U.S. patent application number 15/158387 was filed with the patent office on 2016-09-01 for dna vaccine for use in pancreatic cancer patients.
The applicant listed for this patent is Vaximm AG. Invention is credited to Heinz Lubenau.
Application Number | 20160250311 15/158387 |
Document ID | / |
Family ID | 48741047 |
Filed Date | 2016-09-01 |
United States Patent
Application |
20160250311 |
Kind Code |
A1 |
Lubenau; Heinz |
September 1, 2016 |
DNA VACCINE FOR USE IN PANCREATIC CANCER PATIENTS
Abstract
The present invention relates to an attenuated mutant strain of
Salmonella comprising a recombinant DNA molecule encoding a VEGF
receptor protein. In particular, the present invention relate to
the use of said attenuated mutant strain of Salmonella in cancer
immunotherapy.
Inventors: |
Lubenau; Heinz; (Neustadt an
der Weinstrasse, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vaximm AG |
Basel |
|
CH |
|
|
Family ID: |
48741047 |
Appl. No.: |
15/158387 |
Filed: |
May 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14409434 |
Dec 18, 2014 |
|
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PCT/EP2013/001882 |
Jun 26, 2013 |
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15158387 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61K 31/7068 20130101;
A61K 2039/542 20130101; C07K 14/71 20130101; A61K 39/0011 20130101;
C12N 1/36 20130101; A61K 2039/852 20180801; C12R 1/42 20130101;
A61K 2039/522 20130101; A61P 35/04 20180101; A61K 2039/53 20130101;
C12N 1/20 20130101; A61K 39/0275 20130101; Y02A 50/484 20180101;
Y02A 50/30 20180101; A61K 2039/523 20130101; A61K 2039/545
20130101; A61K 39/001109 20180801; A61K 2039/57 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12R 1/42 20060101 C12R001/42; C12N 1/20 20060101
C12N001/20; C12N 1/36 20060101 C12N001/36; C07K 14/71 20060101
C07K014/71; A61K 31/7068 20060101 A61K031/7068 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2012 |
EP |
12004995.2 |
Claims
1. An attenuated mutant strain of Salmonella typhi comprising at
least one copy of a recombinant DNA molecule comprising an
expression cassette encoding a VEGF receptor protein, particularly
a VEGF receptor protein selected from the group consisting of human
VEGFR-2 and a homolog thereof that shares at least about 80%
homology therewith, particularly wherein human VEGFR-2 has the
amino acid sequence as found in SEQ ID NO 1, for use as a
vaccine.
2. A DNA vaccine comprising the attenuated mutant strain of
Salmonella typhi of claim 1, wherein the attenuated mutant strain
of Salmonella typhi is Salmonella typhi Ty21a, and wherein the
expression cassette is a eukaryotic expression cassette, for use in
cancer immunotherapy.
3. The DNA vaccine of claim 2, wherein the DNA vaccine comprises
the attenuated Salmonella typhi strain Ty21a transformed by a
plasmid that contains a DNA encoding the VEGFR-2 protein of SEQ ID
NO 1 for use in an anti-angiogenic cancer immunotherapy.
4. The DNA vaccine of claim 3, wherein the plasmid is the 7580 bp
pVAX10.VR2-1 as depicted in FIG. 2 and has the sequence as found in
SEQ ID NO 3 and the DNA vaccine is designated VXM01.
5. The DNA vaccine of claim 2, wherein the cancer is pancreatic
cancer.
6. The DNA vaccine of claim 5, wherein said pancreatic cancer is
stage IV or locally advanced pancreatic cancer.
7. The DNA vaccine of claim 2, wherein the cancer includes
metastases.
8. The DNA vaccine of claim 2, wherein the treatment is accompanied
by chemotherapy and/or radiotherapy.
9. The DNA vaccine of claim 8, wherein the chemotherapeutic agent
is gemcitabine.
10. The DNA vaccine of claim 8, wherein the immunotherapeutic
treatment with the vaccine is carried out during the chemotherapy
treatment cycle.
11. The DNA vaccine of claim 2, wherein the vaccine is administered
orally.
12. The DNA vaccine of claim 2, wherein the single dose of the
vaccine is 1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10, or
1.times.10.sup.11 colony forming units (CFU).
13. The DNA vaccine for use according to claim 9, wherein the
single dose of the vaccine is less than 1.times.10.sup.9 CFU,
particularly wherein the single dose of the vaccine is from
1.times.10.sup.8 to 1.times.10.sup.9 CFU.
14. The DNA vaccine for use according to claim 9, wherein the
single dose of the vaccine is less than 1.times.10.sup.8 CFU,
particularly wherein the single dose of the vaccine is from
1.times.10.sup.5 to 1.times.10.sup.8 CFU, more particularly wherein
the single dose of the vaccine is from 1.times.10.sup.6 to
1.times.10.sup.7 CFU.
15. The DNA vaccine of claim 2, wherein the single dose comprises
from about 10.sup.5 to about 10.sup.11, particularly form about
10.sup.6 to about 10.sup.10, more particularly from about 10.sup.6
to about 10.sup.9, more particularly from about 10.sup.6 to about
10.sup.8, most particularly from about 10.sup.6 to about 10.sup.7
colony forming units (CFU).
16. The DNA vaccine VXM01, comprising the attenuated Salmonella
typhi strain Ty21a transformed by a plasmid that contains a DNA
encoding the VEGFR-2 protein of SEQ ID NO 1, wherein the plasmid is
a 7580 bp plasmid DNA and comprises the cDNA of VEGFR-2 that is
under the control of the CMV promoter, the kanamycin resistance
gene, and the pMB1 ori, and is designated as pVAX10.VR2-1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/409,434, filed Dec. 18, 2014, which is a
national stage application under 35 U.S.C. .sctn.371 of
International Application No. PCT/EP2013/001882, filed Jun. 26,
2013, which in turn claims priority to European Application No.
12004995.2, filed Jul. 5, 2012, the content of each of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to an attenuated mutant strain
of Salmonella comprising a recombinant DNA molecule encoding a VEGF
receptor protein. In particular, the present invention relates to
the use of said attenuated mutant strain of Salmonella in cancer
immunotherapy.
BACKGROUND OF THE INVENTION
[0003] Attenuated derivatives of Salmonella enterica are attractive
vehicles for the delivery of heterologous antigens, such as tumor
antigens or tumor stroma antigens, to the mammalian immune system.
S. enterica strains can potentially be delivered via mucosal routes
of immunization, i.e. orally or nasally, which offers advantages of
simplicity and safety compared to parenteral administration.
Furthermore, Salmonella strains elicit strong humoral and cellular
immune responses at the level of both systemic and mucosal
compartments. Batch preparation costs are relatively low and
formulations of live bacterial vaccines are highly stable.
Attenuation can be accomplished by deletion of various genes,
including virulence, regulatory and metabolic genes.
[0004] Several Salmonella typhimurium strains attenuated by aro
mutations have been shown to be safe and effective delivery
vehicles for heterologous antigens in animal models.
[0005] Approaches of delivering DNA constructs encoding antigens,
in particular VEGF receptor proteins, via live attenuated
Salmonella typhimurium strains into mouse target cells are
described in WO 03/073995. Niethammer et al. (Nature Medicine 2002,
8(12), 1369) describes an attenuated S. typhimurium aroA strain
SL7207 harboring an expression vector encoding the murine vascular
endothelial growth factor receptor 2 (VEGFR-2 or FLK-1) and its use
as cancer vaccine.
[0006] There is however only one attenuated Salmonella enterica
serovar strain, namely Salmonella enterica serovar typhi Ty21a
(short: S. typhi Ty21a), which has been accepted for use in
humans.
[0007] This well-tolerated, live oral vaccine against typhoid fever
was derived by chemical mutagenesis of the wild-type virulent
bacterial isolate S. typhi Ty2 and harbors a loss-of-function
mutation in the galE gene, as well as other less defined mutations.
It has been licensed as typhoid vaccine in many countries after it
was shown to be efficacious and safe in field trials.
[0008] Angiogenesis contributes to solid tumor growth and
metastasis. Compounds like bevacizumab and others, for example
small molecules such as sunitinib and axitinib that specifically
target the tumor neovasculature have shown efficacy in a range of
tumor indications (Powles et al., Br J Cancer 2011, 104(5):741-5);
Rini et al., Lancet 2011, 378:1931-1939).
[0009] Tumor neovasculature is lined with endothelial cells that
overexpress vascular endothelial growth factor receptor (VEGFR) 2
and are readily accessible via the blood stream. The genetic
stability of these cells and their ability to support hundreds of
tumor cells per endothelial cell make them a prime target for
anti-cancer therapy, be it via antibodies, tyrosine kinase
inhibitors, or vaccines (Augustin, Trends Pharmacol Sci 1998,
19:216-222). Recently, T-cell based immunotherapy has gained some
clinical success in prostate cancer and validated the potential of
anti-cancer vaccination which was often demonstrated pre-clinically
(Sharma et al., Nat Rev Cancer 2011, 11:805-812). Activating the
immune system against cancer cells faces multiple challenges. For
example, cancerous lesions are often polyclonal and cancer cells
have the propensity to mutate. Antigen specific therapy often only
results in a selection of non-antigen bearing cells. Further
hurdles include tumor encapsulation and loss or down-regulation of
MHC molecules. Vaccination approaches that target the tumor
neovasculature should in theory overcome those hurdles.
OBJECTS OF THE INVENTION
[0010] In view of the prior art, it is an object of the present
invention to provide a novel safe oral VEGF receptor targeting
cancer vaccine. Such a VEGF receptor targeting cancer vaccine would
offer major advantages for improving the treatment options for
cancer patients.
SUMMARY OF THE INVENTION
[0011] The present invention combines anti-angiogenic therapy and
vaccination, targeting VEGFR-2 using a new vaccine (VXM01), which
is an attenuated and reengineered bacterial strain Salmonella typhi
Ty21a comprising a plasmid that encodes the VEGF receptor protein
2.
[0012] VXM01 represents a novel strategy by targeting not a tumor
cell-resident antigen, but a tumor stroma-resident antigen,
overexpressed by non-malignant endothelial cells of the tumor
neovasculature.
[0013] By targeting genetically stable and easily accessible
endothelial cells, this product aims to overcome limitations
encountered previously by vaccines targeting tumor cells directly,
such as tumor-cell heterogeneity, MHC-loss, immunosuppression on a
cellular level and tumor encapsulation as well as physiological
barriers such as the blood brain barrier. Furthermore, since the
therapeutic target is independent of the tumor type, the vaccine
may potentially be active against a variety of different solid
malignancies. The product represents a patient-independent,
"off-the-shelf" oral vaccine, which can be stored and distributed
to the clinical sites for use. While anti-angiogenic therapy,
either via small molecules or via antibodies, has already been
proven to be effective, the approach according to the present
invention differs significantly by activating the patient's own
immune system against the tumor neovasculature and is as such
potentially creating a T-cell memory effect that provides long-term
efficacy. Studies with bevacizumab in colon and ovarian cancer
suggest that continued anti-angiogenic pressure is required to
maintain beneficial treatment effects in the long term (Allegra et
al., J Clin Oncol 2011, 29:11-16; Burger et al., N Engl J Med 2011,
365:2473-2483; Perren et al., N Engl J Med 2011,
365:2484-2496).
[0014] To the inventors' knowledge, this is the first clinical
trial of an oral cancer vaccine. In addition, this vaccine has the
potential to be effective against multiple tumor types.
[0015] It has been shown by this first clinical trial that the
vaccine according to this invention (VXM01) is highly effective in
eliciting an immune response that influences significantly the
tumor growth in the patient. It is remarkable and surprising that
this immune response can be triggered by very low doses of orally
administered VXM01. The vaccine is effective at doses starting
already with 1.times.10.sup.5 or 1.times.10.sup.6 to
1.times.10.sup.7 colony forming units (CFU). First results indicate
that vaccination with VXM01 may lead to the breakdown of existing
tumor vasculature and may support the development of an immune
memory against proliferating endothelial cells.
[0016] The vaccine is effective in monotherapy as well as in a
combination therapy with standard chemotherapeutic agents,
radiotherapy and/or biological cancer therapy.
[0017] In the current clinical trial the stage IV patients were
treated in advance and/or simultaneously with the chemotherapeutic
agent gemcitabine and VXM01. The treatment with VXM01 is however
also effective, if the patients have developed a resistance to
chemotherapy (chemo-refractory patients).
