Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR GENERATING IMMUNITY TO ANTIGEN
FIELD OF THE INVENTION
[0001] The present invention relates to methods of developing immunity against
an
antigen using an expression vector that expresses a secretable fusion protein
comprising an
antigen fused to CD40 ligand. The methods also relate to an immunization
scheme of
priming with the expression vector and boosting with a protein antigen. The
invention also
relates to an approach for producing the vector and the protein antigen
simultaneously in a
production cell system.
BACKGROUND OF THE INVENTION
[0002] The following discussion of the background of the invention is merely
provided to
aid the reader in understanding the invention and is not admitted to describe
or constitute
prior art to the present invention. This application claims priority to U.S.
application serial
nos. 60/529,016, filed December 1 l, 2003, which is incorporated herein in its
entirety
including the drawings. Applications related to this application are
PCT/LTS03/36237 filed
11/12/03 entitled "adenoviral vector vaccine" and U.S. provisional patent
applications
60/524,925 (filed November 24, 2003), 60/525,552 (filed November 25, 2003),
and
60/529,015 (filed December 11, 2003), all of which are incorporated herein in
their entirety
including the drawings.
[0003] The activation of antigen presenting cells (APCs) which includes the
dendritic cells
(DCs), followed by loading of the antigen presenting cell with relevant
antigens, is a requisite
step in the generation of a T cell dependent immune response against cancer
cells. Once
activated and loaded with tumor antigens, DCs migrate to regional lymph nodes
(LNs) to
present antigens to T cells. Very cormnonly, these APCs express insufficient
amounts of
surface activation molecules which are required for optimal activation and
expansion of T
cell clones competent to recognize tumor antigens. See Shortman, et al., Stem
Cells 15:409-
419, 1997.
[0004] Antigen presentation to naive T cells, in the absence of costimulatory
molecule
expression on the surface of the APC, leads to anergy of the T cells. See
Steinbrink, et al.
Blood 99: 246-2476, 2002. Moreover, cross-presentation by DCs without CD4+ T
cell help
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also results in peripheral deletion of Ag-specific T cells in regional LNs.
See I~usuhara, et
al., Eur J Immunol 32:1035-1043, 2002. In contrast, in the presence of CD4+ T
cell help,
DCs acquire functional ability to cross-prime T cells, resulting in clonal
expansion of effector
T cells. See Gunner, et al., Semin Immunol 13:291-302, 2001. This CD4+ T cell
help can be
replaced with CD40-CD40 ligand (CD40L) interactions. See Luft, et al. Int
Immunol 14:367-
380, 2002. CD40L is a 33-kDa type II membrane protein and a member of the TNF
gene
family and is transiently expressed on CD4+ T cells after TCR engagement. See
Skov, et al. J
hnmunol. 164: 3500-3505, 2000.
[0005] The ability of DGs to generate anti-tumor immune responses in vivo has
been
documented in a number of animal tumor models. See Paglia, et al. J Exp Med
183: 317-322,
1996; Zitvogel, et al., J Exp Med. 183: 87-97, 1996. However, DC-mediated
induction of
immunity represents a major therapeutic challenge. It is considered difficult
to ensure that
the antigen presenting cells express appropriate adhesion molecules and
chemokine receptors
to attract DCs to secondary lymphoid organs for priming T cells. See Fong, et
al. Jlmmuraol.
166: 4254-4259, 2001; Markowicz, et al. J Clin Invest. 85: 955-961, 1990; Hsu,
et al. Nat
Med. 2: 52-58, 1996; Nestle, et al. Nat Med. 4: 328-332, 1998; Murphy, et al.,
Prostate 38:
73-78, 1999; Dhodapkar, et al. J Clih Invest. 104: 173-180, 1999.
SUMMARY OF THE INVENTION
[0006] hl a first aspect, the invention relates to the use of a fusion protein
in developing an
immune response to an antigen. In a preferred embodiment, an immune response
to an
antigen is obtained by administering an expression vector encoding a
secretable fusion
protein. The vector includes a transcription unit encoding a secretable fusion
protein which
contains the antigen and CD40 ligand. The fusion protein is also administered
before,
concurrently or after administration of the vector. Preferably, the fusion
protein is
administered after the vector.
[0007] In one approach, the sequence encoding the antigen in the fusion
protein
transcription unit is 5' to sequence encoding the CD40 ligand. In another
approach, the
sequence encoding the CD40 ligand in the fusion protein transcription unit is
5' to sequence
encoding the antigen. In a preferred embodiment, the CD40 ligand lacks all or
a portion of
its transmembrane domain.
2
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[0008] The antigen may be any antigen to which an immune response may be
generated in
an individual. In preferred embodiments, the antigen is a tumor antigen; the
tumor antigen is
the E6 or E7 protein of human papilloma virus; the tumor antigen is a mucin
antigen, which
may be selected from the group consisting of MUC 1, MUC2, MUC3A, MIJC3B,
MLTC4,
MUCSAC, MIJCSB, MUC6, MUC7, MUCB, MLTC9, MUC12, MUC13, MUC15, and
MUC16; the mucin antigen is from MUCl; the human epidermal growth factor (EGF)
like
receptor (e.g., HERl, HER2, HER3 and HER4), the antigen is an infectious agent
antigen;
the infectious agent antigen is a viral antigen; the infectious agent viral
antigen is from
human papilloma virus; the viral antigen is the E6 or E7 protein of human
papilloma virus.
[0009] In another aspect, the invention provides methods of treating an
individual with
cancer that expresses a tumor antigen. The method includes administering the
expression
vector which includes a transcription unit encoding a secretable fusion
protein that contains
the tumor antigen and CD40 ligand. The fusion protein is also administered
before,
concurrently or after administration of the vector. Preferably, the fusion
protein is
administered after the vector.
[0010] In a further aspect, the invention provides a method of generating
immunity in a
subject to an infectious agent. The method includes administering the
expression vector
which includes a transcription unit encoding a secretable fusion protein that
contains the
infectious agent antigen and CD40 ligand. The fusion protein is also
administered before,
concurrently or after administration of the vector. Preferably, the fusion
protein is
administered after the vector.
[0011] In yet a further aspect, the invention relates to an approach for
producing the vector
and the fusion protein together in the same host production cell system. In a
preferred
embodiment, the fusion protein is expressed from the same vector used to
generate immunity
by vaccination. In this way, both the vector and the fusion protein can be
produced
simultaneously through a single production system.
[0012] In preferred embodiments, the expression vector may be a viral
expression vector
or a non-viral expression vector; the expression vector may be an adenoviral
vector; the
vector may be advantageously administered subcutaneously; the vector may be
administered
on a subsequent occasions) to increase the immune response; a signal sequence
may be
placed upstream of the fusion protein for secretion of the fusion protein;
immunity against the
antigen may be long lasting and involve generation of cytotoxic CD8+ T cells
against antigen
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expressing cells and the production of antibody to the antigen; the
transcription unit may
include sequence that encodes a linker between the antigen and the CD40
ligand; suitable
linkers may vary in length and composition; the expression vector may include
a human
cytomegalovirus promoter/enhancer for controlling transcription of the
transcription unit; and
the CD40 ligand may be a human CD40 ligand.
[0013] Abbreviations used herein include "Ad" (adenoviral); "sig" (signal
sequence); and
"ecd" (extracellular domain).
[0014] These and other embodiments are described in detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the nucleotide sequence encoding human MUC1 (SEQ ID NO:1)
[0016] FIG. 2 shows the amino acid sequence of human MUC1 (SEQ ID NO:2).
[0017] FIG. 3 shows the level of interferon gamma produced in an ELISA spot
assay
using spleen cells from MUC-1 transgenic animals (hMLTC-l.Tg ) primed with
adenoviral
expression vector Ad-I~/ecdhMUC1-~Ct~TmCD40L and boosted subcutaneously with
either
expression vector or the mature fusion protein ecdhMUCl-OCt~TmCD40L. The
various
treatment groups include protein boost seven days after two weekly vector
injections (T1),
two weeks after two weekly vector injections (T2), one week after one vector
injection (T3),
and two weelcs after one vector injection (T4). In T5, two subcutaneous
protein injections
(administered two weeks apart) were given starting 7 days after a single
vector injection. In
T1, two vector injections were given without protein.
[0018] FIG. 4 shows the level of T cell cytotoxicity from MUC-1 transgenic
animals
(hMLTC-l.Tg ) primed with adenoviral expression vector Ad-I~lecdhMUCl-
OCt~TmCD40L
and boosted subcutaneously with either expression vector or the mature fusion
protein
ecdhMUCl-~CtOTmCD40L. The various treatment groups are as described in FIG. 3.
[0019] FIG. 5 shows the level of antibody against fusion protein ecdhMIJCl-
~Ct~TmCD40L in serum of MLTC-1 transgenic animals (hMUC-l .Tg ) primed with
adenoviral expression vector Ad-K/ecdhMUCl-OCt4TmCD40L and boosted
subcutaneously
with either expression vector or the mature fusion protein ecdhMLJCl-
4CtdTmCD40L. The
various treatment groups are as described in FIG. 3. Antibodies were detected
in an ELISA.
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Microwell plates coated with the fusion protein ecdhMIJCl-4CtOTmCD40L were
reacted
with serum, washed and bound mouse antibody detected using rat anti-mouse
antibody
conjugated to horseradish peroxidase.
[0020] FIG. 6 shows the level growth of MUC1 expressing tumor cells
(LL2/LL2hMUC-
1) in MUC-1 transgenic animals (hMUC-l.Tg ) administered adenoviral expression
vector
Ad-K/ecdhMLTCl-~CtOTmCD40L versus one or two subsequent administrations of
fusion
protein ecdhMIJCl-OCtOTmCD40L.
[0021] FIG. 7 demonstrates tumor prevention in animals immunized with Ad-sig-
ecdhMLTC-1/OCt~TmCD40L vector and ecdhMUC-1/~CtOTm CD40L protein. VVV =
three Ad-sig-ecdhMUC-1/OCt~Tm CD40L vector subcutaneous injections
administered on
days 1, 7 and 21; PPP = three ecdhMUC-1/OCtOTm CD40L protein subcutaneous
injections
administered on days 1, 7 and 21; or VPP = a single Ad-sig-ecdliMZJC-1/~CtOTm
CD40L
vector subcutaneous injection followed at days 7 and 21 by ecdliMTJC-1/~Ct~Tm
CD40L
protein subcutaneous injections. One week later (day 28), mice were injected
subcutaneously
with five hundred thousand LL2/LLIhMCTC-1 lung cancer cells. Two weeks later
(day 42),
500,000 of the LL2/LLIhMUC-1 tumor cells were administered intravenously to
test mice
via the tail vein. Multiple administrations of vector alone or vector followed
by boosting
with protein was effective in preventing the establislunent of human tumors in
mice.
[0022] FIG. 8 demonstrates the levels of hMUC-1 specific antibodies in
vaccinated test mice
at 63 days following the start of the vaccination. VVV = three Ad-sig-ecdhMUC-
1/OCtOTm
CD40L vector subcutaneous injections administered on days 1, 7 and 21; PPP =
three
ecdhMLJC-1/~CtOTm CD40L protein subcutaneous injections administered on days
1, 7 and
21; or VPP = a single Ad-sig-ecdhMUC-1/OCt~Tm CD40L vector subcutaneous
injection
followed at days 7 and 21 by ecdhMUC-1/~Ct~Tm CD40L protein subcutaneous
injections.
The schedule of one Ad-sig-ecdhMUC-1/deltaCtdeltaTmCD40L vector subcutaneous
injection followed by two successive ecdhMUC-1/deltaCtdeltaTmCD40L protein
subcutaneous inj ections at 7 and 21 days following the vector inj ection
induced the highest
levels of hMUC-1 specific antibodies.
[0023] FIG. 9 demonstrates subcutaneous tumor therapy (post establishment) in
animals
immunized with Ad-sig-ecdhMUC-1/~Ct~TmCD40L vector and ecdhMUC-1/~Ct~Tm
CD40L protein. VVV = three Ad-sig-ecdhMUC-1/~CtOTm CD40L vector subcutaneous
injections administered on days 5, 12 and 26; PPP = three ecdhMUC-1/~Ct~Tm
CD40L
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protein subcutaneous injections administered on days 5, 12 and 26; or VPP = a
single Ad-sig-
ecdhMUC-1/OCtOTm CD40L vector subcutaneous injection followed at days 12 and
26 by
ecdhNIUC-1/OCtOTm CD40L protein subcutaneous injections. Subcutaneous tumor
(500,000 of the LL2/LLIhMUC-1) was administered on day 1 and vaccinations were
carried
out at day 5. Tumor was administered i.v. on day 40 and tumor development
(subcutaneous
and lung) evaluated at day 54.
[0024] FIG. 10 demonstrates lung metastatic tumor nodule therapy (post
establishment) in
the animals treated as described in FIG. 9. Left panel: The results were
similar to the
subcutaneous tumor prevention with schedule VVV and VPP most effective. Right
panel: the
combination of one vector injection followed by two protein injections (VPP)
completely
suppressed the growth of established lung nodules of the hMUC-1 positive
cancer cells.
