Note: Descriptions are shown in the official language in which they were submitted.
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
IMMUNOGENIC CD91 LIGAND-ANTIGENIC MOLECULE COMPLEXES AND
FUSION PROTEINS
This application claims the benefit of U.S. Provisional Application No.
60/444,773,
filed on February 4, 2003, which is incorporated by reference herein in its
entirety.
This invention was made with government support under Grant No. CA/A184479
awarded by the National Institutes of Health. The government has certain
rights in the
invention.
1. INTRODUCTION
The present invention relates to complexes and fusion proteins comprising a
CD91
ligand and an antigenic molecule, for use in the treatment or prevention of a
disease.
2. BACKGROUND OF THE INVENTION
CD91, also known as LDL receptor related protein, is structurally related to
other
receptors in the LDL receptor gene family. Like other members of the LDL
receptor gene
family, CD91 has various functions besides lipid metabolism, including
homeostasis of
proteinases and proteinase inhibitors, cellular entry of toxins and viruses,
lysosomal enzyme
activation, cellular signal transduction and neurotransmission. Herz and
Srtickland, 2001, J.
Clin. Invest. 108(6):779-84.
CD91 is primarily expressed in liver, brain and placenta. The extracellular
domain
of the human receptor comprises six 50-amino acid EGF repeats and 31
complement repeats
of approximately 40-42 amino acids. The complement repeats are organized, from
the
amino to the carboxy-terminus, into clusters of 2, 8, 10 and 11 repeats,
called Cluster I, II,
III and IV (Herz et al., 1988, EMBO J. 7:4119-4127). One study points to
Cluster II (Cl-
II), which contains complement repeats 3-10 (CR3-10), as the major ligand
binding portion
of the receptor (Horn et al., 1997, J. Biol. Chem. 272:13608-13613).
CD91 binds to at least 30 different ligands with high affinity, including
ApoE,
lipoprotein lipase, hepatic lipase, factor IXa, factor VIIIa, factor
VIIa/TFPI, MlVlf-13
(matrix metaloproteinase-13), M1VVIP-9 (matrix metaloproteinase-9),
spingolipid activator
protein, pregnancy zone protein, PAI-1 (plasminogen activator inhibitor-1),
antithrombin
III, tissue factor pathway inhibitor (TFPI), Pseudomonas exotoxin A, heparin
cofactor II,
APP (amyloid precursor protein), thrombospondin-l, thrombospondin-2,
lactoferrin,
rhinovirus, RAP (alpha-2 macroglobulin receptor associated protein) and HIV-
Tat protein.
-1-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
A description of CD91 and CD91 ligands is provided by Herz and Strickland,
2001, J. Clin.
Invest.108:779-784, which is incorporated by reference herein.
The receptor binding domains of some CD91 ligands have been identified and are
listed in section 4.3.
3. SUMMARY OF THE INVENTION
The present invention relates to novel complexes and fusion proteins
comprising a
CD91 ligand and an antigenic molecule. The complexes and fusion proteins of
the
invention can induce an immune response against the antigenic molecule and are
useful in
the treatment or prevention of a disease. The invention also relates to
methods of producing
the complexes and fusion proteins, as well as compositions of complexes or
fusion proteins.
The present invention also provides methods of treating or preventing a
disease in a subject
using said complexes and fusion proteins and methods of inducing an immune
response in a
subj ect using said complexes and fusion proteins.
The following CD91 ligands, listed by way of example and not limitation, are
suitable for use in the invention: ApoE, lipoprotein lipase, hepatic lipase,
factor IXa, factor
VIIIa, factor VIIa/TFPI, MMP-13, M1V11'-9, spingolipid activator protein,
pregnancy zone
protein, PAI-1, antithrombin III, Pseudomohas exotoxin A, tissue factor
pathway inhibitor
(TFPI), heparin cofactor II, APP, thrombospondin-l, thrombospondin-2,
lactoferrin,
rhinovirus, R.AP and HIV-Tat protein.
In one aspect of the invention, a purified complex is provided comprising a
CD91
ligand and an antigenic molecule. In one embodiment, the complex is
immunogenic, i.e.,
when the complex is introduced into a suitable host, the host develops an
immune response
against the antigenic molecule of the complex. In another embodiment, the
antigenic
molecule is not an antibody or a derivative thereof comprising a binding
region thereof. In
one embodiment, the CD91 ligand is noncovalently associated with the antigenic
molecule.
In another embodiment, the CD91 ligand is cross-linked to the antigenic
molecule. In yet
another embodiment, the complex is the product of a method comprising
complexing said
CD91 ligand and said antigenic molecule in vitro. In another embodiment, the
antigenic
molecule is a protein. In a further embodiment, the antigenic molecule is
derived from an
antigenic cell. In another embodiment, the complex is free of biological cells
and vesicles.
In another embodiment, the CD91 ligand is derived from a mammal. In a further
embodiment, the CD91 ligand is derived from a human. In another embodiment,
the
antigenic molecule is derived from a mammal. In another embodiment the
antigenic
-2-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
molecule is derived from a human. In a yet a further embodiment the CD91
ligand and the
antigenic molecule are derived from the same subject. In another embodiment,
the CD91
ligand and the antigenic molecule are derived from different subjects.
In another aspect of the invention, a purified complex is provided comprising
a
fragment of a CD91 ligand and an antigenic molecule. In a particular
embodiment, the
complex is immunogenic. In a preferred embodiment, the fragment comprises a
receptor
binding domain.
In another embodiment of the invention, a purified fusion protein is provided
comprising an antigenic protein fused via a peptide bond to a CD91 ligand. In
another
embodiment, the fusion protein is immunogenic. In a further embodiment, the
complex is
free of biological cells and vesicles.
In another embodiment of the invention, a purified fusion protein is provided
comprising an antigenic protein fused via a peptide bond to a fragment of a
CD91 ligand.
In a particular embodiment, the fusion protein is immunogenic. In another
embodiment, the
antigenic protein is not an antibody or a part thereof. In a preferred
embodiment, the
fragment comprises a receptor binding domain.
In another embodiment, the invention provides a composition comprising a
plurality
of purified complexes. In a further embodiment, each complex comprises a
different
antigenic molecule. In another embodiment, the population of complexes of CD91
ligands
bound to antigenic molecules is purified to apparent homogeneity, as viewed on
an SDS-
PAGE gel.
In another aspect of the invention, a pharmaceutical composition is provided
comprising an amount of purified complex effective for treatment or prevention
of a disease
or a disorder. In a particular embodiment, the complex of the pharmaceutical
composition
is immunogenic. In another embodiment, the pharmaceutical composition further
comprises a pharmaceutically acceptable carrier.
Another embodiment of the invention provides a method for preparing
immunogenic, in vitro complexes of a CD91 ligand associated with one or more
antigenic
molecules. In one embodiment, the method comprises a) incubating a CD91 ligand
or
fragment thereof and one or more antigenic molecules under conditions and for
a length of
time sufficient for formation of complexes of the CD91 ligand non-covalently
bound to the
antigenic molecules; and b) isolating said complexes. In a further embodiment
of this
method, the complexes are free of biological cells and vesicles. In yet a
further
embodiment, the CD91 ligand is purified.
-3-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
In a further aspect of the invention, a method of treating or preventing a
disease in a
subject is provided. In one embodiment, the method comprises administering to
the subject
an amount of a purified complex effective to treat or prevent a disease. In a
further
embodiment, the CD91 ligand is noncovalently associated with the antigenic
molecule. In
another embodiment, the CD91 ligand is cross-linked to the antigenic molecule.
In another
aspect of the invention, the complex is free of biological cells and vesicles.
In yet a further
embodiment, the antigenic molecule displays the amtigenicity of a cancer-
associated or
cancer-specific antigen, or of an antigen that causes or is associated with a
disease,
respectively.
Another embodiment of the invention provides a method of treating or
preventing a
disease in a subject comprising administering to the subject an amount of a
purified fusion
protein comprising an antigenic protein fused via a peptide bond to a CD91
ligand effective
to treat or prevent a disease. In a further embodiment, the fusion protein is
free of
biological cells and vesicles. In another embodiment, the antigenic molecule
displays the
antigenicity of a cancer-associated or cancer-specific antigen, or of an
antigen that causes or
is associated with a disease, respectively.
The invention further provides a recombinant cell transformed with a) a first
nucleic
acid comprising a first nucleotide sequence that is operably linked to a first
promoter and
that encodes a CD91 ligand, and b) a second nucleic acid comprising a second
nucleotide
sequence that is operably linked to a second promoter and encodes an antigenic
molecule,
such that the CD91 ligand and the antigenic molecule are expressed within the
cell and non-
covalently associate with each other to form a complex that in sufficient
amount is capable
of eliciting an immune response to the antigenic molecule. In one embodiment,
the cell is a
human cell.
In another aspect of the invention, a recombinant cell is provided that is
transformed
with a nucleic acid comprising a nucleotide sequence that is operably linked
to a promoter,
wherein said nucleotide sequence encodes a fusion protein comprising an
antigenic protein
fused via a peptide bond to a CD91 ligand. In a particular embodiment, the
fusion protein
is immunogenic.
In one embodiment, the invention provides pharmaceutical compositions
comprising
recombinant cells and pharmaceutically acceptable carriers.
In another aspect of the invention, a method for preparing in vitro complexes
of a
CD91 ligand associated with one or more antigenic molecules comprising cross-
linking the
-4-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
CD91 ligand to one or more antigenic molecules using a cross-linking agent is
provided. In
another embodiment, the complex is immunogenic.
Another aspect of the invention provides a method for preparing a fusion
protein
comprising a) culturing a recombinant cell transformed with a nucleic acid
comprising a
nucleotide sequence that is operably linked to a promoter, wherein said
nucleotide sequence
encodes a fusion protein comprising an antigenic protein fused via a peptide
bond to a
CD91 ligand, under conditions such that the fusion protein is expressed by the
cell and b)
recovering the fusion protein from the cells. In a particular embodiment, the
fusion protein
is immunogenic.
In another embodiment of the invention, the complexes of the invention can be
used
in combination with other therapies in the treatment or prevention of a
disease or disorder.
Another aspect of the invention provides a method of inducing an immune
response
against an antigenic molecule in a subject comprising administering to the
subject an
effective amount of a purified complex comprising said antigenic molecule and
a CD91
ligand. In one embodiment, the CD91 ligand and antigenic molecule are non-
covalently
bound to each other. In another embodiment, the CD91 ligand and antigenic
molecule are
cross-linked to each other. In another embodiment, the complex is free of
biological cells
and vesicles. In another embodiment, the immune response to the antigen is
induced by
enhancing uptake of said antigenic molecule by antigen presenting cells.
Another aspect of the invention provides a method of inducing an immune
response
against an antigenic protein in a subject comprising administering to the
subject an effective
amount of purified fusion protein comprising said antigenic protein fused via
a peptide
bond to a CD91 ligand. In another embodient, the fusion protein is free of
biological cells
and vesicles. In another embodiment, the immune response is induced by
enhancing uptake
of said antigenic protein by antigen presenting cells.
4. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides complexes and fusion proteins of CD91 ligands
and
antigenic molecules, compositions of said fusion proteins and complexes,
methods of
preparing said fusion proteins and complexes, methods of treating or
preventing a disease in
a subject by immunizing with said fusion proteins and complexes, and methods
of inducing
an immune response to an antigenic molecule using the complexes and fusion
proteins of
the invention.
-5-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
In certain embodiments, the compositions and formulations of the present
invention
are administered to a subject to prevent a disease, including inhibiting the
onset or
development of the disease in a subject not having the disease, and inhibiting
the
progression of a disease in an asymptomatic subject. Preferably, the complexes
and or
fusion proteins of the invention in such compositions and formulations are
purified.
The term "disease" means any abnormal physical or mental condition, or a
condition
of a living animal or plant body or of one of its parts that impairs normal
functioning.
Types of disease include, but are not limited to cancer, infectious diseases,
neurodegenerative diseases, endocrinelmetabolic diseases and vascular
diseases.
In other embodiments, the compositions and formulations of the present
invention
are administered to a subject that has been diagnosed with a disease or is
suspected of
having a disease. According to the present invention, treatment of a disease
encompasses
the treatment of subjects already diagnosed as having a disease; the delay of
the onset or
evolution or aggravation or deterioration of the symptoms or signs of a
disease; and/or
promoting regression of a disease in symptomatic subjects.
The term "CD91 ligand" means a molecule that binds to CD91.
The term "antigenic molecule" means any molecule containing an antigenic
determinant, i. e., capable of being bound by an antibody or recognized by a T
cell in the
context of MHC Class I, Class II molecules, or CD 1 molecules. Antigenic
molecules can
be, but not limited to, carbohydrates, lipids, polypeptides and peptides
(polypeptides and
peptides collectively referred to as "proteins"). The term "antigenic
molecule" does not
encompass an antibody or a derivative thereof comprising a binding region
thereof. In one
embodiment, an antigenic molecule comprises one or more antigenic determinants
(epitopes) of a single protein. In another embodiment, an antigenic molecule
comprises
multiple antigenic determinants, at least one antigenic determinant from each
of two or
more proteins. In another embodiment, an antigenic molecule comprises one or
more
antigenic determinants (epitopes) of a single naturally occurring protein. In
another
embodiment, an antigenic molecule comprises multiple antigenic determinants,
at least one
antigenic determinant from each of two or more naturally occuring proteins. In
another
embodiment, the antigenic molecule comprises one or more antigenic
determinants from a
first naturally occurring protein and one or more antigenic determinants from
a second
naturally occurring protein, i. e., it can be a fusion protein of at least a
portion of two
different naturally occurring proteins.
-6-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
The term "immunogenic" in reference to a substance means that the substance,
when
introduced into a suitable host, can cause the production by the host of an
immune response
(antibody or cell-mediated) against the substance.
Complexes between CD91 ligands and antigenic molecules can be produced by any
of a number of methods. For example, antigenic molecules associated with a
disease can be
obtained by recombinant or synthetic methods, or can be isolated and purified
from
recombinant cells. The antigenic molecules can also be isolated from cells
removed from a
subject or cells in culture. Complexes between antigenic molecules and CD91
ligands can
be formed by covalent or non-covalent association of antigenic molecules with
CD91
ligands. Preferably, the complexes of the invention are used in purified form,
preferably to
apparent homogeneity as viewed on an SDS-PAGE gel, or to at least 60%, 70%,
80%, or
90% of total protein.
In addition, complexes may be formed in vitro using a variety of methods,
described
herein. Methods for preparing such CD91 ligand-antigenic molecule complexes
are
described in detail in Sections 4.3 to 4.5, below.
CD91 ligand-antigenic peptide complexes and fusion proteins may be used as
vaccines against various diseases. Without being bound by any particular
theory, such
complexes or fusion proteins may act by eliciting a B-cell and/or T-cell
response in patients
with such disorders. Methods for the use of such CD91 ligand-antigenic
molecule
complexes and fusion proteins as vaccines against various diseases are
described in Section
4.6 in detail herein.
4.1 SOURCES OF ANTIGE1~1IC MOLECULES
According to the invention, the complexes comprise antigenic molecules
complexed
to CD91 ligands. In one embodiment, the antigenic molecule is prepared by
synthetic
means. In another embodiment the antigenic molecule is prepared by recombinant
methods. In yet another embodiment, the antigenic molecules are from a
preparation of
proteins from an antigenic cell of interest. In yet another embodiment, the
antigenic
molecules are prepared from tissue which displays the pathologic or histologic
changes
associated with a disease. In another embodiment of the invention, the
antigenic molecules
are prepared from body fluid or any other biological source.
The compositions of the invention also comprise complexes of CD91 ligands and
antigenic peptides that are prepared by first, generating a population of
peptides from a
_7_
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
preparation of proteins of the antigenic cells of interest, and then
complexing the peptides to
CD91 ligands.
In various specific embodiments, where it is desired to maximize and preserve
the
diversity of antigenic proteins and peptides for use as antigenic molecules in
the complexes
of the invention, the methods used for preparing a protein preparation of
antigenic cells do
not selectively remove or retain any particular protein or peptide from other
proteins and
peptides in the antigenic cell. Even in certain embodiments when cytosolic
proteins or
membrane-derived proteins are used, the methods used to make the preparations
do not
selectively remove or retain any particular protein of the cytosol or of the
membranes.
Therefore, the majority of the proteins present in the cytosol or in the
membranes are also
present in the respective preparations of antigenic proteins and peptides from
antigenic
cells. In preferred embodiments, substantially the entire repertoire of
antigenic proteins and
peptides of the antigenic cells, and substantially all the antigenic proteins
and peptides in
the cytosol or in the membranes are present in the complexing reaction and
form complexes
with CD91 ligands.
Antigenic proteins or antigenic fragments thereof may be used as antigenic
molecules. Optionally, the proteins or fragments may be purified. Antigenic
epitopes of
proteins may optionally be screened using any method known in the art. Such
techniques
include, but are not limited to, methods that are based on algorithmic
identification, peptide
elution and cell-based binding assay techniques. Protein sequences may be
analyzed to
identify antigen-specific epitopes that meet criteria such as conservancy,
binding,
population coverage and immunogenicity. Potential epitopes can be identified
by, e.g. a
hydrophilicity analysis (see e.g., Hopp and Woods, 1981, Proc. Natl. Acad.
Sci. USA
78:3824).
Another such process for epitope identification is described in Sette et al.,
2002,
Curr Opin Investig Drugs 3:132-9. Briefly, computer algorithms analyze the
amino acid
sequence of all known antigens associated with the target indication for the
presence of
peptides which contain epitope motifs and which meet sequence conservancy
requirements
(epitopes from variable regions of the antigen are avoided). Peptides meeting
the
requirements of the computer screen are synthesized and in vitro HLA binding
assays are
performed. Peptides are evaluated for superfamily binding (assessing their
ability to bind
broadly within a family of HLA molecules). Identifying epitopes which bind
broadly within
superfamilies ensures broad population coverage for the ultimate vaccine.
Peptides are
_g_
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
then tested for immunogenicity, both in vivo in mice which express human MHC
and in
vitro against infected or transfected cells.
Using such screening techniques allows identification of the specific amino
acid
sequence or sequences of a protein which elicit an immune response. Disease
relevant
epitopes may be tested and validated via functional T-cell assays, ensuring
their clinical
relevance, e.g., screened using T-cells from humans, confronted with the
disease and who
developed a protective natural immune response. Epitopes may be modified,
e.g., by
changing one or more amino acid residues, to enhance/optimize immunogenicity.
See e.g.,
U.S. Patent 6,037,135, herein expressly incorporated by reference in its
entirety. The
invention provides complexes comprising these antigenic epitopes and CD91
ligands.