[0018] In one aspect, the present invention relates to an
attenuated mutant strain of Salmonella typhi comprising at least
one copy of a recombinant DNA molecule comprising an expression
cassette encoding a VEGF receptor protein for use as a vaccine.
[0019] In particular embodiments, the VEGF receptor protein is
selected from the group consisting of human VEGFR-2 and a homolog
thereof that shares at least about 80% homology therewith.
[0020] In particular embodiments, the VEGF receptor protein is
human VEGFR-2 having the amino acid sequence as found in SEQ ID NO
1.
[0021] In another aspect, the present invention relates to a DNA
vaccine comprising the attenuated mutant strain of Salmonella typhi
of the present invention.
[0022] In particular embodiments, the attenuated mutant strain of
Salmonella typhi is Salmonella typhi Ty21a.
[0023] In particular embodiments, the expression cassette is a
eukaryotic expression cassette.
[0024] In particular embodiments, the DNA vaccine of the present
invention is for use in cancer immunotherapy.
[0025] In particular embodiments, the DNA vaccine comprises the
attenuated Salmonella typhi strain Ty21a transformed by a plasmid
that contains a DNA encoding the VEGFR-2 protein of SEQ ID NO
1.
[0026] In particular embodiments, the DNA vaccine is for use in an
anti-angiogenic cancer immunotherapy.
[0027] In particular embodiments, the plasmid is the 7580 bp
pVAX10.VR2-1 as depicted in FIG. 2 and has the sequence as found in
SEQ ID NO 3 and the DNA vaccine is designated VXM01.
[0028] In particular embodiments, the cancer is pancreatic
cancer.
[0029] In particular embodiments, said pancreatic cancer is stage
IV or locally advanced pancreatic cancer.
[0030] In particular embodiments, the cancer includes
metastases.
[0031] In particular embodiments, the treatment is accompanied by
chemotherapy and/or radiotherapy.
[0032] In particular embodiments, the chemotherapeutic agent is
gemcitabine.
[0033] In particular embodiments, the immunotherapeutic treatment
with the vaccine is carried out during the chemotherapy treatment
cycle.
[0034] In particular embodiments, the vaccine is administered
orally.
[0035] In particular embodiments, the single dose of the vaccine is
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10, or
1.times.10.sup.11 colony forming units (CFU).
[0036] In particular embodiments, the single dose of the vaccine is
less than 1.times.10.sup.9 CFU. In particular embodiments, the
single dose of the vaccine is from 1.times.10.sup.8 to
1.times.10.sup.9 CFU.
[0037] In particular embodiments, the single dose of the vaccine is
less than 1.times.10.sup.8 CFU. In particular embodiments, the
single dose of the vaccine is from 1.times.10.sup.5 to
1.times.10.sup.8 CFU, more particularly the single dose of the
vaccine is from 1.times.10.sup.6 to 1.times.10.sup.7 CFU.
[0038] In particular embodiments, the single dose comprises from
about 10.sup.5 to about 10.sup.11, particularly form about 10.sup.6
to about 10.sup.10, more particularly from about 10.sup.6 to about
10.sup.9, more particularly from about 10.sup.6 to about 10.sup.8,
most particularly from about 10.sup.6 to about 10.sup.7 colony
forming units (CFU).
[0039] In another aspect, the present invention relates to the DNA
vaccine VXM01, comprising the attenuated Salmonella typhi strain
Ty21a transformed by a plasmid that contains a DNA encoding the
VEGFR-2 protein of SEQ ID NO 1, wherein the plasmid is a 7580 bp
plasmid DNA and comprises the cDNA of VEGFR-2 that is under the
control of the CMV promoter, the kanamycin resistance gene, and the
pMB1 ori, and is designated as pVAX10.VR2-1.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention may be understood more readily by
reference to the following detailed description of the invention
and the examples included therein.
[0041] In one aspect, the present invention relates to an
attenuated mutant strain of Salmonella typhi comprising at least
one copy of a recombinant DNA molecule comprising an expression
cassette encoding a VEGF receptor protein.
[0042] According to the invention, the attenuated mutant strain of
Salmonella typhi functions as the bacterial carrier of the
recombinant DNA molecule comprising an expression cassette encoding
a VEGF receptor protein for the delivery of said recombinant DNA
molecule into a target cell.
[0043] In the context of the present invention, the term
"attenuated" refers to a bacterial strain of reduced virulence
compared to the parental bacterial strain, not harboring the
attenuating mutation. Attenuated bacterial strains have preferably
lost their virulence but retained their ability to induce
protective immunity. Attenuation can be accomplished by deletion of
various genes, including virulence, regulatory, and metabolic
genes. Attenuated bacteria may be found naturally or they may be
produced artificially in the laboratory, for example by adaptation
to a new medium or cell culture or they may be produced by
recombinant DNA technology.
[0044] In the context of the present invention, the term "mutant
strain" refers to a bacterial strain harboring a mutation in its
genome. In this context, the term "mutation" refers to a change in
the nucleic acid sequence, including point mutations, insertions,
deletions, translocations and inversions.
[0045] In the context of the present invention, the term
"comprises" or "comprising" means "including, but not limited to".
The term is intended to be open-ended, to specify the presence of
any stated features, elements, integers, steps or components, but
not to preclude the presence or addition of one or more other
features, elements, integers, steps, components or groups thereof.
The term "comprising" thus includes the more restrictive terms
"consisting of" and "essentially consisting of". In one embodiment
the term "comprising" as used throughout the application and in
particular within the claims may be replaced by the term
"consisting of".
[0046] In the context of the present invention, the term
"recombinant DNA molecule" refers to an engineered DNA construct,
preferably composed of DNA pieces of different origin. The
recombinant DNA molecule can be a linear nucleic acid, or
preferably, a circular recombinant DNA plasmid generated by
introducing an open reading frame encoding a VEGF receptor protein
into an expression vector plasmid.
[0047] In the context of the present invention, the term
"expression cassette" refers to a nucleic acid unit comprising at
least a VEGF receptor protein under the control of regulatory
sequences controlling its expression. The expression cassette
comprised in the attenuated mutant strain of Salmonella can
preferably mediate transcription of the included open reading frame
encoding a VEGF receptor protein in a target cell. Expression
cassettes typically comprise a promoter, at least one open reading
frame and a transcription termination signal.
[0048] VEGF receptor proteins are endothelial cell-specific
receptor-tyrosine kinases that can be bound by the ligand vascular
endothelial growth factor (VEGF) which causes them to dimerize and
become activated through transphosphorylation. The VEGF family of
growth factors (Kd 75-760 pM) encompasses 6 family members, VEGF-A
(also known as VEGF) through E and PLGF (placental growth factor,
also known as PGF or PIGF-2). VEGF growth factors regulate growth
and differentiation of multiple components of the vascular system,
especially blood and lymph vessels. There are three main subtypes
of VEGFR, VEGFR-1 (or FLT1), VEGFR-2 (or KDR, FLK1) and VEGFR-3 (or
FLT4). Membrane-bound VEGF receptors have an extracellular portion
consisting of 7 immunoglobulin-like domains, a single transmembrane
spanning region and an intracellular portion containing a split
tyrosine-kinase domain. VEGFR transcripts give also rise to
alternative splice variants that encode soluble VEGF receptor
proteins.
[0049] VEGFR-2, also known as kinase-insert-domain-containing
receptor (KDR), appears to mediate almost all of the known cellular
responses to VEGF. For example, the role of VEGF in angiogenesis
appears to be mediated through the interaction of this protein with
VEGFR-2. VEGFR-2 is a 1356 amino acid long, 200-230 kDa molecular
weight high-affinity receptor for VEGF, as well as for VEGF-C and
VEGF-D. Identified in humans through the screening of endothelial
cDNA for tyrosine kinase receptors, VEGFR-2 shares 85% sequence
identity with the previously discovered mouse fetal liver kinase 1
(Flk-1). VEGFR-2 is normally expressed in endothelial and
hematopoietic precursors, as well as in endothelial cells, nascent
hematopoietic stem cells and the umbilical cord stroma. However, in
quiescent adult vasculature, VEGFR-2 mRNA appears to be down
regulated.
[0050] The extracellular domain of VEGFR-2 contains 18 potential
N-linked glycosylation sites. VEGFR-2 is initially synthesized as a
150 kDa protein and rapidly glycosylated to a 200 kDa intermediate
form, and then further glycosylated at a slower rate to a mature
230 kDa protein which is expressed on the cell surface.
[0051] The amino acid sequence of the human VEGFR-2 encoding cDNA
sequence cloned into the pVAX10.VR2-1 plasmid is presented in FIG.
1.
[0052] In particular embodiments, the attenuated mutant strain of
Salmonella typhi of the present invention is for use as a
medicament.
[0053] In particular embodiments, the attenuated mutant strain of
Salmonella typhi of the present invention is for use as a
vaccine.
[0054] In the context of the present invention, the term "vaccine"
refers to an agent which is able to induce an immune response in a
subject upon administration. A vaccine can preferably prevent,
ameliorate or treat a disease. A vaccine in accordance with the
present invention comprises an attenuated mutant strain of
Salmonella typhi, preferably S. typhi Ty21a. The vaccine in
accordance with the present invention further comprises at least
one copy of a recombinant DNA molecule comprising an expression
cassette, preferably a eukaryotic expression cassette, encoding a
VEGF receptor protein, preferably selected from human VEGFR-2 or a
protein that shares at least about 80% sequence identity
therewith.
[0055] The live attenuated Salmonella mutant strain according to
the present invention comprising a recombinant DNA molecule
encoding a VEGF receptor protein can be used as a vehicle for the
oral delivery of this recombinant DNA molecule. Such a delivery
vector comprising a DNA molecule encoding a heterologous antigen,
such as a VEGF receptor protein, is termed DNA vaccine.
[0056] Genetic immunization might be advantageous over conventional
vaccination. The target DNA can be detected for a considerable
period of time thus acting as a depot of the antigen. Sequence
motifs in some plasmids, like GpC islands, are immunostimulatory
and can function as adjuvants furthered by the immunostimulation
due to LPS and other bacterial components. Live bacterial vectors
produce their own immunomodulatory factors such as
lipopolysaccharides (LPS) in situ which may constitute an advantage
over other forms of administration such as microencapsulation.
Moreover, the use of the natural route of entry proves to be of
benefit since many bacteria, like Salmonella, egress from the gut
lumen via the M cells of Peyer's patches and migrate eventually
into the lymph nodes and spleen, thus allowing targeting of
vaccines to inductive sites of the immune system.
[0057] Furthermore, attenuated derivatives of Salmonella enterica
are attractive as vehicles for the delivery of heterologous
antigens the mammalian immune system, because S. enterica strains
can potentially be delivered via mucosal routes of immunization,
i.e. orally or nasally, which offers advantages of simplicity and
safety compared to parenteral administration. Furthermore,
Salmonella strains elicit strong humoral and cellular immune
responses at the level of both systemic and mucosal
compartments.
[0058] In particular embodiments, the attenuated mutant strain of
Salmonella typhi is Salmonella typhi Ty21a. The vaccine strain
Ty21a has been demonstrated to date to have an excellent safety
profile. Upon exit from the gut lumen via the M cells, the bacteria
are taken up by phagocytic cells, such as macrophages and dendritic
cells. These cells are activated by the pathogen and start to
differentiate, and probably migrate, into the lymph nodes and
spleen. Due to the attenuating mutations, bacteria of the S. typhi
Ty21a strain are not able to persist in these phagocytic cells but
die at this time point. The recombinant DNA molecules are released
and subsequently transferred into the cytosol of the phagocytic
immune cells, either via a specific transport system or by
endosomal leakage. Finally, the recombinant DNA molecules enter the
nucleus, where they are transcribed, leading to VEGF receptor
expression in the cytosol of the phagocytic cells. Specific
cytotoxic T cells against the VEGFR receptor protein are induced by
the activated antigen presenting cells (APCs).
[0059] There is no data available to-date indicating that S. typhi
Ty21a is able to enter the bloodstream systemically. The live
attenuated Salmonella typhi Ty21a vaccine strain thus allows
specific targeting of the immune system while exhibiting an
excellent safety profile.
[0060] In particular embodiments, the VEGF receptor protein is
selected from the group consisting of human VEGFR-2 and a homolog
thereof that shares at least about 80% homology therewith.
[0061] In this context, the term "about" or "approximately" means
within 80% to 120%, alternatively within 90% to 110%, including
within 95% to 105% of a given value or range.