[0025] FIG. 11 compares various boosting strategies following a single
subcutaneous
administration of Ad-sig-ecdhMUC-1/ecdCD40L vector on the ability of animals
to resist
development of a MUC-1 expressing tumor. ecdhMUC-1/ecdCD40L protein in
bacterial
extract; ecdhMUC-1 linked to the keyhole limpet hemocyaninin (I~LH), with or
without
incomplete Freund's adjuvant; PBS (phosphate buffered saline); and control
bacterial extract
(bacterial host strain not infected with Ad-sig-ecdhMUC-1/ecdCD40L vector.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In accordance with one aspect of the invention, a method is provided
for
generating an immune response against an antigen using an expression vector.
The vector
includes a transcription unit encoding a secretable fusion protein containing
an antigen and
CD40 ligand. In a preferred embodiment, the transcription unit includes from
the amino
terminus, a secretory signal sequence, an antigen, a linker and a secretable
form of CD40
ligand. In preferred embodiments, the secretable form of CD40 ligand laclcs
all or
substantially all of its transmembrane domain
[0027] In a preferred approach, the individual is first administered the
vector on one or
more occasions to generate a primary immune response. The fusion protein is
also
administered in an effective amount after administration of vector to boost
the immune
response to the antigen above that obtained with vector administration alone.
[0028] The term "in an effective amount" in reference to administering the
fusion protein
is an amount that generates an increased immune response over that obtained
using the
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expression vector alone. A time interval between administrations is generally
required for
optimal results. An increase in the immune response may be measured as an
increase in T
cell activity or antibody production (see e.g., FIGs. 3-5). Generally, at
least one week
between vector administration and protein boosting is effective although a
shorter interval
may be possible. An effective spacing between administrations may be from 1
week to 12
weeks or even longer. Multiple boosts may be given which may be separated by
from 1-12
weeks or even longer periods of time.
[0029] The use of the fusion protein to boost the immune response avoids
having to
repetitively administer the expression vector which might generate
hypersensitivitiy to
multiple injections. The antigen portion of the fusion protein is preferably
the fusion protein
which is encoded by the transcription unit of the expression vector used in
the initial
administration. However, the antigen portion of the fusion protein may differ
from the
encoded antigen provided that there is at least one shared antigenic
determinant or epitope
common to the antigen of the expression vector and that of the fusion protein
used for
boosting.
[0030] The fusion protein may be prepared in a mammalian cell line system,
which is
complementary to the vector. Example in the case of adenovirus, the cell line
system can be
293 cells that contain the Early Region 1 (El) gene and can support the
propagation of the
E1-substituted recombinant adenoviruses. When the adenoviral vectors infect
the production
cells, the viral vectors will propagate themselves following the viral
replication cycles.
However, the gene of interest that is carried by the viral vector in the
expression cassette will
express during the viral propagation process. This can be utilized for
preparation of the
fusion protein encoded by the vector in the same system for production of the
vector. The
production of both the vector and the fusion protein will take place
simultaneously in the
production system. The vector and protein thus produced can be further
isolated and purified
via different processes.
(0031] The fusion protein may be administered parenterally, such as
intravascularly,
intravenously, intraarterially, intramuscularly, subcutaneously, or the like.
Administration
can also be orally, nasally, rectally, transdermally or inhalationally via an
aerosol. The
protein boost may be administered as a bolus, or slowly infused. The protein
boost is
preferably administered subcutaneously.
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[0032] The fusion protein boost may be formulated with an adjuvant to enhance
the
resulting immune response. As used herein, the term "adjuvant" means a
chemical that, when
administered with the vaccine, enhances the imm~.me response to the vaccine.
An adjuvant is
distinguished from a carrier protein in that the adjuvant is not chemically
coupled to the
immunogen or the antigen. Adjuvants are well known in the art and include, for
example,
mineral oil emulsions (U.S. Pat. No. 4,608,251, supra) such as Freund's
complete or Freund's
incomplete adjuvant (Freund, Adv. Tuberc. Res. 7:130 (1956); Calbiochem, San
Diego
Calif.), aluminum salts, especially aluminum hydroxide or ALLOHYDROGEL
(approved for
use in humans by the U.S. Food and Drug Administration), muramyl dipeptide
(MDP) and its
analogs such as [Thrl ]-MDP (Byers and Allison, Vaccine 5:223 (1987)),
monophosphoryl
lipid A (Johnson et al., Rev. Infect. Dis. 9:5512 (1987)), and the like.
[0033] The fusion protein can be administered in a microencapsulated or a
macroencapsulated form using methods well known in the art. Fusion protein can
be
encapsulated, for example, into liposomes (see, for example, Garcon and Six,
J. Immunol.
146:3697 (1991)), into the inner capsid protein of bovine rotavirus (Redmond
et al., Mol.
Immunol. 28:269 (1991)) into immune stimulating molecules (ISCOMS) composed of
saponins such as Quil A (Morein et al., Nature 308:457 (1984)); Morein et al.,
in
T_mmunological Adjuvants and Vaccines (G. Gregoriadis al. eds.) pp.153-162,
Plenum Press,
NY (1987)) or into controlled-release biodegradable microspheres composed, for
example, of
lactide-glycolide compolymers (O'Hagan et al., Immunology 73:239 (1991);
O'Hagan et al.,
Vaccine 11:149 (1993)).
[0034] The fusion protein also can be adsorbed to the surface of lipid
microspheres
containing squalene or squalane emulsions prepared with a PLURONIC block-
copolymer
such as L-121 and stabilized with a detergent such as TWEEN 80 (see Allison
and Byers,
Vaccines: New Approaches to Ilnmunological Problems (R. Ellis ed.) pp. 431-
449,
Butterworth-Hinemann, Stoneman N.Y. (1992)). A microencapsulated or a
macroencapsulated fusion protein can also include an adjuvant.
[0035] The fusion protein also may be conjugated to a carrier or foreign
molecule such as
a Garner protein that is foreign to the individual to be administered the
protein boost. Foreign
proteins that activate the immune response and can be conjugated to a fusion
protein as
described herein include proteins or other molecules with molecular weights of
at least about
20,000 Daltons, preferably at least about 40,000 Daltons and more preferably
at least about
60,000 Daltons. Carrier proteins useful in the present invention include, for
example, GST,
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hemocyanins such as from the keyhole limpet, serum albumin or cationized serum
albumin,
thyroglobulin, ovalbumin, various toxoid proteins such a tetanus toxoid or
diptheria toxoid,
immunoglobulins, heat shoclc proteins, and the like.
[0036] Methods to chemically couple one protein to another (carrier) protein
are well
known in the art and include, for example, conjugation by a water soluble
carbodiimide such
as 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride, conjugation by
a
homobifunctional cross-linker having, for example, NHS ester groups or sulfo-
NHS ester
analogs, conjugation by a heterobifunctional cross-linker having, for example,
and NHS ester
and a maleimide group such as sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-
carboxylate and, conjugation with gluteraldehyde (see, for example, Hermanson,
Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996)); see, also,
U.S. Pat.
Nos. 4,608,251 and 4,161;519).
[0037] The term "vector" which contains a transcription unit (aka. "expression
vector") as
used herein refers to viral and non-viral expression vectors that when
administered ifa vivo
can enter target cells and express an encoded protein. Viral vectors suitable
for delivery ih
vivo and expression of an exogenous protein are well known and include
adenoviral vectors,
adeno-associated viral vectors, retroviral vectors, herpes simplex viral
vectors, and the like.
Viral vectors are preferably made replication defective in normal cells. See
U.S. Patent no.
6,669,942; 6,566,128; 6,794,188; 6,110, 744; 6,133,029.
[0038] As used herein, the term "cells" is used expansively to encompass any
living cells
such as mammalian cells, plant cells, eukaryotic cells, prokaryotic cells, and
the like.
[0039] The term "adenoviral expression vector" as used herein, refers to any
vector from
an adenovirus that includes exogenous DNA inserted into its genome which
encodes a
polypeptide. The vector must be capable of replicating and being packaged when
any
deficient essential genes are provided in trans. An adenoviral vector
desirably contains at
least a portion of each terminal repeat required to support the replication of
the viral DNA,
preferably at least about 90°/~ of the full ITR sequence, and the DNA
required to encapsidate
the genome into a viral capsid. Many suitable adenoviral vectors have been
described in the
art. See U.S. Patent nos. 6,440,944 and 6,040,174 (replication defective El
deleted vectors
and specialized packaging cell lines). A preferred adenoviral expression
vector is one that is
replication defective in normal cells.
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[0040] Adeno-associated viruses represent a class of small, single-stranded
DNA viruses
that can insert their genetic material at a specific site on chromosome 19.
The preparation
and use of adeno-associated viral vectors for gene delivery is described in
U.S. Patent no.
5,658,785. '
[0041] Non-viral vectors for gene delivery comprise various types of
expression vectors
(e.g., plasmids) which are combined with lipids, proteins and other molecules
(or
combinations of thereof) in order to protect the DNA of the vector during
delivery. Fusigenic
non-viral particles can be constructed by combining viral fusion proteins with
expression
vectors as described. Kaneda, Curr Drug Targets (2003) 4(8):599-602.
Reconstituted HVJ
(hemagglutinating virus of Japan; Sendai virus)-liposomes can be used to
deliver expression
vectors or the vectors may be incorporated directly into inactivated HVJ
particles without
liposomes. See I~aneda, Cuff Drug Targets (2003) 4(8):599-602. DMRIE/DOPE
lipid
mixture are useful a velucle for non-viral expression vectors. See U.S.
6,147,055.
Polycation-DNA complexes also may be used as a non-viral gene delivery
vehicle. See
Thomas et al., Appl Micr-obiol Biotechnol (2003) 62(1):27-34.
[0042] The term "transcription unit" as it is used herein in connection with
an expression
vector means a stretch of DNA that is transcribed as a single, continuous mRNA
strand by
RNA polymerase, and includes the signals for initiation and termination of
transcription. For
example, in one embodiment, a transcription unit of the invention includes
nucleic acid that
encodes from 5' to 3,' a secretory signal sequence, an antigen and CD40
ligand. The
transcription unit is in operable linkage with transcriptional and/or
translational expression
control elements such as a promoter and optionally any upstream or downstream
enhancer
element(s). A useful promoter/enhancer is the cytomegalovirus (CMV) immediate-
early
promoter/enhancer. See U.S. Patents no. 5,849,522 and 6,218,140.
[0043] The term "secretory signal sequence" (aka. "signal sequence," "signal
peptide,"
leader sequence," or leader peptide") as used herein refers to a short peptide
sequence,
generally hydrophobic in charter, including about 20 to 30 amino acids which
is synthesized
at the N-terminus of a polypeptide and directs the polypeptide to the
endoplasmic reticulum.
The secretory signal sequence is generally cleaved upon translocation of the
polypeptide into
the endoplasmic reticulum. Eukaryotic secretory signal sequences are preferred
for directing
secretion of the exogenous gene product of the expression vector. A variety of
suitable such
sequences are well known in the art and include the secretory signal sequence
of human
to
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growth hormone, immunoglobulin kappa chain, and the like. In some embodiments
the
endogenous tumor antigen signal sequence also may be used to direct secretion.
[0044] The term "antigen" as used herein refers broadly to any antigen to
which an
individual can generate an irmnune response. "Antigen" as used herein refers
broadly to
molecule that contains at least one antigenic determinant to which the immune
response may
be directed. The immune response may be cell mediated or humoral or both.
[0045] As is well known in the art, an antigen may be protein in nature,
carbohydrate in
nature, lipid in nature, or nucleic acid in nature, or combinations of these
biomolecules. An
antigen may include non-natural molecules such as polymers and the like.
Antigens include
self antigens and foreign antigens such as antigens produced by another animal
or antigens
from an infectious agent. Infectious agent antigens may be bacterial, viral,
fungal, protozoan,
and the like.
[0046] The term "tumor associated antigen" (TAA) as used herein refers to a
protein
which is present on tumor cells, and on normal cells during fetal life (onco-
fetal antigen),
after birth in selected organs, or on many normal cells, but at much lower
concentration than
on tumor cells. A variety of TAA have been described. An exemplary TAA is a
mucin such
as MUC1, described in further detail below or the HER2 (neu) antigen also
described below.
In contrast, tumor specific antigen (TSA) (aka. "tumor-specific
transplantation antigen or
TSTA) refers to a protein absent from normal cells. TSAs usually appear when
an infecting
virus has caused the cell to become immortal and to express a viral
antigen(s).
[0047] A~z exemplary viral TSA is the E6 or E7 proteins of HPV type 16. TSAs
not
induced by viruses include idiotypes the immunoglobulin idiotypes associated
with ~B cell
lymphomas or the T cell receptor (TCR) on T cell lymphomas.