In a specific embodiment of the invention, where it is desired to treat or
prevent
cancer, a tumor specific or tumor associated antigen may be used as the
antigenic molecule
in the complexes or fusion proteins of the invention. Any such antigens known
in the art
may be used in the complexes and fusion proteins of the invention, in addition
to the
following tumor antigens, listed by way of example and not limitation: 707-AP
(707 alanine
proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen
recognized by T
cells 4), BAGE (B antigen; ~3-catenin/m, ,Q-catenin/mutated), Bcr-abl
(breakpoint cluster
region-Abelson), CAMEL (CTL-recognized antigen on melanoma) , CAP-1
(carcinoembryonic antigen peptide - 1), GASP-8 (caspase-8), CDC27m (cell-
division cycle
27 mutated), CDK4/m (cycline-dependent kinase 4 mutated), CEA
(carcinoembryonic
antigen), CT (cancer/testis antigen), Cyp-B (cyclophilin B), DAM
(differentiation antigen
melanoma), ELF2M (elongation factor 2 mutated), ETV6-AMLl (Ets variant gene
6/acute
myeloid leukemia 1 gene ETS), 6250 (glycoprotein 250), GAGE (G antigen), GnT-
V (N-
acetylglucosaminyltransferase V), Gp100 (glycoprotein 100 kD), HAGE (helicose
antigen),
HER-2/neu (human epidermal receptor-2/neurological), HLA-A *0201 ~R170I
(arginine (R)
to isoleucine (I) exchange at residue 170 of the a helix of the Q2-domain in
the HLA-A2
gene), HPV-E7 (human papilloma virus E7), HSP70-2M (heat shock protein 70 - 2
mutated), HST-2 (human signet ring tumor - 2), hTERT or hTRT (human telomerase
reverse transcriptase), iCE (intestinal carboxyl esterase), KIA.A0205, LAGE (L
antigen),
LDLR/FUT (low density lipid receptor/GDP-L-fucose), ,Q-D-galactosidase 2-a L-
fucosyltransferase, MAGE (melanoma antigen), MART-1/Melan-A (melanoma antigen
recognized by T cells-1/Melanoma antigen A), MC1R melanocortin 1 receptor,
Myosin/m
(myosin mutated), MUC1 (mucin 1 ), MUM-l, -2, -3 ( melanoma ubiquitous mutated
1, 2,
3), NA88-A (NA cDNA clone of patient M88), NY-ESO-1 (New York - esophagus 1),
P15
-9-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
(protein 15), p190 minor bcr-abl (protein of 190 KD bcr-ably, Pml/RARa
(promyelocytic
leukaemialretinoic acid receptor a), PRAMS (preferentially expressed antigen
of
melanoma), PSA (prostate-specific antigen), PSM (prostate-specific membrane
antigen),
RAGE (renal antigen RUl or RU2 or renal ubiquitous 1 or 2), SAGE (sarcoma
antigen),
SART-1 or SART-3 (squamous antigen rejecting tumor 1 or 3), TEL/AMLI
(translocation
Ets-family leukemia/acute myeloid leukemia 1), TPI/m (triosephosphate
isomerase
mutated), TRP-1 (tyrosinase related protein 1, or gp75), TRP-2 (tyrosinase
related protein
2), TRP-2/INT2 (TRP-2/intron 2) and WT1 (Wilins' tumor gene).
In a specific embodiment of the invention, where it is desired to treat or
prevent an
infectious disease, a molecule comprising one or more epitopes of an
infectious agent (e.g.,
viral antigen, bacterial antigen, etc.) that is the causative agent of the
disease is used.
Preferably, where it is desired to treat or prevent a viral disease, a
molecule comprising
epitope(s) of a virus is used; where it is desired to treat or prevent a
bacterial infection, a
molecule comprising epitope(s) of bacteria is used; where it is desired to
treat or prevent, a
protozoal infection, a molecule comprising epitope(s) of protozoa is used; and
where it is
desired to treat or prevent a parasitic infection, a molecule comprising
epitope(s) of
parasites is used.
Preferably, where it is desired to treat or prevent a neurodegenerative or
amyloid
disease, a molecule comprising epitope(s) of an antigenic molecule associated
with a
neurodegenerative disease, or epitope(s) of an antigenic molecule associated
with an
amyloid disease, including but not limited to a fibril peptide or protein, is
used as the
antigenic molecule of the invention. For example, such a neurodegenerative
disease-
associated antigenic molecules may be a molecule associated with Alzheimer's
Disease,
age-related loss of cognitive function, senile dementia, Parkinson's disease,
amyotrophic
lateral sclerosis, Wilson's Disease, cerebral palsy, progressive supranuclear
palsy, Guam
disease, Lewy body dementia, prion diseases, spongiform encephalopathies,
Creutzfeldt-
Jakob disease, polyglutamine diseases, Huntington's disease, myotonic
dystrophy,
Freidrich's ataxia, ataxia, Gilles de la Tourette's syndrome, seizure
disorders, epilepsy,
chronic seizure disorder, stroke, brain trauma, spinal cord trauma, AIDS
dementia,
alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorder,
hypertension,
neuropsychiatric disorder, schizophrenia, or schizoaffective disorder.
Antigenic molecules
that are suitable for in vitro complexing methods are disclosed in PCT
publication no.
WO O1/52~90, dated July 26, 2001, which is incorporated by reference herein in
its entirety,
and include, but are not limited to, ~3-amyloid or a fragment thereof, an
oligomeric A(3
-10-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
complex or a fragment thereof, an ApoE4-A~3 complex, tau protein or an
fragment thereof,
amyloid precursor protein or a fragment thereof, a mutant amyloid precursor
protein or a
fragment thereof, presenillin or a fragment thereof, a mutant of presenillin
or a fragment
thereof, a synuclein or a fragment thereof, or a prion protein or a fragment
thereof, and the
antigenic derivatives of any of the foregoing proteins or fragments thereof.
Amyloid
disease associated antigenic molecules may be molecules associated with
diseases
characterized by the extracellular deposition of protein and/or peptide
fibrils which form
amyloid deposits or plaques, including but not limited to type II diabetes and
amyloidoses
associated with chronic inflammatory or infectious disease states and
malignant neoplasms,
e.g., myeloma. Certain amyloid disease such as Alzheimer's disease and prion
diseases,
e.g., Creutzfeldt Jacob disease, are neurodegenerative diseases.
In another embodiment, for the treatment or prevention of an autoimmune
disease,
the antigenic molecule of the invention is related to an autoimmune disease.
These
autoimmune diseases include, but are not limited to, insulin-dependent
diabetes mellitus
(i.e., IDDM, or autoimmune diabetes), multiple sclerosis, systemic lupus
erythematosus,
Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed
connective
tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune
thyroiditis,
idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves'
disease,
myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia
purpura,
rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility,
Goodpasture's
disease, bullous pemphigoid, discoid lupus, ulcerative colitis, and dense
deposit disease.
Thus, for example, a cytokine can be an antigenic molecule.
Preferably, where it is desired to treat or prevent an endocrine or metabolic
disease,
molecules comprising epitopes of antigenic molecules associated with endocrine
or
metabolic diseases are used as the antigenic molecules of the invention. Thus,
for example,
cholesteryl ester transfer protein can be an antigenic molecule.
Preferably, where it is desired to treat or prevent vascular diseases (e.g.,
cardiovascular disease), molecules comprising epitopes of antigenic molecules
associated
with vascular diseases are used as the antigenic molecules of the invention.
Thus, for
example, angiotensin II can be an antigenic molecule in the treatment or
prevention of
vascular diseases.
-11-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
4.1.1 PREPARATIONS OF ANTIGENIC MOLECULES FROM
ANTIGENIC CELLS OF INTEREST
Antigenic proteins used in the complexes of the invention can be obtained from
antigenic cells of interest.
Since whole cancer cells, infected cells or other antigenic cells are used in
one
embodiment of the present methods, in such embodiment, it is not necessary to
isolate or
characterize or even know the identities of these antigenic proteins in
advance of using such
methods. The source of the antigenic cells may be selected, depending on the
nature of the
disease with which the antigens are associated. In one embodiment of the
invention, any
tissues, or cells isolated from a cancer, including cancer that has
metastasized to multiple
sites, can be used as an antigenic cell in the present method. For example,
leukemic cells
circulating in blood, lymph or other body fluids can also be used, solid tumor
tissue (e.g.,
primary tissue from a biopsy) can be used. In a specific embodiment, antigenic
cells can be
cancer cells or preneoplastic cells. The transition from non-neoplastic cell
growth to
neoplasia commonly consists of hyperplasia, metaplasia, and dysplasia (for
review of such
abnormal growth conditions (See Robbins and Angell, 1976, Basic Pathology, 2d
Ed., W.B.
Saunders Co., Philadelphia, pp. 68-79). A non-limiting list of cancers, the
cells of which
can be used herein is provided in Section 4.13.
Cell lines derived from cancer tissues, cancer cells, infected cells, or cells
that
displays the pathologic or histologic changes associated with a disease can
also be used as
antigenic cells. Cancer or infected tissues, cells, or cell lines of human
origin are
preferred. Cancer cells, infected cells, or antigenic cells can be identified
and isolated by
any method known in the art. For example, cancer cells or infected cells can
be identified
by morphology, enzyme assays, proliferation assays, or the presence of
pathogens or
cancer-causing viruses. If the characteristics of the antigenic molecule of
interest are
known, antigenic cells can also be identified or isolated by any biochemical
or
immunological methods known in the art. For example, cancer cells or infected
cells can be
isolated by surgery, endoscopy, other biopsy techniques, affinity
chromatography, and
fluorescence activated cell sorting (e.g., with fluorescently tagged antibody
against an
antigen express by the cells). Antigenic cells that display similar
antigenicity have one or
more antigenic determinants in common against which an immune response in a
subject is
desired (e.g., for therapeutic or prophylactic purposes).
If the number of antigenic cells obtained from a subject is insufficient, the
cells may
be cultured in vitro by standard methods to expand the number of cells prior
to use in the
-12-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
present methods. There is no requirement that a clonal or homogeneous or
purified
population of antigenic cells be used. A mixture of cells can be used provided
that a
substantial number of cells in the mixture contain the antigens of interest.
In a specific
embodiment, the antigenic cells andlor immune cells are purified.
For the treatment or prevention of a type of cancer, the methods of the
invention
provide compositions of a CD91 ligand complexed to an antigenic molecule
displaying the
antigenicity of a tumor or cancer antigen of the same type of cancer, e.g., an
antigen
overexpressed in tumor or cancer cells relative to nontumor or noncancerous
cells ("tumor
associated antigens"), or an antigen expressed in tumor or cancer cells and
not expressed in
nontumor or noncancerous cells ("tumor specific antigen"). As used herein, the
term "the
same type of cancer" refers to cancer of the same tissue type, or metastasized
from cancer
of the same tissue type. In one embodiment, the antigenic molecules are
antigenic peptides
derived from cancer cells, preferably human cancers, e.g., tumor specific
antigens and
tumor associated antigens. The peptides can be generated by proteolytic
digestion of
proteins (e.g., cytosolic and/or membrane-derived proteins) from cancer cells,
or antigenic
cells that share antigenic determinants with or display similar antigenicity
as the cancer
cells.
In another embodiment, the antigenic molecules are antigenic peptides derived
from
cells infected by a pathogen or infectious agent that causes the infectious
disease, or the
pathogen which includes but is not limited to, a virus, bacterium, fungus,
protozoan,
parasite, etc. Preferably, the pathogen is one that infects humans. For
example, for the
treatment or prevention of infectious diseases, the methods of the invention
provide
compositions of CD91 ligands complexed to antigenic molecules that display the
antigenicity of an antigen of an infectious agent that causes an infectious
disease or of an
antigen that is associated with or causes an infectious disease. The antigenic
peptides are
generated by proteolytic digestion of (e.g., cytosolic and/or membrane-
derived) proteins
obtained from infected cells, antigenic cells that share antigenic
determinants with or
display similar antigenicity as the infected cells, or the pathogens including
viral
particles. The antigenic peptides can also be generated from antigenic cells
that display the
antigenicity of an agent (pathogen) that causes the infectious disease, or a
variant of such
agent. Infectious agents that can infect cells which can be used herein is
provided in
Section 4.14.
In yet another embodiment, any pathogen or infectious agent that can cause an
infectious disease can be used to infect a cell, and the infected cell used as
an antigenic cell
-13-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
for the preparation of antigenic peptides. Variants of a pathogen or
infectious agent, such as
but limited to replication-defective variants, non-pathogenic or attenuated
variants, non-
infectious variants, can also be used as an antigenic cell for this purpose.
For example,
many viruses, bacteria, fungi, parasites and protozoans that can be cultured
ih vitro or
isolated from infected materials can serve as a source of antigenic cells.
Methods known in
the art for propagating such pathogens including viral particles can be used.
In order to prepare pathogen-infected cells, uninfected cells of a cell type
susceptible
to infection by the pathogen or infectious agent that causes the disease can
be infected ira
vitro. Depending on the mode of transmission and the biology of the pathogen
or infectious
agent, standard techniques can be used to facilitate infection by the pathogen
or infectious
agent, and propagation of the infected cells. For example, influenza viruses
may be used to
infect normal human fibroblasts; and mycobacteria may be used to infect normal
human
Schwann cells. In various embodiments, variants of an infectious agent, such
as
replication-defective viruses, non-pathogenic or attenuated mutants, or
temperature-
sensitive mutants can also be used to infect or transform cells to generate
antigenic cells for
the preparation of antigenic peptides. If large numbers of a pathogen are
needed to infect
cells, or if pathogens are used directly as antigenic cells, any method known
in the art can
be used to propagate and grow the pathogens. Such methods will depend on the
pathogen,
and may not involve infecting a host. For example, many techniques axe known
in the art
for growing pathogenic bacteria, fungi and other non-viral microorganisms in
culture,
including large scale fermentation.
In another embodiment, any cell or tissue that displays the pathologic or
histologic
changes associated with a disease can be used as antigenic cell for the
preparation of
antigenic peptides. For example, neurons that display the pathologic changes
associated
with Alzheimer's disease, such as plaque formation, are suitable as antigenic
cells.
In another embodiment of the invention for the treatment or prevention of a
disease,
the methods of the invention provide compositions of CD91 ligands complexed to
antigenic
molecules displaying the antigenicity of an antigen that causes or is
associated with the
disease. In a specific embodiment, the antigenic molecules are from cells
which display the
pathologic or histologic changes associated with the disease.
In a specific embodiment of the invention, if a gene encoding an antigenic
protein is
available, normal cells of the appropriate cell type from the intended
recipient may be
transformed or transfected ih vitro with an expression construct comprising a
nucleic acid
molecule encoding such antigen, such that the antigen is expressed in the
recipient's cells.
-14-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Optionally, more than one such antigen may be expressed in the recipient's
cell in
this fashion, as will be appreciated by those skilled in the art, any
techniques known, such
as those described in Ausubel et al., 1989, Current Protocols in Molecular
Biology, Greene
Publishing Associates and Wiley Interscience, New York, may be used to perform
the
transformation or transfection and subsequent recombinant expression of the
antigen gene
in recipient's cells.
In one embodiment of the invention, a protein preparation is provided which is
derived from a pathogen, a cancer cell, an infected cell or a cell that
displays the pathologic
or histologic changes associated with a disease. For example, for the
treatment of cancer,
the protein preparations are prepared, postoperatively, from tumor cells
obtained from a
cancer patient. In another embodiment of the present invention, one or more
antigenic
proteins of interest are synthesized in cell lines modified by the
introduction of recombinant
expression systems that encode such antigens, and such cells are used to
prepare the
proteins. The proteins can be obtained from one or more cellular fraction(s),
for example,
the cytosol of the antigenic cells, or they can be extracted or solubilized
from the
membranes or cell walls of the antigenic cells. Any technique known in the art
for cell
lysis, fractionation of cellular contents, and protein enrichment or isolation
can be
used. See, for example, Current Protocols in Immunology, vol. 2, chapter 8,
Coligan et al.
(ed.), John Wiley & Sons, Inc.; Pathogenic and Clinical Microbiology: A
Laboratory
Manual by Rowland et al., Little Brown & Co., June 1994; which are
incorporated herein
by reference in their entireties. Depending on the techniques used to
fractionate the cellular
contents, a cellular fraction comprises at least 20, 50, 100, 500, 1,000,
5,000, 10,000, or
20,000 different proteins.
As used herein, the term "protein preparation" refers to a mixture of proteins
obtained from antigenic cells, a cellular fraction of antigenic cells, or
virus particles. The
proteins can be obtained from a cellular fraction, such as the cytosol. The
proteins can also
be non-cytosolic proteins (e.g., those from cell walls, cell membranes or
organelles), or
both. Cellular fractions may include but are not limited to cytosolic
fractions, membrane
fractions, and organelle fractions, such as nuclear, mitochondrial, lysosomal,
and
endoplasmic reticulum-derived fractions. The protein preparations can be
obtained from
non-recombinant or recombinant cells. The term "antigenic proteins" as used
herein also
encompasses antigenic polypeptides and antigenic peptides that may be present
in the
preparation. The protein preparation obtained from the antigenic cells or
cellular fractions
thereof or virus particles can optionally be purified from other non-
proteinaceous materials
-15-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
to various degrees by techniques known in the art. The protein preparation may
comprise at
least 50%, SS%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 97%, 98%, 99% of the
different proteins and peptides present in the antigenic cells or virus
particles or a fraction
of the antigenic cells.
In a specific embodiment, the protein preparations have not been subjected to
any
method of preparation that selectively removes or retains one or more
particular proteins)
from the other proteins in the antigenic cells.
In a specific embodiment, the protein preparation is the total cell lysate
which is not
fractionated andlor purified, and may contain other non-proteinaceous
materials of the
cells. In another specific embodiment, the protein preparation is total
protein in a cellular
fraction, which has not been subjected to further fractionation or
purification, and may
contain other non-proteinaceous materials of the cells. In yet another
embodiment, the
protein preparation is the total protein in a preparation of viral particles.
In specific
embodiments, the protein preparation comprises total cellular protein, total
cytosolic
proteins, or total membrane-bound proteins of antigenic cell(s). In various
embodiments,
the protein preparation comprises at least 20, 50, 100, 500, 1,000, 5,000,
10,000, or 20,000
different proteins. A plurality of different antigenic proteins are present in
a protein
preparation of antigenic cells. Moreover, the proteins in the protein
preparation may be
subjected to a step of protease digestion prior to ih vitf°o complexing
to CD91
ligands. Alternatively, the proteins in the protein preparation are not
subjected to a step of
protease digestion prior to in vitro complexing to CD91 ligands.
To make a protein preparation of antigenic cells or virus particles, the
lysing of
antigenic cells or disruption of cell walls, cell membranes, or viral particle
structure can be
performed using standard protocols known in the art. In various embodiments,
the
antigenic cells can be lysed, for example, by mechanical shearing, sonication,
freezing and
thawing, adjusting the osmolarity of the medium surrounding the cells, or a
combination of
techniques. In less preferred embodiments, the antigenic cells can be lysed by
chemicals,
such as detergents.
Once the cells are lysed, it is desirable to remove cellular debris, materials
that are
non-proteinaceous or materials that do not contain cytosolic, and/or membrane-
derived
proteins (including proteins in the membranes of organelles). Removal of these
components can be accomplished by techniques such as low speed centrifugation
or
filtration. After removing cellular debris and intact cells, a high speed
centrifugation step
can be used to separate the cytosolic proteins which are in the supernatant,
and the
-16-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
membrane-derived proteins which are collected in the pellet. Standard
procedures
commonly known in the art allows the further isolation of the membrane-derived
proteins
from the pellet. Standard techniques commonly known in the art can be used to
extract
viral proteins from viral particles. In certain embodiments, the methods used
do not or are
not designed to selectively remove or retain any one or more particular
proteins) from
other proteins that are present in the antigenic cell, in the cytosol or in
the membranes.
In other embodiments, optionally, the proteins from the antigenic cells can be
separated by their general biochemical and/or biophysical properties, such as
size, charge,
or combinations thereof. Any techniques known in the art can be used to
perform the
separation. Selected fractions of the proteins/peptides that comprise at least
20, 50, 100,
500, 1,000, 5,000, 10,000, or 20,000 different proteins or that comprise at
least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 97%, 98%, 99% of the different proteins
present in the antigenic cells or a cellular fraction thereof, or virus
particles, are used to
form complexes to a CD91 ligand.
An exemplary, but not limiting, method that may be used to make a protein
preparation comprising cytosolic proteins is as follows:
Cells, which may be tumor cells derived from a biopsy of the patient
or tumor cells cultivated ih vitY~, or cell infected with a pathogenic agent,
are
suspended in 3 volumes of 1X Lysis buffer comprising 30mM sodium
bicarbonate pH 7.5, 1mM PMSF, incubated on ice for 20 minutes and then
the hypotonically-swollen cells are homogenized in a dounce homogenizer
until >95% cells are lysed. As an alternative to shearing, cells can be
sonicated, on ice, until >99% cells are lysed as determined by microscopic
examination. When sonication is used, cells are suspended in a buffer such
as phosphate buffered saline (PBS) which may comprises 1 mM PMSF,
before sonication.
