr Ala Cys Phe 645 650 655Val Ser Asn Leu Ala Thr Gly Arg Asn
Asn Ser Ile Val Lys Ser Ile 660 665 670Thr Val Ser Ala Ser Gly Thr
Ser Pro Gly Leu Ser Ala Gly Ala Thr 675 680 685Val Gly Ile Met Ile
Gly Val Leu Val Gly Val Ala Leu Ile 690 695 70051678PRThomo sapiens
51Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln1
5 10 15Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro
Thr 20 25 30Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala
Glu Gly 35 40 45Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His
Leu Phe Gly 50 55 60Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn
Arg Gln Ile Ile65 70 75 80Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr
Pro Gly Pro Ala Tyr Ser 85 90 95Gly Arg Glu Ile Ile Tyr Pro Asn Ala
Ser Leu Leu Ile Gln Asn Ile 100 105 110Ile Gln Asn Asp Thr Gly Phe
Tyr Thr Leu His Val Ile Lys Ser Asp 115 120 125Leu Val Asn Glu Glu
Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu 130 135 140Pro Lys Pro
Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys145 150 155
160Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr
165 170 175Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg
Leu Gln 180 185 190Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn
Val Thr Arg Asn 195 200 205Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln
Asn Pro Val Ser Ala Arg 210 215 220Arg Ser Asp Ser Val Ile Leu Asn
Val Leu Tyr Gly Pro Asp Ala Pro225 230 235 240Thr Ile Ser Pro Leu
Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn 245 250 255Leu Ser Cys
His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe 260 265 270Val
Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn 275 280
285Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser
290 295 300Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val
Tyr Ala305 310 315 320Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn
Ser Asn Pro Val Glu 325 330 335Asp Glu Asp Ala Val Ala Leu Thr Cys
Glu Pro Glu Ile Gln Asn Thr 340 345 350Thr Tyr Leu Trp Trp Val Asn
Asn Gln Ser Leu Pro Val Ser Pro Arg 355 360 365Leu Gln Leu Ser Asn
Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr 370 375 380Arg Asn Asp
Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Glu Leu Ser385 390 395
400Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp
405 410 415Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly
Val Asn 420 425 430Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro
Ala Gln Tyr Ser 435 440 445Trp Leu Ile Asp Gly Asn Ile Gln Gln His
Thr Gln Glu Leu Phe Ile 450 455 460Ser Asn Ile Thr Glu Lys Asn Ser
Gly Leu Tyr Thr Cys Gln Ala Asn465 470 475 480Asn Ser Ala Ser Gly
His Ser Arg Thr Thr Val Lys Thr Ile Thr Val 485 490 495Ser Ala Glu
Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro 500 505 510Val
Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln 515 520
525Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser
530 535 540Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu
Phe Asn545 550 555 560Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys
Gly Ile Gln Asn Ser 565 570 575Val Ser Ala Asn Arg Ser Asp Pro Val
Thr Leu Asp Val Leu Tyr Gly 580 585 590Pro Asp Thr Pro Ile Ile Ser
Pro Pro Asp Ser Ser Tyr Leu Ser Gly 595 600 605Ala Asn Leu Asn Leu
Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln 610 615 620Tyr Ser Trp
Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu625 630 635
640Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe
645 650 655Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys
Ser Ile 660 665 670Thr Val Ser Ala Ser Gly 67552702PRTartificial
sequenceexemplary variant CEA protein sequence 52Met Glu Ser Pro
Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln1 5 10 15Arg Leu Leu
Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr 20 25 30Thr Ala
Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly 35 40 45Lys
Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly 50 55
60Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile65
70 75 80Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr
Ser 85 90 95Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Val Gln
Asn Ile 100 105 110Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val
Ile Lys Ser Asp 115 120 125Leu Val Asn Glu Glu Ala Thr Gly Gln Phe
Arg Val Tyr Pro Glu Leu 130 135 140Pro Lys Pro Ser Ile Ser Ser Asn
Asn Ser Lys Pro Val Glu Asp Lys145 150 155 160Asp Ala Val Ala Phe
Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr 165 170 175Leu Trp Trp
Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln 180 185 190Leu
Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn 195 200
205Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg
210 215 220Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp
Ala Pro225 230 235 240Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser
Gly Glu Asn Leu Asn 245 250 255Leu Ser Cys His Ala Ala Ser Asn Pro
Pro Ala Gln Tyr Ser Trp Phe 260 265 270Val Asn Gly Thr Phe Gln Gln
Ser Thr Gln Glu Leu Phe Ile Pro Asn 275 280 285Ile Thr Val Asn Asn
Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser 290 295 300Asp Thr Gly
Leu Asn Arg Thr Thr Val Thr Thr Val Thr Val Tyr Ala305 310 315
320Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val
Glu
325 330 335Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln
Asn Thr 340 345 350Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro
Val Ser Pro Arg 355 360 365Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu
Thr Leu Leu Ser Val Thr 370 375 380Arg Asn Asp Val Gly Pro Tyr Glu
Cys Gly Ile Gln Asn Glu Leu Ser385 390 395 400Val Asp His Ser Asp
Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp 405 410 415Asp Pro Thr
Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn 420 425 430Leu
Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser 435 440
445Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile
450 455 460Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln
Ala Asn465 470 475 480Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val
Lys Thr Ile Thr Val 485 490 495Ser Ala Glu Leu Pro Lys Pro Ser Ile
Ser Ser Asn Asn Ser Lys Pro 500 505 510Val Glu Asp Lys Asp Ala Val
Ala Phe Thr Cys Glu Pro Glu Ala Gln 515 520 525Asn Thr Thr Tyr Leu
Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser 530 535 540Pro Arg Leu
Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn545 550 555
560Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser
565 570 575Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu
Tyr Gly 580 585 590Pro Asp Thr Pro Ile Val Ser Pro Pro Asp Ser Ser
Tyr Leu Ser Gly 595 600 605Ala Asn Leu Asn Leu Ser Cys His Ser Ala
Ser Asn Pro Ser Pro Gln 610 615 620Tyr Ser Trp Arg Ile Asn Gly Ile
Pro Gln Gln His Thr Gln Val Leu625 630 635 640Phe Ile Ala Lys Ile
Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe 645 650 655Val Ser Asn
Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile 660 665 670Thr
Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr 675 680
685Val Gly Leu Met Ile Gly Val Leu Val Gly Val Ala Leu Ile 690 695
700539PRTartificial sequencestatistical motif 53Ser Tyr Phe Pro Glu
Ile Thr His Ile1 5545PRTartificial sequencestatistical motif 54Met
His Cys Asx Asn1 5554PRTartificial sequencestatistical motif 55Phe
Ile Met Met1564PRTartificial sequencefurin sensitive linker 56Arg
Glu Lys Arg1574PRTartificial sequencelinker 57Arg Leu Arg Ala1
* * * * *
References