[0048] An exemplary viral TSA is the E6 or E7 proteins of HPV type 16. HPV can
cause
a variety of epithelial lesions of the skin and genital tract. HPV related
diseases of the genital
tract constitute the second leading cause of cancer death among women in the
world. These
include genital warts, cervical intraepithelial neoplasia (CII~ and cancer of
the cervix. The
HPV type most cormnonly associated with high grade CIN and cervical cancer is
HPV type
16. The majority of cervical cancers express the non-structural HPV16-derived
gene
products E6 and E7 oncoproteins. In HPV-induced cervical cancer model , the
E6/E7
oncoproteins are required for maintenance of the malignant phenotype and their
expression
correlates with the transforming potential of HPV 16. In addition to using E6
or E7 as the
11
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tumor antigen, one may use an antigenic fragment of these proteins instead. An
antigenic
fragment may be determined by testing the immune response with portions of the
molecule
such as are predicted to carry an epitope using well known computer
alogorithms (e.g. Hopp
and Woods hydrophobicity analysis).
[0049] TSAs not induced by viruses can be idiotypes of the immunoglobulin on B
cell
lymphomas or the T cell receptor (TCR) on T cell lymphomas. Tumor-associated
antigens
(TAA) are more cormnon than TSA.
[0050] Both TAA and TSA may be the immunological target of an expression
vector
vaccine. Unless indicated otherwise, the term "tumor antigen" is used herein
to refer
collectively to TAA and TSA.
[0051] The term "mucin " as used herein refers to any of a class of high
molecular weight
glycoproteins with a high content of clustered oligosaccharides O-
glycosidically linked to
tandem repeating peptide sequences which are rich in threonine, serine and
proline. Mucin
plays a role in cellular protection and, with many sugars exposed on the
extended structure,
effects multiple interactions with various cell types including leukocytes and
infectious
agents. Mucin antigens also include those identified as CD227, Tumor-
associated epithelial
membrane antigen (EMA), Polymorphic epithelial mucin (PEM), Peanut- reactive
urinary
mucin (PUM), episialin, Breast carcinoma-associated antigen DF3, H23 antigen,
mucin 1,
Episialin, Tumor-associated mucin, Carcinoma-associated mucin. Also included
are CA15-3
antigen, M344 antigen, Sialosyl Lewis Antigen (SLA), CA19-9, CA195 and other
mucin
antigen previously identified by monoclonal antibodies (e.g., see U.S. Patent
no. 5,849,876).
The term mucin does not include proteoglycans which are glycoproteins
characterized by
glycosaminoglycan chains covalently attached to the protein backbone.
[0052] At least 15 different mucins have been described including MUCl, MUC2,
MUC3A, MUC3B, MUC4, MUCSAC, MUCSB, MUC6, MUC7, MUCB, MUC9, MUC12,
MUC 13, MUC 15, and MUC 16 (these may also be designated with a hyphen between
"MUC" and the number). The nucleotide sequence and amino acid sequence of
these mucins
are known. The NCBI and Swiss Prot accession nos. for each of these mucins are
as follows:
MUC1 (NCBI NM002456, Swiss Prot P15941), MUC2, (NCBI NM002457, Swiss Prot
Q02817) MUC3A (NCBI AF113616, Swiss Prot Q02505), MUC3B (NCBI AJ291390, Swiss
Prot Q9H195), MUC4 (NCBI NM138299, Swiss Prot Q99102), MUCSAC (NCBI
AF043909, Swiss Prot Q8WWQ5), MUCSB (Swiss Prot Q9HC84), MUC6 (NCBI U97698,
12
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Swiss Prot Q8N8I1), MUC7 (NCBI L42983, Swiss Prot Q8TAX7), MUC8 (NCBI U14383,
Swiss Prot Q12964), MUC9 (NCBI U09550, Swiss Prot Q12889), MUC12 (Swiss Prot
Q9UKN1), MUC13 (NCBI NM017648, Swiss Prot Q9H3R2), MUC15 (NCBI NM145650,
Swiss Prot Q8WW41), and MUC16 (NCBI AF361486, Swiss Prot Q8WXI7; alca CA125).
[0053] There are two structurally and functionally distinct classes of mucins:
secreted gel-
forming mucins (MUC2, MUCSAC, MUCSB, and MUC6) and transmembrane mucins
(MUCl, MUC3A, MUC3B, MUC4, MUC12, MUC17). The products of some MUC genes
do not fit well into either class (MUC7, MUC8, MUC9, MUC 13, MUC 15, MUC 16).
[0054] The characteristics of particular mucins as TAA in particular cancers
is supported
by alterations in expression and structure in association with pre-neoplastic
and neoplastic
lesions (Filipe MI: Invest Cell Pathol 1979, 2:195-216; Filipe MI, Acta Med
Port 1979,
1:351-365). For instance, normal mucosa of the stomach is characterized by the
expression
of MUC1, MUCSA/C, MUC6 mRNA and the encoded immunoreactive protein. Also, high
levels of MUC2, MUC3 mucin mRNA and encoded immunoreactive protein are
associated
with intestinal metaplasia. Gastric cancer exhibits markedly altered secretory
mucin mRNA
levels compared with adjacent normal mucosa, with decreased levels of MUGS and
MUC6
mRNA and increased levels of MUC3 and MUC4 mRNA. High levels of MUC2 and MUC3
mRNA and protein are detectable in the small intestine, and MUC2 is the most
abundant
colonic mucin.
[0055] Mucins represent diagnostic markers for early detection of pancreatic
cancer and
other cell types. Studies have shown, that ductal adenocarcinomas (DACs) and
tumor cell
lines commonly overexpress MUC1 mucin . See Andrianifahanana et al., Clin
Cancer Res
2001, 7:4033-4040). This mucin was detected only at low levels in the most
chronic
pancreatitis and normal pancreas tissues but is overexpressed in all stages of
pancreatic
cancers. The de yaovo expression of MUC4 in pancreatic adenocarcinoma and cell
lines has
been reported (Hollingsworth et al., Int J Cancer 1994, 57:198-203 ). MUC4
mRNA
expression has been observed in the majority of pancreatic adenocarcinoma and
established
pancreatic cancer cell lines but not in normal pancreas or chronic
pancreatitis tissues. MUC
4 expression also has been associated with lung cancer (see Nguyen et al. 1996
Tumor Biol.
17:176-192). MUGS is associated with metastases in non-small cell lung cancer
(see Yu et
al., 1996 Int. J. Cancer 69:457-465). MUC6 is overexpressed and MUCSAC is de
novo
expressed in gastric and invasive DACs (I~im et al., Gastroenterology 2002,
123:1052-1060).
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MUC7 has been reported as a marker for invasive bladder cancer (see Retz et
al. 1998 Cancer
Res. 58:5662-5666)
[0056] Expression of the MUC2 secreted gel-forming mucin is generally
decreased in
colorectal adenocarcinoma, but preserved in mutinous carcinomas, a distinct
subtype of
colon cancer associated with microsatellite instability. MUC2 is increased in
laryngeal
cancer (Jeamion et al. 2001 Otolaryngol Head Neck Surg. 124:199-202). Another
secreted
gel-forming mucin, MUCSAC, a product of normal gastric mucosa, is absent from
normal
Colon, but frequently present in colorectal adenomas and colon cancers.
[0057] MUC1, also known as episialin, polymorphit epithelial mucin (PEM),
mucin like
cancer associated antigen (MCA), CA27.29, peanut-reactive urinary mucin (PUM),
tumor-
associated epithelial mucin, epithelial membrane antigen (EMA), human milk fat
globule
(HMFG) antigen, MUC1/REP, MUC1/SEC, MUC1/Y, CD227, is the most well known of
the
mucins. The gene encoding MUC1 maps to 1q21-q24. The MUC1 gene contains seven
exons and produces several different alternatively spliced variants. The
tandem repeat
domain is highly O-glycosylated and alterations in glycosylation have been
shown in
epithelial cancer cells.
[0058] MUC 1 mRNA is polymorphic in size. There are presently nine isoforms of
MUC 1
based on alternate splicing (isoform no.: NCBI accession no.; 1: ID P15941-1,
2: ID P15941-
2, 3: ID P15941-3, 4: ID P15941-4, 5: P15941-5, 6: ID P15941-6, 7: ID P15941-
7, 8: ID
P15941-8, and 9: ID P15941-9).
[0059] MUC1 isoform 1 (aka. MUC1/REP) is a polynorphic, type I transmembrane
protein containing: 1) a large extracellular domain, primarily consisting of a
20-amino acid
(aa) repeat motif (a region known as Variable Number (30 - 100) of tandem
repeats - VNTR);
2) a transmembrane domain; and 3) a 72-as cytoplasmic tail. During
biosynthesis, the
MUC1/REP protein is modified to a large extent, and a considerable number of O-
linked
sugar moieties confer mucin-like characteristics on the mature protein. Soon
after translation,
MLJC1/REP is cleaved into two products that form a tightly associated
heterodimer complex
composed of a large extracellular domain, linked noncovalently to a much
smaller protein
including the cytoplasmic and transmembrane domains. The extracellular domain
can be
shed from the cell. Using Swiss Prot P15941 as a reference (see FIG. 1), the
extracellular
domain (ecm) of MUC1 isoform 1 represents amino acids 24 to 1158, the
transmembrane
domain represents 1159-1181, and the cytoplasmit domain represents 1182-1255.
The SEA
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domain represents is 1034-1151 and represents a C-terminal portion of what is
referred to as
the extracellular domain. The SEA domain of a mucin is generally a target for
proteolytic
cleavage, yielding two subunits, the smaller of which is associated with the
cell membrane.
[0060] MUC1 isoform 5 (aka MUCl/SEC) is a form of MUC1 that is secreted by
cells. It
has an extracellular domain that is identical to that of isofonn 1 (MUC1/REP),
but lacks a
transmembrane domain for anchoring the protein to a cell membrane. MUC 1
isoform 7 (aka
MUCl/Y) contains the cytoplasmic and transmembrane domains observed in
isoforms 1
(MUC1/REP) and 5 (MUC1/SEC), but has an extracellular domain that is smaller
than
MUC1, lacking the repeat motif and its flanking region (see Baruch A. et al.,
1999 Cancer
Res. 59, 1552-1561). Isoform 7 behaves as a receptor and binds the secreted
isofonn 5.
Binding induces phosphorylation of isofonn 7 and alters cellular morphology
and initiates
cell signaling through second messenger proteins such as GRB2, (see Zrihan-
Licht S. et al.,
1995 FEBS Lett. 356, 130-136). It has been shown that !3-catenin interacts
with the
cytoplasmic domain of MUC1 (Yamamoto M. et al., 1997 J. Biol. Chem. 272, 12492-
12494).
[0061] MUCl is expressed focally at low levels on normal epithelial cell
surfaces. See
15. Greenlee, et al., Caface~ Statistics CA Cancey-J. 50, 7-33 (2000); Ren, et
al., J. Biol.
Chem. 277, 17616-17622 (2002); Kontani, et al., B~. J. Cahcer~ 84, 1258-1264
(2001);
Rowse, et al., Carace~ Res. 58, 315 (1998). MUCl is overexpressed in
carcinomas of the
breast, ovary, pancreas as well as other carcinomas (see also Gendler S.J. et
al, 1990 J. Biol.
Chem. 265, 15286-15293). A correlation is found between acquisition of
additional copies of
MIJC1 gene and high mRNA levels (p < 0.0001), revealing the genetic mechanism
responsible for MLTC1 gene overexpression, and supporting the role of MUC1
gene dosage in
the pathogenesis of breast cancer (Bieche I. et al.,. 1997 Cancer Genet.
Cytogenet. 98, 75-80).
MLJC1 mucin, as detected immunologically, is increased in expression in colon
cancers,
which correlates with a worse prognosis and in ovarian cancers.
[0062] High level expression of the MUC1 antigen plays a role in neoplastic
epithelial
mucosal cell development by disrupting the regulation of anchorage dependent
growth
(disrupting E-cadherin function), which leads to metastases. See Greenlee, et
al., Cancer
Statistics CA Cancer J. 50, 7-33 (2000); Ren, et al. J. Biol. Chem. 277, 17616-
17622 (2002).
Non-MHC-restricted cytotoxic T cell responses to MUC1 have been reported in
patients with
breast cancer. See Kontani et al., Br. J. Cancef° 84, 1258-1264 (2001).
Human MUC1
transgenic mice ("MUC-l.Tg") have been reported to be unresponsive to
stimulation with
human MUC1 antigen. See Rowse, et al., Cafacef~ Res. 58, 315 (1998). Human
MUC1
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transgenic mice are useful for evaluating the development of immunity to MUC1
as a self
antigen.
[0063] MUC1 protein and mRNA have been found in the ER-positive MCF-7 and BT-
474
cells as well as in the ER-negative MDA-MB-231 and SK-BR-3 BCC cells. The mRNA
Transcript level was lugher in ER+ than in ER- cell lines. MUC1 reacts with
intracellular
adhesion molecule-1 (ICAM-1). At least six tandem repeats of MUC1 are needed
(Regimbald et al., 1996 Cancer Res. 56,4244-4249). The tandem repeat peptide
of MUC1
from T-47D BCC was found to be highly O-glycosylated with 4.8 glycosylated
sites per
repeat, which compares to 2.6 sites per repeat for the mucin from milk.