[0062] In the context of the present invention, the term "protein
that has at least about 80% sequence identity with human VEGFR-2"
refers to a protein that differs in the amino acid sequence and/or
the nucleic acid sequence encoding the amino acid sequence of human
VEGFR-2. The protein may be of natural origin, e.g. a homolog of
VEGFR-2 of a different species, or an engineered protein, e.g. an
engineered VEGFR-2 derivative. It is known that the usage of codons
is different between species. Thus, when expressing a heterologous
protein in a target cell, it may be necessary, or at least helpful,
to adapt the nucleic acid sequence to the codon usage of the target
cell. Methods for defining and constructing derivatives of a given
protein are well-known to any one of ordinary skill in the art.
[0063] The protein that shares at least about 80% sequence identity
with human VEGFR-2 may contain one or more mutations comprising an
addition, a deletion and/or a substitution of one or more amino
acids. According to the teaching of the present invention, said
deleted, added and/or substituted amino acids may be consecutive
amino acids or may be interspersed over the length of the amino
acid sequence of the protein that shares at least about 80%
sequence identity with human VEGFR-2. According to the teaching of
the present invention, a number of amino acids may be added,
deleted, and/or substituted, as long as the sequence identity with
human VEGFR-2 is at least about 80%. In particular embodiments, the
sequence identity with human VEGFR-2 is at least about 80%, at
least about 85%, at least about 90%, or most particularly at least
about 95%. Methods and algorithms for determining sequence identity
including the comparison of the parental protein and its
derivatives having deletions, additions and/or substitutions
relative to a parental sequence, are well-known to the practitioner
of ordinary skill in the art. On the DNA level, the nucleic acid
sequences encoding the protein that has at least about 80% sequence
identity with human VEGFR-2 may differ to a larger extent due to
the degeneracy of the genetic code.
[0064] In particular embodiments, the VEGF receptor protein is
human VEGFR-2 having the amino acid sequence as found in SEQ ID NO
1.
[0065] In another aspect, the present invention relates to a DNA
vaccine comprising the attenuated mutant strain of Salmonella typhi
of the present invention.
[0066] In particular embodiments, the attenuated mutant strain of
Salmonella typhi is Salmonella typhi Ty21a. The attenuated S. typhi
Ty21a strain is an active component of Typhoral L.RTM., also known
as Vivotif.RTM. (manufactured by Berna Biotech Ltd., a Crucell
Company, Switzerland). It is currently the only licensed live oral
vaccine against typhoid fever. This vaccine has been extensively
tested and has proved to be safe regarding patient toxicity as well
as transmission to third parties (Wandan et al., J. Infectious
Diseases 1982, 145:292-295). The vaccine is licensed in more than
40 countries. The Marketing Authorization number of Typhoral L.RTM.
is PL 15747/0001 dated 16 Dec. 1996. One dose of vaccine contains
at least 2.times.10.sup.9 viable S. typhi Ty21a colony forming
units and at least 5.times.10.sup.9 non-viable S. typhi Ty21a
cells.
[0067] One of the biochemical properties of the Salmonella typhi
Ty21a bacterial strain is its inability to metabolize galactose.
The attenuated mutant strain is also not able to reduce sulfate to
sulfite which differentiates it from the wild type Salmonella Ty2
strain. With regard to its serological characteristics, the
Salmonella typhi Ty21a strain contains the 09-antigen which is a
polysaccharide of the outer membrane of the bacteria and lacks the
05-antigen which is in turn a characteristic component of
Salmonella typhimurium. This serological characteristic supports
the rationale for including the respective test in a panel of
identity tests for batch release.
[0068] In particular embodiments, the expression cassette is a
eukaryotic expression cassette. In the context of the present
invention, the term "eukaryotic expression cassette" refers to an
expression cassette which allows for expression of the open reading
frame in a eukaryotic cell. It has been shown that the amount of
heterologous antigen required to induce an adequate immune response
may be toxic for the bacterium and result in cell death,
over-attenuation or loss of expression of the heterologous antigen.
Using a eukaryotic expression cassette that is not expressed in the
bacterial vector but only in the target cell may overcome this
toxicity problem and the protein expressed may exhibit a eukaryotic
glycosylation pattern.
[0069] A eukaryotic expression cassette comprises regulatory
sequences that are able to control the expression of an open
reading frame in a eukaryotic cell, preferably a promoter and
polyadenylation signal. Promoters and polyadenylation signals
included in the recombinant DNA molecules comprised by the
attenuated mutant strain of Salmonella of the present invention are
preferably selected to be functional within the cells of the
subject to be immunized. Examples of suitable promoters, especially
for the production of a DNA vaccine for humans, include but are not
limited to promoters from cytomegalovirus (CMV), such as the strong
CMV immediate early promoter, Simian virus 40 (SV40), Mouse Mammary
Tumor Virus (MMTV), Human Immunodeficiency Virus (HIV), such as the
HIF Long Terminal Repeat (LTR) promoter, Moloney virus, Epstein
Barr Virus (EBV), and from Rous Sarcoma Virus (RSV) as well as
promoters from human genes such as human actin, human myosin, human
hemoglobin, human muscle creatine, and human metallothionein. In a
particular embodiment, the eukaryotic expression cassette contains
the CMV promoter. In the context of the present invention, the term
"CMV promoter" refers to the strong immediate-early cytomegalovirus
promoter.
[0070] Examples of suitable polyadenylation signals, especially for
the production of a DNA vaccine for humans, include but are not
limited to the bovine growth hormone (BGH) polyadenylation site,
SV40 polyadenylation signals and LTR polyadenylation signals. In a
particular embodiment, the eukaryotic expression cassette included
in the recombinant DNA molecules comprised by the attenuated mutant
strain of Salmonella of the present invention comprises the BGH
polyadenylation site.
[0071] In addition to the regulatory elements required for
expression of the VEGF receptor protein, like a promoter and the
polyadenylation signal, other elements can also be included in the
recombinant DNA molecule. Such additional elements include
enhancers. The enhancer can be, for example, the enhancer of human
actin, human myosin, human hemoglobin, human muscle creatine and
viral enhancers such as those from CMV, RSV and EBV.
[0072] Regulatory sequences and codons are generally species
dependent, so in order to maximize protein production, the
regulatory sequences and codons are preferably selected to be
effective in the species to be immunized. The person skilled in the
art can produce recombinant DNA molecules that are functional in a
given subject species.
[0073] In particular embodiments, the DNA vaccine of the present
invention is for use in cancer immunotherapy.
[0074] In particular embodiments, the DNA vaccine comprises the
attenuated Salmonella typhi strain Ty21a transformed by a plasmid
that contains a DNA encoding the VEGFR-2 protein of SEQ ID NO
1.
[0075] In particular embodiments, the DNA vaccine is for use in an
anti-angiogenic cancer immunotherapy.
[0076] In particular embodiments, the recombinant DNA molecule
comprises the kanamycin antibiotic resistance gene, the pMB1 ori,
and a eukaryotic expression cassette encoding human VEGFR-2 or a
protein that shares at least 80% sequence homology therewith, under
the control of a CMV promoter. In particular embodiments, human
VEGFR-2 has the nucleic acid sequence as found in SEQ ID NO 2.
[0077] The eukaryotic Cytomegalovirus (CMV) immediate-early
promoter ensures efficient translation of the VEGFR-2 protein in
the host cell, and the prokaryotic origin of replication (ori)
mediates multiplication within the bacterial host.
[0078] In particular embodiments, the recombinant DNA molecule is
derived from commercially available pVAX1.TM. expression plasmid
(Invitrogen, San Diego, Calif.). This expression vector was
modified by replacing the high copy pUC origin of replication by
the low copy pMB1 origin of replication of pBR322. The low copy
modification was made in order to reduce the metabolic burden and
to make the construct more stable. Details of the plasmid
pVAX10.VR2-1 construct are depicted in FIG. 2. The generated
expression vector backbone was designated pVAX10. Inserting human
VEGFR-2 of the nucleic acid sequence as found in SEQ ID NO 2 into
this expression vector backbone yielded the expression plasmid
pVAX10.VR2-1.
[0079] The expression plasmid pVAX10.VR2-1 is schematically
depicted in FIG. 2. In particular embodiments, the plasmid is the
7580 bp pVAX10.VR2-1 as depicted in FIG. 2 and has the sequence as
found in SEQ ID NO 3 and the DNA vaccine is designated VXM01. VXM01
is an oral cancer vaccine consisting of an attenuated strain of
Salmonella enterica serovar typhi Ty21a carrying at least one copy
of a plasmid DNA, pVAX10.VR2-1, encoding a eukaryotic expression
cassette of the human Vascular Endothelial Growth Factor-Receptor 2
(VEGFR-2).
[0080] In particular embodiments, the cancer is pancreatic
cancer.
[0081] In particular embodiments, said pancreatic cancer is stage
IV or locally advanced pancreatic cancer.
[0082] In particular embodiments, the cancer includes
metastases.
[0083] In particular embodiments, cancer immunotherapy further
comprises administration of one or more further attenuated mutant
strain(s) of Salmonella comprising at least one copy of a
recombinant DNA molecule comprising an expression cassette encoding
a tumor antigen and/or a tumor stroma antigen. In particular
embodiments, said one or more further mutant strain(s) of
Salmonella is/are Salmonella typhi Ty21a comprising a eukaryotic
expression cassette. In particular embodiments, said one or more
further strain(s) of Salmonella comprise(s) an attenuated mutant
strain of Salmonella encoding human WT1.
[0084] Combining the attenuated mutant strain of Salmonella of the
present invention with a second attenuated mutant strain comprising
a DNA molecule encoding a second tumor stroma antigen or a tumor
antigen may have synergistic antitumor effects. In particular,
simultaneous targeting of the tumor and the tumor stroma may
minimize the risk of tumor escape. Combining VEGFR-2 based
immunotherapy with WT1 based cancer immunotherapy may prove
especially effective, since WT1 overexpressing human cells and the
tumor vasculature are attacked at the same time.
[0085] In particular embodiments, the attenuated mutant strain of
Salmonella is co-administered with said one or more further
attenuated mutant strain(s) of Salmonella.
[0086] In the context of the present invention, the term
"co-administration" or "co-administer" means administration of two
different attenuated mutant strains of Salmonella within three
consecutive days, more particularly within two consecutive days,
more particularly on the same day, more particularly within 12
hours. Most particularly, in the context of the present invention,
the term "co-administration" refers to simultaneous administration
of two different attenuated mutant strains of Salmonella.
[0087] In particular embodiments, the treatment is accompanied by
chemotherapy and/or radiotherapy and/or biological cancer therapy.
For cure of cancer, complete eradication of cancer stem cells may
be essential. For maximal efficacy, a combination of different
therapy approaches may be beneficial.
[0088] In the context of the present invention, the term
"biological cancer therapy" or "cancer immunotherapy" refers to the
stimulation of the patient's immune system to attack malignant
tumor cells or the tumor stroma. Biological cancer therapy
approaches include delivery of tumor antigens, delivery of
therapeutic antibodies as drugs, administration of
immunostimulatory cytokines and administration of immune cells.
[0089] Chemotherapeutic agents that may be used in combination with
the attenuated mutant strain of Salmonella of the present invention
may be, for example: amifostine (ethyol), cabazitaxel, cisplatin,
dacarbazine (DTIC), dactinomycin, docetaxel, mechlorethamine,
streptozocin, cyclophosphamide, carrnustine (BCNU), lomustine
(CCNU), doxorubicin (adriamycin), doxorubicin lipo (doxil), folinic
acid, gemcitabine (gemzar), daunorubicin, daunorubicin lipo
(daunoxome), procarbazine, ketokonazole, mitomycin, cytarabine,
etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine,
vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere),
aldesleukin, asparaginase, busulfan, carboplatin, cladribine,
camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38),
dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide,
idarubicin, mesna, interferon alpha, interferon beta, irinotecan,
mitoxantrone, topotecan, leuprolide, megestrol, melphalan,
mercaptopurine, oxaliplatin, plicamycin, mitotane, pegaspargase,
pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen,
teniposide, testolactone, thioguanine, thiotepa, uracil mustard,
vinorelbine, chlorambucil and combinations thereof.
[0090] Most preferred chemotherapeutic agents according to the
invention in combination with VXM01 are cabazitaxel, carboplatin,
oxaliplatin, cisplatin, cyclophosphamide, docetaxel, gemcitabine,
doxorubicin, paclitaxel (taxol), irinotecan, vincristine,
vinblastine, vinorelbin, folinic acid, 5-fluorouracil and
bleomycin, especially gemcitabine.
[0091] In particular embodiments, the chemotherapeutic agent is
gemcitabine.