The lysate is centrifuged at 1,000 x g for 10 minutes to remove intact
cells, nuclei and other cellular debris. The resulting supernatant is
recentrifuged at about 100,000 x g for about one hour, and the supernatant
recovered. The 100,000 x g supernatant may be dialyzed for 36 hours at
4°C
(three times, 100 times volumes each time) against PBS or other suitable
buffer, to provide the soluble cytosolic proteins of the present invention. If
necessary, insoluble material in the preparation may be removed by filtration
or low-speed centrifugation.
-17-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
An exemplary, but not limiting, method that may be used to make a protein
preparation comprising membrane-derived proteins is as follows:
Cells, which may be tumor cells derived from a biopsy of the patient
or tumor cells cultivated in vitro, or cells infected with a pathogenic agent,
are suspended in 3 volumes of 1X Lysis buffer comprising 30mM sodium
bicarbonate pH 7.5, lxnM PMSF, incubated on ice for 20 minutes and then
the hypotonically-swollen cells are homogenized in a Bounce homogenizer
until >95% cells are lysed. As an alternative to shearing, cells can be
sonicated, on ice, until >99% cells are lysed as determined by microscopic
examination. When sonication is used, cells are suspended in a buffer such
as phosphate buffered saline (PBS) which may comprises 1 mM PMSF,
before sonication.
The lysate is then centrifuged at 100,000 x g for 10 minutes to collect
the cell membranes. Membrane-derived proteins can be dislodged from the
lipid bilayer and isolated from the 100,000g pellet (where the membrane-
derived proteins are located) by resuspending the pellet in 5 volumes of PBS
containing 1 % sodium deoxycholate (without Ca2+ and Mg2+) and incubated
on ice for 1 h. The resulting suspension is centrifuged for 30 min at 20,OOOg
and the resulting supernatant harvested and dialyzed against several changes
of PBS (without Ca2+ and Mg2+) to remove the detergent. The resulting
dialysate is centrifuged for 90 min at 100,000g and the supernatant purified
further. Then calcium and magnesium are both added to the supernatant to
give final concentrations of 2mM. If necessary, insoluble material in the
preparation may be removed by filtration or low-speed centrifugation.
In a specific embodiment, the population of cytosolic and/or membrane-derived
proteins obtained from antigenic cells can be complexed to a CD91 ligand
directly without
protease treatment or any further extraction or selection processes.
Alternatively, the
proteins can be subjected to protease treatment prior to complexing.
According to the invention, the cytosolic and membrane-derived proteins
obtained
from antigenic cells can be optionally digested to generate antigenic
peptides. In one
embodiment, either the cytosolic or the membrane-derived proteins are used in
the
digestion. In another embodiment, the cytosolic and membrane-derived proteins
are
combined in the digestion reaction to generate antigenic peptides. In
preferred
embodiments, the protein preparations that are used in the protease digestion
have not been
-18-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
subjected to any methods) of preparation that selectively remove or retain one
or more
particular proteins) from the other proteins in the antigenic cells, or the
cytosol or
membranes of the antigenic cells.
Various proteases or proteolytic enzymes can be used in the invention to
produce
from a protein preparation of antigenic cells a population of peptides which
comprises
antigenic peptides. The enzymatic digestions can be performed either
individually or in
suitable combinations with any of the proteolytic enzymes that are well known
in the art
including, but not limited to, trypsin, Staphylococcal peptidase I (also known
as protease
V8), chymotrypsin, pepsin, cathepsin G, thermolysin, elastase, and papain.
Trypsin is a
highly specific serine proteinase that cleaves on the carboxyl-terminal side
of lysines and
arginines. Due to the limited number of cleavage sites, it is expected to
leave many MHC-
binding epitopes intact. Staphylococcal peptidase I, a serine proteinase, has
specificity for
cleavage after glutamic and aspartic acid residues. A digestion can be carried
out with a
single protease or a mixture of proteases. The proteases or proteolytic
enzymes used are
incubated under conditions suitable for the particular enzyme. Preferably, the
enzyme is
purified. Non-enzymatic methods, such as cyanogen bromide cleavage, can also
be used
for generating peptides. The protein preparation to be digested can be
aliquoted into a
plurality of reactions each using a different enzyme, and the resulting
peptides may
optionally be pooled together for use. It may not be necessary to completely
digest the
proteins in the enzymatic reactions. These reactions results in the generation
of a diverse
and different set of peptides for each protein that is present in the protein
preparation. The
production of different peptide sets allows for a greater probability of
generating antigenic
peptides that are capable of inducing an immune response to the antigens in
the protein
preparation when they are complexed to a CD91 ligand. In a preferred
embodiment, the
protein preparation to be digested is aliquoted into two separate reactions
and two different
proteolytic enzymes are used to produce two different sets of peptides of the
proteins
present in the protein preparation. Depending on the proteins, enzymes and
reaction
conditions, undigested proteins may remain in the reactions. In a preferred
embodiment,
trypsin and Staphylococcal peptidase I are used separately to digest the
protein preparation.
In another preferred embodiment, the proteolytic enzymes used in the invention
exhibit similar activities as the proteolytic activities that are found in the
proteasome. The
proteasome is responsible for the extralysosomal, endocatalytic degradation of
cytosolic and
nuclear proteins which are mis-folded or damaged in a cell. The proteasome can
degrade
proteins completely to yield single amino acids, can generate optimal major
-19-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
histocompatibility complex class I (MHC I)-binding epitopes, and can generate
longer
peptide precursors which could potentially undergo further trimming elsewhere
in the cell
to yield cytotoxic T cell epitopes. Cleavage preferences of the proteasome is
on the
carboxyl (COOH)-side of basic, acidic, and hydrophobic amino acids. Three
known
proteolytic enzymatic activities that are present in the proteasome are
chymotrypsin-like
activity, trypsin-like activity, and peptidylglutamylpeptide-hydrolyzing
activity (Uebel and
Tampe, 1999, Curr. Opin. Immunol. 11:203-208). As such, enzymes having such
activities
and specificities can be used separately or in combination to digest the
protein
preparation. In a preferred embodiment, trypsin, chymotrypsin, and/or
peptidylglutamylpeptide-hydrolase are used.
The resulting peptide digestions comprise antigenic peptides, non-antigenic
peptides, and single amino acid residues. The reactions may also comprise
undigested or
incompletely digested antigenic proteins. The proteolytic enzymatic digestions
of the
invention are monitored in order to generate peptides that fall within a
desirable range of
lengths. In a preferred embodiment, the peptides generated are from about 7 to
about 20
amino acid residues. Most antigenic peptides that are presented to T cells by
MHC class I
and class II fall within this range. In various embodiments, the population of
peptides
comprises peptides having a size range of 6 to 21, 8 to 19, 10 to 20, or at
least 7, 8, 9, 10,
1 l, 12, 15, 20, 25, 30, 35, 40, 45, or 50, amino acid residues. In preferred
embodiments, the
antigenic peptides have 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
or 35 amino acid
residues. To monitor the progression of protein digestion, a test reaction can
be performed
where small aliquots of a protein digestion are taken out of the reaction and
monitored for
the progression of digestion through either tricine-polyacrylamide gel
electrophoresis
("tricine-PAGE"), high performance liquid chromatography ("HPLC"), or mass
spectrometry, or any other method known in the art to determine the size of
peptides. Using
such a test reaction, a determination can be made as to when peptide fragments
of a
particular size range will be generated at a particular enzymatic
concentration. Other
variables of the reaction that can be manipulated include the amount of
protein in the
reaction, the temperature, the duration of incubation, the presence of
cofactors, etc.
Once the proper conditions are established for the generation of peptide
fragments
of a particular size range from a type of antigenic cell, the enzymatic
reaction conditions
can be duplicated to generate antigenic peptides which can be pooled. It is
preferred that
the enzymatic digestion is terminated before the peptides are complexed to a
CD91
ligand. In one embodiment of the invention, inhibitors can be used for
terminating an
-20-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
enzymatic digestion. Enzymatic inhibitors that can be used in the invention
include, but are
not limited to, PMSF, bestatin, amastatin, leupeptin, and cystatin, depending
on which
enzymes are used in the protein digestion. Inhibitors for most proteases are
well known in
the art. Alternatively, another method of terminating an enzymatic digestion
is by physical
removal of the enzyme from the reaction. This can be done by attaching the
enzyme of
choice to a solid phase, such as a resin or a material that can easily be
removed from the
reaction by well known methods such as centrifugation or filtration. The
protein preparation
is allowed to contact or flow across the solid phase for a period of time.
Such immobilized
enzymes can be purchased commercially or can be produced by procedures for
immobilizing enzymes that are well known in the art.
At the end of the digestion reaction, the peptides can optionally be separated
from
low molecular weight materials, such as dipeptides, or single amino acid
residues, in the
preparation. For example, the peptides can be isolated by centrifugation
through a
membrane, such as the Centriprep-3. Optionally, the peptides can be separated
by their
general biochemical and/or biophysical properties, such as size, charge, or
combinations
thereof. Any techniques known in the art can be used to perform the separation
resulting in
digested protein preparation comprising at least 50, 100, 500, 1,000, 5,000,
10,000, 20,000,
50,000, or 100,000 different peptides.
In another embodiment of the invention, peptides that are endogenously present
in
antigenic cells can be used in the invention either alone or in combination
with the peptides
generated by the proteolytic digestion of the cytosolic and membrane-derived
proteins. According to the invention, such peptides that are isolated directly
from a protein
preparation of antigenic cells can be complexed to CD91 ligands.
In specific embodiments, either the cytosolic or the membrane-derived proteins
are
used in the isolation process. In another specific embodiment, the cytosolic
and membrane-
derived proteins are combined in the isolation process. In preferred
embodiments, the
protein preparations that are used in the isolation have not been subjected to
any methods)
of preparation that selectively remove or retain one or more particular
proteins) from the
other proteins in the antigenic cells, or the cytosol or membranes of the
antigenic
cells. Preferably, the protein preparation comprises comprise at least 20, 50,
100, 500,
1,000, 5,000, 10,000, or 20,000 different proteins or that comprise at least
50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95% 97%, 98%, 99% of the different proteins
present in
the antigenic cells or a cellular fraction thereof, or virus particles.
-21 -
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
In various embodiments, the method comprise treating the protein preparation
to
ATP, guanidium hydrochloride, and/or exposing the protein preparation to
acidic conditions
such that antigenic peptides in the protein preparation can be eluted. Many
different acids
can be used, including but not limited to, trifluoroacetic acid. Methods known
in the art
such as those described in Marston and Hartley, 1990, Meth. Enzymol. 182:264-
276 for
dissociating protein aggregates can be used.
In a particular embodiment, the isolation process comprises exposing a protein
preparation of antigenic cells with ATP, for example, at room temperature for
one hour,
and/or treating a protein preparation of antigenic cells with trifluoroacetic
acid (TFA), for
example 0.1% TFA. The treatment preferably comprises sonicating the protein
preparation
in 0.1 % TFA. In a most preferred embodiment, a protein preparation is first
exposed to
ATP, followed by sonication in 0.1 % TFA. Various protease inhibitors can be
used in the
invention prior to cell lysis and the isolation process. For example, a
mixture of 14 protease
inhibitors can be used: phenylinethylsulfonyl fluoride (PMSF) 2 mM,
ethylenediaminetetreacedic acid (EDTA) 1 mM, ethylene glycolbis(P-aminoethyl
ether)N,N,N',N'-tetraacetic acid (EGTA) 1 mM, (all obtained from Sigma, St.
Louis, MO),
and Antipain 20 mg/ml, Bestatin 5 mg/ml, Chemostatin 20 ptg/nil, E64 20
Jig/ml,
Leupeptine 1 ttglml, Pepstatine 1 gg/ml, Pefabloc 40 Ag/ml, and Apoprotein 10
tkg/rnl (all
obtained from Boehringer Mannheim, Indianapolis, IN). The peptides resulting
from the
protein preparation comprise antigenic peptides and non-antigenic peptides of
a variety of
sizes ranging from at least 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45,
or 50, amino acid
residues. In preferred embodiments, the peptides have 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17,
18, 19, 20, or 35 amino acid residues. At the end of the process, the peptides
are preferably
recovered by separating from the proteins in the preparation prior to
complexing with a
CD91 ligand. For example, the peptides can be recovered by centrifugation
through a
membrane, such as the Centriprep-3, by drying under vacuum, or by reverse
phase
chromatography, e.g., fractionation in a BioCad20 microanalytiocal HPLC Poros
RH2
column (Perseptive Biosystems, Cambridge, MA), equilibrated with 0.1% TFA in
water
and elution by acetonitrile. Accordingly, antigenic peptides that are
endogenously present
in antigenic cells and that are isolated directly from a protein preparation
can be complexed
to a CD91 ligand. Alternatively, a mixed population of peptides comprising
peptides that
are endogenously present in antigenic cells and peptides from digested
cytosolic and
membrane-derived proteins, can be complexed to a CD91 ligand.
-22-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
4.1.2 SYNTHETIC PRODUCTION OF ANTIGENIC PROTEINS
Once the nucleotide sequence or amino acid sequence of an antigenic protein
has
been determined or obtained, the peptide can be produced, either by
recombinant techniques
or by synthetic methods. The antigenic peptide may be synthesized using
conventional
peptide synthesis or any of a number of other protocols well known in the art.
For example,
a peptide can be synthesized by use of a peptide synthesizer. Either the
entire protein can
be synthesized, or an antigenic determinant thereof, preferably the portion of
the protein
that contains the mutant or variant amino acid(s).
Peptides may be synthesized by solid-phase peptide synthesis using procedures
similar to those described by Merrifield, 1963, J. Am. Chem. Soc. 85:2149.
During
synthesis, N-a protected amino acids having protected side chains are added
stepwise to a
growing polypeptide chain linked by its C-terminal and to an insoluble
polymeric support
i.e., polystyrene beads. The peptides are synthesized by linking an amino
group of an N-a
deprotected amino acid to an a carboxyl group of an N-a protected amino acid
that has
been activated by reacting it with a reagent such as dicyclohexylcarbodiimide.
The
attachment of a free amino group to the activated carboxyl leads to peptide
bond
formation. The most commonly used N-a protecting groups include Boc which is
acid
labile and Fmoc which is base labile. Details of appropriate chemistries,
resins, protecting
groups, protected amino acids and reagents are well known in the art (see
Atherton et al.,
1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and
Bodanszky,
1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag).
In addition, analogs and derivatives antigenic proteins can be chemically
synthesized. Furthermore, if desired, nonclassical amino acids or chemical
amino acid
analogs can be introduced as a substitution or addition into the sequence of
the antigenic
proteins. Non-classical amino acids include but are not limited to the D-
isomers of the
common amino acids, a amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino
butyric
acid, ~y Abu, E-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-
amino
propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,
citrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, ~i-alanine,
fluoro-amino acids, designer amino acids such as (3-methyl amino acids, Ca
methyl amino
acids, Na methyl amino acids, and amino acid analogs in general.
Purification of the resulting peptides is accomplished using conventional
procedures, such as preparative HPLC using gel permeation, partition andlor
ion exchange
chromatography. The techniques, choice of appropriate matrices and buffers are
well
- 23 -
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
known in the art (Atherton et al., 1989, Solid Phase Peptide Synthesis: A
Practical
Approach, IRI, Press). Preferably, the synthetic peptide prepared is 50%, 55%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 95% ,96%, 97%, 98%, 99% or 100% pure.
4.1.3 RECOMBINANT PRODUCTION OF ANTIGENIC PROTEINS
As an alternative to synthetic production, antigenic protein can produced by
recombinant means. Once the nucleotide sequence encoding an antigenic protein
has been
identified, the nucleotide sequence, or a fragment thereof, can be obtained
and cloned into
an expression vector for recombinant expression. The expression vector can
then be
introduced into a host cell for propagation of the antigen. Methods for
recombinant
production of antigenic proteins are described in detail herein.
Any recombinant antigenic protein useful in the complexes of the invention is
preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ,96%, 97%, 98%,
99%
or 100% pure. The DNA rnay be obtained by DNA amplification or molecular
cloning
directly from a tissue, cell culture, or cloned DNA (e.g., a DNA "library")
using standard
molecular biology techniques (see, e.g., Methods in Enzymology, 1987, volume
154,
Academic Press; Sambrook et al. 1989, Molecular Cloning - A Laboratory Manual,
2nd
Edition, Cold Spring Harbor Press, New York; and Current Protocols in
Molecular Biology,
Ausubel et al. (eds.), Greene Publishing Associates and Wiley Interscience,
New York, each
of which is incorporated herein by reference in its entirety). Clones derived
from genomic
DNA may contain regulatory and intron DNA regions in addition to coding
regions; clones
derived from cDNA will contain only exon sequences. Whatever the source, the
antigen
gene should be cloned into a suitable vector for propagation of the gene.
In a preferred embodiment, DNA can be amplified from genomic or cDNA by
polymerase chain reaction (PCR) amplification using primers designed from the
known
sequence of a related or homologous antigen. PCR is used to amplify the
desired sequence
in DNA clone or a genomic or cDNA library, prior to selection. PCR can be
carried out,
e.g., by use of a thermal cycler and Taq polymerise (Gene Amp~). The
polymerise chain
reaction (PCR) is commonly used for obtaining genes or gene fragments of
interest. For
example, a nucleotide sequence encoding an antigenic protein of any desired
length can be
generated using PCR primers that flank the nucleotide sequence encoding the
peptide-
binding domain. Alternatively, an antigenic protein gene sequence can be
cleaved at
appropriate sites with restriction endonuclease(s) if such sites are
available, releasing a
fragment of DNA encoding the antigenic protein gene, or an antigenic
derivative thereof. If
-24-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
convenient restriction sites are not available, they may be created in the
appropriate
positions by site-directed mutagenesis and/or DNA amplification methods known
in the art
(see, for example, Shankarappa et al., 1992, PCR Method Appl. 1:277-278). The
DNA
fragment that encodes the antigenic protein is then isolated, and ligated into
an appropriate
expression vector, care being taken to ensure that the proper translation
reading frame is
maintained.
In an alternative embodiment, for the molecular cloning of an antigenic
protein gene
from genomic DNA, DNA fragments are generated to form a genomic library. Since
some
of the sequences encoding related protein antigens are available and can be
purified and
labeled, the cloned DNA fragments in the genomic DNA library may be screened
by
nucleic acid hybridization to a labeled probe (Benton and Davis, 1977, Science
196:180;
Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA
fragments with substantial homology to the probe will hybridize. It is also
possible to
identify an appropriate fragment by restriction enzyme digestion(s) and
comparison of
fragment sizes with those expected according to a known restriction map.
Alternatives to isolating the antigenic protein genomic DNA include, but are
not
limited to, chemically synthesizing the gene sequence itself from a known
sequence or
synthesizing a cDNA to the mRNA which encodes the antigenic protein. For
example,
RNA for cDNA cloning of the antigenic protein gene can be isolated from cells
which
express the antigenic protein. A cDNA library may be generated by methods
known in the
art and screened by methods, such as those disclosed for screening a genomic
DNA
library. If an antibody to the antigenic protein is available,the antigenic
protein may be
identified by binding of a labeled antibody to the antigenic protein
synthesizing clones.
Other specific embodiments for the cloning of a nucleotide sequence encoding
an
antigenic protein, are presented as examples but not by way of limitation, as
follows:
In a specific embodiment, nucleotide sequences encoding an antigenic protein
can
be identified and obtained by hybridization with a probe comprising a
nucleotide sequence
encoding the antigenic protein under conditions of low to medium stringency.
By way of
example and not limitation, procedures using such conditions of low stringency
are as
follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A.
78:6789-6792).
Filters containing DNA are pretreated for 6 h at 40°C in a solution
containing 35%
formamide, SX SSC, 50 mM Tris-HCl (pH 7.5), S mM EDTA, 0.1% PVP, 0.1% Ficoll,
1%
BSA, and 500 ,ug/ml denatured salmon sperm DNA. Hybridizations are carried out
in the
same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2%
BSA, 100
- 25 -
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
~.g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm 3aP-
labeled
probe is used. Filters are incubated in hybridization mixture for 18-20 h at
40°C, and then
washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-
HCl (pH 7.4), 5 mM
EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and
incubated an
additional 1.5 h at 60°C. Filters are blotted dry and exposed for
autoradiography. If
necessary, filters are washed for a third time at 65-68°C and reexposed
to film. Other
conditions of low stringency which may be used are well known in the art
(e.g., as
employed for cross-species hybridizations).