[0064] The term "mucin antigen" as used herein refers to the full length mucin
or a
portion of a mucin that contains an epitope characterized in being able to
elicit cellular
immunity using a MUC-CD40L expression vector administered ifZ vivo as
described herein.
A "mucin antigen" includes one or more epitopes from the extracellular domain
of a mucin
such as one or more of the tandem repeat motifs associated with the VNTR, or
the SEA
region. A mucin antigen may contain the entire extracellular domain. Also
included within
the meaning of "mucin antigen" are variations in the sequence including
conservative amino
acid changes and the like which do not alter the ability of the antigen to
elicit an immune
response that crossreacts with a native mucin sequence.
[0065] The VNTR consists of variable numbers of a tandemly repeated peptide
sequences
which differ in length (and composition) according to a genetic polymorphism
and the nature
of the mucin. The VNTR may also include 5' and 3' regions which contain
degenerate
tandem repeats. For example, in MUC1, the number of repeats varies from 21 to
125 in the
northern European population. In the U.S. the most infrequent alleles contains
41 and 85
repeats, while more common alleles have 60-84 repeats. The MUC1 repeat has the
general
repeating peptide sequence PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 3). Underlying
the MUC1 tandem repeat is a genetic sequence polymorphism at three positions
shown
bolded and underlined (positions 2, 3 and 13). The concerted replacement DT-
DES
(sequence variation 1) and the single replacements P-~Q (sequence variation
2), P-~A
(sequence variation 3), and PST (sequence variation 4) have been identified
and vary with
position in the domain (see Engelmann et al., 2001 J. Biol. Chem. 276:27764-
27769). The
most frequent replacement DT DES occurs in up to 50% of the repeats. Table 1
shows some
exemplary tandem repeat sequences.
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Table 1: Mucin Tandem Repeat Sequences
Mucin Tandem Repeat (SEQ ID NO:) Mucin source
MUC1 PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 3) Mammary
PDNKPAPGSTAPPAHGVTSA (SEQ ID NO: 33) Pancreatic
MUC2 PTTTPPITTTTTVTPTPTPTGTQT (SEQ ID NO: Intestinal
4)
Tracheobronchial
MLTC3 HSTPSFTSSITTTETTS (SEQ ID NO: 5) Intestinal
Gall Bladder
MUC4 TSSASTGHATPLPVTD (SEQ ID NO: 6) Colon
Tracheobronchial
MUCSAC TTSTTSAP (SEQ ID NO: 7) Gastric
Tracheobronchial
MUCSB SSTPGTAHTLTMLTTTATTPTATGSTATP (SEQ Tracheobronchial
ID NO: 8)
Salivary
MLTC7 TTAAPPTPSATTPAPPSSSAPG (SEQ ID NO: Salivary
9)
MUCB TSCPRPLQEGTPGSRAAHALSRRGHRVHELPTS Tracheobronchial
SPGGDTGF (SEQ ID NO: 10)
[0066] Although a mucin antigen as used herein may comprise only a single
tandem
repeat sequence motif, it should be understood that the immune response will
generally be
stronger and more efficiently generated if the vector encodes multiple such
repeats. The
invention vector preferably encodes mucin tandem repeats from 2-4, more
preferably from 5-
9, even more preferably from 10-19, yet even more preferably from 20-29, still
more
preferably from 30-39, and still yet more preferably from 40-50. Tandem
repeats greater than
50 are possible and may include the number of such repeats found in natural
mucins.
[0067] A mucin antigen as this term is used herein also may encompass tandem
repeats
from different types of mucins. For example, an expression vector may encode
tandem
repeats from two different mucins, e.g., MUC1 and MUC2. Such a vector also may
encode
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multiple forms of the SEA domain as well or a combination of tandem repeats
and one or
more SEA domains.
[0068] A secretable form of an antigen is one that lacks all or substantially
all of its
transmembrane domain, if present in the mature protein. For example, in the
case of a mucin,
the transmembrane domain, if present, is generally about 24 amino acids in
length and
functions to anchor the mucin or a fragment of the mucin in the cell membrane.
A secretable
form of MUC1 in which all of the transmembrane domain has been deleted is MUCl
missing
residues 1159-1181. A mucin (or antigen) missing substantially all of the
transmembrane is
one where the domain comprises 6 residues or less of sequence at one end of
the
transmembrane domain, more preferably less than about 4 residues of sequence
at one end of
the transmembrane domain, even more preferably less than about 2 residues of
sequence on
one end of the transmembrane domain, and most preferably 1 residue or less on
one end of
the transmembrane domain. In a preferred embodiment, the vaccine vector
transcription unit
encodes a secretable form of a mucin (or antigen) lacking the entire
transmembrane domain.
A mucin that lacks substantially all of the transmembrane domain rendering the
mucin
secretable is one that contains no more than six residues of sequence on one
end of the
domain. The extracellular domain of a hmnan mucin such as MUC1 is denoted
herein as
"ecdhMUC 1."
[0069] It should be understood that a mucin which lacks a functional
transmembrane
domain may still include all or a portion of the cytoplasmic domain and all or
a portion of the
SEA region, if present.
[0070] A source of DNA encoding the various mucins, and mucin antigens may be
obtained from mucin expressing cell lines using a commercial cDNA synthesis
kit and
amplification using a suitable pair of PCR primers that can be designed from
the published
mucin DNA sequences. For example, MUC1 or MUC2 encoding nucleic acid may be
obtained from CRL-1500 cells, available from the American Type Culture
Collection.
Mucin encoding DNA also may be obtained by amplification from RNA or cDNA
obtained
or prepared from human or other animal tissues. For DNA segments that are not
that large,
the DNA may be synthesized using an automated oligonucleotide synthesizer.
[0071] The term "linlcer" as used herein with respect to the transcription
unit of the
expression vector refers to one or more amino acid residues between the
carboxy terminal
end of the antigen and the amino terminal end of CD40 ligand. The composition
and length
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of the linker may be determined in accordance with methods well known in the
art and may
be tested for efficacy. See e.g. Arai et al., design of the linkers which
effectively separate
domains of a bifunctional fusion protein. Protein Engineering, Vol. 14, No. 8,
529-532,
August 2001. The linker is generally from about 3 to about 1 S amino acids
long, more
preferably about 5 to about 10 amino acids long, however, longer or shorter
linkers may be
used or the linker may be dispensed with entirely. Longer linkers may be up to
about 50
amino acids, or up to about 100 amino acids. A short linker of less than 10
residues is
preferred when the mucin antigen is N-terminal to the CD40 ligand.
[0072] The term "CD40 ligand" (CD40L) as used herein refers to a full length
or portion
of the molecule known also as CD154 or TNFS. CD40L is a type II membrane
polypeptide
having a cytoplasmic domain at its N-terminus, a transmembrane region and then
an
extracellular domain at its C-terminus. Unless otherwise indicated the full
length CD40L is
designated herein as "CD40L," "wtCD40L" or "wtTmCD40L." The form of CD40L in
which the cytoplasmic domain has been deleted is designated herein as
"~CtCD40L." The
form of CD40L where the transmembrane domain has been deleted is designated
herein as
"4TmCD40L." The form of CD40L where both the cytoplasmic and transmembrane
domains have been deleted is designated herein as "OCt~TmCD40L." The
nucleotide and
amino acid sequence of CD40L from mouse and human is well known in the art and
can be
found, for example, in U.S. Patent No. 5,962,406 (Armitage et al.). Also
included within the
meaiung of CD40 ligand are variations in the sequence including conservative
amino acid
changes and the like which do not alter the ability of the ligand to elicit an
immune response
to a mucin in conjunction the fusion protein of the invention.
[0073] Murine CD40L (mCD40L) is 260 amino acids in length. The cytoplasmic
(Ct)
domain of mCD40L extends approximately from position 1-22, the transmembrane
domain
extends approximately from position 23-46, while the extracellular domain
extends
approximately from position 47-260.
[0074] Human CD40L (hCD40L) is 261 amino acids in length. The cytoplasmic
domain
of hCD40L extends approximately from position 1-22, the transmembrane domain
extends
approximately from position 23-46, while the extracellular domain extends
approximately
from position 47-261.
[0075] The phrase "CD40 ligand is missing all or substantially all of the
transmembrane
domain rendering CD40 ligand secretable" as used herein refers to a
recombinant form of
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CD40 ligand that can be secreted from a cell. The transmembrane domain of
CD40L which
contains about 24 amino acids in length, functions to anchor CD40 ligand in
the cell
membrane. CD40L from wluch all of the transmembrane domain has been deleted is
CD40
ligand lacking residues 23-46. CD40 ligand missing substantially all of the
transmembrane is
one that retains 6 residues or less of sequence at one end of the
transmembrane domain, more
preferably less than about 4 residues of sequence at one end of the
transmembrane domain,
even more preferably less than about 2 residues of sequence on one end of the
transmembrane domain, and most preferably 1 residue or less on one end of the
transmembrane domain. Thus, a CD40L that lacks substantially all of the
transmembrane
domain rendering the CD40L secretable is one that retains no more than six
residues of
sequence on one end of the domain. Such as CD40L would contain, in addition to
the
extracellular domain and optionally the cytoplasmic domain, and no more than
amino acids
41-46 or 23-28 located in the transmembrane domain of CD40L. In a preferred
embodiment,
the vaccine vector transcription unit encodes a secretable form of CD40
containing less than
10% of the transmembrane domain. More preferably, CD40L contains no
transmembrane
domain.
[0076] It should be understood that a CD40L which lacks a functional
transmembrane
domain may still include all or a portion of the cytoplasmic domain. Likewise,
a CD40L
which lacks a functional transmembrane domain may include all or a substantial
portion of
the extracellular domain.
[0077] As used herein, an expression vector and fusion protein boost is
administered as a
vaccine to induce immunity to a tumor antigen. The expression vector and
protein boost may
be formulated as appropriate with a suitable pharmaceutically acceptable
carrier.
Accordingly, the vectors or protein boost may be used in the manufacture of a
medicament or
pharmaceutical composition. Expression vectors and the fusion protein may be
formulated as
solutions or lyophilized powders for parenteral administration. Powders may be
reconstituted
by addition of a suitable diluent or other pharmaceutically acceptable carrier
prior to use.
Liquid formulations may be buffered, isotonic, aqueous solutions. Powders also
may be
sprayed in dry form. Examples of suitable diluents are normal isotonic saline
solution,
standard 5% dextrose in water, or buffered sodium or ammonium acetate
solution. Such
formulations are especially suitable for parenteral administration, but may
also be used for
oral administration or contained in a metered dose inhaler or nebulizer for
insufflation. It
CA 02548347 2006-06-06
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may be desirable to add excipients such as polyvinylpyrrolidone, gelatin,
hydroxy cellulose,
acacia, polyethylene glycol, mannitol, sodium chloride, sodium citrate, and
the like.
[0078] Alternately, expression vectors and the fusion protein may be prepared
for oral
administration. Pharmaceutically acceptable solid or liquid carriers may be
added to enhance
or stabilize the composition, or to facilitate preparation of the vectors.
Solid carriers include
starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or
stearic acid, talc,
pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil,
olive oil, saline and
water. The carrier may also include a sustained release material such as
glyceryl
monostearate or glyceryl distearate, alone or with a wax. The amount of solid
Garner varies
but, preferably, will be between about 20 mg to about 1 g per dosage unit.
When a liquid
carrier is used, the preparation may be in the form of a syrup, elixir,
emulsion, or an aqueous
or non-aqueous suspension.
[0079] Expression vectors and the fusion protein may be formulated to include
other
medically useful drugs or biological agents. The vectors also may be
administered in
conjunction with the administration of other drugs or biological agents useful
for the disease
or condition that the invention compounds are directed.
[0080] As employed herein, the phrase "an effective amount," refers to a dose
sufficient to
provide concentrations high enough to generate (or contribute to the
generation of) an
immune response in the recipient thereof. The specific effective dose level
for any particular
subj ect will depend upon a variety of factors including the disorder being
treated, the severity
of the disorder, the activity of the specific compound, the route of
administration, the rate of
clearance of the viral vectors, the duration of treatment, the drugs used in
combination or
coincident with the viral vectors, the age, body weight, sex, diet, and
general health of the
subject, and like factors well known in the medical arts and sciences. Various
general
considerations taken into account in determining the "therapeutically
effective amount" are
known to those of skill in the art and are described, e.g., in Gilman et al.,
eds., Goodman And
Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press,
1990; and
Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton,
Pa., 1990. For
administration of vectors, the range of particles per administration typically
if from about 1 X
10~ to 1 X 1011, more preferably 1 X 108 to 5 X 101°, and even more
preferably 5 X 108 to 2
X 101°. A vector can be administered parenterally, such as
intravascularly, intravenously,
intraarterially, intramuscularly, subcutaneously, or the like. Administration
can also be
orally, nasally, rectally, transdermally or inhalationally via an aerosol. The
vectors may be
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administered as a bolus, or slowly infused. The vector is preferably
administered
subcutaneously.