[0092] It may be also favorable dependent on the occurrence of
possible side effects to include treatment with antibiotics or
anti-inflammatory agents.
[0093] Should adverse events occur that resemble hypersensitivity
reactions mediated by histamine, leukotrienes, or cytokines,
treatment options for fever, anaphylaxis, blood pressure
instability, bronchospasm, and dyspnoea are available. Treatment
options in case of unwanted T-cell derived auto-aggression are
derived from standard treatment schemes in acute and chronic graft
vs. host disease applied after stem cell transplantation.
Cyclosporin and glucocorticoids are proposed as treatment
options.
[0094] In the unlikely case of systemic Salmonella typhi Ty21a type
infection, appropriate antibiotic therapy is recommended, for
example with fluoroquinolones including ciprofloxacin or ofloxacin.
Bacterial infections of the gastrointestinal tract are to be
treated with respective agents, such as rifaximin.
[0095] In particular embodiments, the attenuated mutant strain of
Salmonella is administered during the chemotherapy or the
radiotherapy treatment cycle or during biological cancer therapy.
In particular embodiments, the immunotherapeutic treatment with the
vaccine is carried out during the chemotherapy treatment cycle.
[0096] In particular embodiments, the attenuated mutant strain of
Salmonella is administered before the chemotherapy or the
radiotherapy treatment cycle or before biological cancer therapy.
This approach may have the advantage that chemotherapy or
radiotherapy can be performed under conditions of enhanced cancer
immunity.
[0097] In particular embodiments, the attenuated mutant strain of
Salmonella is administered after the chemotherapy or the
radiotherapy treatment cycle or after biological cancer
therapy.
[0098] In particular embodiments, the vaccine is administered
orally. Oral administration is simpler, safer and more comfortable
than parenteral administration. Adverse effects of parenteral,
subcutaneous or intradermal administration may be overcome by oral
administration of the DNA vaccine of the present invention. The
attenuated mutant strain of Salmonella of the present invention may
however also be administered by any other suitable route.
Preferably, a therapeutically effective dose is administered to the
subject, and this dose depends on the particular application, the
type of malignancy, the subject's weight, age, sex and state of
health, the manner of administration and the formulation, etc.
Administration may be single or multiple, as required.
[0099] The attenuated mutant strain of Salmonella of the present
invention may be provided in the form of a solution, a suspension,
lyophilisate, or any other suitable form. It may be provided in
combination with pharmaceutically acceptable carriers, diluents,
and/or excipients. Agents for adjusting the pH value, buffers,
agents for adjusting toxicity, and the like may also be included.
In the context of the present invention, the term "pharmaceutically
acceptable" refers to molecular entities and other ingredients of
pharmaceutical compositions that are physiologically tolerable and
do not typically produce untoward reactions when administered to a
mammal (e.g., human). The term "pharmaceutically acceptable" may
also mean approved by a regulatory agency of a Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in mammals, and, more particularly,
in humans.
[0100] The vaccine of the present invention is surprisingly
effective at relatively low doses. In particular embodiments, the
single dose of the vaccine is about 1.times.10.sup.5, about
1.times.10.sup.6, about 1.times.10.sup.7, about 1.times.10.sup.8,
about 1.times.10.sup.9, about 1.times.10.sup.10, or about
1.times.10.sup.11 colony forming units (CFU). Administration of low
doses of this live bacterial vaccine minimizes the risk of
excretion and thus of transmission to third parties.
[0101] In this context, the term "about" or "approximately" means
within a factor of 3, alternatively within a factor of 2, including
within a factor of 1.5 of a given value or range.
[0102] In particular embodiments, the single dose of the vaccine is
less than about 1.times.10.sup.9 CFU. In particular embodiments,
the single dose of the vaccine is from 1.times.10.sup.8 to
1.times.10.sup.9 CFU.
[0103] In particular embodiments, the single dose of the vaccine is
less than about 1.times.10.sup.8 CFU. In particular embodiments,
the single dose of the vaccine is from 1.times.10.sup.5 to
1.times.10.sup.8 CFU, more particularly the single dose of the
vaccine is from 1.times.10.sup.6 to 1.times.10.sup.7 CFU.
[0104] In particular embodiments, the single dose comprises from
about 10.sup.5 to about 10.sup.11, particularly form about 10.sup.6
to about 10.sup.10, more particularly from about 10.sup.6 to about
10.sup.9, more particularly from about 10.sup.6 to about 10.sup.8,
most particularly from about 10.sup.6 to about 10.sup.7 colony
forming units (CFU).
[0105] In particular embodiments, the attenuated mutant strain of
Salmonella is for use in individualized cancer immunotherapy.
Individualized cancer immunotherapy may comprise the step of
assessing the tumor stroma antigen expression pattern and/or the
tumor antigen expression pattern of a patient. Individualized
cancer immunotherapy may also comprise the step of assessing
pre-immune responses against a tumor stroma antigen or a tumor
antigen, preferably the pre-immune response against VEGFR-2. In
line with this, pre-existing immune responses against VEGFR-2 were
shown to strongly correlate with positive clinical responses of
VXM01, in particular with a decrease in tumor perfusion.
[0106] VXM01 can be used--either by itself or in combination with
other Salmonella typhi Ty21a based cancer vaccines comprising
eukaryotic expression systems--for the treatment of various cancer
types. In particular embodiments, VXM01 may be used for
individualized patient specific cancer treatment. For that purpose,
the patient's stromal and/or tumor antigen expression pattern may
be assessed in a first step for example by companion diagnostics
targeting the patient's specific stromal and/or tumor antigen
pattern. Alternatively, pre-existing immune responses against
stromal and/or tumor antigens may be assessed. Depending on the
patient's stromal and/or tumor antigen expression pattern, VMX01
may be administered either alone or in combination with one or more
suitable further Salmonella typhi Ty21a based cancer vaccine(s)
comprising eukaryotic expression systems. Combinations of VXM01
with one or more further Salmonella typhi Ty21a based cancer
vaccine(s) may however also be administered as fixed combinations.
These cocktails combining two or more Salmonella typhi Ty21a based
cancer vaccines can be composed of separate off the shelf products.
The combinations, either fixed or individualized, may contain VXM01
as anti-angiogenic basis therapy.
[0107] In another aspect, the present invention relates to the DNA
vaccine VXM01, comprising the attenuated Salmonella typhi strain
Ty21a transformed by a plasmid that contains a DNA encoding the
VEGFR-2 protein of SEQ ID NO 1, wherein the plasmid is a 7580 bp
plasmid DNA and comprises the cDNA of VEGFR-2 that is under the
control of the CMV promoter, the kanamycin resistance gene, and the
pMB1 ori, and is designated as pVAX10.VR2-1.
SHORT DESCRIPTION OF FIGURES AND TABLES
[0108] FIG. 1: Amino acid sequence of VEGFR-2 cDNA cloned into
plasmid pVAX10.VR2-1
[0109] FIG. 2: Plasmid map of pVAX10.VR2-1
[0110] FIG. 3: Dose escalating design of vaccine VXM01
[0111] FIG. 4: Overall study scheme
[0112] FIG. 5: VXM01 specific T cell responses
[0113] FIG. 6: Effects of VXM01 on tumor perfusion
[0114] FIG. 7: Effects of VXM01 on VEGF A serum levels
[0115] FIG. 8: Effects of VXM01 on collagen IV serum levels
[0116] FIG. 9: Effects of VXM01 on blood pressure
[0117] FIG. 10: VXM01-induced anti-carrier immunity
[0118] FIG. 11: Analysis cascade of VXM01 excretion
[0119] FIG. 12: VXM01 excretion
[0120] Table 1: Patient selection criteria
[0121] Table 2: VMX01 specific T cell responses
EXAMPLES
Example 1
Salmonella typhi Ty21a Strain Preparation and Plasmid
Preparation
[0122] The first step in the preparation of the research seed lot
(RSL) consisted of the isolation of the attenuated Salmonella typhi
Ty21a strain followed by the transformation of the attenuated
bacteria with the plasmid DNA (pVAX10.VR2-1).
[0123] Liquid culture medium was inoculated with a Salmonella typhi
Ty21a isolate and the liquid culture was then plated onto an agar
medium for the purpose of isolating single bacterial colonies.
Single colonies were isolated and grown in liquid culture medium.
Two cultures, namely VAX.Ty21-1 and VAX.Ty21-2, were then
formulated with glycerol, aliquoted (1 ml) and stored at
-75.degree. C..+-.5.degree. C. pending use. Identity of each of the
two cultures was further confirmed.
[0124] The principle of plasmid synthesis was based on double
strand in vitro gene synthesis with the following steps: [0125] The
whole pVAX10-VR2.1 plasmid sequence of 7.58 kb was subdivided (by
software analysis) in 5 sections of .about.1.5 kb. Each section was
subdivided into 40-50 bp oligonucleotides each having overlapping
regions between oligonucleotides of both strands [0126] The in
vitro synthesized oligonucleotides were then phosphorylated by
incubation with T4 polynucleotide kinase [0127] After the annealing
process of overlapping oligonucleotides under appropriate
conditions, the Taq DNA ligase enzyme connected the aligned
oligonucleotides [0128] Upon completion of the ligation step, PCR
was performed using primers annealed at outward positions, to
increase the yield of the ligated plasmid fragments (.about.1.5 kb)
[0129] A preparative agarose gel electrophoresis was performed to
isolate the PCR products [0130] The isolated PCR products were
cloned into TOPO vectors (Invitrogen K#4575-40) and transformed
into TOP10 E. coli cells for propagation [0131] After TOPO plasmid
isolation, a restriction and sequence verification was performed
[0132] The isolated aligned fragments were assembled via overlap
PCR. This process was followed by linearly assembling the
pVAX10.VR2-1 plasmid [0133] After XhoI restriction digest (single
restriction site is present in the pVAX10.VR2-1 plasmid, see FIG.
2) and covalent binding via T4 ligase, E. coli was transformed with
the circular plasmid for propagation [0134] After final plasmid
sequence verification, the pVAX10.VR2-1 plasmid was transformed
into the S. typhi Ty21a bacterial strain.
[0135] The plasmid pVAX10.VR2-1 was thus successfully synthesized
(no deviation to reference sequence). This plasmid was further used
to transform the S. typhi Ty21a bacterial strain.
Example 2
VXM01--Phase I Clinical Trial; Study Description
[0136] This phase I trial examined the safety, tolerability, and
immunological and clinical responses to VXM01. The randomized,
placebo-controlled, double blind dose-escalation study included 45
patients with locally advanced or stage IV pancreatic cancer. The
patients received four doses of VXM01 or placebo in addition to
gemcitabine as standard of care. Doses from 10.sup.6 CFU up to
10.sup.10 CFU of VXM01 were evaluated in the study. An independent
data safety monitoring board (DSMB) was involved in the
dose-escalation decisions. In addition to safety as primary
endpoint, the VXM01-specific immune reaction, as well as clinical
response parameters were evaluated.
Preclinical Efficacy Assessment:
[0137] The efficacy and safety of this approach in animals has been
validated multiple times by the inventors. Further experiments by
the inventors showed an activity of this vaccine in two different
models of pancreatic cancer.
[0138] VXM01, the vaccine used in this trial, is a humanized
version of the anti-VEGFR-2 vaccine previously tested in mice. It
encodes the human full-length VEGFR-2 and uses the licensed
Salmonella typhi strain Ty21a instead of Salmonella typhimurium as
a carrier. The vaccine is assumed to lead to VEGFR-2 protein
expression in monocytes and dendritic cells after entry of VXM01 in
the Peyer's patches via M cells of the gut, and internalization by
antigen-presenting cells followed by translation of the encoded
DNA.
Preclinical Safety Assessment:
[0139] Preclinical toxicity studies in mice included, but were not
restricted to a single dose toxicity study in mice conducted with
the human vaccine VXM01. As VXM01 is specific for the human host,
the study of the human vaccine in mice focused on possible effects
of process-related impurities and related signs and symptoms of
possible relevance for cardiovascular, respiratory, or central
nervous system impairment. In order to investigate the toxicity
profile of an anti-VEGFR-2 T-cell response, a repeated dose
toxicity study was conducted using the murine analog construct of
VXM01 which induced a dose-dependent T-cell response in mice. In
accordance to the inventors' previous observations, no
treatment-related deaths and no toxicologically important clinical
signs were observed throughout these studies, which were conducted
according to Good Laboratory Practice (GLP).