Any technique for mutagenesis known in the art can be used to modify
individual
nucleotides in a DNA sequence, for purpose of making amino acid substitutions)
in the
expressed peptide sequence, or for creating/deleting restriction sites to
facilitate further
manipulations. Such techniques include but are not limited to, chemical
mutagenesis, in
vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem.
253:6551),
oligonucleotide-directed mutagenesis (Smith, 1985, Ann. Rev. Genet. 19:423-
463; Hill et
al., 1987, Methods Enzymol. 155:558-568), PCR-based overlap extension (Ho et
al., 1989,
Gene 77:51-59), PCR-based megaprimer mutagenesis (Sarkar et al., 1990,
Biotechniques
8:404-407), etc. Modifications can be confirmed by double stranded
dideoxynucleotide
DNA sequencing.
4.1.3.1 HOST-VECTOR SYSTEMS
Nucleotide sequences encoding an antigenic protein can be inserted into the
expression vector for propagation and expression in recombinant cells. An
expression
construct, as used herein, refers to a nucleotide sequence encoding an
antigenic protein
operably associated with one or more regulatory regions which allows
expression of the
antigenic protein in an appropriate host cell. "Operably-associated" refers to
an association
in which the regulatory regions and the antigenic protein sequence to be
expressed are
joined and positioned in such a way as to permit transcription, and
ultimately, translation of
the protein sequence. A variety of expression vectors may be used for the
expression of
proteins, including, but not limited to, plasmids, cosmids, phage, phagemids,
or modified
viruses. Examples include bacteriophages such as lambda derivatives, or
plasmids such as
pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene).
Typically, such
expression vectors comprise a functional origin of replication for propagation
of the vector
in an appropriate host cell, one or more restriction endonuclease sites for
insertion of the
antigenic protein gene sequence, and one or more selection markers.
-26-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Vectors based on E. coli are the most popular and versatile systems for high
level
expression of foreign proteins (Makrides, 1996, Microbiol. Rev. 60:512-538).
Non-limiting
examples of prokaryotic expression vectors may include the 7~gt vector series
such as Agtl l
(Huynh et al., 1984 in "DNA Cloning Techniques", Vol. I: A Practical Approach
(D.
Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series
(Studier et al., 1990,
Methods Enzymol., 185:60-89). Non-limiting examples of regulatory regions that
can be
used for expression in E. coli may include but not limited to lac, trp, lpp,
phoA, recA, tac,
M'L, and phage T3 and T7 promoters (Makrides, 1996, supra).
However, a potential drawback of a prokaryotic host-vector system is the
inability to
perform many of the post-translational processing events of mammalian cells.
Thus, a
eukaryotic host-vector system is preferred, a mammalian host-vector system is
more
preferred, and a human host-vector system is the most preferred. The
regulatory regions
necessary for transcription of an antigenic protein or polypeptide can be
provided by the
expression vector. A translation initiation codon (ATG) may also be provided
to express a
nucleotide sequence encoding an antigenic protein that lacks an initiation
codon. In a
compatible host-construct system, cellular proteins required for
transcription, such as RNA
polymerase and transcription factors, will bind to the regulatory regions on
the expression
construct to effect transcription of the antigenic protein sequence in the
host organism. The
precise nature of the regulatory regions needed for gene expression may vary
from host cell
to host cell. Generally, a promoter is required which is capable of binding
RNA polymerase
to initiate the transcription of an operably-associated nucleic acid sequence.
Such
regulatory regions may include those 5'-non-coding sequences involved with
initiation of
transcription and translation, such as a TATA box, cap site, a CART box,
transcription
factor binding sites, enhancer elements, and the like. The non-coding region
3' to the
coding sequence may contain transcriptional termination regulatory sequences,
such as
terminators and polyadenylation sites.
Both constitutive and inducible regulatory regions may be used for expression
of the
antigenic protein. It may be desirable to use inducible promoters when the
conditions
optimal for growth of the recombinant cells and the conditions for high level
expression of
the antigenic protein are different. Examples of useful regulatory regions are
provided in
the next section below.
For expression of antigenic proteins in mammalian host cells, a variety of
regulatory
regions can be used, for example, the SV40 early and late promoters, the
cytomegalovirus
(CMV) immediate early promoter, and the Rous sarcoma virus long terminal
repeat (RSV-
-27-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
LTR) promoter. Inducible promoters that may be useful in mammalian cells
include but are
not limited to those associated with the metallothionein II gene, mouse
mammary tumor
virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the ~3-
interferon gene,
and the Hsp70 gene (Williams et al., 1989, Cancer Res. 49:2735-42 ; Taylor et
al., 1990,
Mol. Cell. Biol. 10:165-75).
The following animal regulatory regions, which exhibit tissue specificity and
have
been utilized in transgenic animals, can also be used in cells of a particular
tissue type of
interest: elastase I gene control region which is active in pancreatic acinar
cells (Swift et al.,
1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.
Biol. 50:399-
409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which
is active
in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin
gene
control region which is active in lymphoid cells (Grosschedl et al., 1984,
Cell 38:647-658;
Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell.
Biol. 7:1436-
1444), mouse mammary tumor virus control region which is active in testicular,
breast,
lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene
control region
which is active in liver (Pinkert et al., 1987, Genes Dev. 1:268-276), alpha-
fetoprotein gene
control region which is active in liver (Krumlauf et al., 1985, Mol. Cell.
Biol. 5:1639-1648;
Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control
region which is
active in the liver (Kelsey et al., 1987, Genes Dev. 1:161-171), beta-globin
gene control
region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-
340; Kollias
et al., 1986, Cell 46: 89-94; myelin basic protein gene control region which
is active in
oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712);
myosin light
chain-2 gene control region which is active in skeletal muscle (Sari, 1985,
Nature 314:283-
286), and gonadotropic releasing hormone gene control region which is active
in the
hypothalamus (Mason et al., 1986, Science 234:1372-1378).
The efficiency of expression of the antigenic protein in a host cell may be
enhanced
by the inclusion of appropriate transcription enhancer elements in the
expression vector,
such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus,
immunoglobulin
genes, metallothionein, ,Q-actin (see Bittner et al., 1987, Methods in
Enzymol. 153:516-544;
Gorman, 1990, Curr. Op. Biotechnol. 1:6-47).
The expression vector may also contain sequences that permit maintenance and
replication of the vector in more than one type of host cell, or integration
of the vector into
the host chromosome. Such sequences may include but are not limited to
replication
origins, autonomously replicating sequences (ARS), centromere DNA, and
telomere
- 28 -
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
DNA. It may also be advantageous to use shuttle vectors that can be replicated
and
maintained in at least two types of host cells.
In addition, the expression vector may contain selectable or screenable marker
genes
for initially isolating or identifying host cells that contain DNA encoding an
antigenic
protein. For long term, high yield production of antigenic proteins, stable
expression in
mammalian cells is preferred. A number of selection systems may be used for
mammalian
cells, including, but not limited, to the Herpes simplex virus thymidine
kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase
(Szybalski and
Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A. 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be
employed in tk,
lzgprt or aprt cells, respectively. Also, antimetabolite resistance can be
used as the basis of
selection for dihydrofolate reductase (dhfr), which confers resistance to
methotrexate
(Wigler et al., 1980, Natl. Acad. Sci. U.S.A. 77:3567; O'Hare et al., 1981,
Proc. Natl. Acad.
Sci. U.S.A. 78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan and
Berg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:2072); neomycin
phosphotransferase (neo),
which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,
1981, J.
Mol. Biol. 150:1); and hygromycin phosphotransferase (hyg), which confers
resistance to
hygromycin (Santerre et al., 1984, Gene 30:147). Other selectable markers,
such as but not
limited to histidinol and ZeocinTM can also be used.
In order to insert the DNA sequence of the antigenic protein into the cloning
site of
a vector, DNA sequences with regulatory functions, such as promoters, must be
attached to
DNA sequences encoding the antigenic protein. To do this, linkers or adapters
providing
the appropriate compatible restriction sites may be ligated to the ends of
cDNA or synthetic
DNA encoding an antigenic protein, by techniques well known in the art (Wu et
al., 1987,
Methods Enzymol. 152:343-349). Cleavage with a restriction enzyme can be
followed by
modification to create blunt ends by digesting back or filling in single-
stranded DNA
termini before ligation. Alternatively, a desired restriction enzyme site can
be introduced
into a fragment of DNA by amplification of the DNA by use of PCR with primers
containing the desired restriction enzyme site.
The expression construct comprising an antigenic protein sequence operably
associated with regulatory regions can be directly introduced into appropriate
host cells for
expression and production of antigenic protein-CD91 ligand complexes without
further
cloning (see, for example, U.S. Patent No. 5,580,859). The expression
constructs may also
contain DNA sequences that facilitate integration of the antigenic protein
nucleotide
-29-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
sequence into the genome of the host cell, e.g., via homologous recombination.
In this
instance, it is not necessary to employ an expression vector comprising a
replication origin
suitable for appropriate host cells in order to propagate and express the
antigenic protein in
the host cells.
Expression constructs containing cloned nucleotide sequence encoding antigenic
polypeptides can be introduced into the host cell by a variety of techniques
known in the art,
including but not limited to, for prokaryotic cells, bacterial transformation
(Hanahan, 1985,
in DNA Cloning, A Practical Approach, 1:109-136), and for eukaryotic cells,
calcium
phosphate mediated transfection (Wigler et al., 1977, Cell 11:223-232),
liposome-mediated
transfection (Schaefer-Bidder et al., 1982, Science 215: 166-168),
electroporation (Wolff et
al., 1987, Proc. Natl. Acad. Sci. 84:3344), and microinjection (Cappechi,
1980, Cell
22:479-488). Co-expression of an antigenic peptide and a CD91 ligand in the
same host
cell can be achieved by essentially the same methods.
For long term, high yield production of properly processed antigenic proteins
or
1 S antigenic protein-CD91 ligand complexes, stable expression in mammalian
cells is
preferred. Cell lines that stably express antigenic proteins or antigenic
protein-CD91 ligand
complexes may be engineered by using a vector that contains a selectable
marker. By way
of example but not limitation, following the introduction of the expression
constructs,
engineered cells may be allowed to grow for 1-2 days in an enriched media, and
then are
switched to a selective media. The selectable marker in the expression
construct confers
resistance to the selection and optimally allows cells to stably integrate the
expression
construct into their chromosomes and to grow in culture and to be expanded
into cell
lines. Such cells can be cultured for a long period of time while antigenic
protein is
expressed continuously.
Any of the cloning and expression vectors described herein may be synthesized
and
assembled from known DNA sequences by techniques well known in the art. The
regulatory regions and enhancer elements can be of a variety of origins, both
natural and
synthetic. Some vectors and host cells may be obtained commercially. Non-
limiting
examples of useful vectors are described in Appendix 5 of Current Protocols in
Molecular
Biology, 1988, ed. Ausubel et al., Greene Publish. Assoc. & Wiley
Interscience, which is
incorporated herein by reference; and the catalogs of commercial suppliers
such as Clontech
Laboratories, Stratagene Inc., and Invitrogen, Inc.
Alternatively, a number of viral-based expression systems may also be utilized
with
mammalian cells to produce antigenic proteins. Vectors using DNA virus
backbones have
-30
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
been derived from simian virus 40 (SV40) (Hamer et al., 1979, Cell 17:725),
adenovirus
(Van Doren et al., 1984, Mol. Cell Biol. 4:1653), adeno-associated virus
(McLaughlin et
al., 1988, J. Virol. 62:1963), and bovine papillomas virus (Zinn et al., 1982,
Proc. Natl.
Acad. Sci. 79:4897). In cases where an adenovirus is used as an expression
vector, the
donor DNA sequence may be ligated to an adenovirus transcription/translation
control
region, e.g., the late promoter and tripartite leader sequence. This chimeric
gene may then
be inserted in the adenovirus genome by in vitro or in vivo recombination.
Insertion in a
non-essential region of the viral genome (e.g., region E1 or E3) will result
in a recombinant
virus that is viable and capable of expressing heterologous products in
infected hosts (see,
e.g., Logan and Shenk, 1984, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659).
Bovine papillomavirus (BPV) can infect many higher vertebrates, including man,
and its DNA replicates as an episome. A number of shuttle vectors have been
developed
for recombinant gene expression which exist as stable, multicopy (20-300
copies/cell)
extrachromosomal elements in mammalian cells. Typically, these vectors contain
a
segment of BPV DNA (the entire genome or a 69°1o transforming
fragment), a promoter
with a broad host range, a polyadenylation signal, splice signals, a
selectable marker, and
"poisonless" plasmid sequences that allow the vector to be propagated in E.
coli. Following
construction and amplification in bacteria, the expression gene construct is
transfected into
cultured mammalian cells, for example, by the techniques of calcium phosphate
coprecipitation or electroporation. For those host cells that do not manifest
a transformed
phenotype, selection of transformants is achieved by use of a dominant
selectable marker,
such as histidinol and 6418 resistance., For example, BPV vectors such as
pBCMGSNeo
and pBCMGHis may be used to express antigenic peptide sequences (Karasuyama et
al.,
Eur. J. Immunol. 18:97-104; Ohe et al., Human Gene Therapy 6:325-33) which may
then
be transfected into a diverse range of cell types for expression of the
antigenic protein.
Alternatively, the vaccinia 7.SK promoter may be used (see, e.g., Mackett et
al.,
1982, Proc. Natl. Acad. Sci. U.S.A. 79:7415-7419; Mackett et al., 1984, J.
Virol. 49:857-
864; Panicali et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:4927-4931) In
cases where a
human host cell is used, vectors based on the Epstein-Barr virus (EBV) origin
(OriP) and
EBV nuclear antigen 1 (EBNA-1; a traps-acting replication factor) may be used.
Such
vectors can be used with a broad range of human host cells, e.g., EBO-pCD
(Spickofsky et
al., 1990, DNA Prot. Eng. Tech. 2:14-18), pDR2 and ~DR2 (available from
Clontech
Laboratories).
-31-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Antigenic proteins may also be made with a retrovirus-based expression system.
In
contrast to transfection, retroviruses can efficiently infect and transfer
genes to a wide range
of cell types including, for example, primary hematopoietic cells. In
retroviruses such as
Moloney marine leukemia virus, most of the viral gene sequences can be removed
and
replaced with nucleic acid sequences encoding the antigenic protein, while the
missing viral
functions can be supplied in trams. The host range for infection by a
retroviral vector can
also be manipulated by the choice of envelope used for vector packaging.
For example, a retroviral vector can comprise a 5' long terminal repeat (LTR),
a 3'
LTR, a packaging signal, a bacterial origin of replication, and a selectable
marker. The
antigenic peptide DNA is inserted into a position between the 5' LTR and 3'
LTR, such that
transcription from the 5' LTR promoter transcribes the cloned DNA. The 5' LTR
comprises a promoter, including but not limited to an LTR promoter, an R
region, a US
region and a primer binding site, in that order. Nucleotide sequences of these
LTR elements
are well known in the art. A heterologous promoter as well as multiple drug
selection
markers may also be included in the expression vector to facilitate selection
of infected cells
(see McLauchlin et al., 1990, Prog. Nucleic Acid Res. and Molec. Biol. 38:91-
135;
Morgenstern et al., 1990, Nucleic Acid Res. 18: 3587-3596; Choulika et al.,
1996, J. Virol
70:1792-1798; Boesen et al., 1994, Biotherapy 6: 291-302; Salmons and
Gunzberg, 1993,
Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in
Genetics
and Devel. 3:110-114).
Other useful eukaryotic host-vector system may include yeast and insect
systems. In yeast, a number of vectors containing constitutive or inducible
promoters may
be used with Saccharomyces ce~evisiae (baker's yeast), Schizosacclaaronayces
pombe
(fission yeast), Pichia pastoris, and Hansenula polymorplza (methylotropic
yeasts). For a
review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. S et
al., Greene
Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expression
and Secretion
Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 1987, Acad.
Press,
N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press,
Wash., D.C.,
Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in
Enzymology,
Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The
Molecular
Biology of the Yeast Sacclaaromyces, 1982, Eds. Strathern et al., Cold Spring
Harbor Press,
Vols. I and II.
In an insect system a baculovirus, Autographa californica nuclear polyhidrosis
virus
(AcNPV), can be used as a vector to express an antigenic peptide in Spodoptera
frugiperda
-32-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
cells. The antigenic protein DNA may be cloned into non-essential regions (for
example
the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for
example the polyhedrin promoter). These recombinant viruses are then used to
infect host
cells in which the inserted DNA is expressed (see, e.g., Smith et al., 1983,
J. Virol. 46:584;
Smith, U.S. Patent No. 4,215,051).
The expression vector must be used with a compatible host cell which may be
derived from a prokaryotic or an eukaryotic organism, including, but not
limited to bacteria,
yeasts, insects, mammals, and humans. Any cell type that is compatible with
the expression
vector may be used, including those that have been cultured in vitro or
genetically
engineered. Host cells may be obtained from normal or affected subjects,
including healthy
humans, patients with cancer or an infectious disease, private laboratory
deposits, public
culture collections such as the American Type Culture Collection, or from
commercial
suppliers.
Preferred mammalian host cells include but are not limited to those derived
from
humans, monkeys and rodents, (see, for example, Kriegler M. in "Gene Transfer
and
Expression: A Laboratory Manual", New York, Freeman & Co. 1990), such as
monkey
kidney cell line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic
kidney line (293, 293-EBNA), or 293 cells subcloned for growth in suspension
culture
(Graham et al., 1977, J. Gen. Virol. 36:59), baby hamster kidney cells (BHK,
ATCC CCL
10), Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, 1980, Proc.
Natl. Acad.
Sci. 77:4216), mouse sertoli cells (Mather, 1980, Biol. Reprod. 23:243-251),
mouse
fibroblast cells (NgI-3T3), monkey kidney cells (CVI ATCC CCL 70), african
green
monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells
(HELA, ATCC CCL 2), canine kidney cells (MDCI~, ATCC CCL 34), buffalo rat
liver
cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75), human
liver
cells (Hep G2, HB 8065), and mouse mammary tumor cells (MMT 060562, ATCC
CCL51). Exemplary cancer cell types used for demonstrating the utility of
recombinant
cells as a cancer vaccine are provided as follows: mouse fibroblast cell line,
NIH3T3,
mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815,
mouse
lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma
cell line,
B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung
carcinoma cell
lines, SCLC#2 and SCLC#7.
The recombinant cells may be cultured under standard conditions of
temperature,
incubation time, optical density, and media composition. Alternatively, a
cells may be
-33-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
cultured under conditions emulating the nutritional and physiological
requirements of a cell
in which the antigenic protein is endogenously expressed.
The antigenic protein, or an antigenic portion thereof, can be purified by any
methods appropriate for the protein, and then used to form complexes with CD91
ligands as
described in section 4.4, below.
4.1.3.2 PURIFICATION METHODS FOR RECOMBINANT
ANTIGENIC PROTEINS
Generally, the recombinant antigenic proteins of the invention can be
recovered and
purified from recombinant cell cultures by known methods, including ammonium
sulfate
precipitation, acid 'extraction, anion or cation exchange chromatography,
phosphocellulose
chromatography, immunoaffinity chromatography, hydroxyapatite chromatography,
and
lectin chromatography.
In one embodiment, the invention provides methods for purification of
recombinant
antigenic proteins by affinity purification. The principle of affinity
chromatography well
known in the art. One approach is based on specific molecular interactions
between an
affinity label present on the antigenic protein and its binding partner. A
second approach,
immunoaffinity chromatography, relies on the immunospecific binding of an
antibody to an
epitope present on the tag.