[0081] As demonstrated herein, vectors encoding tumor associated antigens can
induce a
protective cellular and humoral immunity against such antigens, including
those to which
tolerance had developed. Although not wishing to be bound by any theory, it is
believed that
the invention vaccines generate upon administration a continual local release
of the fusion
protein composed of the secretable form of the antigen linked to a secretory
form of CD40
ligand. As demonstrated herein this facilitates DCs maturation, promoting the
development
of effective antigen-specific immunity. It is also demonstrated herein that
the secretable
fusion protein encoding the extracellular domain of human MUC 1 and the marine
CD40L
lacking a transmembrane and cytoplasmic domain (i.e. ecdhMUCl-4Ct~TmCD40L)
produced from an adenoviral vector dramatically enhanced the potency of the
cellular
immune response to MUC 1 expressing tumor cells. Although not wishing to be
bound by
any theory, it is believed that subcutaneous injection of the Ad-K-ecdhIVIUCl-
OCtOTmCD40L vector elicited strong MLTC1 specific CD8+ T cell-mediated
immunity,
which prevents the engraftment of cancer cells which express the MUC1 tumor
associated
antigen.
[0082] The immunity generated against the antigens using the invention methods
is long
lasting. As used herein, the term long lasting means that immunity elicited by
the antigen
encoded by the vector can be demonstrated for up to 6 months from the last
administration,
more preferably for up to 8 months, more preferably for up to one year, more
preferably up to
1.5 years, and more preferably for at least two years.
[0083] In one embodiment, immunity to a mucin TAA can be generated by
producing a
fusion protein that comprises the extracellular domain of MUC1 fused the amino-
terminal
end of the CD40 ligand from which the transmembrane and cytoplasmic domains
were
deleted. Construction of such vector is disclosed in the Examples. As was
observed herein,
subcutaneous administration of this adenoviral vector mucin vaccine induced a
very robust
and long lasting CD8+ cytotoxic T cell lynphocyte dependent systemic immune
response
against cancer cells which carry the MUC 1 antigen. The mucin vaccine induced
the
production of memory cells, which underlie the long lasting immunity.
[0084] It was observed that vaccination of mice with the adenoviral vector Ad-
sig-
ecdhMLJCl/ecdmCD40L induced an immune response which suppressed the growth of
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human MUC1 (hMUCl) antigen positive tumor cells in 100% of mice transgenic for
hMUCl
(i.e. these mice are anergic to the hMUCl antigen prior to the vector
injection. See Rowse, et
al., Cafzcer Res. 58, 315 (1998). The immune response to the Ad- sig-
ecdhMUCl/ecdmCD40L vector lasted up to a year and was shown to be antigen
specific.
These results demonstrated that the Ad- sig-ecdhMTJCl-ecd/ecdCD40L vector can
be used
for treating epithelial malignancies that express the MUC 1.
[0085] Subcutaneous injection of the adenoviral MUC1 expression vector
increased the
level of hMUCl specific T cells in the spleens of injected hMUCl transgenic
mice by 250
fold. The transgenic mice were anergic to the hMUCl antigen prior to the
vector injection.
Thus, vector injection overcame the anergy, inducing a CD8+ T cell dependent
systemic Thl
immune response that was antigen specific, and HLA restricted. The ability to
overcome
anergy as observed for vaccination with the adenoviral MUC1 expression vector,
was not
observed when transgenic mice were vaccinated with purified ecdhMCTCl/ecdCD40L-
HIS
protein.
[0086] Although not wishing to be bound by any theory, it is believed that the
cells
infected in the vicinity of the site of subcutaneous injection of the vector
release the tumor
antigen/CD40 ligand secretory which is taken up by antigen presenting cells
(e.g. DCs) in the
vicinity of the infected cells. The internalized tumor antigen would be
digested in the
proteosome with the resultant tumor antigen peptides trafficking to the
endoplasmic
reticulum where they would bind to Class I MHC molecules. Eventually, the DCs
would
present the tumor antigen on the surface in the Class I MHC molecule.
Activated, tumor
antigen-loaded antigen presenting cells would migrate to lymphocyte bearing
secondary
organs such as the regional lymph nodes or the spleen. During the two weeks of
continuous
release of the tumor antigeuCD40 fusion protein, CD8 cytotoxic T cell
lymphocytes
competent to recognize and kill cells, which carried the tumor associated
antigens, would be
expanded in the lymph nodes and spleen by the presence of the activated and
antigen loaded
dendritic cells. The continuous nature of the stimulation and the expansion of
the tumor
antigen specific cytotoxic T cells by the continuous release from the vector
infected cells is
believed to generate an immune response which would be greater in magnitude
than is
possible using a vector which carried a tumor antigen/CD40 ligand which is non-
secretory.
[0087] The methods of the present invention, therefore, can be used to
generate immunity
to an antigen which is a self antigen in an individual. For example, a vector
that encodes a
mucin antigen from MUC1 can be used to generate CD8+ immunity in a human where
the
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MUC1 mucin antigen is a self antigen. The invention methods also cam be used
to overcome
a state of immunological anergy to an antigen which is a self antigen.
[0088] The following examples serve to illustrate the present invention. These
examples
are in no way intended to limit the scope of the invention.
EXAMPLES
1. Construction of adenoviral expression vectors
[0089] The transcription unit, sig-ecdhMUCl-~CtOTmCD40L of the adenoviral
vector
encodes a signal sequence (from an Ig kappa chain) followed by the
extracellular domain of
human MUC 1 which is connected via a linker to a fragment of the CD40 ligand
(human or
mouse) which contains the extracellular domain without the transmembrane or
cytoplasmic
domains. The fusion protein was engineered to be secreted from vector infected
cells by the
addition of the kappa chain signal sequence to the amino-terminal end of the
fusion protein.
[0090] The amino acid sequence of human MUC-1 and the encoding nucleotide
sequence
are shown in FIGS. 2 and 1, respectively. The encoded MUC1 protein represents
1255 amino
acids encoded by nucleotides 74 to 3,841 of SEQ ID NO: 1. The first 23 amino
acids
(encoded by 74 to 142 of SEQ ID NO:1) represent the MUC1 signal sequence which
is
removed from the mature mucin. The extracellular domain represents about 1135
amino
acids from positions 24 to 1158 (encoded by nucleotides 143 to 3547). The
tandem repeat
region represents approximately 900 amino acids. Amino acids 74 to 126
(encoded by 229 to
451 of SEQ ID NO:1) represents a 5' degenerate tandem repeat region, amino
acids 127 to
945 represents the tandem repeat region (encoded by 452 to 2,908 of SEQ ID NO:
1) while
amino acids 946 to 962 represent a 3' degenerate tandem repeat region (encoded
by 2809 to
2959 of SEQ ID N0:1). The SEA domain represents amino acids 1034 to 1151, the
transmembrane domain represents 1159 to 1181, and the cytoplasmic domain
represents 1182
to 1255 (see SEQ ID N0:2).
[0091] The transcription unit was introduced into the E1 gene region of the
adenoviral
vector backbone. After the adenoviral vector particles were generated in HEK
293 cells, the
vector DNA was purified by cesium chloride gradient centrifugation. The
presence of the
signal peptide in the adenoviral vector was confirmed by restriction enzyme
analysis and by
DNA sequencing.
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[0092] A transcription unit that included DNA encoding the signal sequence of
the mouse
IgG kappa chain gene upstream of DNA encoding human MUC-1 ("sig-ecdhMUC-1")
was
generated by PCR using plasmid pcDNA3-hMUC-1 (gift of Finn O.J., University of
Pittsburgh School of Medicine) and the following primers: DNA encoding the
mouse IgG
kappa chain METDTLLLWVLLLWVPGSTGD (single letter amino acid code) (SEQ ID
NO: 11) was prepared by PCR amplification (SEQ ID NOs: 12 ,13 and 14) to
generate the
full 21 amino acid mouse IgG kappa chain signal sequence (the start codon
"ATG" is shovcm
bolded in SEQ ID N0:12).
' - CCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG-3'
(SEQ ID NO: 12)
5'- TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TC-3'
(SEQ ID N0:13)
5'- TG CTC TGG GTT CCA GGT TCC ACT GGT GAC GAT G -3'
(SEQ ID NO: 14)
5'- GGT TCC ACT GGT GAC GAT GTC ACC TCG GTC CCA GTC-3'(SEQ ID NO:15)
(forward primer for MUC-1 repeat region)
5'- GAGCTCGAG ATT GTG GAC TGG AGG GGC GGT G-3'
(SEQ ID NO: 16) (reverse primer for MUC-1 repeat region)
sig-ecdhMUC-1 with the upstream kappa signal sequence was generated by four
rounds of
PCR amplification (1St round: primers SEQ ID NOs 15 and 16; 2"d round: primer
SEQ ID
NOs 14 and 16; 3rd round: primer SEQ ID NOs 13 and 16; 4th round: primer SEQ
ID NOs 12
and 16). The sig-ecdhMUC-1 encoding DNA was cloned into the pcDNATM 3.1 TOPO
vector (Invitrogen, San Diego, CA) forming pcDNA-sig-ecdhMUC-1.
[0093] pShuttle -OCt~TmCD40L (no signal sequence and marine CD40L) was
prepared
as follows: Plasmid pDC406-mCD40L was purchased from the American Type Culture
Collection. A pair of PCR primers (SEQ ID NOs: 17 and 1 S) was designed to
amplify the
mouse CD40 ligand from position 52 to 260 (i.e., without the cytoplasmic and
transmembrane domains) and include sequence encoding a linker (indicated as "+
spacer ") at
the 5' end of the amplicon.
Mouse OCt~TmCD40L+ spacer forward primer (MCD40LSPF) (CD40L sequence
italicized;
cloning site underlined and bolded):
5'-CCGCTCGAGAACGACGCACAAGCACCAAAATCAAAGGTCGAAG
AGGAAGTA -3' (SEQ ID NO: 17).
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Mouse CD40L reverse primer (MCD40LR; cloning site underlined)
5'-GCGGGCC CGCGGCCGCCGCTAG TCTAGA GAG TTT GAG TAA GCC AAA AGA
TGA G-3'
(SEQ ID NO: 18)
[0094] The forward primer MCD40LSPF encodes a 10 residue spacer (LENDAQAPKS;
single letter code; SEQ ID NO: 19) to be located between the mucin and the
CD40 ligand
(mCD40L) of the transcription unit. PCR performed using the forward and
reverse primers
(SEQ ID NOs 17 and 18) and plasmid pDC406-mCD40L as the template resulted in
PCR
fragment "space+OCt~TMCD40L", which was inserted into the plasmid pcDNA-sig-
ecdhMZJCl after restriction endonuclease digestion with XbaI (TCTAGA) and Xho
I
(CTCGAG). This vector is designated pcDNA-sig- ecdhMLTCl/OCt~TmCD40L. A vector
was produced that was otherwise the same except that it encoded full length
CD40L rather
than the truncated form. This vector was made using a CD40 forward primer that
annealed to
the starting codons of marine CD40L. This vector is designated pShuttleCD40L
(no signal
sequence).
[0095] The sig-ecdhMLJCl/~Ct~TmCD40L encoding DNA was cut from the
pCDNA3TOP0 vector using HindIII-XbaI restriction and inserted into pShuttle-
CMV (see
Murphy et al., Prostate 38: 73-78, 1999) downstream of the CMV promoter. The
plasmid is
designated pShuttle-sig-ecdhMUCl-OCtOTmCD40L. Thus, the transcription unit sig-
ecdhMLTCl-~CtOTmCD40L encodes the mouse IgG kappa chain secretory signal
followed
by the extracellulax domain of human MUC1 followed by a 10 amino acid linker
with
(NDAQAPK; SEQ ID NO: 19) followed by marine CD40 ligand residues 52-260.
[0096] In some vectors, the mouse HSF1 trimer domain was added between the
ecdhMUCl encoding DNA and OCtOTm CD40L by PCR using plasmid pcDNA-sig-
ecdhMUCl/OCtOTmCD40L and the following primers:
5'-AAC AAG CTC ATT CAG TTC CTG ATC TCA CTG GTG GGATCC AAC GAC
GCA CAA GCA CCA AAA TC-3'
(SEQ ID NO: 20).
5'- AGC CTT CGG CAG AAG CAT GCC CAG CAA CAG AAA GTC GTC AAC AAG
CTC ATT CAG TTC CTG-3'
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(SEQ ID NO: 21).
5' AAT GAG GCT CTG TGG CGG GAG GTG GCC AGC CTT CGG CAG AAG CAT G-
3'
(SEQ ID NO: 22).
5'GAT ATC CTC AGG CTC GAG AAG GAC GGA CAA GCA CCA AAA GAG AAT GAG
GCT CTG TGG CGG G-3'
(SEQ ID NO: 23).