[0140] The vector Salmonella typhi Ty21a used here is a live,
attenuated bacterial carrier that allows for the oral delivery of
the vaccine VXM01. It is itself an approved vaccine against typhoid
fever (Vivotif.RTM., Crucell, formerly Berna Biotech Ltd.,
Switzerland) that has been extensively tested and has demonstrated
its safety regarding patient toxicity as well as transmission to
third parties (Wandan et al., J. Infectious Diseases 1982,
145:292-295). VXM01 is classified as a gene transfer medicinal
product and subject to the respective guidance and regulations.
Study Descriptions and Objectives:
[0141] The conducted study was a monocenter, placebo controlled,
double blind dose escalation study of the experimental vaccine
VXM01 in patients with inoperable or stage IV pancreatic cancer.
The vaccine was given as add-on to a standard of care gemcitabine
treatment.
[0142] The objectives were to examine the safety and tolerability,
and immunological and clinical responses to the investigational
anti-VEGFR-2 vaccine VXM01, as well as to identify the maximum
tolerated dose (MTD) of VXM01. The MTD is defined as the highest
dose level at which less than two of up to six patients under VXM01
treatment experience a dose-limiting toxicity (DLT).
[0143] Primary endpoints for safety and tolerability were as
follows: Number of DLTs defined as any adverse event (AE) related
to study drug of grade 4 or higher, or grade 3 or higher for
gastrointestinal fistula, diarrhea, gastrointestinal perforation,
multi-organ failure, anaphylaxis, any auto-immune disorder,
cytokine-release syndrome, intestinal bleeding, renal failure,
proteinuria, thromboembolic events, stroke, heart failure, or
vasculitis according to the National Cancer Institute Common
Terminology Criteria for Adverse Events (CTCAE).
[0144] Secondary endpoints, which assess the efficacy of the
experimental vaccine to elicit a specific immune response to
VEGFR-2, included the number of immune positive patients.
[0145] A further secondary endpoint was the clinical response:
Tumor staging according to the response evaluation criteria in
solid tumors (RECIST), overall response rate, progression free
survival, overall survival, and changes in tumor perfusion. Tumor
perfusion was determined by dynamic contrast-enhanced Magnetic
Resonance Imaging (DCE-MRI) on a 1.5 Tesla system (Magnetom Aera,
Siemens, Erlangen, Germany).
[0146] VXM01 had been manufactured according to Good Manufacturing
Practice (GMP) and was given in a buffered solution. The placebo
control consisted of isotonic sodium chloride solution.
Patient Selection and Clinical Study Design:
[0147] The study included a maximum of 45 patients with either
locally advanced and inoperable or stage IV pancreatic cancer. The
eligibility criteria are summarized in Table 1.
TABLE-US-00001 Inclusion Criteria 1. Written informed consent,
signed and dated 2. Locally advanced, inoperable and stage IV
pancreatic cancer patients according to UICC based on diagnostic
imaging using computer-tomography (CT) or histological examinations
3. Male or post-menopausal female 4. Age .gtoreq.18 years 5.
Chemotherapy naive within 60 days before screening visit except
gemcitabine treatment 6. Karnofsky index >70 7. Life expectancy
>3 months 8. Adequate renal, hepatic, and bone marrow function
9. Absolute neutrophil count >1500/.mu.L 10. Hemoglobin >10
g/dL 11. Platelets >75000/.mu.L 12. Prothrombin time and
international normalized ratio (INR) <1.5 times upper limit of
normal (ULN) (except under anticoagulant treatment) 13. Aspartate
aminotransferase <4 times ULN 14. Alanine aminotransferase <4
times ULN 15. Total bilirubin <3 times ULN 16. Creatinine
clearance estimated according to Cockcroft-Gault > 30 mL/min 17.
Proteinuria <1 g protein on 24 h urine collection Exclusion
Criteria 18. State after pancreas resection (complete or partial)
19. Resectable disease 20. Drug trial participation within 60 days
before screening visit 21. Other previous or current malignancy
except basal or squamous cell skin cancer, in situ cervical cancer,
or any other cancer from which the patient has been disease- free
for <2 years 22. Prior vaccination with Ty21a State after
pancreas resection (complete or partial) 23. Resectable disease 24.
Drug trial participation within 60 days before screening visit 25.
Other previous or current malignancy except basal or squamous cell
skin cancer, in situ cervical cancer, or any other cancer from
which the patient has been disease- free for <2 years 26. Prior
vaccination with Ty21a 27. Cardiovascular disease defined as:
Uncontrolled hypertension (systolic blood pressure >160 mmHg or
diastolic blood pressure >100 mmHg) Arterial thromboembolic
event within 6 months before randomization including: Myocardial
infarction Unstable angina pectoris Cerebrovascular accident
Transient ischemic attack 28. Congestive heart failure New York
Heart Association grade III to IV 29. Serious ventricular
arrhythmia requiring medication 30. Clinically significant
peripheral artery disease > grade 2b according to Fontaine 31.
Hemoptysis within 6 months before randomization 32. Esophageal
varices 33. Upper or lower gastrointestinal bleeding within 6
months before randomization 34. Significant traumatic injury within
4 weeks before randomization 35. Non-healing wound, bone fracture
or any history of gastrointestinal ulcers within three years before
inclusion, or positive gastroscopy within 3 months before inclusion
36. Gastrointestinal fistula 37. Thrombolysis therapy within 4
weeks before randomization 38. Bowel obstruction within the last 30
days before screening visit 39. Liver cirrhosis .gtoreq. grade B
according to Child-Pugh Score-Classification 40. Presence of any
acute or chronic systemic infection 41. Radiotherapy within 4 weeks
before randomization 42. Major surgical procedures, or open biopsy
within 4 weeks before randomization 43. Fine needle aspiration
within 7 days before randomization 44. Chronic concurrent therapy
within 2 weeks before and during the double-blind study period
with: Corticosteroids (except steroids for adrenal failure) or
immunosuppressive agents Antibiotics Bevacizumab Any epidermal
growth factor receptor inhibitor 45. Chemotherapy except
gemcitabine before Day 10 Multi-drug resistant gram- negative germ
46. Pregnancy 47. Lactation 48. Inability to comply with study
and/or follow-up procedures 49. History of other disease, metabolic
dysfunction, physical examination finding, or clinical laboratory
finding giving reasonable suspicion of a disease or condition that
contraindicates the use of an investigational drug or that might
affect the interpretation of the study results or render the
patient at high risk for treatment complications 50. Women of
childbearing potential 51. Any history of drug hypersensitivity 52.
Any condition which results in an undue risk for the patient during
the study participation according to the investigator
[0148] A total of 371 patients were screened for the study. 326
patients were ineligible because of excluded medical therapies
(179), preexisting medical conditions (129) in patient's medical
history and personal reasons (18). 45 patients were enrolled,
randomized and completed successfully the 10 days in-house study
phase at the Clinical Research Unit at the University Clinics of
Heidelberg (KliPS), in line with the study protocol. Demographic
baseline disease characteristics of the patients were not
significantly different in the two groups, but time since diagnosis
was longer in the VXM01 group (8 vs. 6 months) and patients in the
VXM01 group had a more advanced tumor stage at the time of
inclusion (CA19.9>1000 in 40% vs. 20% and metastatic disease in
83% vs. 53%).
[0149] Male and postmenopausal female patients were enrolled in
this study. However, differences between the two genders were not
investigated. The average survival time of the patients
participating in this trial was under 6 months. However, the
follow-up period for the patients as defined per protocol was up to
24 months. The study treatment was given first-line as an add-on to
standard of care. Taking further into account other factors, among
them the multiple primary and secondary pharmacodynamic preclinical
studies, the risk-benefit analysis was assumed to have a favorable
result for the patient population selected.
[0150] The starting dose consisted of a solution containing
10.sup.6 colony forming units (CFU) of VXM01 or placebo. This VXM01
dose was chosen for safety reasons and was assumed to be below the
minimal effective dose to elicit an immune response. For
comparison, one dose of Typhoral.RTM., the licensed vaccine against
typhoid fever, contains 2.times.10.sup.9 to 6.times.10.sup.9 CFU of
Salmonella typhi Ty21a, equivalent to approximately thousand times
the VXM01 starting dose. The dose was escalated in factor-of-ten
logarithmic steps, which appears to be justified for a live
bacterial vaccine. The dose escalating design is depicted in FIG.
3.
[0151] Complying with guidelines for first in human trials, the
patients of one dose group were treated in cohorts. The first
administration of VXM01 in any dose group was given to one patient
only accompanied by one patient receiving placebo. The second
cohort of each dose group consisted of two patients receiving VXM01
and one patient receiving placebo. This staggered administration
with one front-runner, i.e. only one patient receiving VXM01 first,
served to mitigate the risks.
[0152] A third cohort of patients (three receiving VXM01 and one
receiving placebo) were included in the 10.sup.8, 10.sup.9, and
10.sup.10 dose groups. This approach minimized exposure to VXM01
doses assumed to be sub-therapeutic. The third cohort and the first
two cohorts of the next higher treatment group were treated in
parallel based on a clearly defined randomization strategy. This
strategy allowed for recruitment of available patients and avoided
selection bias for patients treated in parallel in the lower and
higher dose group. In the 10.sup.6 and 10.sup.7 dose groups, a
third cohort of patients was included only if one patient out of
the initial three patients receiving VXM01 of the respective dose
group experienced a DLT and required confirmation by a decision of
the Data Safety Monitoring Board (DSMB).
[0153] All patients completed the seven day vaccination course of 4
doses every second day in line with the protocol without any dose
reduction. Because of no observed dose limiting toxicities (DLT)
the maximum tolerated dose was not reached. VXM01 was well
tolerated at all dose levels. AEs and SAEs where equally
distributed among both groups and there were no obvious signs for
dose-dependent side effects among the groups.
[0154] The environmental risk inherent to an oral vaccine is the
potential of excretion to the environment and subsequent
vaccination of people outside the target population. All study
patients were confined in the study site (KliPS) for the period
during which vaccinations took place plus three additional days.
All feces of study patients were collected and incinerated. Body
fluids and feces samples were investigated for VXM01 shedding.
Fecal excretion of VXM01 was observed in two patients, one in the
10.sup.9 and one in the 10.sup.10 dose group. VXM01 excretion in
feces in both patients was transient at one occasion after the
first or second administration, respectively, and disappeared
without antibiotic treatment. In other body fluids, no excretion
was determined.
[0155] Hygienic precautions were applied to protect study personnel
from accidental uptake. Study personnel was trained specifically
for this aspect of the study.
[0156] Patients were only discharged from hospital, if they tested
negative for excretion of the vaccine after the last administration
of the study drug. In case a patient tested positive for excretion
after the last administration, an antibiotic decontamination of the
gastrointestinal tract was conducted before the patient was
discharged. Excretion was followed up until results were negative.
These measures appear to have been justified and sufficient to
protect the environment and study personnel from exposure to VXM01
until the shedding profile had been elucidated.
[0157] VXM01 was applied in parallel to the gemcitabine background
therapy as shown in FIG. 4 (overall study scheme). In brief,
gemcitabine was given on days 1, 8, and 15 of a 28 days
chemotherapy cycle. The vaccine was given four times on days 1, 3,
5, and 7, starting three days after the last dose of gemcitabine.
The double blinded phase of the study ended 31 days after the last
patient had received the last administration.
[0158] For this phase I trial (advanced or stage IV pancreatic
cancer patients) a patient population with dismal prognosis and the
relatively gentle standard of care with regard to immunosuppression
was chosen. Co-regimes of the chemotherapeutic agent gemcitabine
with tumor vaccination may be synergistic. In addition, specific
T-cell activation was measured in this patient setting
demonstrating effectiveness of the vaccine VXM01. A placebo control
was included in the present trial, in order to gain further
knowledge on specific safety issues related to the active vaccine
vs. the background treatment. In addition, the pooled placebo
patients served as a sound comparator for assessing specific immune
activation and other signs of clinical efficacy. If and when moving
into phase II, a different patient entity with a longer life
expectancy can be envisaged depending on the observed safety
profile. Such studies will also include tumor types that have shown
to be more susceptible to anti-angiogenic treatment.
Example 3
VXM01 Specific T-Cell Responses
[0159] Responses to VXM01 were assessed by monitoring the
frequencies of VEGFR2 specific T-cells in peripheral blood of VXM01
and placebo treated patients, detected by INF.gamma. ELISpot, at
different time points prior during and post vaccination.