Described below are several methods based on specific molecular interactions
of a
tag and its binding partner. Protein A affinity chromatography, a method that
is generally
applicable to purifying recombinant antigenic proteins that are fused to the
constant regions
of immunoglobulin, is a well known technique in the art. Staphylococcus
protein A is a 42
kD polypeptide that binds specifically to a region located between the second
and third
constant regions of heavy chain immunoglobulins. Because of the Fc domains of
different
classes, subclasses and species of immunoglobulins, affinity of protein A for
human Fc
regions is strong, but may vary with other species. Subclasses that are less
preferred
include human IgG-3, and most rat subclasses. For certain subclasses, protein
G (of
Streptococci) may be used in place of protein A in the purification. Protein-A
sepharose
(Pharmacia or Biorad) is a commonly used solid phase for affinity purification
of
antibodies, and can be used essentially in the same manner for the
purification of an
antigenic protein of interest fused to an immunoglobulin Fc fragment.
Antigenic protein of
interest present in cell lysate or, if secreted by the cell, in the
supernatant, binds specifically
to protein A on the solid phase, while the contaminants are washed away. Bound
antigenic
-34-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
protein of interest can be eluted by various buffer systems known in the art,
including a
succession of citrate, acetate and glycine-HCl buffers which gradually lowers
the pH. This
method is less preferred if the recombinant cells also produce antibodies
which will be
copurified with the antigenic protein. See, for example, Langone, 1982, J.
hnrnunol. Meth.
51:3; Wilchek et al., 1982, Biochem. Intl. 4: 629; Sjobring et al., 1991, J.
Biol. Chem.
26:399; page 617-618, in Antibodies A Laboratory Manual, edited by Harlow and
Lane,
Cold Spring Harbor laboratory, 1988.
Alternatively, a polyhistidine tag may be used, in which case, the antigenic
protein
can be purified by metal chelate chromatography. The polyhistidine tag,
usually a sequence
of six histidines, has a high affinity for divalent metal ions, such as nickel
ions (Ni2+), which
can be irmnobilized on a solid phase, such as nitrilotriacetic acid matrices.
Polyhistidine
has a well characterized affinity for Nia+-NTA-agarose, and can be eluted with
either of two
mild treatments: imidazole (0.1-0.2 M) will effectively compete with the resin
for binding
sites; or lowering the pH just below 6.0 will protonate the histidine side-
chains and disrupt
the binding. The purification method comprises loading the cell culture
supernatant onto
the Ni2+-NTA-agarose column, washing the contaminants through, and eluting the
antigenic
protein of interest with imidazole or weak acid. Ni2+-NTA-agarose can be
obtained from
commercial suppliers such as Sigma (St. Louis) and Qiagen. Antibodies that
recognize the
polyhistidine tag are also available which can be used to detect and quantify
the antigenic
protein.
Another exemplary affinity label that can be used is the glutathione-S-
transferase
(GST) sequence, originally cloned from the helminth, Schistosoma japohicum. In
general,
an antigenic protein-GST fusion expressed in a prokaryotic host cell, such as
E. coli, can be
purified from the cell culture supernatant by absorption with glutathione
agarose beads,
followed by elution in the presence of free reduced glutathione at neutral pH.
Denaturing
conditions are not required at any stage during purification, and therefore,
it may be
desirable co-purification of CD91 ligands and antigenic proteins, for use in
the loading of
immobilized CD91 ligands with antigenic proteins. Moreover, since GST is known
to form
dimers under certain conditions, dimeric antigenic proteins may be obtained.
See, Smith,
1993, Methods Mol. Cell Bio. 4:220-229.
Another useful affinity label that can be used is the maltose binding protein
(MBP)
of E. coli, which is encoded by the malE gene. The secreted protein-MBP
present in the
cell supernatant binds to amylose resin while contaminants are washed away.
The bound
-35-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
antigenic protein-MBP is eluted from the amylose resin by maltose. See, e.g.,
Guan et al.,
1987, Gene 67:21-30.
The second approach for purifying antigenic proteins is applicable to affinity
labels
that contain an epitope for which polyclonal or monoclonal antibodies are
available. Various methods known in the art for purification of protein by
immunospecific
binding, such as immunoaffinity chromatography, and immunoprecipitation, can
be
used. See, e.g., Chapter 13 in Antibodies A Laboratory Manual, edited by
Harlow and
Lane, Cold Spring Harbor laboratory, 1988; and Chapter 8, Sections I and II,
in Current
Protocols in Immunology, ed. by Coligan et al., John Wiley, 1991; the
disclosure of which
are both incorporated by reference herein.
4.2 SOURCES OF CD91 LIGANDS
CD91 ligands suitable for the complexes and fusion proteins of the invention
can be
isolated from the cells and/or serum of a subject by various methods described
in the art
(see, e.g., Bensadoun et al., Methods in Molecular Biology, Vol. 109, Humana
Press Inc.,
Totowa, NJ, (Doolittle and Reue eds.), pages 145-50).
Amino acid sequences and nucleotide sequences of many CD91 ligands are
generally available in sequence databases, such as GenBank. Computer programs,
such as
Entrez, can be used to browse the database, and retrieve any amino acid
sequence and
genetic sequence data of interest by accession number. These databases can
also be
searched to identify sequences with various degrees of similarities to a query
sequence
using programs, such as FASTA and BLAST, which rank the similar sequences by
alignment scores and statistics.
For example, the following is a list of GenBank accession numbers for some
CD91
ligands: ApoE (GenBank Accession Number: LPHUE), Lipoprotein lipase (GenBank
Accession Number: LIHUL), Hepatic lipase (GenBank Accession Number: AAC34206),
Factor IXa (GenBank Accession Number: I~FHLI), Factor VIIIa (GenBank Accession
Number: P00451, GenBank Accession Number: EZHL)), Factor VIIa/TFPI (GenBank
Accession Number: KFHU7, GenBank Accession Number: P10646), MMP-13 (GenBank
Accession Number: P45452), MMP-9 (GenBank Accession Number: CAC07541),
Pregnancy Zone Protein (GenBank Accession Number: 529738), PAI-1, Antithrombin
III
(GenBank Accession Number: AAB40025), Tissue factor pathway inhibitor
(TFPI)(GenBank Accession Number: KFHU7), Heparin cofactor II (GenBank
Accession
Number: NP_000176), Thrombospondin-1 (GenBank Accession Number: NP 003237),
-36-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Thrombospondin-2 (GenBank Accession Number: NP_003238), Lactoferrin (GenBank
Accession Number: AAN84790) and HIV-Tat protein (GenBank Accession Number:
BAA12992, GenBank Accession Number: BAA13000)
Once the nucleotide sequence of a CD91 ligand has been identified, CD91
ligands
can be produced by any method known in the art, such as the recombinant
methods
described above (Section 4.1.3) for the production of antigenic proteins. CD91
ligand-
encoding cDNA or genomic DNA may be obtained from any species, for example, by
PCR
amplification. Oligonucleotide primers representing known nucleic acid
sequences of
related CD91 ligands can be used as PCR primers. One can choose to synthesize
several
different degenerate primers for use in the PCR reactions. It is also possible
to vary the
stringency of hybridization conditions used in priming the PCR reactions, to
allow for
greater or lesser degrees of nucleotide sequence similarity between the known
CD91 ligand
nucleotide sequence and the nucleic acid homolog being isolated. For cross
species
hybridization, low stringency conditions are preferred. For same species
hybridization,
moderately stringent conditions are preferred. After successful amplification,
the sequence
encoding a CD91 ligand may be cloned and sequenced. If the size of the coding
region of
the CD91 ligand gene being amplified is too large to be amplified in a single
PCR, several
PCR covering the entire gene, preferably with overlapping regions, may be
carned out, and
the products of the PCR ligated together to form the entire coding sequence.
Alternatively,
if a segment of a CD91 ligand gene is amplified, that segment may be cloned,
and utilized
as a probe to isolate a complete cDNA or genomic clone. The CD91 ligand gene
may then
be cloned into an appropriate expression vector, and be produced, propogated,
isolated and
purified according to methods for recombinant production of proteins, such as
those
described for recombinant production of antigenic proteins described in
Section 4.1.3,
above.
An alternative to producing CD91 ligands by recombinant techniques is peptide
synthesis. For example, a CD91 ligand, or a fragment thereof, can be
synthesized using
methods known in the art, such as those described for the synthesis of
antigenic proteins, in
Section 4.1.2, above. In this case, conventional peptide synthesis may be
used, or other
synthetic protocols well known in the art.
Purification of the resulting CD91 ligand, or fragment thereof, may then be
accomplished using conventional procedures, such as preparative HPLC using gel
permeation, partition and/or ion exchange chromatography. The choice of
appropriate
matrices and buffers are well known in the art.
-37-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
4.3 COMPLEXES AND FUSION PROTEINS COMPRISING A CD91
LIGAND FRAGMENT AND AN ANTIGENIC MOLECULE
The invention contemplates complexes and fusion proteins comprising a CD91
ligand fragment and an antigenic molecule. Preferably the CD91 ligand fragment
contains
a receptor binding domain. Receptor binding domains have been identified for
various
CD91 ligands (see, e.g., Swertfeger and Hui, 2001, J. Biol. Chem. 276:25043
and
Weisgraber et al., 1983, J. Biol. Chem. 258:12348 (receptor binding domain for
Apo E at
amino acid residues 141-155); Williams et al., 1994, J. Biol. Chem. 269:8653
and Krapp
et al., 1995, J. Lipid Res. 36:2362-73 (receptor binding domain for
lipoprotein lipase at
amino acid residues 313-448); Krapp et al., 1996, J. Lipid Res. 37:926-36
(receptor binding
domain lipoprotein lipase at amino acid residues 378-448); Nykjaer et al.,
1994, J. Biol.
Chem. 269:31747-55 (receptor binding domain for lipoprotein lipase at amino
acid residue
378-423); Krapp et al., 1996, J. Lipid Res. 37:926-36 (sequences within the
carboxyl-
terminal 73 amino acids are important for surface binding of human hepatic
lipase, and four
basic amino acids [K4~z, R473~ Ra7a~ located at the carboxyl terminal region
may play a
critical role); Bassel-Duby et al., 1992, J. Biol. Chem. 267:9668, Grobmyer et
al., 1993, J.
Biol. Chem. 268: 13291, Nykjaer et al., 1994, J. Biol. Chem. 269:25668, Larsen
et al.,
1989, Blood 73:1842, Camani et al., 1994, J. Biol. Chem. 269:5770 (receptor
binding
domain for tPA at amino acid residues 4-50 (fibronectin fingerlike domain), 51-
87 (EGF
domain), 4-87 (finger + EGF domain)); Nykjaer et al., 1994, J. Biol. Chem.
269:25668
(receptor binding domain for uPA a chain at amino acid residues 50-131
(kringle domain)
and ~3 chain at amino acid residues 159-411 (serine protease domain);
Warshawsky et al.,
1995, Biochem. 34:3403, Warshawsky et al., 1993, J. Biol. Chem. 268:22046 and
Warshawsky et al., 1994, J. Biol. Chem. 269:3325 (receptor binding domain for
RAP at
amino acid residues 1-114, 12-107, 200-319 and 115-319); Liu et al., 2000,
Nat. Med.
6(12):1380-87 (receptor binding domain for HIV TAT, core domain at amino acid
residues
37-48 and basic domain at amino acid residues 48-57); Mikhailenko et al.,
1997, J. Biol.
Chem. 272:6784-91 and Chen et al., 1996, Biochem. J., 318:959-963 (receptor
binding
domain for thrombospondin-1, N-terminal heparin binding domain (mouse) at
amino acid
residues 1-214); Neels et al., 2000, Blood 96:3459-65 (CD91 does not bind
factor IX
zymogen--it only binds the active species; factor IXa binds to CD91 at a site
outside of the
active site); Sarafanov et al., 2001, J. Biol. Chem. 276:11970-79 (receptor
binding domain
for factor VIIIa, A2 domain at amino acid residues 373-740); Saenko et al.,
1999, J. Biol.
Chem. 274:37685-92 (receptor binding domain for factor VIIIa, A2 domain at
amino acid
-38-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
residues 484509); Lenting et al., 1999, J. Biol. Chem. 274:23734-39 (receptor
binding
domain for factor VIIIa, light chain-C2 domain at amino acid residues 2173-
2332);
Iakhiaev et al., 1999, J. Biol. Chem. 274:36995-37003 and Hamik et al., 1999,
J. Biol.
Chem. 274:4962-69 (receptor binding domain for TFPI at C terminal domain);
Steffansson
et al., 1998, J. Biol. Chem. 273:6358-66, Horn et al., 1996, Fibrinolysis
10:21, Rodenburg
et al., 1998, Biochem. J. 329:55-63 and Horn et al., 1998, Thromb. Haemost.
80:822-8
(receptor binding domain for PAI-I, heparin binding domain (basic residues);
high affinity
site generated in PAI-1 when in complex with a proteinase); Jensen et al.,
1996, Biochim.
Biophys. Acta. 1293:254-8 and Arbelaez et al., 1997, Protein Exp. Purif.
10:301-8
(receptor binding domain for pregnancy zone protein (PZP) at C-terminal
fragment);
Nielsen et al., 1996, J. Biol. Chem. 27 1:12909-12, VanLeuven et al., 1986, J.
Biol. Chem.
261:11369-73 and Sottrup-Jensen et al., 1986, FEBS Lett 205:20-24 (receptor
binding
domain for cY2M, 20 kDa C-terminal stretch at amino acid residues 1314-1451 (
human
a2M)); Meilinger et al., 1999, J. Biol. Chem. 274:38091-96 (receptor binding
domain for
complement C3 present in activated C3 (thioester cleaved forms)); and
Kinoshita et al.,
2001, J. Neurosci. 21:8354-61 and Kounnas et al., 1995, Cell 82:331-40
(receptor binding
domain for APP on extracellular and intracellular domains; isoforms containing
a Kunitz
proteinase inhibitor (KPI) domain). The preceding references are incorporated
by reference
in their entirety.
Many assays are known in the art for identifying receptor binding domains of
molecules of interest. Any such assay can be used to identify suitable CD91
ligand
fragments for use in the invention. Once the binding domain is identified, a
fragment of the
CD91 ligand can be made or constructed using any method known in the art,
including, by
way of example and not limitation, proteolytic cleavage, and recombinant or
synthetic
techniques.
One such method for identifying a binding domain of a CD91 ligand comprises
constructing mutants of the CD91 ligand (for example, the mutants may contain
a deletion,
truncation or point mutation). The mutants are then assayed for their ability
to interact with
a receptor which is known to bind to the CD91 ligand. In this manner, the
binding domain
of the ligand can be identified.
Another such method for identifying a binding domain of a CD91 ligand
comprises
assaying for the ability of a particular fragment or epitope of a CD91 ligand
to modulate
receptor-ligand binding events.
-39-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Another example of a method for identifying a binding domain of a CD91 ligand
comprises the use of an antibody that binds to a particular epitope on the
CD91 ligand. The
CD91 ligand is first exposed to the antibody, and then the CD91 ligand is
assayed for its
ability to bind a receptor of interest.
Any method known in the art for detecting receptor binding may be used (see,
e.g.,
Stanford and Horton, 2002, Receptors: Structure and Function, A Practical
Approach"
Oxford University Press and Current Protocols in Molecular Biology, Ausubel et
al. (eds.),
Greene Publishing Associates and Wiley Interscience, New York, which are both
incorporated by reference herein).
Specifically, cell-free assays known in the art may be used to detect receptor-
ligand
binding such as those described by Sjolander and Urbaniczky, 1991. Anal. Chem.
63:2338-
2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705, which are
incoporated by
reference herein. Other cell free assays that may be used include assays in
which an
immobilized protein is tested for its ability to bind to a putative cognate
protein (e.g.,
ELISA and affinity matrices), and two hybrid or three hybrid assays (see,
e.g., U.S. Patent
No. 5,283,317; Zervos et al., 1993, Cell 72:223-232; Madura et al., 1993, J.
Biol. Chem.
268:12046-12054; Bartel et al., 1993, Biotechniques 14:920-924; Iwabachi et
al., 1993,
Oncogene 8:1693-1696; and PCT W094/10300; the preceding disclosures are hereby
incorporated by reference).
In addition, any cell based assay known in the art may be used to detect
receptor-
ligand interactions. In such an assay, a receptor protein or biologically
active portion
thereof is contacted with either 1) mutant ligand molecules, 2) fragments or
epitopes of
ligand, or 3) ligand molecules in the presence of an antibody, or fragments or
epitopes of
ligand. The receptor-ligand binding interaction is then monitored, for
example, by cellular
phenotype or a receptor mediated cellular response (e.g., uptake of ligand or
modulation of
signal transduction).
4.4 IN VITRO COMPLEXING
In an alternative embodiment, complexes of CD91 ligands with antigenic
molecules
are produced in vitro. Imrnunogenic CD91 ligand-antigenic molecule complexes
can be
generated ira vitro by covalent or non-covalent coupling of a CD91 ligand with
an antigenic
molecule. As described in Section 4.1 above, antigenic molecules may be
isolated from
various sources, chemically synthesized, or produced recombinantly. CD91
ligands may
also be prepared by a variety of methods, as described in Section 4.2, above.
After isolation
-40-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
of CD91 ligands and antigenic molecules, complexes are produced ira vitro.
Procedures for
forming such CD91 ligand-antigenic molecule complexes and preferred, exemplary
protocols for covalently and noncovalently forming such complexes are provided
herein. Such methods can be readily adapted for medium or large scale
production of the
immunotherapeutic or prophylactic vaccines of the invention.
4.4.1 FORMATION OF NON-COVALENT CD91 LIGAND-
ANTIGENIC MOLECULE COMPLEXES
Any method known in the art can be used for preparing non-covalent complexes
of
the invention. The formation of non-covalent immunogenic complexes of CD91
ligands
and antigenic molecules can be achieved under conditions that favor complex
formation.
The following example describes a method for preparing non
covalent immunogenic complexes of CD91 ligands and antigenic molecules:
CD91 ligands are prepared, as described in Section 4.2, above. The
antigenic molecules and the CD91 ligand are mixed together. The mixture is
then incubated in a suitable buffer. The preparations are then centrifuged
through filter to remove any unbound peptide. If the CD91 ligand is bound
to a solid phase, the CD91 ligand-antigenic molecule complexes formed can
be washed free of unbound antigenic molecules prior to eluting the CD91
ligand-antigenic molecule complexes off the solid phase. The association of
the antigenic molecules with the CD91 ligands can be assayed by any
method known in the art.
The following example describes a preferred method for preparing non-
covalent immunogenic complexes of CD91 ligands and antigenic molecules:
CD91 ligands are prepared, as described in Section 4.2, above. The
antigenic molecules (100~.g) and the pretreated CD91 ligand (l~,g-lmg) are
mixed together to give an approximately 5:1 antigenic molecule : CD91
ligand molar ratio. The mixture is then incubated for 15 minutes to 3 hours
at 4° to 45°C in a suitable binding buffer such as one
containing 20mM
sodium phosphate, pH 7.2, 350mM NaCI, 3mM MgCl2, 1mM phenyl methyl
sulfonyl fluoride (PMSF), and 1-10 mM ADP. The preparations are
centrifuged through a Centricon 10 assembly (Millipore) to remove any
unbound peptide. If the CD91 ligand is bound to a solid phase, the CD91
ligand-antigenic molecule complexes formed can be washed free of unbound
-41 -
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
antigenic molecules prior to eluting the CD91 ligand-antigenic molecule
complexes off the solid phase. The association of the antigenic molecules
with the CD91 ligands can be assayed by SDS-PAGE.
Following complexing, the immunogenic CD91 ligand-antigenic molecule
complexes can optionally be assayed in vivo or in vitro using, for example,
the methods
described in Section 4.7, below.
In another embodiment of the invention, a linker molecule may be used to non-
covalently tether a CD91 ligand to an antigenic molecule. A linker molecule is
a bispecific
molecule in which one moiety of the linker molecule binds a CD91 ligand, while
another
moiety of the linker molecule binds an antigenic molecule. Such linker
molecules are
described in PCT WO O1/7~772, which is incorporated by reference herein. In a
particular
embodiment, the linker molecule is covalently linked to an antigenic molecule
and non-
covalently linked to a CD91 ligand. In another embodiment, the linker molecule
is non-
covalently linked to an antigenic molecule and non-covalently linked to a CD91
ligand. In
yet another embodiment, the linker molecule is non-covalently linked to an
antigenic
molecule and covalently linked to a CD91 ligand.