5'-GCGGGCC CGCGGCCGCCGCTAG TCTAGA GAG TTT GAG TAA GCC AAA AGA
TGA G-3'
(SEQ ID NO: 18).
[0097] HSF1/dCtOTm CD40L with the trimer domain sequence was generated by four
rounds of PCR amplification (1St round: primers SEQ ID NOs 23 and 18; 2"a
round: primer
SEQ ID NOs 22 and 18; 3rd round: primer SEQ ID NOs 21 and 18; 4th round:
primer SEQ ID
NOs 20 and 18). The HSF1/~Ct~Tm CD40L encoding DNA was cloned into pcDNA-sig-
hMUC-1 restriction sites XbaI (TCTAGA) and Xho I (CTCGAG). The sequence
between
MUCl and mCD40L is as follows:
LEN D A Q A P K E N E A L W R E V A S F R Q K H A Q Q Q K V V
N K L I ~ F L I S L V G S N D A Q A P K S (SEQ ID NO: 24), wherein the
underlined segment is the trimer sequence which is bonded by the linker
LENDAQAPK
(SEQ ID N0:25) and NDAQAPKS (SEQ ~ N0:26) .
[0098] In some vectors, a His tag encoding sequence was added to the end of
the OCt4Tm
CD40L and was generated by PCR using Plasmid pDC406-mCD40L (purchased from the
American Type Culture Collection) and the following primers:
5'- CCG CTCGAG AACGACGCACAAGCACCAAA.ATCAAAGGTCGAAGAGGAA
GTA -3' (SEQ ID NO: 2,7) (forward primer)
5'-ATG GTG ATG ATG ACC GGT ACG GAG TTT GAG TAA GCC AAA AGA TGA
GAA GCC-3' (SEQ ID NO: 28) (reverse primer)
5'-GTGC TCTAGA TCA GAATTC TG GTG ATG GTG ATG ATGI ACC GGT ACG
GAG -3' (SEQ ID NO: 29) (poly His region encoded by nucleotides in the box)
[0099] Vector /OCt~Tm CD40L/His with the His tag sequence was generated by 2
rounds
of PCR amplification (1st round: primers 1 +2; 2"a round: primer 1+3). The
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/OCt~TmCD40L/His encoding DNA was cloned into pcDNA-sig-ecdhMLJC-1 restriction
sites XbaI (TCTAGA) and Xho I (CTCGAG).
[00100] The recombinant adenoviral vectors were generated using the AdEasy
vector
system (Stratagene, San Diego, CA). Briefly the resulting plasmid pShuttle-sig-
ecdhMUCl-
OCtOTmCD40L, and other control adenoviral vectors were linearized with Pme I
and co-
transformed into E. coli strain BJ5183 together with pAdEasy-1, the viral DNA
plasmid.
Recombinants were selected with kanamycin and screened by restriction enzyme
analysis.
The recombinant adenoviral construct was then cleaved with Pac I to expose its
Inverted
Terminal Repeats (ITR) and transfected into 293A cells to produce viral
particles. The titer
of recombinant adenovirus was determined by the Tissue culture Infectious Dose
(TCIDso)
method.
[00101] Primers for amplifying human ~CtOTmCD40L+ spacer using a human CD40
ligand cDNA template are set forth below.
Human ~CtOTmCD40L+ spacer forward primer (HCD40LSPF) (CD40L sequence
italicized):
S'- CCG
CTCGAGAACGACGCACAAGCACCAAAATCAGTGTATCTTCATAGAAGGTTGGACA
AG-3' (SEQ ID NO: 30)
Human CD40L reverse primer (HCD40LR)
5'-CCCTCTAGA TCAGAGTTTGAGTAAGCCAAAGGAC-3' (SEQ ID NO: 31)
[00102] These primers will amplify a OCt~TmCD40L+spacer which encodes 47-261
of
human CD40L. The forward primer HCD40LSPF encodes a 10 residue spacer
(LENDAQAPKS; single letter code; SEQ ID NO: 19) to be located between the
tumor
antigen and the CD40 ligand (hCD40L) of the transcription unit. PCR performed
using the
forward and reverse primers (SEQ ID NOs 30 and 31) and Plasmid pDC406-hCD40L
as the
template results in PCR fragment "space+~CtOTmCD40L(human)," which is inserted
into
the plasmid pcDNA-sig-ecdhMUCl after restriction endonuclease digestion with
XbaI
(TCTAGA) and Xho I (CTCGAG). The sig-ecdhMZJCl /~CtOTmCD40L (human) encoding
DNA was cut from the pCDNA3TOP0 using HindIII-XbaI restriction and inserted
into
pShuttle-CMV (see Murphy et al., Prostate 38: 73-78, 1999) downstream of the
CMV
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promoter. This vector is designated pShuttle sig-ecdhMUCl/OCtOTmCD40L(human).
Modification of pShuttle sig-ecdhMLJCl/OCt~TmCD40L(human) to include the
ecdhMUCl
upstream of the human CD40 ligand sequence was accomplished essentially as
described
above for the marine CD40 ligand encoding vectors. Thus, the transcription
unit sig-
ecdl~MTJC1-~CtOTmCD40L(human) encodes the kappa secretory signal followed by
the
extracellular domain of human MUC1 followed by a 10 amino acid linker
(NDAQAPK; SEQ
ID N0:19) followed by human CD40 ligand residues 47-261.
[00103] In an alternative approach, DNA encoding the human growth hormone
signal
sequence MATGSRTSLLLAFGLLCLPWLQEGSA (single letter amino acid code) (SEQ ID
NO: 32) could be used in place of the kappa chain signal sequence.
Z. Overcoming Anergy to MUC1 in MUC1 transgenic mice
a) Cytokine production of adenoviral infected DCs
[00104] Bone marrow derived DCs was harvested from hMUC-.Tg transgenic mice at
48
hours after exposure to the adenoviral vectors. The cells were exposed to
vector at MOI 100,
and plated in 24-well plates at 2 ~ 105 cells/ml. After incubation for 24
hours at 37°C,
supernatant fluid (1m1) was harvested and centrifuged to remove debris. The
level of marine
IL-12 or IFN-gamma released into the culture medium was assessed by enzyme-
linked
irmnunoadsorbent assay (ELISA) using the mouse IL-12 p70 or IFN-gamma R & D
Systems
kits.
[00105] Bone marrow derived DCs contacted with the Ad-sig-ecdmMUCl-~Ct~TCD40L
(marine) vector showed significantly increased the levels of interferon gamma
and IL-12
cytokines from DCs harvested from the hMUC-.Tg transgenic mice at 48 hours
after
exposure to the vector. In contrast, virtually no cytokines were detected from
restimulated
DC's from animals immunized with an adenoviral vector that encoded the
extracellular
domain of hMUCl but without fusion to a secretable form of CD40L. These
results indicate
that the ecdhMUCl/ecdmCD40L (marine) fusion protein forms functional trimers
and binds
to the CD40 receptor on DCs.
b) Evaluation of trimer formation by ecdhMUCl-HSFl-
OCt4TmCD40L fusion protein expressed from Ad-sig-ecdhMUCl-
HSFl-~Ct4TmCD40L-HIS
[00106] Trimerization of ecdhMUCl-HSF1-~CtOTmCD40L-HIS fusion protein was
evaluated following release from cells transformed with Ad-sig-ecdhMUCl-HSF1-
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OCtOTmCD40L-HIS vector. The expressed fusion protein was purified from the
supernatant
of 293 cells exposed to the vector using a His Tag purification kit.
Nondenaturing gel
electrophoresis showed a molecular weight consistent with trimer formation.
c) Effect of Ad-sig-ecdhMUCl-~CtOTmCD40L vector injection on
establishment of MUC1 expressing cancer cells.
[00107] hMIJC-l.Tg mice injected subcutaneously with the Ad-sig-ecdhMUCl-
OCtOTmCD40L (marine) vector were resistant to engraftment by the hMUCl
positive
LL2/LLIhMUCI mouse cancer cells. Control animals not injected with vector were
not
resistant to the growth of the same cells. Also, hMUC-l.Tg mice injected with
the Ad-sig-
ecdhMIJCl/ecdCD40L (marine) vector were not resistant to engraftment by
parental cell line
(LL2/LLl), which does not express MLTC1.
[00108] hMUC-l.Tg mice injected intravenously with ecdhMUCl- OCtOTmCD40L
(marine) protein were not resistant to engraftment by the hMUCl positive
LL2/LLIhMUCI
mouse cancer cells. Furthermore, hMUC-l.Tg mice injected with Ad-sig-ecdhMUCl-
~CtOTmCD40L (marine) vector lived longer than did control vector injected mice
subsequently administered the LL2/LLIhMIJCI cell line.
3. Cellular Mechanisms Underlying Breakdown of Anergy
a) Cytokine Release from Vaccinated vs. Non Vaccinated Mice.
[00109] A population of splenic CD8+ T lymphocytes was obtained seven days
following
Ad-sig-ecdllMUC1-~CtOTmCD40L (marine) vector administration was obtained by
depleting CD4+ T lymphocytes using CD4+ antibody coated magnetic beads. The
isolated
CD8+ T lymphocytes released over 2,000 times the level of interferon gamma as
did CD8+ T
cells from MUC-l.Tg mice administered a control vector (without MUC1).
b) Cytotoxicity Assay
[00110] Splenic T cells collected from hMUC-l.Tg mice 7 days following
administration
of Ad-sig-ecdhMUCl-~CtOTmCD40L (marine) vector were cultured with hMUCl
antigen
positive LL2/LLIhMUCI cancer cells ira vitro for 7 days. The stimulated
splenic T cells
were mixed in varying ratios with either the hMUCl positive LL2/LLIhMIJCI
cells or the
hMUCl negative LL2/LL1 cancer cells. The results showed that T cells from Ad-
sig-
ecdhMLJCl-OCt~TmCD40L (marine) vector vaccinated mice were cytotoxic only for
the
cancer cells expressing hMUCl.
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c) Ad-sig-ecdhMUCl-dCtOTmCD40L vector Injection
Overcomes Resistance to Expansion of hMUCl Specific T Cells.
[00111] DCs obtained iyz vitro from bone marrow cells were exposed to the Ad-
sig-
ecdhMUCl-OCt~TmCD40L (marine) vector for 48 hours. Splenic CD8+ T cells,
obtained
from hMLTC-l.Tg transgenic mice 7 days following no vector injection or
subcutaneous
injection with the Ad- sig-ecdhMUCl-~CtOTmCD40L (marine) vector, were mixed in
a 1/1
ratio with the Ad- sig-ecdhMLTCl/ecdCD40L (marine) vector-infected DCs. The
ERKl/EK2
proteins, the endpoint of the Ras/MAPI~ signaling pathway, were phosphorylated
in the
CD8+ T cells isolated from Ad- sig-ecdhMUCl-OCtOTmCD40L vector injected hMCTC-
l.Tg
transgenic mice following 45 minutes of in vitro exposure to Ad- sig-
ecdhIVIUCl-
~CtOTmCD40L (marine) vector infected DCs. In contrast no increase in
phosphorylation of
ERKl and ERK2 proteins was seen in CD8 positive T cells from unvaccinated hMUC-
l.Tg
mice. These results demonstrate that CD8 positive T cells from MIJC-l.Tg
transgenic mice
vaccinated with the Ad- sig-ecdhMUCl-OCt4TmCD40L (marine) vector were no
longer
anergic to MUC 1.
4. Production of the fusion protein and vector
[00112] The tumor antigen fusion protein was produced directly from an
adenoviral vector
that carries the expression cassette of the fusion gene encoding the fusion
protein. The
production cells (e.g. 293 cell line) at 80% confluency in growth medium were
infected with
the viral vector at the ratio of 10-100 viral particles per cell. The infected
cells were further
cultured for 48-72 hours, when the viral vectors propagated in the cells and
the tumor antigen
fusion proteins were expressed in the cells and secreted into culture media.
The infected cells
were collected when 70-90% of them showed cytopathic effect (CPE). The cell
culture
media was collected separately. Cell lysates were prepared through 3-time
freeze-and-thaw
cycles. The viral particles were isolated via the standard procedure (19). The
tumor antigen
fusion proteins were purified through affinity chromatograph from the
collected cell media
5. Amplification of the immune response by protein boosting
[00113] The relative value of protein boosting with the tumor antigen fusion
protein versus
boosting with the adenoviral expression vector was evaluated.
[00114] hMUC-l.Tg a~.limals were primed by subcutaneous administration of Ad-
I~/ecdhMLTCl-OCt~TmCD40L vector as described. The protein boost constituted 10
micrograms of ecdhMTJC-1/ecdCD40L fusion protein injected subcutaneously. The
time of
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protein boosting and comparison with vector was evaluated in various treatment
groups
shown in table 2.
Table 2: Immunization
Schedule
Testing Group Week 1 Week 2 Week 3 Week
4
Control Vector Vector Nothing Nothing
Treatment 1 Vector Vector Protein Nothing
(T1)
Treatment 2 Vector Vector Nothing Protein
(T2)
Treatment 3 Vector Protein Nothing Nothing
(T3)
Treatment 4 Vector Nothing Protein Nothing
(T4.)