[0160] Firstly, T-cells and peptide pulsed DC were added to wells
coated with anti-INFy antibodies. After a period of incubation,
cells were removed with secreted INF.gamma. left binding with the
coat antibodies. Then detection antibody was added to detect the
bound INF.gamma., and after a signal amplification, the final yield
could be viewed as "color spots" representing single activated and
specific T-cells.
[0161] Positivity of ELISpot samples was graded according to
predefined rules defining signal increase resulting in grade 0 to 3
per sample:
No increase: grade 0 Clear increase but <3.times.: grade 1
.gtoreq.3.times. but <5.times. increase: grade 2
.gtoreq.5.times. increase: grade 3
[0162] The ELISpot immune response of study patients is depicted in
Table 2:
TABLE-US-00002 TABLE 2 VEGFR-2 specific T-cell response (Patients
w/grading score .gtoreq. 3) 10.sup.6 10.sup.7 10.sup.8 10.sup.9
10.sup.10 CFUs/ CFUs/ CFUs/ CFUs/ CFUs/ Placebo admin admin admin
admin admin 1/11 2/6 3/5 1/6 0/6 2/6
[0163] The results of the ELISpot immune response of study patients
is graphically depicted in FIG. 5.
Example 4
Effects on Tumor Perfusion
[0164] Tumor perfusion was evaluated by contrast media transit time
(Ktrans) during dynamic contrast enhanced magnetic resonance
imaging (DCEMRI) to characterize treatment response. Dynamic
Contrast-Enhanced T1-weighted imaging was performed. DCE-MRI was
assessed on a 1.5 Tesla System (Magnetom Aera, Siemens, Erlangen,
Germany) on day 0, 38 and 3 months after treatment. Dynamic
contrast-enhanced MR-imaging was performed with VIBE (volumetric
interpolated breath-hold) sequences. For that purpose, a dose of 8
ml Gadovist was injected.
[0165] For every examination, regions of interest were manually
drawn within the tumor-tissue followed by pixel-by-pixel analysis
using a Siemens software package (Tissue 4D). ROI-modeling was
based on the Tofts model with assumed T10 (1000 ms) and Parker AIF.
For the estimation of tumor-perfusion Ktrans was regarded as
primary endpoint.
[0166] Mean changes in tumor perfusion were -9% in the VXM01 group
(n=26) vs. +18% in the placebo group (n=11). A greater than 33%
drop in tumor perfusion was detected in 35% of evaluable VXM01
treated patients vs. 10% in the placebo group. The strongest
responders were further analyzed in a subgroup analysis. Maximum
average effects were detected at the d38 time point. The effects of
various doses of VXM01 on tumor perfusion are graphically depicted
in FIG. 6.
Example 5
Biomarkers of Angiogenesis
[0167] In order to further characterize the VEGFR-2 specific T-cell
mediated, anti-angiogenic activity of VXM01, accompanying changes
in angiogenesis biomarkers VEGF A, human collagen IV and blood
pressure were monitored.
VEGF A:
[0168] VEGF A was measured in human serum samples by ELISA using a
commercial assay kit (ELISA Kit Quantikine Human VEGF A
Immunoassay, R&D Systems, Cat.-No.: DVE00). The assay was used
as described in the package insert and as modified as part of this
study plan according to the foregoing validation study
580.132.2786.
[0169] This assay employs the quantitative sandwich enzyme
immunoassay technique. A monoclonal antibody specific for human
VEGF A had been pre-coated onto a microplate. Standards, quality
controls (commercially obtained) and samples were pipetted into the
wells and any VEGF A present was bound by the immobilized antibody.
Calibrator, quality control samples, and samples were analyzed as
duplicates. After washing away any unbound substances, an
enzyme-linked polyclonal antibody specific for VEGF A was added to
the wells. Following a wash to remove any unbound antibody-enzyme
reagent, a substrate solution was added to the wells and color
developed in proportion to the amount of VEGF A bound in the
initial step. The color development was stopped and the intensity
of the color was measured using a spectrophotometric microtiter
plate reader at 450 nm. A standard curve was generated by plotting
the absorbance versus the respective VEGF A concentration for each
standard. The concentration of VEGF A in the sample was determined
directly from this curve.
[0170] VEGF-A serum levels increased in the VXM01 group by 235% on
both d38 and m3 vs. 17% and 31% in the placebo group (p=0.05 at
m3). The quantification of VEGF A in patient serum samples is
graphically depicted in FIG. 7.
Collagen IV:
[0171] Human Collagen IV was measured in human serum samples by
ELISA using a commercial assay kit (Human Collagen IV ELISA, Serum,
KAMIYA BIOMEDICAL COMPANY, Cat.-No.: KT-035). The assay was used as
described in the package insert and as modified as part of this
study report according to the foregoing validation study
580.132.3645.
[0172] The human Collagen IV ELISA was a solid phase one-step
sandwich ELISA. Collagen IV in the sample was bound simultaneously
by a solid phase monoclonal antibody and a monoclonal
antibody-enzyme conjugate, each directed at different antigenic
sites. This resulted in the collagen IV molecule being sandwiched
between the solid phase and enzyme-labeled antibodies. After
removing unbound enzyme-labeled antibody and sample, the plate was
incubated with chromogenic substrate (TMB). The resultant color
development was directly proportional to the amount of collagen IV
in the sample.
[0173] Serum levels of collagen IV increased on d38 and m3 in
average by 7% and 22%, respectively, in the VXM01 group vs. changes
of 2% and -7% in the placebo group (p=0.02 at m3). The
quantification of collagen IV in patient serum samples is
graphically depicted in FIG. 8.
Blood Pressure:
[0174] Blood pressure (systolic and diastolic) and pulse rate as
pharmacodynamic markers of anti-angiogenic efficacy were measured
after 5 minutes rest in supine position. Average systolic blood
pressure changes were +3.6 mmHg and +3.9 mmHg in the treatment
group vs. -8.8 mHg and 9.1 mmHg under placebo (p=0.08 at d38).
Effects on average blood pressure after the first vaccination dose
(up to day 38) are graphically depicted in FIG. 9.
Example 6
Anti-Carrier Immunity
[0175] In order to assess immune responses to the bacterial
vehicle, anti-Salmonella typhi IgG and IgM immunoglobulins were
detected by ELISA using two commercial assay kits (Salmonella typhi
IgG ELISA, Cat. No. ST0936G and Salmonella typhi IgM ELISA, Cat.
No. ST084M; Calbiotech. Inc., 10461 Austin Dr, Spring Valley,
Calif. 91978, USA). These assays were qualitative assays. The
assays were used as described in the package inserts respectively
App. I/1) and as modified as part of the study plan according to
the foregoing validation study 580.132.2785.
[0176] Both assays employed the enzyme-linked immunosorbent assay
technique. Calibrator, negative control, positive control and
samples were analyzed as duplicates. Diluted patient serum
(dilution 1:101) was added to wells coated with purified antigen.
IgG or IgM specific antibody, if present, bound to the antigen. All
unbound materials were washed away and the enzyme conjugate was
added to bind to the antibody-antigen complex, if present. Excess
enzyme conjugate was washed off and substrate was added. The plate
was incubated to allow for hydrolysis of the substrate by the
enzyme. The intensity of the color generated was proportional to
the amount of IgG or IgM specific antibody in the sample. The
intensity of the color was measured using a spectrophotometric
microtiter plate reader at 450 nm. The cut off was calculated as
follows:
Calibrator OD.times.Calibrator Factor (CF).
[0177] The antibody index of each determination was determined by
dividing the OD value of each sample by cut-off value.
[0178] Antibody Index Interpretation:
TABLE-US-00003 <0.9 No detectable antibody to Salmonella typhi
IgG or IgM by ELISA 0.9-1.1 Borderline positive >1.1 Detectable
antibody to Salmonella typhi IgG or IgM by ELISA
[0179] The number of patients with detectable anti-Salmonella typhi
IgG immunoglobulins are depicted in FIG. 10.
Example 7
Excretion
[0180] The shedding of bacteria in stool and body fluids, tears,
saliva, urine and blood was monitored in the study VXM01-01-DE
according to methods validated transferred as formerly validated
according to GLP at an established central service laboratory
(Huntingdon Life Sciences, Huntingdon, UK). Shedding and
biodistribution in body fluids of VXM01 were determined by plate
and enrichment cultivation. Identity of the VXM01 carrier bacterium
was determined by serological agglutination and PCR methods.
[0181] Test samples (blood, tears, urine, saliva and faeces) were
collected at the site in Heidelberg and same-day delivery of
post-vaccination samples took place to MicroMol GmbH located in
Karlsruhe, Germany. The bacterial vector shedding and
biodistribution analysis cascade was designed to detect live CFU of
VXM01 or horizontal plasmid transfer. It consists of two separate
analysis branches (Branch I and Branch II):
Branch I: Plating method to detect any horizontal plasmid transfer
Branch II: Liquid enrichment culture to detect live CFU of
VXM01
[0182] The analysis cascade is followed by a matrix decision in
order to determine the excretion of live bacteria VXM01 or
observation of a horizontal plasmid transfer. The cascade is
outlined in FIG. 11.
Analysis Branch I for Detection of Horizontal Plasmid Transfer:
[0183] Day 0: Plating of the 5 test samples on 3 TSA (+kanamycin)
plates each, incubation over night at 37.degree. C. [0184] Day 1:
Visual discrimination between VXM01 (Ty21a) and non-VXM01
morphotypes on the selective plates. Selection of non-VXM01
morphotypes (9 colonies each), streaking on agar plates
(+kanamycin) for agglutination and parallel liquid culture
(+kanamycin) for each pooled morphotype for PCR analysis the
following day [0185] Day 2: PCR of each liquid morphotype pool
Analysis Branch II for Detection of VXM01:
[0185] [0186] Day 0: Preparation of liquid enrichment cultures
(+kanamycin) for each of the 5 test samples [0187] Day 1: Direct
PCR on each liquid enrichment culture. Streaking of each enrichment
culture on agar plates (+kanamycin) for serological analysis the
following day in case PCR is positive for plasmid [0188] Day 2:
Serological confirmation of presence of VXM01 (Ty21a)
[0189] Due to the fact that PCR of any test sample would not be
discriminative between live CFU and/or free-floating plasmid or
Ty21a genomic DNA and as agglutination cannot be applied directly
on test samples, PCR as well as agglutination methods were used as
second-line methods after plating method was applied. Identified
live colonies grown on kanamycin-containing plates were further
characterized by these methods. Only the plating method enables
discrimination between live and dead cells (either VXM01 or foreign
non-VXM01 plasmid transformants due to horizontal plasmid
transfer).
[0190] Fecal excretion of VXM01 was observed in two patients, one
in the 10.sup.9 and one in the 10.sup.10 dose group. VXM01
excretion in feces in both patients was transient at one occasion
after the first or second administration, respectively, and
disappeared without antibiotic treatment. The numbers of VMX01
excreting patients in the various dose groups are graphically
depicted in FIG. 12.
[0191] In summary, VXM01 has the potential to target a variety of
tumor types and to overcome multiple hurdles encountered by other
present cancer vaccine approaches. A tempting vision is the
possibility of combining the vaccine of the present invention with
a multitude of other anti-cancer and immune-modulatory agents. The
results of the here presented study motivate the inventors to move
forward this highly interesting approach.