4.4.2 FORMATION OF COVALENT CD91 LIGAND-ANTIGENIC
MOLECULE COMPLEXES
As an alternative to non-covalent complexes, antigenic molecules covalently
attached to CD91 ligands may be used as vaccines to elicit an immune response.
To prepare such covalent CD91 ligand-antigenic molecule complexes, CD91
ligands and antigenic molecules are prepared, as described in Sections 4.2 and
4.1,
respectively. The CD91 ligand and the antigenic molecule are then covalently
coupled
using any method known in the axt.
In one embodiment, CD91 ligands may be covalently coupled to antigenic
molecules by chemical cross-linking. Chemical cross-linking agents, such as
aldehydes,
ketones and glyoxals, and their use are well known in the art and are suitable
for use in the
invention. Cross-linking agents, including but not limited to, protein A,
glutaraldehyde,
carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA),
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) can be used. For
example, in
one embodiment of the invention, glutaraldehyde may be used to cross-link a
CD91 ligand
and an antigenic molecule. In addition, other methods for covalent attachment
of proteins
-42-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
known in the art can be used in the invention, such as photo cross-linking
(See Current
Protocols in Molecular Biology, Ausubel et al. (eds.), Greene Publishing
Associates and
Wiley Interscience, New York and Binder et al., 2000, Nature Imrnunol. 1:151-
155, which
are incorporated by reference herein).
4.5 CD91 LIGAND-ANTIGENIC MOLECULE FUSION PROTEINS
In another embodiment, recombinant fusion proteins, comprising a CD91 ligand
fused via a peptide bond to an antigenic protein, may be used to treat or
prevent cancer or
an infectious disease. To produce such a recombinant fusion protein, an
expression vector
is constructed using nucleic acid sequences encoding a CD91 ligand fused to
sequences
encoding an antigenic protein, using recombinant methods known in the art,
such as those
described in Section 4.1.3, above.
CD91 ligand-antigenic molecule fusions are then expressed and isolated. In one
embodiment, the N-terminal portion of a CD91 ligand or CD91 ligand fragment is
fused to
the C-terminal portion of an antigenic molecule. In another embodiment, the C-
terminal
portion of a CD91 ligand or CD91 ligand fragment is fused to the N-terminal
portion of an
antigenic molecule. Such fusion proteins can be used to elicit an immune
response (Suzue
et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:13146-Sl). By specifically
designing the
antigenic protein portion of the molecule, such fusion proteins can be used to
induce an
immune response and in immunotherapy against target diseases or disorders.
4.5.1 RECOMBINANT EXPRESSION AND PRODUCTION OF
CD91 LIGAND- ANTIGENIC MOLECULE FUSION
PROTEINS
To produce CD91 ligand-antigenic molecule fusion proteins, a nucleotide
sequence
encoding a CD91 ligand or a CD91 ligand fragment and an antigenic molecule can
be
introduced into cells including, but not limited to, epithelial cells,
endothelial cells,
keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T
lymphocytes,
B lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
megakaryocytes,
granulocytes; various stem or progenitor cells, in particular hematopoietic
stem or
progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood,
peripheral
blood, fetal liver, etc. The choice of cell type depends on the type of
antigenic protein
being expressed, and can be determined by one of skill in the art.
- 43 -
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Preferably, the cells used in the methods of the invention are of mammalian
origin. Mammals contemplated by this aspect of the invention include humans,
companion
animals (e.g., dogs and cats), livestock animals (e.g., sheep, cattle, goats,
pigs and horses),
laboratory animals (e.g., mice, rats and rabbits), and captive or free wild
animals.
In various embodiments, any cells, preferably human cells, can be used in the
present methods for producing CD91 ligand-antigenic protein fusion proteins.
Introduction
of gene constructs encoding the CD91 ligand and the antigenic molecule can be
carried out
by any method known in the art, including gene therapy art, such as but not
limited to
transfection, electroporation, microinjection, infection with a viral or
bacteriophage vector
containing the nucleic acid sequence encoding the CD91 ligand and antigenic
protein, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer,
spheroplast
fusion, etc. Numerous techniques are known in the art for the introduction of
foreign genes
into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618;
Cohen et al.,
1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29: 69-92) may
be used in
accordance with the present invention, provided that the necessary
developmental and
physiological functions of the recipient cells are not disrupted. The
technique should
provide for the stable transfer of the nucleic acid sequence encoding the CD91
ligand and
antigenic protein to the cell, so that the sequence is expressible and
preferably heritable and
expressible by its cell progeny.
In addition, the methods for recombinant production described in section 4.1.3
and
its subsections hereinabove can be used.
4.6 THERAPEUTIC AND PROPHYLACTIC USES OF CD91 LIGAND-
ANTIGENIC MOLECULE COMPLEXES AND FUSION PROTEINS
The present invention encompasses methods for treatment and prevention of
cancer
and other diseases using complexes or fusion proteins of CD91 ligands and
antigenic
molecules. The present invention also provides methods of inducing an immune
response
to an antigenic molecule using the complexes and fusion proteins of the
invention.
In one embodiment, an immunogenic amount of complex or fusion protein of a
CD91 ligand and an antigenic molecule, wherein the antigenic molecule displays
the
antigenicity of an antigen that causes or is associated with a disease, can be
used in the
treatment or prevention of the disease. In a specific embodiment, the CD91
ligand and the
antigenic molecule of the complex are noncovalently linked. In another
embodiment, the
CD91 ligand and the antigenic molecule of the complex are chemically cross-
linked.
-44-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
In a preferred aspect of the invention, the purified CD91 ligand-antigenic
molecule
complex or fusion protein vaccines may have particular utility in the
treatment of human
cancer or other diseases. It is appreciated, however, that the vaccines
developed using the
principles described herein will be useful in treating diseases of other
mammals, for
example, farm animals including cattle, horses, sheep, goats, and pigs, and
household pets
including cats and dogs.
The invention also provides a method of treating a disease or disorder
amenable to
treatment by induction of an immune response against an antigenic molecule
comprising
administering to a subject having the disease or disorder an immunogenic
amount of a
purified complex comprising the antigenic molecule noncovalently bound to, or
cross-
linked to, a CD91 ligand.
The invention also provides a method of treating a disease or disorder
amenable to
treatment by induction of an immune response against an antigenic molecule
comprising
administering to a subject having the disease or disorder an immunogenic
amount of a
purified fusion protein comprising the antigenic molecule fused via a peptide
bond to a
CD91 ligand.
4.6.1 INDUCING AN IMMUNE RESPONSE TO AN ANTIGEN
The complexes and fusion proteins of the invention can be used to induce an
immune response to an antigenic molecule in a subject.
Without being bound by any particular theory, it is believed that an immune
response to an antigenic molecule is induced by the complexes and fusion
proteins of the
invention by enhancing the uptake of the antigenic molecule by antigen
presenting cells,
including macrophages, dendritic cells and B cells.
4.6.2 PASSIVE IMMUNOTHERAPY
CD91 ligand-antigenic molecule complexes and fusion proteins can also be used
for
passive immunotherapy against cancer or other diseases. Passive immunity is
the short-
term protection of a host, achieved by the administration of pre-formed
antibody (e.g., in
purified form or by administering serum containing a pre-formed
antibody(ies)), directed
against an antigen or pathogenic organism of interest, e.g., a tumor or viral
antigen. For
example, CD91 ligand-antigenic molecule complexes or fusion proteins may be
used to
elicit an immune response in a subject, the sera removed from the subject and
used for
-45-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
treatment or prevention of a disease in a subject having a disease caused by
the presence of
a common antigen.
4.7 DETERMINATION OF IMMUNOGENICITY OF CD91 LIGAND-
ANTIGENIC MOLECULE COMPLEXES AND FUSION PROTEINS
Optionally, CD91 ligand-antigenic molecule complexes and fusion proteins can
be
assayed for immunogenicity using any method known in the art. By way of
example but
not limitation, one of the following three procedures can be used.
4.7.1 MLTC ASSAY
Briefly, mice are injected with the CD91 ligand-antigenic molecule complex or
fusion protein, using any convenient route of administration. As a negative
control, other
mice are injected with CD91 ligand-antigenic molecule complexes or fusion
proteins not
associated with the antigen of interest, or cells containing CD91 ligand-
antigenic molecule
complexes or fusion proteins not associated with the antigen of interest.
Cells containing
the antigen of interest may act as a positive control for the assay. The mice
are injected
twice, 7-10 days apart. Ten days after the last immunization, the spleens are
removed and
the lymphocytes released. The released lymphocytes may be re-stimulated
subsequently in
vitro by the addition of dead cells that expressed the antigen of interest.
For example, 8x106 immune spleen cells may be stimulated with 4x104 mitomycin
C treated or'y irradiated (5-10,000 rads) cells containing the antigen of
interest (or cells
transfected with an appropriate gene, as the case may be) in 3m1 RPMI medium
containing
10% fetal calf serum. In certain cases 33% secondary mixed lymphocyte culture
supernatant may be included in the culture medium as a source of T cell growth
factors
(See, Glasebrook, et al., 1980, J. Exp. Med. 151:876). To test the primary
cytotoxic T cell
response after immunization, spleen cells may be cultured without stimulation.
In some
experiments spleen cells of the immunized mice may also be re-stimulated with
antigenically distinct cells, to determine the specificity of the cytotoxic T
cell response.
Six days later the cultures are tested for cytotoxicity in a 4 hour SICr-
release assay
(see Palladino et al., 1987, Cayace~ Res. 47:5074-5079 and Blachere, et al.,
1993, J.
Immunotherapy 14:352-356). In this assay, the mixed lymphocyte culture is
added to a
target cell suspension to give different effectoraarget (E:T) ratios (usually
1:1 to 40:1). The
target cells are prelabelled by incubating 1x106 target cells in culture
medium containing 20
mCi SlCrlml for one hour at 37°C. The cells are washed three times
following
-46-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
labeling. Each assay point (E:T ratio) is performed in triplicate and the
appropriate controls
incorporated to measure spontaneous SICr release (no lymphocytes added to
assay) and
100% release (cells lysed with detergent). After incubating the cell mixtures
for 4 hours,
the cells are pelletted by centrifugation at 200g for 5 minutes. The amount of
SICr released
into the supernatant is measured by a gamma counter. The percent cytotoxicity
is measured
as cpm in the test sample minus spontaneously released cpm divided by the
total detergent
released cpm minus spontaneously released cpm.
In order to block the MHC class I cascade a concentrated hybridoma supernatant
derived from K-44 hybridoma cells (an anti-MHC class I hybridoma) is added to
the test
samples to a final concentration of 12.5%.
4.7.2 CD4+ T CELL PROLIFERATION ASSAY
Primary T cells are obtained from spleen, fresh blood, or CSF and purified by
centrifugation using FICOLL-PAQUE PLUS (Pharmacia, Upsalla, Sweden)
essentially as
described by Kruse and Sebald, 1992, EMBO J. 11: 3237-3244. The peripheral
blood
mononuclear cells are incubated for 7-10 days with a lysate of cells
expressing an antigen of
interest. Antigen presenting cells may, optionally be added to the culture 24
to 48 hours
prior to the assay, in order to process and present the antigen in the lysate.
The cells are
then harvested by centrifugation, and washed in RPMI 1640 media (GibcoBRL,
Gaithersburg, Md.). 5x104 activated T cells/well (PHA-blasts) are in RPMI 1640
media
containing 10% fetal bovine serum, 10 mM HEPES, pH 7.5, 2 mM L-glutamine, 100
units/ml penicillin G, and 100 pg/ml streptomycin sulphate in 96 well plates
for 72 hrs at
37°C., pulsed with 1 ~uCi 3H-thymidine (DuPont NEN, Boston, Mass.)/well
for 6 hrs,
harvested, and radioactivity measured in a TOPCOUNT scintillation counter
(Packard
Instrument Co., Meriden, Conn.).
4.7.3 ANTIBODY RESPONSE ASSAY
In one embodiment of the invention, the immunogenicity of a CD91 ligand-
antigenic molecule complex or fusion protein is determined by measuring
antibodies
produced in response to the vaccination with the complex or fusion protein, by
an antibody
response assay, such as an enzyme-linked immunosorbent assay (ELISA) assay.
Methods
for such assays are well known in the art (see, e.g., Section 2.1 of Current
Protocols in
Immunology, Coligan et al. (eds.), John Wiley and Sons, Inc. 1997). In
one~mode of the
embodiment, microtitre plates (96-well Immuno Plate II, Nunc) are coated with
50 ~,1/well
-47-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
of a 0.75 pg/ml solution of a purified, non-CD91 ligand-complexed form of the
antigen
used in the vaccine (e.g. A(342) in PBS at 4°C for 16 hours and at
20°C for 1 hour. The
wells are emptied and blocked with 200 ,ul PBS-T-BSA (PBS containing 0.05%
(v/v)
TWEEN 20 and 1% (w/v) bovine serum albumin) per well at 20°C for 1
hour, then washed
3 times with PBS-T. Fifty,ul/well of plasma or CSF from a vaccinated animal
(such as a
model mouse or a human patient) is applied at 20°C for 1 hour, and the
plates are washed 3
times with PBS-T. The anti-antigenic molecule antibody activity is then
measured
calorimetrically after incubating at 20°C for 1 hour with SOp,I/well of
sheep anti-mouse or
anti-human immunoglobulin, as appropriate, conjugated with horseradish
peroxidase
(Arnersham) diluted 1:1,500 in PBS-T-BSA and (after 3 further PBS-T washes as
above)
with 50 ,ul of an o-phenylene diamine (OPD)-H202 substrate solution. The
reaction is
stopped with 150 ~l of 2M H2S04 after 5 minutes and absorbance is determined
in a
Kontron SLT-210 photometer (SLT Lab-instr., Zurich, Switzerland) at 492 nm
(ref. 620
nm).
4.8 DOSAGE REGIMENS
For treatment of cancer, dosages of CD91 ligand-antigenic molecule complexes
and
fusion proteins may be extrapolated from prior art methods established in
experimental
tumor models (Blachere et al., 1993, J. hnmunotherapy 14:352-356). For other
diseases,
dosages may be extrapolated from prior art methods established in an
appropriate
experimental disease model. Extrapolation to human dosages is based on body
weight and
surface area. For example, prior art methods of extrapolating human dosage
based on body
weight can be carried out as follows: since the conversion factor for
converting the mouse
dosage to human dosage is Dose Human per kg = Dose Mouse per kg x 12 (See
Freireich,
E.J., et al., 1966, Cancer Chemotherap. Rep. 50:219-244), the effective dose
of CD91
ligand-antigenic molecule complexes or fusion proteins in humans weighing 70kg
should
be lmg/kg-12 x 70, i.e., about 6mg (5.8mg).
Drug doses are also given in milligrams per square meter of body surface area
because this method rather than body weight achieves a good correlation to
certain
metabolic and excretionary functions (Shirkey, 1965, JAMA 193:443). Moreover,
body
surface area can be used as a common denominator for drug dosage in adults and
children
as well as in different animal species as indicated below in Table 1
(Freireich et al., 1966,
Cancer Chemotherap. Rep. 50:219-244).
-48-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
TABLE 1
REPRESENTATIVE
SURFACE AREA
TO WEIGHT
RATIOS (km) FOR VARIOUS SPECIESI
Species Body Weight Surface Area km Factor
~Sqm~
Mouse 0.02 0.0066 3.0
Rat 0.15 0.025 5.9
Monke 3-00 0.24 12
Dog 8-00 0.40 20
Human, Child20 0.80 25
Adult 60 1.6 37
Example:To express a mg/kg dose in any given species as the equivalent mg/sq m
dose,
multiply the dose by the appropriate km factor. In adult human, 100mg/kg is
equivalent to 100 mg/kg x 37 kg/sq m = 3700 mg/sq m.
As an alternative to the standard extrapolation, the dosage range for the
complexes
and fusion proteins of the invention is l,ug to 5 mg, 1 ~,g to 1 mg, or 5 ~Cg
to 500 ~.g, with a
preferred range being 10 ~,g to 250 ,ug. Representative dosages include 10
~,g, 25 ,ug, 50 ,ug,
75 ,ug, 100 ~,g, 150 ,ug, 200 ,ug and 250 ~,g.
The doses recited above are preferably given once weekly for a period of about
4-6
weeks, and the mode or site of administration is preferably varied with each
administration. In a preferred example, intradermal administrations are given,
with each
site of administration varied sequentially. Thus, by way of example and not
limitation, the
first injection may be given subcutaneously on the left arm, the second on the
right arm, the
third on the left belly, the fourth on the right belly, the fifth on the left
thigh, the sixth on the
right thigh, etc. The same site may be repeated after a gap of one or more
injections. Also,
split injections may be given. Thus, for example, half the dose may be given
in one site and
the other half on an other site on the same day.
Alternatively, the mode of administration is sequentially varied, e.g., weekly
injections are given in sequence intradermally, intramuscularly, intravenously
or
intraperitoneally.
After 4-6 weeks, further injections are preferably given at two-week intervals
over a
period of time of one month. Later inj ections may be given monthly. The pace
of later
injections may be modified, depending upon the patient's clinical progress and
responsiveness to the immunotherapy.
Freireich, et al., 1966, Cancer Chemotherap. Rep. 50:219-244.
-49-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
For prevention, the subject is immunized by administration of the vaccine
formulation, in at least one dose, and preferably two or more doses. However,
the subject
may be administered as many doses as is required to maintain a state of
immunity. For
example, boosters given at regular intervals, i.e., at six months or yearly,
may be desirable
in order to sustain immunity at an effective level.
4.9 VACCINE FORMULATION
Complexes and fusion proteins of CD91 ligands and antigenic molecules purified
by
the methods of the invention may be formulated into pharmaceutical
preparations (vaccines)
for administration to mammals for treatment or prevention of cancer or other
diseases. Drug solubility and the site of absorption are factors which should
be considered
when choosing the route of administration of a therapeutic agent. CD91 ligand-
antigenic
molecule complexes of the invention may be administered using any desired
route of
administration, including but not limited to, e.g., intradermally,
subcutaneously,
intravenously or intramuscularly, although intradermally is preferred.
Advantages of
intradermal administration include use of lower doses and rapid absorption,
respectively. Mucosal routes of administration include, but are not limited
to, oral, rectal
and nasal administration. Preparations for mucosal administrations are
suitable in various
formulations as described below. The route of administration can be varied
during a course
of treatment.
The invention also contemplates pharmaceutical compositions (vaccines)
comprising a CD91 ligand-antigenic molecule complex or fusion protein and an
adjuvant. The complex or fusion protein is preferably purified. More than one
adjuvant can
be used in pharmaceutical compositions of the invention. Some adjuvants that
may be used
in the invention include, but are not limited to: heat shock proteins, saponin
adjuvants (e.g.,
QS-21), alpha 2 macroglobulin, lipopolysaccharide (LPS), immunostimulatory
oligonucleotides which include CpG oligonucleotides, and complexes of heat
shock
proteins and antigenic molecules, such as peptides, or the like. Furthermore,
the following
patents and printed publications disclose immunostimulatory oligonucleotides
which
include CpG oligonucleotides that can be used in the compositions of the
invention: U.S.
Patents 6,207,646; 6,339,068; 6,239,116; 6,429,199; and PCT Patent
publication, WO
01/22972, WO 00/06588, by Krieg et al.; WO 01/83503; WO 01/55370; and WO
01/12804
by Agrawal; WO 02/052002 by Fearon et al.; WO 01/35991 by Tuck et al.; WO
01/12223
by Van Nest; WO 98/55495; WO 99/62923 by Schwartz; U.S. Patent 6,406,705 by
Davis et
-50-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
al.; and PCT Patent publication WO 02/26757 by Kandimalla et al., all of the
forgoing are
incorporated herein by reference in their entireties. In a preferred
embodiment, a
combination of saponin adjuvants (e.g., QS-21) and immunostimulatory
oligonucleotides
(e.g., a CpG oligonucleotide) are used.