Treatment 5 Vector Protein Nothing Protein
(TS)
Negative ControlNothing Nothing Nothing Nothing
[00115] Spleen cells from the different groups were isolated and evaluated by
the
ELISPOT assay for interferon gamma positivity. As seen in FIG 3. two
subcutaneous protein
inj ections at a 14 day interval beginning one week after the initial vector
inj ection showed the
greatest elevation of the frequency of positive T cells as compared to no
treatment or
compared with one or two vector injections without protein boost. The next
highest elevation
of the frequency of interferon gamma positive T cells was with the T3 group
(one protein
injection 7 days following the initial vector injection).
[00116] Cytotoxic T cells development in the various immunization groups was
also
evaluated (FIG. 4). Spleen cells from the various treatment groups were
stimulated ira vitro
for 5 days with a hMUC-1 positive cell line (LL1/LL2hMUC-1 ). CDR T cells were
isolated
and mixed with the target cells (LLl/LL2hMLJC-1) in a 50/1 ratio. Cytotoxic
activity
generally followed the ELISPOT assay results, with the TS group showing the
greatest
increase levels of LLl/LL2hMLJC-1 specific cytotoxic T cell activity. The
level of
cytotoxicity seen with T cells from the TS group was nine fold that seen with
the negative
control group.
[00117] Serum from the animals in the various treatment groups were evaluated
for anti-
ecdhMUCl-OCt~TmCD40L specific antibodies in an ELISA. Briefly, microwells
coated
with the ecdhMUCl-dCtOTmCD40L protein were incubated with test mouse serum,
washed
and bound mouse antibody identified using a secondary rat anti-mouse antibody
conjugated
to horseradish peroxidase.
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[00118] FIG. 5 shows a dramatic increase in the level of antibodies to the
ecdhMIJCl-
OCt~TmCD40L fusion protein generated by the treatment with one vector
injection and two
protein injections spaced at a 14 day interval. The increase in the anti-
ecdhMUCl-
~CtdTmCD40L antibodies following the TS treatment was 2 fold greater than with
any of the
other treatment group.
[00119] The results from these assays demonstrate that protein boosting is
superior to
vector boosting in generating cytotoxic T cell activity against tumor antigen
expressing cells
as well as antibody responses to the tumor antigen. The overall best results
with protein
boosting were obtained using a single injection of adenoviral expression
vector followed one
week later with a subcutaneous protein boost, which is repeated two weeks
later by another
protein boost.
[00120] Antibodies in serum from vaccinated hMLJC-1.Tg mice were evaluated for
binding
to cancer biopsy tissue specimens. Tissue microarrays containing normal breast
and breast
cancer tissue sections were obtained commercially. Tissue was contacted with
serum from
transgenic mice immunized with Ad-K/ecdhMUC-1//dCtOTm CD40L vector and boosted
later with ecdhMUC-1/l4Ct~Tm CD40L protein. The arrays were washed and then
exposed
to a horseradish peroxidase (HRP) secondary antibody which recognizes mouse
IgG
antibody. As a control, the serum was exposed first to a hMLTC-1 peptide from
the antigenic
repeat of the hMUC-1 domain (same as used for the protein boost).
[00121] Serum from the vaccinated mice bound to the breast epithelial cells
from biopsy
specimens of cancerous epithelial cells. No binding to the intervening
fibroblast or stromal
cells were observed. Serum from normal mice showed no reaction.
[00122] Serum from hMUC-l.Tg mice vaccinated with the Ad-sig-hMUC-1/ecdCD40L
followed by two subsequent administrations of protein sc-hMUC-1/ecdCD40L
reacted with
biopsy specimens from human prostate cancer on tissue microarray slides.
[00123] To determine specificity of the serum generated antibodies for the
hMUC-1 repeat,
serum from vaccine immunized animals described above was mixed with increasing
amounts
of a peptide containing the amino acid sequence from the hMITC-1 repeat. The
mixture was
then applied to the microarray slides and evaluated for reactivity. A peptide
with the same
amino acids as the hMLTC-1 repeat but with the sequence scrambled ("scrambled
peptide")
was added to serum from vaccinated animals as a control. The hMLTC-1 peptide
blocked
binding of the antibodies in vaccinated serum to the breast cancer epithelial
cells. No
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blocking was seen for the scrambled peptide. These suggests demonstrate that
the vector
prime/protein boost vaccination induced a hMUC-1 specific humoral response
reactive with
MUC-1 expressed by biopsy specimens of human breast cancer epithelial cells.
[00124] Tumor immunity in protein boosted mice was evaluated. hMUC-1.Tg
animals
were primed by subcutaneous administration of Ad-K/ecdhMUCl-~Ct~TmCD40L vector
as
described or were immunized with one or two administrations of the ecdhMUCl-
OCtOTmCD40L fusion protein. Animals were then challenged with LL2/LLIhMUC-1
tumor
cells.
[00125] FIG. 6 shows that mice vaccinated with the Ad-K/ecdhMUCl-OCtdTmCD40L
vector survived longer than 120 days (solid bold line), whereas all mice not
vaccinated with
the Ad-sig-ecdhMUC-1/ecdCD40L vector died by 50 days (broken line). These
results show
that the vector injections induced a suppression of the growth of the
LL2/LLIhMUC-1 cell
line in the hMLTC-l.Tg mice.
[00126] The specificity of tumor growth suppression for the hMIJC-1 antigen
was
evaluated by comparing rejection of the LL2/LLIhMUC-1 cell line (which is
positive for the
hMUC-1 antigen) with the LL2/LLl cell line, which is otherwise identical
except for the
absence of the hMUC-1 antigen. The results showed subcutaneous injection of
the
adenoviral vector completely suppressed the growth of the LL2/LLIhMUC-1 cell
line but did
not the same cells which do not express MTJC-1.
[00127] Tumor growth suppression was evaluated using combinations of vector
and protein
administration. Three combinations of Ad-sig-ecdhMLJC-1/ecdCD40L vector and
ecdhMUC-1/ecdCD40L protein were administered to hMLJC-l.Tg mice before
challenge
with LL2/LLIhMUC-1 tumor cells. VVV = three Ad-sig-ecdhMUC-1/OCt~Tm CD40L
vector subcutaneous injections administered on days 1, 7 and 21; PPP = three
ecdhMUC-
1/4CtOTm CD40L protein subcutaneous injections administered on days l, 7 and
21; or VPP
= a single Ad-sig-ecdhMUC-1/OCtOTm CD40L vector subcutaneous injection
followed at
days 7 and 21 by ecdhMUC-l I~CtOTm CD40L protein subcutaneous injections. See
FIG. 7
for further details. The mice were challenged one week later with a
subcutaneous injection of
five hundred thousand LL2/LLIhMLTC-1 lung cancer cells, and two weelcs later
with an
intravenous injection of 500,000 LL2/LLIhMLJC-1 tumor cells. The size of the
subcutaneous tumor nodules at day were measured by caliper at multiple time
points to
determine the effect of the various vaccine schedules on the growth of the
LL2/LLIhMUC-1
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cells as subcutaneous nodules. The metasteses were measured by total lung
weight following
sacrifice.
[00128] FIG. 7 shows that three injections of the fusion protein (PPP) without
a preceding
Ad-sig-ecdhMCTC-1/ecdCD40L vector injection failed to induce complete
resistance to the
development of the subcutaneous LL2/LLIhMUC-1 tumor. In contrast, the schedule
of three
successive vector injections (VVV) or one vector injection followed by two
protein injections
(VPP) completely suppressed the appearance of the subcutaneous LL2/LLIhMUC-1
tumor.
[00129] The levels of hMUC-1 specific antibodies in these mice at 63 days
following the
start of the vaccination were measured (FIG. 8). The schedule of a single
vector injection
followed by two successive fusion protein boosts (VPP) induced the highest
levels of hMUC-
1 specific antibodies, schedule VVV was intermediate, and schedule VPP was
virtually
ineffective. Thus, cancer therapy in these animals related somewhat inversely
to the antibody
response.
[00130] A tumor treatment (post establishment) protocol was also evaluated. hl
this
schedule, subcutaneous tumor (500,000 of the LLZ/LLIhMUC-1) was administered
on day 1.
The three schedules (PPP, VPP and VVV) were accomplished on days 5, 12 and 26.
Tumor
was administered i.v. on day 35 and tmnor development (subcutaneous and lung)
evaluated at
day 49. Further details are found in the legend to FIG. 9.
[00131] As shown in FIG. 9, the combination of one vector injection followed
by two
protein injections (VPP) completely suppressed the growth of established
subcutaneous
hMUC-1 positive cancer cell tumor. Three successive vector administrations
(VVV) had a
small therapeutic affect while three successive protein injections (PPP) had
little to no effect.
[00132] The growth of metastatic lung nodules in the pretreatment and post-
treatment (pre-
establislunent) cancer models is shown in FIG. 10. The pretreatment results in
FIG. 10, left
hand panel show that three successive fusion protein injections (PPP) did not
appear to
suppress lung nodule growth. In contrast, schedule VVV and schedule VPP
appeared to
completely suppress the engraftment of the lung cancer in the lungs of the
vaccinated
animals.
[00133] The post treatment results in FIG. 10, right hand panel show that the
combination
of one vector injection followed by two protein injections (VPP) completely
suppressed the
growth of established lung nodules of the hMLJC-1 positive cancer cells. In
contrast, three
CA 02548347 2006-06-06
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successive vector administrations (VW) and three successive protein injections
(PPP)
showed some therapeutic effect but less than for the VPP protocol.
[00134] These results suggest that the best overall cancer therapy schedule is
the VPP
schedule, involving a single injection of Ad-sig-ecdhMZTC-1/ecdCD40L vector
followed in
one week by two successive subcutaneous injections, spaced two weeks apart, of
the
ecdhMUC-1/ecdCD40L protein. This protocol is characterized by induction of
antibody
(humoral immunity) and T cell immunity (cellular immunity) to the mucin
antigen.
[00135] Boosting with ecdMUC-1/ecdCD4.OL soluble protein versus other soluble
proteins
following a primary administration of the adenoviral expression vector
encoding the same
protein was evaluated in hMUC-1.Tg animals challenged with MUC-1 expressing
tumor
(LL2/LLIhMUC-1 cell line). Animals were boosted with a bacterial extract
containing
ecdMUC-1/ecdCD40 (from a bacterial host strain infected with Ad-sig-ecdMUC-
1/ecdCD40L vector); ecdMUC-1 linked to the keyhole limpet hemocyaninin (KLH),
with or
without incomplete Freund's adjuvant; PB S; and control bacterial extract
(from a bacterial
host strain not infected with Ad-sig-ecdMUC-1/ecdCD40L vector). The tumor
cells were
given 7 days following the completion of the 2nd protein boost. The results
shown in FIG.
12 indicate that boosting with ecdMUC-1/ecdCD40L soluble protein was superior
to all other
approaches.
6. Construction of Adenoviral vectors encoding HPV E7 - CD40 ligand
fusion protein.
[00136] Methods of generating immunity by administering and adenoviral vector
expressing a transcription unit fusion protein constituting E7 linked to a
secretable form of
CD40 ligand was recently reported. Ziang et al., "An adenoviral vector cancer
vaccine that
delivers a tumor-associated antigen/CD40-ligand fusion protein to dendritic
cells" Proc. Natl.
Acad. Sci (LJSA) published November 25, 2003, 10.1073/pnas.2135379100 (vol.
100(25):15101).
[00137] The transcription unit included DNA encoding the signal peptide from
the HGH
gene upstream of DNA encoding the full length HPV type 16 E7 protein upstream
of
OCtOTmCD40L. DNA encoding the human growth hormone signal sequence
MATGSRTSLLLAFGLLCLPWLQEGSA (single letter amino acid code) (SEQ ID NO: 32)
36
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was prepared by annealing phosphorylated oligonucleotides (SEQ ID NOs:33 and
34).to
generate the full 26 amino acid HGH sequence with Bgl II and Notl overhangs.
Growth hormone signal upper strand (coding sequence in italics):
5'-GATCT CCACC ATG GCT ACA GGC TCC CGG ACG TGC CTG CTC CTG GCT TTT
GGC CTG CTC TGC CTG CCC TGG CTT CAA GAG GGC AGT GCC GGC -3' (SEQ ID
NO: 33)
Growth hormone signal lower strand:
3'-A GGTGG TAC CGA TGT CCG AGG GCC TGC AGG GAC GAG GAC CGA AAA
CCG GAC GAG ACG GAC GGG ACC GAA GTT CTC CCG TCA CGG CCGCCGG -5'.
(SEQ ID N0:34)
[00138] Synthetic HGH signal sequence was prepared by annealing the above
upper and
lower strand oligos. The oligos were dissolved in 50 ~1 H20 (about 3 mg/ml). 1
~1 from
each oligo (upper and lower strand) was added to 48 ~,1 annealing buffer (100
mM potassium
acetate, 30 mM HEPES-I~OH pH 7.4, and 2 mM Mg-acetate) incubated at 4 minutes
at 95°C,
minutes at 70°C and slowly cooled to about 4°C. The annealed DNA
was phosphorylated
using T4 PNI~ (polynucleotide lcinase) under standard conditions.