Sequence CWU 1
1
311356PRTHomo sapiens 1Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu
Trp Leu Cys Val Glu 1 5 10 15 Thr Arg Ala Ala Ser Val Gly Leu Pro
Ser Val Ser Leu Asp Leu Pro 20 25 30 Arg Leu Ser Ile Gln Lys Asp
Ile Leu Thr Ile Lys Ala Asn Thr Thr 35 40 45 Leu Gln Ile Thr Cys
Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro 50 55 60 Asn Asn Gln
Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser 65 70 75 80 Asp
Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn 85 90
95 Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser
100 105 110 Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile
Ala Ser 115 120 125 Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu
Asn Lys Asn Lys 130 135 140 Thr Val Val Ile Pro Cys Leu Gly Ser Ile
Ser Asn Leu Asn Val Ser 145 150 155 160 Leu Cys Ala Arg Tyr Pro Glu
Lys Arg Phe Val Pro Asp Gly Asn Arg 165 170 175 Ile Ser Trp Asp Ser
Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile 180 185 190 Ser Tyr Ala
Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser 195 200 205 Tyr
Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr 210 215
220 Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu
225 230 235 240 Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn
Val Gly Ile 245 250 255 Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His
Gln His Lys Lys Leu 260 265 270 Val Asn Arg Asp Leu Lys Thr Gln Ser
Gly Ser Glu Met Lys Lys Phe 275 280 285 Leu Ser Thr Leu Thr Ile Asp
Gly Val Thr Arg Ser Asp Gln Gly Leu 290 295 300 Tyr Thr Cys Ala Ala
Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 305 310 315 320 Phe Val
Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met 325 330 335
Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala 340
345 350 Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn
Gly 355 360 365 Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His
Val Leu Thr 370 375 380 Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn
Tyr Thr Val Ile Leu 385 390 395 400 Thr Asn Pro Ile Ser Lys Glu Lys
Gln Ser His Val Val Ser Leu Val 405 410 415 Val Tyr Val Pro Pro Gln
Ile Gly Glu Lys Ser Leu Ile Ser Pro Val 420 425 430 Asp Ser Tyr Gln
Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr 435 440 445 Ala Ile
Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu 450 455 460
Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr 465
470 475 480 Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly
Asn Lys 485 490 495 Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu
Gly Lys Asn Lys 500 505 510 Thr Val Ser Thr Leu Val Ile Gln Ala Ala
Asn Val Ser Ala Leu Tyr 515 520 525 Lys Cys Glu Ala Val Asn Lys Val
Gly Arg Gly Glu Arg Val Ile Ser 530 535 540 Phe His Val Thr Arg Gly
Pro Glu Ile Thr Leu Gln Pro Asp Met Gln 545 550 555 560 Pro Thr Glu
Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser 565 570 575 Thr
Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro 580 585
590 Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr
595 600 605 Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn
Asp Ile 610 615 620 Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp
Gln Gly Asp Tyr 625 630 635 640 Val Cys Leu Ala Gln Asp Arg Lys Thr
Lys Lys Arg His Cys Val Val 645 650 655 Arg Gln Leu Thr Val Leu Glu
Arg Val Ala Pro Thr Ile Thr Gly Asn 660 665 670 Leu Glu Asn Gln Thr
Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys 675 680 685 Thr Ala Ser
Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn 690 695 700 Glu
Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg 705 710
715 720 Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr
Thr 725 730 735 Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu
Ala Phe Phe 740 745 750 Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu
Glu Ile Ile Ile Leu 755 760 765 Val Gly Thr Ala Val Ile Ala Met Phe
Phe Trp Leu Leu Leu Val Ile 770 775 780 Ile Leu Arg Thr Val Lys Arg
Ala Asn Gly Gly Glu Leu Lys Thr Gly 785 790 795 800 Tyr Leu Ser Ile
Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu His 805 810 815 Cys Glu
Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp 820 825 830
Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val 835
840 845 Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Arg
Thr 850 855 860 Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser
Glu His Arg 865 870 875 880 Ala Leu Met Ser Glu Leu Lys Ile Leu Ile
His Ile Gly His His Leu 885 890 895 Asn Val Val Asn Leu Leu Gly Ala
Cys Thr Lys Pro Gly Gly Pro Leu 900 905 910 Met Val Ile Val Glu Phe
Cys Lys Phe Gly Asn Leu Ser Thr Tyr Leu 915 920 925 Arg Ser Lys Arg
Asn Glu Phe Val Pro Tyr Lys Thr Lys Gly Ala Arg 930 935 940 Phe Arg
Gln Gly Lys Asp Tyr Val Gly Ala Ile Pro Val Asp Leu Lys 945 950 955
960 Arg Arg Leu Asp Ser Ile Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly
965 970 975 Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu
Ala Pro 980 985 990 Glu Asp Leu Tyr Lys Asp Phe Leu Thr Leu Glu His
Leu Ile Cys Tyr 995 1000 1005 Ser Phe Gln Val Ala Lys Gly Met Glu
Phe Leu Ala Ser Arg Lys 1010 1015 1020 Cys Ile His Arg Asp Leu Ala
Ala Arg Asn Ile Leu Leu Ser Glu 1025 1030 1035 Lys Asn Val Val Lys
Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile 1040 1045 1050 Tyr Lys Asp
Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro 1055 1060 1065 Leu
Lys Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr 1070 1075
1080 Ile Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile
1085 1090 1095 Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile
Asp Glu 1100 1105 1110 Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg
Met Arg Ala Pro 1115 1120 1125 Asp Tyr Thr Thr Pro Glu Met Tyr Gln
Thr Met Leu Asp Cys Trp 1130 1135 1140 His Gly Glu Pro Ser Gln Arg
Pro Thr Phe Ser Glu Leu Val Glu 1145 1150 1155 His Leu Gly Asn Leu
Leu Gln Ala Asn Ala Gln Gln Asp Gly Lys 1160 1165 1170 Asp Tyr Ile
Val Leu Pro Ile Ser Glu Thr Leu Ser Met Glu Glu 1175 1180 1185 Asp
Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu 1190 1195
1200 Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala
1205 1210 1215 Gly Ile Ser Gln Tyr Leu Gln Asn Ser Lys Arg Lys Ser
Arg Pro 1220 1225 1230 Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu
Glu Glu Pro Glu 1235 1240 1245 Val Lys Val Ile Pro Asp Asp Asn Gln
Thr Asp Ser Gly Met Val 1250 1255 1260 Leu Ala Ser Glu Glu Leu Lys
Thr Leu Glu Asp Arg Thr Lys Leu 1265 1270 1275 Ser Pro Ser Phe Gly
Gly Met Val Pro Ser Lys Ser Arg Glu Ser 1280 1285 1290 Val Ala Ser
Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly 1295 1300 1305 Tyr
His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Glu Glu 1310 1315
1320 Ala Glu Leu Leu Lys Leu Ile Glu Ile Gly Val Gln Thr Gly Ser
1325 1330 1335 Thr Ala Gln Ile Leu Gln Pro Asp Ser Gly Thr Thr Leu
Ser Ser 1340 1345 1350 Pro Pro Val 1355 24071DNAHomo sapiens
2atgcagagca aggtgctgct ggccgtcgcc ctgtggctct gcgtggagac ccgggccgcc
60tctgtgggtt tgcctagtgt ttctcttgat ctgcccaggc tcagcataca aaaagacata
120cttacaatta aggctaatac aactcttcaa attacttgca ggggacagag
ggacttggac 180tggctttggc ccaataatca gagtggcagt gagcaaaggg
tggaggtgac tgagtgcagc 240gatggcctct tctgtaagac actcacaatt
ccaaaagtga tcggaaatga cactggagcc 300tacaagtgct tctaccggga
aactgacttg gcctcggtca tttatgtcta tgttcaagat 360tacagatctc
catttattgc ttctgttagt gaccaacatg gagtcgtgta cattactgag
420aacaaaaaca aaactgtggt gattccatgt ctcgggtcca tttcaaatct
caacgtgtca 480ctttgtgcaa gatacccaga aaagagattt gttcctgatg
gtaacagaat ttcctgggac 540agcaagaagg gctttactat tcccagctac
atgatcagct atgctggcat ggtcttctgt 600gaagcaaaaa ttaatgatga
aagttaccag tctattatgt acatagttgt cgttgtaggg 660tataggattt
atgatgtggt tctgagtccg tctcatggaa ttgaactatc tgttggagaa
720aagcttgtct taaattgtac agcaagaact gaactaaatg tggggattga
cttcaactgg 780gaataccctt cttcgaagca tcagcataag aaacttgtaa
accgagacct aaaaacccag 840tctgggagtg agatgaagaa atttttgagc
accttaacta tagatggtgt aacccggagt 900gaccaaggat tgtacacctg
tgcagcatcc agtgggctga tgaccaagaa gaacagcaca 960tttgtcaggg
tccatgaaaa accttttgtt gcttttggaa gtggcatgga atctctggtg
1020gaagccacgg tgggggagcg tgtcagaatc cctgcgaagt accttggtta
cccaccccca 1080gaaataaaat ggtataaaaa tggaataccc cttgagtcca
atcacacaat taaagcgggg 1140catgtactga cgattatgga agtgagtgaa
agagacacag gaaattacac tgtcatcctt 1200accaatccca tttcaaagga
gaagcagagc catgtggtct ctctggttgt gtatgtccca 1260ccccagattg
gtgagaaatc tctaatctct cctgtggatt cctaccagta cggcaccact
1320caaacgctga catgtacggt ctatgccatt cctcccccgc atcacatcca
ctggtattgg 1380cagttggagg aagagtgcgc caacgagccc agccaagctg
tctcagtgac aaacccatac 1440ccttgtgaag aatggagaag tgtggaggac
ttccagggag gaaataaaat tgaagttaat 1500aaaaatcaat ttgctctaat
tgaaggaaaa aacaaaactg taagtaccct tgttatccaa 1560gcggcaaatg
tgtcagcttt gtacaaatgt gaagcggtca acaaagtcgg gagaggagag
1620agggtgatct ccttccacgt gaccaggggt cctgaaatta ctttgcaacc
tgacatgcag 1680cccactgagc aggagagcgt gtctttgtgg tgcactgcag
acagatctac gtttgagaac 1740ctcacatggt acaagcttgg cccacagcct
ctgccaatcc atgtgggaga gttgcccaca 1800cctgtttgca agaacttgga
tactctttgg aaattgaatg ccaccatgtt ctctaatagc 1860acaaatgaca
ttttgatcat ggagcttaag aatgcatcct tgcaggacca aggagactat
1920gtctgccttg ctcaagacag gaagaccaag aaaagacatt gcgtggtcag
gcagctcaca 1980gtcctagagc gtgtggcacc cacgatcaca ggaaacctgg
agaatcagac gacaagtatt 2040ggggaaagca tcgaagtctc atgcacggca
tctgggaatc cccctccaca gatcatgtgg 2100tttaaagata atgagaccct
tgtagaagac tcaggcattg tattgaagga tgggaaccgg 2160aacctcacta
tccgcagagt gaggaaggag gacgaaggcc tctacacctg ccaggcatgc
2220agtgttcttg gctgtgcaaa agtggaggca tttttcataa tagaaggtgc
ccaggaaaag 2280acgaacttgg aaatcattat tctagtaggc acggcggtga
ttgccatgtt cttctggcta 2340cttcttgtca tcatcctacg gaccgttaag
cgggccaatg gaggggaact gaagacaggc 2400tacttgtcca tcgtcatgga
tccagatgaa ctcccattgg atgaacattg tgaacgactg 2460ccttatgatg
ccagcaaatg ggaattcccc agagaccggc tgaagctagg taagcctctt
2520ggccgtggtg cctttggcca agtgattgaa gcagatgcct ttggaattga
caagacagca 2580acttgcagga cagtagcagt caaaatgttg aaagaaggag
caacacacag tgagcatcga 2640gctctcatgt ctgaactcaa gatcctcatt
catattggtc accatctcaa tgtggtcaac 2700cttctaggtg cctgtaccaa
gccaggaggg ccactcatgg tgattgtgga attctgcaaa 2760tttggaaacc
tgtccactta cctgaggagc aagagaaatg aatttgtccc ctacaagacc
2820aaaggggcac gattccgtca agggaaagac tacgttggag caatccctgt
ggatctgaaa 2880cggcgcttgg acagcatcac cagtagccag agctcagcca
gctctggatt tgtggaggag 2940aagtccctca gtgatgtaga agaagaggaa