Other suitable adjuvants that can be used in the invention can be found in A
Compendium of Vaccine Adjuvants and Excipients (2"a Edition), Vogel, F.,
Powell, M.,
and Alving, C., in Vaccine Design - The Subunit and Adjuvant Approach, Powell,
M.,
Newman, M., Burdman, J., Editors, Plenum Press, New York, 1995, pp. 141-227,
and 2"a
Meeting on Novel Adjuvants Currently In/Close to Human Clinical Testing, World
Health
Organi i ation - Organization Mondiale de la Sante Foundation Merieux, Annecy,
France, S-
7 June 2000, Kenney, R., Rabinovich, N.R., Pichyangkul, S., Price, V., and
Engers, H.,
Vaccine, 20 (2002) 2155-63. All of which are incorporated herein by reference.
In a specific embodiment, a complex or fusion protein of the invention is
administered separately from one or more adjuvants. Tn one embodiment, a
complex or
fusion protein of the invention and an adjuvant are administered in a sequence
and within a
time interval such that the complex or fusion protein and an adjuvant can act
together to
provide an increased benefit that is greater than either administered alone.
In a specific
embodiment, the complex or fusion protein and an adjuvant are administered
sufficiently
close in time so as to provide the desired therapeutic or prophylactic
outcome. Each can be
administered simultaneously and/or separately, in any appropriate form and by
any suitable
route. In one embodiment, the complex or fusion protein and an adjuvant are
administered
by different routes of administration. In an alternate embodiment, each is
administered by
the same route of administration. The complex or fusion protein and an
adjuvant can be
administered at the same or different sites, e.g. arm and leg.
In specific embodiments, a complex or fusion protein of the invention and an
adjuvant are administered less than 1 hour apart, at about 1 hour apart, 1
hour to 2 hours
apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours
apart, 5 hours to
6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to
9 hours apart, 9
hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours
apart, no more than
24 hours apart or no more than 48 hours apart. In other specific embodiments,
a complex
or fusion protein of the invention and an adjuvant are administered 2 to 4
days apart, 4 to 6
days apart, 1 week a part, 1 to 2 weeks apart, 2 to 4 weeks apart, one moth
apart, 1 to 2
months apart, or 2 or more months apart. In preferred embodiments, a complex
or fusion
protein of the invention and an adjuvant are administered in a time frame
where both are
-51-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
still active. One skilled in the art would be able to determine such a time
frame by
determining the half life of each administered component.
Compositions comprising CD91 ligand-antigenic molecule complexes or fusion
proteins formulated in a compatible pharmaceutical Garner may be prepared,
packaged, and
labeled for treatment of the indicated cancer or infectious disease. In
preferred aspects, an
amount of CD91 ligand-antigenic molecule complex or fusion protein is
administered to a
human that is in the range of about 2 to 150 pg, preferably 20 to 200 ,ug,
most preferably
about 5 pg, given once weekly for about 4-6 weeks, intradermally with the site
of
administration varied sequentially.
If the complex or fusion protein is water-soluble, then it may be formulated
in an
appropriate buffer, for example, phosphate buffered saline or other
physiologically
compatible solutions. Alternatively, if the resulting complex has poor
solubility in aqueous
solvents, then it may be formulated with a non-ionic surfactant such as Tween,
or
polyethylene glycol. Thus, the CD91 ligand-antigenic molecule complexes and
fusion
proteins and their physiologically acceptable solvates may be formulated for
administration
by inhalation or insufflation (either through the mouth or the nose) or oral,
buccal,
parenteral, rectal administration.
For oral administration, the pharmaceutical preparation may be in liquid form,
for
example, solutions, syrups or suspensions, or may be presented as a drug
product for
reconstitution with water or other suitable vehicle before use. Such liquid
preparations may
be prepared by conventional means with pharmaceutically acceptable additives
such as
suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated
edible fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily
esters, or fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-
hydroxybenzoates or sorbic acid). The pharmaceutical compositions may take the
form of,
for example, tablets or capsules prepared by conventional means with
pharmaceutically
acceptable excipients such as binding agents (e.g., pregelatinized maize
starch, polyvinyl
pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline
cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium
stearate, talc or
silica); disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g.,
sodium lauryl sulphate). The tablets may be coated by methods well-known in
the art.
Preparations for oral administration may be suitably formulated to give
controlled
release of the complexes. Such compositions may take the form of tablets or
lozenges
formulated in conventional manner.
-52-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
In a specific embodiment, the complexes and compositions of the present
invention
are administered intrathecally by an implant be placed in or near the lesioned
area. Suitable
implants include, but are not limited to, gelfoam, wax, liposome or
microparticle-based
implants. Such compositions are preferably used when it is desired to achieve
sustained
release of the CD91 ligand-antigenic molecule complexes.
For administration by inhalation, the complexes and fusion proteins may be
conveniently delivered in the form of an aerosol spray presentation from
pressurized packs
or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other
suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined by
providing a valve to
deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in
an inhaler or
insufflator may be formulated containing a powder mix of the complexes and a
suitable
powder base such as lactose or starch.
The complexes and fusion proteins may be formulated for parenteral
administration
by injection, e.g., by bolus injection or continuous infusion. Formulations
for injection may
be presented in unit dosage form, e.g., in ampoules or in mufti-dose
containers, with an
added preservative. The compositions may take such forms as suspensions,
solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory agents such
as
suspending, stabilizing and/or dispersing agents. Alternatively, the active
ingredient may
be in powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water,
before use.
The complexes and fusion proteins may also be formulated in rectal
compositions
such as suppositories or retention enemas, e.g., containing conventional
suppository bases
such as cocoa butter or other glycerides.
In addition to the formulations described previously, the complexes and fusion
proteins may also be formulated as a depot preparation. Such long acting
formulations may
be administered by implantation (for example, subcutaneously or
intramuscularly) or by
intramuscular injection. Thus, for example, the complexes may be formulated
with suitable
polymeric or hydrophobic materials (for example, as an emulsion in an
acceptable oil) or
ion exchange resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble
salt. Liposomes and emulsions are well known examples of delivery vehicles or
carriers for
hydrophilic drugs.
The compositions may, if desired, be presented in a pack or dispenser device
which
may contain one or more unit dosage forms containing the active ingredient.
The pack may
- 53
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
for example comprise metal or plastic foil, such as a blister pack. The pack
or dispenser
device may be accompanied by instructions for administration. Compositions
comprising a
compound of the invention formulated in a compatible pharmaceutical carrier
may also be
prepared, placed in an appropriate container, and labeled for treatment of an
indicated
condition.
The invention also provides kits for carrying out the therapeutic regimens of
the
invention. Such kits comprise in one or more containers therapeutically or
prophylactically
effective amounts of CD91 ligand-antigenic molecule complexes or fusion
proteins in
pharmaceutically acceptable form (preferably purified), optionally with a
pharmaceutically
acceptable carrier. The kit optionally further comprises in the same or
different container
an adjuvant, e.g., a saponin. The CD91 ligand-antigenic molecule complexes or
fusion
proteins in a vial of a kit of the invention may be in the form of a
pharmaceutically
acceptable solution, e.g., in combination with sterile saline, dextrose
solution, or buffered
solution, or other pharmaceutically acceptable sterile fluid. Alternatively,
the complex or
fusion protein may be lyophilized or desiccated; in this instance, the kit
optionally further
comprises in a container a pharmaceutically acceptable solution (e.g., saline,
dextrose
solution, etc.), preferably sterile, to reconstitute the complex or fusion
protein to form a
solution for injection purposes. Kits directed to the treatment or prevention
of cancer or
infectious disease optionally further comprise in a separate container,
antigen presenting
cells (APCs), which may be sensitized. If the APCs are not sensitized, the kit
may further
comprise a purified antigenic molecule for sensitizing the APCs. The APCs are
preferably
purified. The invention also contemplates kits which, in addition to the
components above,
also include one or more therapeutic or preventive compositions known in the
art in the
treatment or prevention of a disease or disorder. Examples of such
compositions, listed by
way of example and not limitation are found in section 4.12, below.
In another embodiment, a kit of the invention further comprises a needle or
syringe,
preferably packaged in sterile form, for injecting the complex or fusion
protein, and/or a
packaged alcohol pad. Instructions are optionally included for administration
of CD91
ligand-antigenic molecule complexes or fusion proteins by a clinician or by
the patient.
4.10 DETERMINATION OF VACCINE EFFICACY
The immunopotency of the complexes and fusion proteins of the invention can be
determined by monitoring the immune response in test animals following
immunization
with the complexes and fusion proteins of the invention, or by use of any
immunoassay
-54-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
known in the art. Generation of a humoral (antibody) response and/or cell-
mediated
immunity, may be taken as an indication of an immune response. Test animals
may include
mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and
eventually human
subj ects.
Methods of introducing the vaccine may include oral, intracerebral,
intradermal,
transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal or any
other standard routes of immunization. The immune response of the test
subjects can be
analyzed by various approaches such as: the reactivity of the resultant immune
serum to the
antigen of interest, as assayed by known techniques, e.g., immunosorbant assay
(ELISA),
immunoblots, radioimmunoprecipitations, etc., or by protection of the
immunized host
against cancer or the infectious disease.
As one example of suitable animal testing of a vaccine protective a disease,
the
vaccine of the invention may be tested in rabbits for the ability to induce an
antibody
response to the antigenic molecule. Male specific-pathogen-free (SPF) young
adult New
Zealand White rabbits may be used. The test group each receives a fixed
concentration of
the vaccine. A control group receives an injection of 1 mM Tris-HCl pH 9.0
without the
antigen molecule.
Blood samples may be drawn from the rabbits every one or two weeks, and serum
analyzed for antibodies to the antigenic molecule. The presence of antibodies
specific for
the antigen may be assayed, e.g., using an ELISA assay.
4.11 MONITORING OF EFFECTS DURING IMMUNOTHERAPY
Optionally, the effect of immunotherapy with CD91 ligand-antigenic molecule
complexes or fusion proteins on progression of a disease can be monitored by
any methods
known to one skilled in the art. In addition, cellular immunity may optionally
be
monitored by methods including but not limited to measuring: a) delayed
hypersensitivity
as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes
in vitro; c)
levels of antigenic molecule.
Delayed hypersensitivity skin tests are also of great value in the overall
immunocompetence and cellular immunity to an antigen. Inability to react to a
battery of
common skin antigens is termed anergy (Sato et al., 1995, Clin. Imlnunol.
Pathol. 74:35-
43).
Proper technique of skin testing requires that the antigens be stored sterile
at 4°C,
protected from light and reconstituted shortly before use. A 25- or 27-gauge
needle ensures
-55-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
intradermal, rather than subcutaneous, administration of antigen. Twenty-four
and 48 hours
after intradermal administration of the antigen, the largest dimensions of
both erythema and
induration are measured with a ruler. Hypoactivity to any given antigen or
group of
antigens is confirmed by testing with higher concentrations of antigen or, in
ambiguous
circumstances, by a repeat test with an intermediate test.
In another optional method, the activity of cytolytic T-lymphocytes can be
assessed
in vitro using the following method. Eight x 106 peripheral blood-derived T
lymphocytes
isolated by the Ficoll-Hypaque centrifugation gradient technique, are
restimulated with
4x104 mitomycinC-treated cells in 3m1 RPMI medium containing 10% fetal calf
serum. In
some experiments, 33% secondary mixed lymphocyte culture supernatant or IL-2,
is
included in the culture medium as a source of T cell growth factors.
In order to measure the primary response of cytolytic T-lymphocytes after
immunization, T cells are cultured without the stimulator cells. In other
experiments, T
cells are restimulated with antigenically distinct cells. After six days, the
cultures are tested
for cytotoxicity in a 4 hour SICr-release assay. The spontaneous SICr-release
of the targets
should reach a level less than 20%. For the anti-MHC class I blocking
activity, a tenfold
concentrated supernatant of W6/32 hybridoma is added to the test at a final
concentration of
12.5% (Heike et al., J. Immunotherapy 15: 165-174).
In immunization procedures, the amount of immunogen to be used and the
immunization schedule will be determined by a physician skilled in the axt and
will be
administered by reference to the immune response and antibody titers of the
subj ect.
4.12 COMBINATION THERAPY
The complexes and fusion proteins of the invention can be used in combination
with
other therapeutic compositions known in the art in the treatment or prevention
of a disease
or disorder, such as immunotherapeutic agents, antineoplastic agents, anti-
viral agents, anti-
fungal agents, antibiotics and anti-inflammatory agents. The complexes or
fusion proteins
may be coadministered with one or more therapeutic compositions known in the
art or may
be administered after or before one or more therapeutic compositions known in
the art. The
invention contemplates combinations of the complexes or fusion proteins of the
invention
and one or more therapeutic compositions know in the art to be useful in
treating or
preventing any of the diseases or disorders listed in sections 4.13-4.18
below. The
administration of the complexes and fusion proteins of the invention can
augment the effect
of anti-cancer agents or anti-infectives, and vice versa. With combination
therapy, additive
-56-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
potency or additive therapeutic effects can be observed. In combinations where
synergistic
outcomes are achieved (where the therapeutic efficacy is greater than
additive), dosages that
alone would be suboptimal, can be employed. The use of combination therapy can
also
provide better therapeutic profiles than the administration of the treatment
modality, or the
complexes and fusion proteins of the invention alone. Additive or synergistic
effects may
allow the dosage andlor dosing frequency of either or both modalities to be
adjusted to
reduce or avoid unwanted or adverse effects. The invention also encompasses
pharmaceutical compositions comprising a CD91 ligand-antigenic molecule
complex or
fusion protein and one or more therapeutic compositions know in the art to be
useful in
treating or preventing any of the diseases or disorders listed in sections
4.13-4.18
below. The invention also encompasses pharmaceutical compositions comprising
an
CD91 ligand-antigenic molecule complex or fusion protein and a reduced dose of
one or
more therapeutic compositions know in the art to be useful in treating or
preventing any of
the diseases or disorders listed in sections 4.13-4.18 below. As used herein,
the phrase
"reduced dose" refers to a dose that is below the normally administered range,
i. e., below
the standard dose as suggested by the Physicians' Desk Reference, 54th Edition
(2000) or a
similar reference.
For example, the complexes or fusion proteins of the invention may be
administered
in combination with one or more chemotherapeutic agent. Such chemotherapeutic
agents
are known in the art and include but are not limited to: methotrexate, taxol,
mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,
ifosfamide,
nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine,
etoposides,
campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,
plicamycin,
mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel,
and docetaxel,
doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel and
paclitaxel,
leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrosoureas such
as carmustine
and lomustine, vinca alkaloids, platinum compounds, mitomycin, gemcitabine,
hexamethylinelamine, topotecan, tyrosine kinase inhibitors, tyrphostins,
GleevecTM
(imatinib mesylate), herbimycin A, genistein, erbstatin, and lavendustin A.
In other embodiments, suitable chemotherapeutics include, but are not limited
to,
methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea,
cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin,
dacarbazine,
procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, S-
fluorouracil, BCNLT,
irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin,
dactinomycin,
-57-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,
paclitaxel, and
docetaxel. Moreover, the anti-cancer agent can be, but is not limited to, a
drug listed in
Table 2.
Alkvlatin~ agents
TABLE 2
Nitrogen mustards: Cyclophosphamide
Ifosfamide
Trofosfamide
Chlorambucil
Nitrosoureas: Carmustine (BCNU)
Lomustine (CCNU)
Alkylsulphonates: Busulfan
Treosulfan
Triazenes: Dacarbazine
Platinum containing compounds:Cisplatin
Carboplatin
Plant Alkaloids
Vinca alkaloids: Vincristine
Vinblastine
Vindesine
Vinorelbine
Taxoids: Paclitaxel
Docetaxol
DNA Tonoisomerase Inhibitors
Epipodophyllins: Etoposide
Teniposide
Topotecan
9-aminocamptothecin
Camptothecin
Crisnatol
mitomycins: Mitomycin C
Anti-metabolites
Anti-folates:
DHFR inhibitors: Methotrexate
Trimetrexate
IIVVIP dehydrogenase Mycophenolic acid
Inhibitors:
Tiazofurin
Ribavirin
EICAR
Ribonuclotide reductaseHydroxyurea
Inhibitors:
Pvrimidine analogs:
Deferoxamine
Uracil analogs: 5-Fluorouracil
Floxuridine
Doxifluridine
-58-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Ratitrexed
Cytosine analogs: Cytarabine (ara C)
Cytosine arabinoside
Fludarabine
Purine analogs: Mercaptopurine
Thioguanine
DNA Antimetabolites:3-HP
2'-deoxy-5-fluorouridine
5-HP
alpha-TGDR
aphidicolin glycinate
ara-C
5-aza-2'-deoxycytidine
beta-TGDR
cyclocytidine
guanazole
inosine glycodialdehyde
macebecin II
pyrazoloimidazole
Hormonal therapies:
Receptor antagonists:
Anti-estrogen: Tamoxifen
Raloxifene
Megestrol
LHRH agonists: Goscrclin
Leuprolide acetate
Anti-androgens: Flutamide
Bicalutamide
Retinoids/Deltoids
Vitamin D3 analogs: EB 1089
CB 1093
KH 1060
Photodynamic therapies:Vertoporfin (BPD-MA)
Phthalocyanine
Photosensitizer Pc4
Demethoxy-hypocrellin A
(2BA-2-DMHA)
Cytokines: Interferon- a
Interferon- 'y
Tumor necrosis factor
Angiogenesis Inhibitors:Angiostatin (plasminogen
fragment)
antiangiogenic antithrombin
III
Angiozyme
ABT-627
Bay 12-9566
Benefin
Bevacizumab
BMS-275291
cartilage-derived inhibitor
(CDI)
-59-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
CAI
CD59 complement fragment
CEP-7055
Col 3
Combretastatin A-4
Endostatin (collagen XVIII
fragment)
Fibronectin fragment
Gro-beta
Halofuginone
Heparinases
Heparin hexasaccharide fragment
HMV833
Human chorionic gonadotropin
(hCG)
IM-862
Interferon alpha/beta/gamma
Interferon inducible protein
(IP-10)
Interleukin-12
Kringle 5 (plasminogen fragment)
Marimastat
Metalloproteinase inhibitors
(TMs)
2-Methoxyestradiol
MMI 270 (CGS 27023A)
MoAb IMC-1 C 11
Neovastat
NM-3
Panzem
PI-88
Placental ribonuclease inhibitor
Plasminogen activator inhibitor
Platelet factor-4 (PF4)
Prinomastat
Prolactin l6kD fragment
Proliferin-related protein (PRP)
PTK 787/ZK 222594
Retinoids
Solimastat
Squalamine
SS 3304
SU 5416
SU6668
SU11248
Tetrahydrocortisol-S
tetrathiomolybdate
thalidomide
Thrombospondin-1 (TSP-1)
TNP-470
-60-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
Transforming growth factor-beta
(TGF-b)
Vasculostatin
Vasostatin (calreticulin fragment)
ZD6126
ZD 6474
farnesyl transferase inhibitors
(FTI)
bisphosphonates
Antimitotic agents: allocolchicine
Halichondrin B
colchicine
colchicine derivative
dolstatin 10
maytansine
rhizoxin
thiocolchicine
trityl cysteine
Others:
Isoprenylation inhibitors:
Dopaminergic neurotoxins:1-methyl-4-phenylpyridinium
ion
Cell cycle inhibitors:Staurosporine
Actinomycins: Actinomycin D
Dactinomycin
Bleomycins: Bleomycin A2
Bleomycin B2
Peplomycin
Anthracyclines: Daunorubicin
Doxorubicin (adriamycin)
Idarubicin
Epirubicin
Pirarubicin
Zorubicin
Mitoxantrone
MDR inhibitors: Verapamil
Ca2+ATPase inhibitors: Thapsigargin
Additional anti-cancer agents that may be used in the methods and compositions
of
the present invention include, but are not limited to: acivicin; aclarubicin;
acodazole
hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone
acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;
asperlin;
azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;
bisantrene
hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium;
bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer;
carboplatin;
carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil;
cirolemycin;
-61-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;
dacarbazine;
dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin;
dezaguanine;
dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin
hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;
edatrexate;
eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;
epirubicin
hydrochloride; erbulozole; esorubicin hydrochloride; estramustine;
estramustine phosphate
sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole
hydrochloride;
fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil;
flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride;
hydroxyurea;
idarubicin hydrochloride; ifosfamide; ilinofosine; interleukin II (including
recombinant
interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon
alfa-nl ; interferon
alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan
hydrochloride;
lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
lometrexol sodium;
lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril;
mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa;
mitindomide;
mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane;
mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin;
oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin
sulfate;
perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin;
plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride;
puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide;
safingol;
safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin;
spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin;
streptozocin;
sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride;
temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa;
tiazofurin;
tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate;
trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil
mustard; uredepa;
vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;
vindesine sulfate;
vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine
tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin;
zorubicin
hydrochloride.