[00139] The HGH signal sequence with Bgl II and Not I overhangs was inserted
via Bgl II
and Not I into pShuttle-E7-~Ct~TmCD40L(no signal sequence) to yield pshuttle-
HGH/E7-
OCt~TmCD40L. pShuttle-E7-OCt~TmCD40L (no signal sequence) was prepared by
inserting HPV-16 E7 upstream of the CD40 ligand sequence as follows: Sequence
encoding
the full HPV-16 E7 protein was obtained by PCR amplifying from the HPV viral
genome
using the following primers:
HPV 16 E 7 forward primer (SEQ m NO: 35)
5'-ATTT GCGGCCGC TGTAATCATGCATGGAGA-3'
HPV E7 reverse primer (SEQ ID NO: 36)
5-CC CTCGAG TTATGGTTTCTGAGAACAGAT-3'
The resulting amplicon was HPV 16 E 7 encoding DNA with 5' end Not I and 3'
end Xho 1
restriction sites. The E7 DNA was inserted into the pShuttle~Ct~TmCD40L
between the
CMV promoter and directly 5' to the spacer of the OCtOTMCD40L sequence using
Not I
(GCGGCCGC) and Xho I (CTCGAG). The plasmid is designated pShuttle-E7-
OCt~TmCD40L (no signal sequence) and was used for insertion of the HGH signal
sequence
upstream of E7 to generate HGH/E7-OCt~TmCD40L as already described. Thus, the
transcription unit HGH/E7-~CtOTmCD40L encodes the HGH secretory signal
followed by
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the full length HPV type 16 E7 followed by a 10 amino acid linker with
(FENDAQAPKS;
SEQ ID NO: 37) followed by marine CD40 ligand residues 52-260.
[00140] A transcription unit that included DNA encoding the signal sequence of
the mouse
IgG kappa chain gene upstream of DNA encoding the full length HPV type 16 E7
protein
("K/E7") was generated by PCR using HPV16 plasmid and the following primers:
(primer 1) 5'-ACG ATG GAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG-3'
(SEQ ID NO: 38)
(primer 2) 5'- TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TC-3'
(SEQ ID NO: 39)
(primer 3) 5'- TG CTC TGG GTT CCA GGT TCC ACT GGT GAC ATG CAT G-3'
(SEQ ID NO: 40);
(primer 4) 5'- TGG GTT CCA GGT TCC ACT GGT GAC ATG CAT GGA G AT ACA
CCT AC-3' (SEQ ID NO: 41); and
(primer 5) 5'- CCG CTC GAG TGG TTT CTG AGA ACA GAT GGG GCA C -3.'
(SEQ ID NO: 42)
K/E7 with the upstream kappa signal sequence was generated by four rounds of
PCR
amplification (1St round: primers 4 +5; 2"a round: add primer 3; 3rd round:
add primer 2; 4tn
round: add primer 1). The KlE7 encoding DNA was cloned into the pcDNATM 3.1
TOPO
vector (Invitrogen, San Diego, CA) forming pcDNA-KlE7.
[00141] A DNA fragment that contained the mouse CD40 ligand from which the
transmembrane and cytoplasmic domain had been deleted (OCtOTmCD40L) was
generated
from a mouse CD40 ligand cDNA Plasmid (pDC406-mCD40L; ATCC) using the
following
PCR primers:
5'-CCG CTCGAG AAC GAC GCA CAA GCA CCA AAA AGC AAG GTC GAA GAG GAA
GTA AAC CTT C-3'(SEQ ID NO: 43); and
5'-CGCGCCGCGCGCTAG TCTAGA GAGTTTGAGTAAGCCAAAAGATGAG-3'(SEQ
ID NO: 44) (high fidelity PCR kit, Roche).
Fragment OCtOTmCD40L was digested with Xba I and XhoI restriction
endonucleases and
then ligated into pcDNA-E7. K/E7-OCtOTmCD40L fragment was cut from the pcDNA
vector and inserted into the pShuttle plasmid using Hind III and Xba I sites
(pShuttle K/E7-
CtOTmCD40L). Thus, the K/E7-~CtOTmCD40L fragment includes the kappa chain
secretory signal followed by the full length HPV type 16 E7 followed by a 10
amino acid
linker (LQNDAQAPKS; SEQ ID NO: 31) followed by marine CD40 ligand residues 52-
260.
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[00142] A vector encoding E7 fused to human CD40 ligand lacking a
transmembrane
domain is prepared by inserting "space+OCt~TmCD40L(human)" (prepared as
described
above) into the plasmid pShuttle-CMV (13) after restriction endonuclease
digestion with
Hind III (AAGCTT) and Xho I (CTCGAG). This vector is designated
pShuttle~CtOTmCD40L(human). Modification of pShuttle~CtOTmCD40L(human) to
include the HPV-16 E7 upstream of the human CD40 ligand sequence was
accomplished
essentially as described above for the marine CD40 ligand encoding vectors.
The resulting
plasmid is designated pShuttle-E7-OCt~TmCD40L(human)(no signal sequence) and
is used
for insertion of the HGH signal sequence upstream of E7 to generate HGH/E7-
OCt~TmCD40L(human). Thus, the transcription unit HGH/E7-OCtOTmCD40L(human)
encodes the HGH secretory signal followed by the full length HPV type 16 E7
followed by a
amino acid linker (FENDAQAPKS; SEQ ID N0:19) followed by human CD40 ligand
residues 47-261.
7. Construction of adenoviral vectors encoding ratHER2(Neu)1CD40L
[00143] The overexpression of the Her-2-Neu (H2N) growth factor receptor in
30% of
breast cancers is associated with increased frequency of recurrence after
surgery, and
shortened survival. Mice transgenic for the rat equivalent of HER2 ("H2N" or
"rH2N") gene
and therefore tolerant of this gene (Muller et al., Cell 54: 105-115, (1998);
Gut et al. P~~c.
Natl. Acad. Sci. USA 89: 10578-10582, (1992)) were used as experimental hosts
for
evaluating immunity in the Ad-sig-rH2N/ecdCD40L vector. In this model, the
mouse is
made transgenic for a normal unactivated rat Her-2-Neu gene under the control
of a
mammary specific transcriptional promoter such as the MMTV promoter. The MMTV
promoter produces overexpression of a non-mutant rat Her-2-Neu receptor, which
is
analogous to what occurs in human breast cancer. This model produces palpable
tumor
nodules in the primary tissue (the breast) at 24 weeks as well as pulmonary
metastases at 32
weeks. The development of breast cancer occurs spontaneously. The cancer
begins focally
as a clonal event in the breast epithelial tissue through a step-wise process
(Id.). Dysplasia
can be detected by 12 weeks of birth. Palpable tumors in the mammary glands
can be
detected at 25 weeks, and metastatic breast cancer in the lung can be
demonstrated in 70% of
mice by 32 weeks (Id.).
[00144] Ad-sig-rH2N/ecdCD40L vector was subcutaneously administered to
transgenic
animals one or two times at 7 day intervals to test if an immune response
could be induced
39
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WO 2005/058950 PCT/US2004/041690
against the rat Her-2-Neu antigen. Two subcutaneous injections of the Ad-sig-
rH2N/ecdCD40L vector induced complete resistance to the growth of the N202
(rH2N
positive) mouse breast cancer cell line, whereas one subcutaneous injection of
the same
vector did not induce sufficient immune response to completely suppress the
growth of the
rH2N positive N202 cell line. ELISPOT assays showed that the administration of
two
subcutaneous injections of the Ad-sig-rH2N/ecdCD40L vector 7 days apart
induced levels of
rH2N specific T cells in the spleens of vaccinated mice which were 10 times
higher than the
levels of rH2N specific T cells induced in mice following one injection of the
Ad-sig-
rH2N/ecdCD40L vector. Finally, the immune resistance induced against the NT2
cells by the
Ad-sig-rH2N/ecdCD40L vector prime vaccination was better than the response
obtained in
transgenic animals vaccinated with irradiated cytokine positive tumor cells
(mitomycin
treated NTW cells which had been transfected with a GMCSF transcription unit).
[00145] The rH2N specific antibody levels were also measured in mice
vaccinated with one
or two subcutaneous injections of the Ad-sig-rH2N/ecdCD40L vector. As shown
below in
Figure 11, the levels of the rH2N specific antibody levels were higher
following two
subcutaneous injections than following a single subcutaneous injection of the
Ad-sig-
rH2N/ecdCD40L vector.
8. Construction of adenoviral vectors encoding huHER2/CD40L
[00146] An adenoviral vector encoding sig ecdhuHER2/CD40L was prepared as
follows.
The mouse IgG kappa chain METDTLLLWVLLLWVPGSTGD (single letter amino acid
code) (SEQ ID NO: 11) was prepared by PCR amplification (SEQ ID NOs: 12, 13
and 45) to
generate the full 21 amino acid mouse IgG kappa chain signal sequence (the
start codon
"ATG" is shown bolded in SEQ ID NO:1~).
5'-CCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG GTA CTG CTG-3'
(SEQ ID NO: 12)
5'- TC CTG CTA TGG GTA CTG CTG CTC TGG GTT CCA GGT TC-3'
(SEQ ID N0:13)
The forward primer
5'-5'- TG CTC TGG GTT CCA GGT TCC ACT GGT GAC GAA CTC -3'(SEQ ID N0:45)
The forward primer for the human HER2 extracellular domain
CA 02548347 2006-06-06
WO 2005/058950 PCT/US2004/041690
5'- TCC ACT GGT GAC GAACTCACCTACCTGCCCACCAATGC-3' (SEQ ID N0:46)
The reverse Primer for the human HER2 extracellular domain
5'- GGAGCTCGAG GGCTGGGTCCCCATCAAAGCTCTC-3' (SEQ )D N0:47)
sig-ecdhHER2 with the upstream kappa signal sequence is generated by four
rounds of PCR
amplification (1St round: primers SEQ ID NOs 46 and 47; 2"a round: primer SEQ
ID NOs 45
and 47; 3rd round: primer SEQ ID NOs 13 and 47; 4th round: primer SEQ ID NOs
12 and 47).
The sig-ecdhHER2 encoding DNA can be cloned into the pcDNATM 3.1 TOPO vector
(Invitrogen, San Diego, CA) forming pcDNA-sig-ecdhHER2. The additional cloning
steps
described for the MLTC-1/CD40 Ligand expression vector are also applicable for
the
HER2/CD40 ligand expression vector.
[00147] This region HER2 extracellular domain to be fused to CD40 ligand
contains two
CTL epitopes; One is an HLA-A2 peptide, K I F G S L A F L (SEQ ID N0:48)
representing amino acids 369-377. This peptide elicited short-lived peptide-
specific
immunity in HER2 expressing cancer patients. See Knutson et al., hnmunization
of cancer
patients with a HER-2/neu, HLA-A2 peptide, Clin Cancer Res. 2002 May;B(5):1014-
8p369-
377. The second epitope is E L T Y L P T N A S (SEQ ID NO: 49) (HER2 residues
63-
71) also was useful in generating immunity to HER2 expressing tumor cells. See
Wang et al.
Essential roles of tumor-derived helper T cell epitopes for an effective
peptide-based tumor
vaccine, Cancer Immun. 2003 Nov 21;3:16. The region of the HER2 ecd also
includes a B
cell epitope P L H N Q E V T A E D G T Q R C E K C S K P C (SEQ ID NO:
50) (HER2 positions 316-339). See Dakappagari et al., Chimeric mufti-human
epidermal
growth factor receptor-2 B cell epitope peptide vaccine mediates superior
antitumor
responses, J hnmunol. 2003 Apr 15;170(8):4242-53.
[00148] All patents and publications mentioned in the specification are
indicative of the
levels of those of ordinary skill in the art to which the invention pertains.
All patents and
publications are herein incorporated by reference to the same extent as if
each individual
publication was specifically and individually indicated to be incorporated by
reference.
[00149] The invention illustratively described herein suitably may be
practiced~in the
absence of any element or elements, limitation or limitations which is not
specifically
disclosed herein. Thus, for example, in each instance herein any of the terms
"comprising,"
"consisting essentially of ' and "consisting of may be replaced with either of
the other two
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CA 02548347 2006-06-06
WO 2005/058950 PCT/US2004/041690
terms. The terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that in the use
of such terms and
expressions of excluding any equivalents of the features shown and described
or portions
thereof, but it is recognized that various modifications are possible within
the scope of the
invention claimed. Thus, it should be understood that although the present
invention has
been specifically disclosed by preferred embodiments and optional features,
modification and
variation of the concepts herein disclosed may be resorted to by those skilled
in the art, and
that such modifications and variations are considered to be within the scope
of this invention
as defined by the appended claims.
[00150] Other embodiments are set forth within the following claims.
42