gctcctgaag atctgtataa ggacttcctg 3000accttggagc atctcatctg
ttacagcttc caagtggcta agggcatgga gttcttggca 3060tcgcgaaagt
gtatccacag ggacctggcg gcacgaaata tcctcttatc ggagaagaac
3120gtggttaaaa tctgtgactt tggcttggcc cgggatattt ataaagatcc
agattatgtc 3180agaaaaggag atgctcgcct ccctttgaaa tggatggccc
cagaaacaat ttttgacaga 3240gtgtacacaa tccagagtga cgtctggtct
tttggtgttt tgctgtggga aatattttcc 3300ttaggtgctt ctccatatcc
tggggtaaag attgatgaag aattttgtag gcgattgaaa 3360gaaggaacta
gaatgagggc ccctgattat actacaccag aaatgtacca gaccatgctg
3420gactgctggc acggggagcc cagtcagaga cccacgtttt cagagttggt
ggaacatttg 3480ggaaatctct tgcaagctaa tgctcagcag gatggcaaag
actacattgt tcttccgata 3540tcagagactt tgagcatgga agaggattct
ggactctctc tgcctacctc acctgtttcc 3600tgtatggagg aggaggaagt
atgtgacccc aaattccatt atgacaacac agcaggaatc 3660agtcagtatc
tgcagaacag taagcgaaag agccggcctg tgagtgtaaa aacatttgaa
3720gatatcccgt tagaagaacc agaagtaaaa gtaatcccag atgacaacca
gacggacagt 3780ggtatggttc ttgcctcaga agagctgaaa actttggaag
acagaaccaa attatctcca 3840tcttttggtg gaatggtgcc cagcaaaagc
agggagtctg tggcatctga aggctcaaac 3900cagacaagcg gctaccagtc
cggatatcac tccgatgaca cagacaccac cgtgtactcc 3960agtgaggaag
cagaactttt aaagctgata gagattggag tgcaaaccgg tagcacagcc
4020cagattctcc agcctgactc ggggaccaca ctgagctctc ctcctgttta a
407137580DNAartificial sequencesequence of expression vector
pVAX10.VR2-1 3tgggcttttg ctggcctttt gctcacatgt tcttgactct
tcgcgatgta cgggccagat 60atacgcgttg acattgatta ttgactagtt attaatagta
atcaattacg gggtcattag 120ttcatagccc atatatggag ttccgcgtta
cataacttac ggtaaatggc ccgcctggct 180gaccgcccaa cgacccccgc
ccattgacgt caataatgac gtatgttccc atagtaacgc 240caatagggac
tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg
300cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
gacggtaaat 360ggcccgcctg gcattatgcc cagtacatga ccttatggga
ctttcctact tggcagtaca 420tctacgtatt agtcatcgct attaccatgg
tgatgcggtt ttggcagtac atcaatgggc 480gtggatagcg gtttgactca
cggggatttc caagtctcca ccccattgac gtcaatggga 540gtttgttttg
gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
600tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
gctctctggc 660taactagaga acccactgct tactggctta tcgaaattaa
tacgactcac tatagggaga 720cccaagctgg ctagcaggat gcagagcaag
gtgctgctgg ccgtcgccct gtggctctgc 780gtggagaccc gggccgcctc
tgtgggtttg cctagtgttt ctcttgatct gcccaggctc 840agcatacaaa
aagacatact tacaattaag gctaatacaa ctcttcaaat tacttgcagg
900ggacagaggg acttggactg gctttggccc aataatcaga gtggcagtga
gcaaagggtg 960gaggtgactg agtgcagcga tggcctcttc tgtaagacac
tcacaattcc aaaagtgatc 1020ggaaatgaca ctggagccta caagtgcttc
taccgggaaa ctgacttggc ctcggtcatt 1080tatgtctatg ttcaagatta
cagatctcca tttattgctt ctgttagtga ccaacatgga 1140gtcgtgtaca
ttactgagaa caaaaacaaa actgtggtga ttccatgtct cgggtccatt
1200tcaaatctca acgtgtcact ttgtgcaaga tacccagaaa agagatttgt
tcctgatggt 1260aacagaattt cctgggacag caagaagggc tttactattc
ccagctacat gatcagctat 1320gctggcatgg tcttctgtga agcaaaaatt
aatgatgaaa gttaccagtc tattatgtac 1380atagttgtcg ttgtagggta
taggatttat gatgtggttc tgagtccgtc tcatggaatt 1440gaactatctg
ttggagaaaa gcttgtctta aattgtacag caagaactga actaaatgtg
1500gggattgact tcaactggga atacccttct tcgaagcatc agcataagaa
acttgtaaac 1560cgagacctaa aaacccagtc tgggagtgag atgaagaaat
ttttgagcac cttaactata 1620gatggtgtaa cccggagtga ccaaggattg
tacacctgtg cagcatccag tgggctgatg 1680accaagaaga acagcacatt
tgtcagggtc catgaaaaac cttttgttgc ttttggaagt 1740ggcatggaat
ctctggtgga agccacggtg ggggagcgtg tcagaatccc tgcgaagtac
1800cttggttacc cacccccaga aataaaatgg tataaaaatg gaatacccct
tgagtccaat 1860cacacaatta aagcggggca tgtactgacg attatggaag
tgagtgaaag
agacacagga 1920aattacactg tcatccttac caatcccatt tcaaaggaga
agcagagcca tgtggtctct 1980ctggttgtgt atgtcccacc ccagattggt
gagaaatctc taatctctcc tgtggattcc 2040taccagtacg gcaccactca
aacgctgaca tgtacggtct atgccattcc tcccccgcat 2100cacatccact
ggtattggca gttggaggaa gagtgcgcca acgagcccag ccaagctgtc
2160tcagtgacaa acccataccc ttgtgaagaa tggagaagtg tggaggactt
ccagggagga 2220aataaaattg aagttaataa aaatcaattt gctctaattg
aaggaaaaaa caaaactgta 2280agtacccttg ttatccaagc ggcaaatgtg
tcagctttgt acaaatgtga agcggtcaac 2340aaagtcggga gaggagagag
ggtgatctcc ttccacgtga ccaggggtcc tgaaattact 2400ttgcaacctg
acatgcagcc cactgagcag gagagcgtgt ctttgtggtg cactgcagac
2460agatctacgt ttgagaacct cacatggtac aagcttggcc cacagcctct
gccaatccat 2520gtgggagagt tgcccacacc tgtttgcaag aacttggata
ctctttggaa attgaatgcc 2580accatgttct ctaatagcac aaatgacatt
ttgatcatgg agcttaagaa tgcatccttg 2640caggaccaag gagactatgt
ctgccttgct caagacagga agaccaagaa aagacattgc 2700gtggtcaggc
agctcacagt cctagagcgt gtggcaccca cgatcacagg aaacctggag
2760aatcagacga caagtattgg ggaaagcatc gaagtctcat gcacggcatc
tgggaatccc 2820cctccacaga tcatgtggtt taaagataat gagacccttg
tagaagactc aggcattgta 2880ttgaaggatg ggaaccggaa cctcactatc
cgcagagtga ggaaggagga cgaaggcctc 2940tacacctgcc aggcatgcag
tgttcttggc tgtgcaaaag tggaggcatt tttcataata 3000gaaggtgccc
aggaaaagac gaacttggaa atcattattc tagtaggcac ggcggtgatt
3060gccatgttct tctggctact tcttgtcatc atcctacgga ccgttaagcg
ggccaatgga 3120ggggaactga agacaggcta cttgtccatc gtcatggatc
cagatgaact cccattggat 3180gaacattgtg aacgactgcc ttatgatgcc
agcaaatggg aattccccag agaccggctg 3240aagctaggta agcctcttgg
ccgtggtgcc tttggccaag tgattgaagc agatgccttt 3300ggaattgaca
agacagcaac ttgcaggaca gtagcagtca aaatgttgaa agaaggagca
3360acacacagtg agcatcgagc tctcatgtct gaactcaaga tcctcattca
tattggtcac 3420catctcaatg tggtcaacct tctaggtgcc tgtaccaagc
caggagggcc actcatggtg 3480attgtggaat tctgcaaatt tggaaacctg
tccacttacc tgaggagcaa gagaaatgaa 3540tttgtcccct acaagaccaa
aggggcacga ttccgtcaag ggaaagacta cgttggagca 3600atccctgtgg
atctgaaacg gcgcttggac agcatcacca gtagccagag ctcagccagc
3660tctggatttg tggaggagaa gtccctcagt gatgtagaag aagaggaagc
tcctgaagat 3720ctgtataagg acttcctgac cttggagcat ctcatctgtt
acagcttcca agtggctaag 3780ggcatggagt tcttggcatc gcgaaagtgt
atccacaggg acctggcggc acgaaatatc 3840ctcttatcgg agaagaacgt
ggttaaaatc tgtgactttg gcttggcccg ggatatttat 3900aaagatccag
attatgtcag aaaaggagat gctcgcctcc ctttgaaatg gatggcccca
3960gaaacaattt ttgacagagt gtacacaatc cagagtgacg tctggtcttt
tggtgttttg 4020ctgtgggaaa tattttcctt aggtgcttct ccatatcctg
gggtaaagat tgatgaagaa 4080ttttgtaggc gattgaaaga aggaactaga
atgagggccc ctgattatac tacaccagaa 4140atgtaccaga ccatgctgga
ctgctggcac ggggagccca gtcagagacc cacgttttca 4200gagttggtgg
aacatttggg aaatctcttg caagctaatg ctcagcagga tggcaaagac
4260tacattgttc ttccgatatc agagactttg agcatggaag aggattctgg
actctctctg 4320cctacctcac ctgtttcctg tatggaggag gaggaagtat
gtgaccccaa attccattat 4380gacaacacag caggaatcag tcagtatctg
cagaacagta agcgaaagag ccggcctgtg 4440agtgtaaaaa catttgaaga
tatcccgtta gaagaaccag aagtaaaagt aatcccagat 4500gacaaccaga
cggacagtgg tatggttctt gcctcagaag agctgaaaac tttggaagac
4560agaaccaaat tatctccatc ttttggtgga atggtgccca gcaaaagcag
ggagtctgtg 4620gcatctgaag gctcaaacca gacaagcggc taccagtccg
gatatcactc cgatgacaca 4680gacaccaccg tgtactccag tgaggaagca
gaacttttaa agctgataga gattggagtg 4740caaaccggta gcacagccca
gattctccag cctgactcgg ggaccacact gagctctcct 4800cctgtttaaa
aggaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
4860gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
cttgaccctg 4920gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
aaattgcatc gcattgtctg 4980agtaggtgtc attctattct ggggggtggg
gtggggcagg acagcaaggg ggaggattgg 5040gaagacaata gcaggcatgc
tggggatgcg gtgggctcta tggcttctac tgggcggttt 5100tatggacagc
aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc
5160cctgcaaagt aaactggatg gctttctcgc cgccaaggat ctgatggcgc
aggggatcaa 5220gctctgatca agagacagga tgaggatcgt ttcgcatgat
tgaacaagat ggattgcacg 5280caggttctcc ggccgcttgg gtggagaggc
tattcggcta tgactgggca caacagacaa 5340tcggctgctc tgatgccgcc
gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 5400tcaagaccga
cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
5460ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
gaagcgggaa 5520gggactggct gctattgggc gaagtgccgg ggcaggatct
cctgtcatct caccttgctc 5580ctgccgagaa agtatccatc atggctgatg
caatgcggcg gctgcatacg cttgatccgg 5640ctacctgccc attcgaccac
caagcgaaac atcgcatcga gcgagcacgt actcggatgg 5700aagccggtct
tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
5760aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
gtgacccatg 5820gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
cttttctgga ttcatcgact 5880gtggccggct gggtgtggcg gaccgctatc
aggacatagc gttggctacc cgtgatattg 5940ctgaagagct tggcggcgaa
tgggctgacc gcttcctcgt gctttacggt atcgccgctc 6000ccgattcgca
gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg
6060cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
tcacaccgca 6120tacaggtggc acttttcggg gaaatgtgcg cggaacccct
atttgtttat ttttctaaat 6180acattcaaat atgtatccgc tcatgagaca
ataaccctga taaatgcttc aataatagca 6240cgtgctaaaa cttcattttt
aatttaaaag gatctaggtg aagatccttt ttgataatct 6300catgaccaaa
atcccttaac gtgagttttc gttccactga gcgtcagacc cccatcagtg
6360accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
gacattaacg 6420cttctggaga aactcaacga gctggacgcg gatgaacagg
cagacatctg tgaatcgctt 6480cacgaccacg ctgatgagct ttaccgcagc
tgcctcgcgc gtttcggtga tgacggtgaa 6540aacctctgac acatgcagct
cccggagacg gtcacagctt gtctgtaagc ggatgccggg 6600agcagacaag
cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
6660acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
tcagagcaga 6720ttgtactgag agtgcaccat atgcggtgtg aaataccgca
cagatgcgta aggagaaaat 6780accgcatcag gcgctcttcc gcttcctcgc
tcactgactc gctgcgctcg gtcgttcggc 6840tgcggcgagc ggtatcagct
cactcaaagg cggtaatacg gttatccaca gaatcagggg 6900ataacgcagg
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
6960ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
aaaaatcgac 7020gctcaagtca gaggtggcga aacccgacag gactataaag
ataccaggcg tttccccctg 7080gaagctccct cgtgcgctct cctgttccga
ccctgccgct taccggatac ctgtccgcct 7140ttctcccttc gggaagcgtg
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 7200tgtaggtcgt
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
7260gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
ttatcgccac 7320tggcagcagc cactggtaac aggattagca gagcgaggta
tgtaggcggt gctacagagt 7380tcttgaagtg gtggcctaac tacggctaca
ctagaaggac agtatttggt atctgcgctc 7440tgctgaagcc agttaccttc
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 7500ccgctggtag
cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
7560ctcaagaaga tcctttgatc 7580
* * * * *