Other anti-cancer drugs that can be used include, but are not limited to: 20-
epi-1,25
dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene;
adecypenol;
-62-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;
amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;
anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;
antarelix;
anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma;
antiestrogen;
antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis
gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine
deaminase;
asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin
3; azasetron;
azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine;
betaclamycin
B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene;
bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;
buthionine sulfoximine;
calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;
capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700;
cartilage
derived inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B;
cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine;
clomifene analogues; clotrimazole; collismycin A; collismycin B;
combretastatin A4;
combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin
8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam;
cypemycin;
cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;
dehydrodidemnin
B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone;
didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-
;
dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron;
doxifluridine;
droloxifene; dronabinol; duocannycin SA; ebselen; ecomustine; edelfosine;
edrecolomab;
eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine
analogue; estrogen
agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole;
fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;
fluasterone;
fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin;
fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;
gelatinase
inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin;
hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone;
ilmofosine;
ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like
growth
factor-1 receptor inhibitor; interferon agonists; interferons; interleukins;
iobenguane;
iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole;
isohomohalicondrin B;
itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;
leinamycin;
-63-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting
factor; leukocyte
alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole;
linear polyamine analogue; lipophilic disaccharide peptide; lipophilic
platinum compounds;
lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
losoxantrone; lovastatin;
loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;
maitansine;
mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix
metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim;
mismatched double
stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide;
mitotoxin
fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal
antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium
cell
wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor
suppressor
1-based therapy; mustard anti-cancer agent; mycaperoxide B; mycobacterial cell
wall
extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin;
nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;
nemorubicin;
neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators;
nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone;
oligonucleotides;
onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;
ormaplatin;
osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel
derivatives; palauamine; palinitoylrhizoxin; pamidronic acid; panaxytriol;
panomifene;
parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate
sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate;
phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin;
piritrexim;
placetin A; placetin B; plasminogen activator inhibitor; platinum complex;
platinum
compounds; platinum-triamine complex; porfimer sodium; porfiromycin;
prednisone;
propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based
immune
modulator; protein kinase C inhibitor; protein kinase C inhibitors,
microalgal; protein
tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors;
purpurins;
pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists;
raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras
inhibitors; ras-GAP
inhibitor; retelliptine demethylated; rhenium Re 1 ~6 etidronate; rhizoxin;
ribozymes; RII
retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1;
ruboxyl;
safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;
semustine;
senescence derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal
-64
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
transduction modulators; single chain antigen binding protein; sizofiran;
sobuzoxane;
sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding
protein;
sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1;
squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide;
stromelysin
inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen
methiodide;
tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;
telomerase inhibitors;
temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine;
thiocoraline; thrombopoietin; thrombopoietin mimetic; thyrnalfasin;
thymopoietin receptor
agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin;
tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell factor;
translation
inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;
triptorelin; tropisetron;
turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;
ubenimex; urogenital
sinus-derived growth inhibitory factor; urokinase receptor antagonists;
vapreotide; variolin
B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin;
vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;
and zinostatin
stimalamer.
The complexes or fusion proteins of the invention may also be administered in
combination with one or more cytokines or immunomodulatory agents. In various
embodiments, the cytokine is selected from the group consisting of IL-lc~ IL-
1(3, IL-2, IL-
3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFNc~ IFN~3,
IFN~y, TNFa, TNF(3,
G-CSF, GM-CSF, TGF-,Q, IL-15, IL-18, GM-CSF, INF-'y, INF-c~ SLC, endothelial
monocyte activating protein-2 (EMAP2), MIP-3c~ MIl'-3,Q, or an MHC gene, such
as HLA-
B7. Addtionally, other exemplary cytokines include other members of the TNF
family,
including but not limited to TNF-a-related apoptosis-inducing ligand (TRAIL),
TNF-a
related activation-induced cytokine (TRANCE), TNF-a related weak inducer of
apoptosis
(TWEAK), CD40 ligand (CD40L), LT-a, LT-,Q, OX40L, CD4OL, Fast, CD27L, CD30L,
4-1BBL, APRIL, LIGHT, TLl, TNFSF16, TNFSF17, and AITR-L, or a functional
portion
thereof. See, e.g., Kwon et al., 1999, Curr. Opin. Imnunol. 11:340-345 for a
general
review of the TNF family.
The complexes or fusion proteins of the invention may also be administered in
combination with one or more antifungal agents. Antifungal agents suitable for
use in the
invention, include but are not limited to: polyenes (e.g., amphotericin b,
candicidin,
mepartricin, natamycin, and nystatin), allylamines (e.g., butenafine, and
naftifine),
-65-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
imidazoles (e.g., bifonazole, butoconazole, chlordantoin, flutrimazole,
isoconazole,
ketoconazole, and lanoconazole), thiocarbamates (e.g., tolciclate, tolindate,
and tolnaftate),
triazoles (e.g., fluconazole, itraconazole, saperconazole, and terconazole),
bromosalicylchloranilide, buclosamide, calcium propionate, chlorphenesin,
ciclopirox,
azaserine, griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin,
siccanin,
tubercidin, and viridin.
Also encompassed by the invention are combination therapies using a CD91
ligand-
antigenic molecule complex or fusion protein with an antibiotic agent.
Antibiotic agents
suitable for use in the invention, include but are not limited to:
aminoglycoside antibiotics
(e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin, neomycin,
neomycin,
undecylenate, netilinicin, paromomycin, ribostamycin, sisomicin, and
spectinomycin),
amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, and
thiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin),
carbacephems (e.g.,
loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g.,
cefaclor,
cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole,
cefpiramide, and
cefpirome), cephamycins (e.g., cefbuperazone, cefinetazole, and cefininox),
monobactams
(e.g., aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef, and
moxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil,
amoxicillin,
bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin,
fenbenicillin,
floxacillin, penamccillin, penethamate hydriodide, penicillin o-benethamine,
penicillin 0,
penicillin V, penicillin V benzathine, penicillin V hydrabamine,
penimepicycline, and
phencihicillin potassium), lincosamides (e.g., clindamycin, and lincomycin),
macrolides
(e.g., azithromycin, carbomycin, clarithomycin, dirithromycin, erythromycin,
and
erythromycin acistrate), amphomycin, bacitracin, capreomycin, colistin,
enduracidin,
enviomycin, tetracyclines (e.g., apicycline, chlortetracycline, clomocycline,
and
demeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans
(e.g.,
furaltadone, and furazolium chloride), quinolones and analogs thereof (e.g.,
cinoxacin,
ciprofloxacin, clinafloxacin, flumequine, and grepagloxacin), sulfonamides
(e.g., acetyl
sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,
sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,
glucosulfone sodium,
and solasulfone), cycloserine, mupirocin and tuberin.
Also encompassed by the invention are combination therapies using a CD91
ligand-
antigenic molecule complex or fusion protein with an antiviral agent. Such
antiviral agents
_66_
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
include but are not limited to: ribavirin, rifampicin, AZT, ddI, ddC,
acyclovir and
ganciclovir.
The invention also contemplates the combination of a CD91 ligand-antigenic
molecule complex or fusion protein and one or more immunoreactive
reagents. Immunoreactive reagents include antibodies, molecules or proteins
engineered to
include (i.e., comprise) the antigen binding portion of an antibody, molecules
or proteins
engineered to include an antigen binding domain that mediates antibody
dependent immune
responses, a peptide or domain that interacts specifically with the antigen of
interest, or any
antigen binding domain that interacts with an antigen/epitope of interest, and
the domain of
the constant region of an antibody that mediates antibody dependent immune
effector cell
responses or processes. Examples of such domains or regions within the Ab
constant
region that can be used in the present invention include those disclosed in
Reddy et al.,
2000, J. Immunol. 164(4):1925-33; Coloma et al., 1997, Nat Biotechnol.
15(2):159-63;
Carayannopoulos et al., 1994, Proc Natl. Aead. Sci. U.S.A. 91(18):8348-52;
Morrison,
1992, Annu Recombinant expression vector Immunol. 10:239-65; Traunecker et
al., 1992,
Int. J. Cancer Suppl., 7:51-2; Gillies et al., 1990, Hum. Antibodies
Hybridomas, 1(1):47-54;
each of which is incorporated herein by reference in its entirety. Examples of
antibodies
useful in the methods and compositions of the invention include, but are not
limited to,
MDX-010 (Medarex, NJ) which is a humanized anti-CTLA-4 antibody currently in
clinic
for the treatment of prostate cancer; SYNAGIS~ (MedTinmune, MD) which is a
humanized
anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment
of patients
with RSV infection; HERCEPTIN~ (Trastuzumab) (Genentech, CA) which is a
humanized
anti-HER2 monoclonal antibody for the treatment of patients with metastatic
breast cancer;
REMICADE~ (infliximab) (Centocor, PA) which is a chimeric anti-TNFa monoclonal
antibody for the treatment of patients with Crone's disease; REOPRO~
(abciximab)
(Centocor) which is an anti-glycoprotein IIb/IIIa receptor on the platelets
for the prevention
of clot formation; ZENAPAX~ (daclizumab) (Roche Pharmaceuticals, Switzerland)
which
is an immunosuppressive, humanized anti-CD25 monoclonal antibody for the
prevention of
acute renal allograft rejection. Other examples are a humanized anti-CD18
F(ab')Z
(Genentech); CDP860 which is a humanized anti-CD18 F(ab')Z (Celltech, UK);
PRO542
which is an anti-HIV gp120 antibody fused with CD4 (Progenics/Genzyme
Transgenics);
Ostavir which is a human anti Hepatitis B virus antibody (Protein Design
Lab/Novartis);
PROTOV1RTM which is a humanized anti-CMV IgGl antibody (Protein Design
Lab/Novartis); MAK-195 (SEGARD) which is a marine anti-TNF-a F(ab')Z (Knoll
-67-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
PharmaBASF); IC14 which is an anti-CD14 antibody (ICOS Pharm); a humanized
anti-
VEGF IgGl antibody (Genentech); OVAREXTM which is a marine anti-CA 125
antibody
(Altarex); PANOREXTM which is a marine anti-17-IA cell surface antigen IgG2a
antibody (Glaxo Wellcome/Centocor); BEC2 which is a marine anti-idiotype (GD3
epitope) IgG antibody (ImClone System);1MC-C225 which is a chimeric anti-EGFR
IgG
antibody (ImClone System); VITAXINTM which is a humanized anti-aV(~3 integrin
antibody (Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03 which is a
humanized anti CD52 IgGl antibody (Leukosite); Smart M195 which is a humanized
anti-
CD33 IgG antibody (Protein Design LablKanebo); RITUXAN~'M which is a chimeric
anti-
CD20 IgGl antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDETM
which is a humanized anti-CD22 IgG antibody (Imrnunomedics); Smart ID10 which
is a
humanized anti-HLA antibody (Protein Design Lab); ONCOLYMTM (Lym-1) is a
radiolabelled marine anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone); ABX-
IL8 is a human anti-IL8 antibody (Abgenix); anti-CD 11 a is a humanized IgGl
antibody
(Genentech/Xoma); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-
114
is a primatized anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALINTM is a
radiolabelled marine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a
humanized
anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody
(IDEC);
IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3
is a
humanized anti-CD3 IgG (Protein Design Lab); SG1.1 is a humanized anti-
complement
factor 5 (CS) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-a
antibody
(CATBASF); CDP870 is a humanized anti-TNF-a Fab fragment (Celltech); IDEC-151
is a
primatized anti-CD4 IgGl antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is
a
human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti-
TNF-a IgG4 antibody (Celltech); LDP-02 is a humanized anti-a4~i7 antibody
(LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody
(Ortho
Biotech); ANTOVATM is a humanized anti-CD40L IgG antibody (Biogen); ANTEGRENTM
is a humanized anti-VLA-4 IgG antibody (Elan); MDX-33 is a human anti-CD64
(Fc~yR)
antibody (Medarex/Centeon); SCH55700 is a humanized anti-IL-5 IgG4 antibody
(Celltech/Schering); SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4
antibodies, respectively, (SmithKline Beecham); rhuMab-E25 is a humanized anti-
IgE IgGl
antibody (Genentech/Norvartis/Tanox Biosystems); ABX-CBL is a marine anti CD-
147
IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody (MedimmuneBio
Transplant); Orthoclone/OKT3 is a marine anti-CD3 IgG2a antibody (ortho
Biotech);
_68_
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
SIMLTLECTTM is a chimeric anti-CD25 IgGl antibody (Novartis Pharm); LDP-O1 is
a
humanized anti-~i2-integrin IgG antibody (LeukoSite); Anti-LFA-1 is a murine
anti CD18
F(ab')a (Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-via antibody
(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VII antibody
(Centocor). Preferred antibodies for use include anti-CTLA-4 antibodies and
anti-41BB
antibodies. The above-listed immunoreactive reagents, as well as any other
imrnunoreactive reagents, may be administered according to any regimen known
to those of
skill in the art, including the regimens recommended by the suppliers of the
immunoreactive reagents.
4.13 TARGET CANCERS
Types of cancers that can be treated or prevented by the compositions and
methods
of the present invention include, but are not limited to, human sarcomas and
carcinomas,
e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lyrnphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer,
ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma,
seminoma, embryonal carcinoma, Wilins' tumor, cervical cancer, testicular
tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute
myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic
and
erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia
and
chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's
disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and
heavy
chain disease.
In a specific embodiment, the cancer is metastatic. In another specific
embodiment,
the patient having a cancer is immunosuppressed by reason of having undergone
anti-cancer
-69-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
therapy (e.g., chemotherapy radiation) prior to administration of the CD91
ligand-antigenic
molecule complexes or fusion proteins.
There are many reasons why immunotherapy as provided by the present invention
is
desired for use in cancer patients. First, if cancer patients are
immunosuppressed, surgery
with anesthesia and subsequent chemotherapy may worsen the immunosuppression.
With
appropriate immunotherapy in the preoperative period, this immunosuppression
may be
prevented or reversed. This could lead to fewer infectious complications and
to accelerated
wound healing. Second, tumor bulk is minimal following surgery and
immunotherapy is
most likely to be effective in this situation. A third reason is the
possibility that tumor cells
are shed into the circulation at surgery and effective immunotherapy applied
at this time can
eliminate these cells.
The preventive and therapeutic compositions and methods of the invention are
directed at enhancing the immunocompetence of the cancer patient either before
surgery, at
or after surgery, and to induce tumor-specific immunity to cancer cells, with
the objective
being inhibition of cancer, and with the ultimate clinical objective being
total cancer
regression and eradication. The methods of the invention can also be used in
individuals at
enhanced risk of a particular type of cancer, e.g., due to familial history or
environmental
risk factors.
4.14 TARGET INFECTIOUS DISEASES
Infectious diseases that can be treated or prevented by the compositions and
methods of the present invention are caused by infectious agents including,
but not limited
to, viruses, bacteria, fungi protozoa and parasites. The invention is not
limited to treating or
preventing infectious diseases caused by intracellular pathogens.
Viral diseases that can be treated or prevented by the methods of the present
invention include, but are not limited to, those caused by hepatitis type A,
hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-
I), herpes
simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,
respiratory syncytial
virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus,
huntavirus,
coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human
immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type
II (HIV-
II).
Bacterial diseases that can be treated or prevented by the methods of the
present
invention are caused by bacteria including, but not limited to, bacteria that
have an
-70-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
intracellular stage in its life cycle, mycobacteria, rickettsia, mycoplasma,
neisseria and
legionella.
Protozoal diseases that can be treated or prevented by the methods of the
present
invention are caused by protozoa including, but not limited to, leishmania,
kokzidioa, and
trypanosorna.
Parasitic diseases that can be treated or prevented by the methods of the
present
invention are caused by parasites including, but not limited to, chlarnydia
and rickettsia.
4.15 TARGET NEURODEGENERATIVE DISEASES
Neurodegenerative disorders that can be prevented or treated using the
compositions
and methods of the invention include disorders relating to the central nervous
system and/or
peripheral nervous system including, but not limited to, cognitive and
neurodegenerative
disorders such as Alzheimer's Disease, age-related loss of cognitive function
and senile
dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's
Disease, cerebral and
progressive supranuclear palsy, Guam disease, Lewy body dementia, prion
diseases, such as
spongiform encephalopathies, e.g., Creutzfeldt-Jakob disease, polyglutamine
diseases, such
as Huntington's disease, myotonic dystrophy, Freidrich's ataxia and other
ataxias, well as
Gilles de la Tourette's syndrome, seizure disorders such as epilepsy and
chronic seizure
disorder, stroke, brain or spinal cord trauma, A>DS dementia, alcoholism,
autism, retinal
ischemia, glaucoma, autonomic function disorders such as hypertension and
sleep disorders,
and neuropsychiatric disorders that include, but are not limited to
schizophrenia,
schizoaffective disorder, attention deficit disorder, dysthymic disorder,
major depressive
disorder, mania, obsessive-compulsive disorder, psychoactive substance use
disorders,
anxiety, panic disorder, as well as unipolar and bipolar affective disorders.
Additional
neuropsychiatric and neurodegenerative disorders include, for example, those
listed in the
American Psychiatric Association's Diagnostic and Statistical manual of Mental
Disorders
(DSM), the most current version of which is incorporated herein by reference
in its entirety.
4.16 TARGET ENDOCRINE/METABOLIC DISEASES
Endocrine/metabolic diseases that can be prevented or treated using the
compositions and methods of the invention include, but are not limited to,
hyperlipidemia,
hypercholesterolemia and general disorders of lipid and/or cholesterol
metabolism.
-71-
CA 02515123 2005-08-04
WO 2004/069207 PCT/US2004/003865
4.17 TARGET VASCULAR DISEASES
Vascular diseases that can be treated or prevented by the compositions and
methods
of the invention include, but are not limited to, hypertensive heart disease,
hypertensive
renal disease, secondary hypertension and atherosclerosis.
4.18 TARGET AUTOIMMUNE DISEASES
Autoimmune diseases that can be treated or prevented by administration of the
antigenic molecules or fusion proteins of the invention include, but are not
limited to,
insulin-dependent diabetes mellitus (i.e., IDDM, or autoimmune diabetes),
multiple
sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma,
polymyositis,
chronic active hepatitis, mixed connective tissue disease, primary biliary
cirrhosis,
pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease,
vitiligo, gluten-
sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune
neutropenia,
idiopathic thrombocytopenia purpura, rheumatoid arthritis, cirrhosis,
pemphigus vulgaris,
autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid
lupus,
ulcerative colitis, and dense deposit disease.
All references cited herein are incorporated herein by reference in their
entirety and
for all purposes to the same extent as if each individual publication or
patent or patent
application was specifically and individually indicated to be incorporated by
reference in its
entirety for all purposes.
Many modifications and variations of this invention can be made without
departing
from its spirit and scope, as will be apparent to those skilled in the art.
The specific
embodiments described herein are offered by way of example only, and the
invention is to
be limited only by the terms of the appended claims along with the full scope
of equivalents
to which such claims are entitled.
_72_
