U.S. patent application number 09/992613 was filed with the patent office on 2002-05-23 for immunotherapeutic stress protein-peptide complexes against cancer.
This patent application is currently assigned to Mount Sinai School of Medicine of New York University. Invention is credited to Srivastava, Pramod K..
Application Number | 20020061316 09/992613 |
Document ID | / |
Family ID | 23226518 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061316 |
Kind Code |
A1 |
Srivastava, Pramod K. |
May 23, 2002 |
Immunotherapeutic stress protein-peptide complexes against
cancer
Abstract
Disclosed is a method for inhibiting the proliferation of a
tumor in a mammal. The method involves the steps of (a) isolating a
stress protein-peptide complex from tumor cells previously removed
from the mammal and (b) administering the isolated stress
protein-peptide complex back to the mammal in order to stimulate in
the mammal an immune response against the tumor from which the
complex was isolated. Stress protein-peptide complexes having
particular utility in the practice of the instant invention include
the Hsp70-peptide, Hsp90-peptide and gp96-peptide complexes.
Inventors: |
Srivastava, Pramod K.;
(Avon, CT) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
Mount Sinai School of Medicine of
New York University
|
Family ID: |
23226518 |
Appl. No.: |
09/992613 |
Filed: |
November 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09992613 |
Nov 14, 2001 |
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09489218 |
Jan 21, 2000 |
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09489218 |
Jan 21, 2000 |
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09061365 |
Apr 16, 1998 |
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09061365 |
Apr 16, 1998 |
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08315892 |
Sep 30, 1994 |
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Current U.S.
Class: |
424/277.1 ;
424/185.1 |
Current CPC
Class: |
A61K 39/0005 20130101;
A61K 2039/622 20130101; A61P 35/04 20180101; A61K 39/00 20130101;
A61P 35/02 20180101; A61K 39/001176 20180801; A61P 37/04 20180101;
A61K 2039/6043 20130101; A61K 38/19 20130101; C07K 14/4702
20130101; A61P 35/00 20180101; A61K 38/19 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/277.1 ;
424/185.1 |
International
Class: |
A61K 039/00 |
Claims
What is claimed is:
1. A method for inhibiting proliferation of a tumor in a mammal,
the method comprising: administering to the mammal harboring the
tumor a composition comprising, (a) an immunogenic stress
protein-peptide complex isolated from a cell derived from the
tumor, said complex being operative to initiate in the mammal an
immune response against said tumor, and (b) a pharmaceutically
acceptable carrier, in an amount sufficient to elicit in the mammal
an immune response against the tumor thereby inhibiting
proliferation of the tumor.
2. The method of claim 1, wherein the stress protein in the complex
is a Hsp70, a Hsp90 or a gp96.
3. The method of claim 1, wherein a peptide in the complex is non
covalently associated with the stress protein.
4. The method of claim 1, wherein administering the complex
initiates an immune response mediated by a T cell.
5. The method of claim 4, wherein administering the complex
initiates an immune response mediated by a cytotoxic T cell.
6. The method of claim 1, wherein the complex is administered to
the mammal in an amount in the range of about 1 to about 1000
micrograms of complex/kg body weight of mammal/administration.
7. The method of claim 6, wherein said amount is in the range of
about 100 to about 250 micrograms of complex/kg body weight of
mammal/administration.
8. The method of claim 1, wherein the complex is administered
repeatedly to the mammal.
9. The method of claim 1, wherein the composition is administered
to the mammal in combination with a cytokine.
10. A method for inhibiting proliferation of a tumor in a mammal,
the method comprising the steps of: (a) providing a tumor cell
excised from the mammal, (b) isolating from the cell an immunogenic
stress protein-peptide complex operative to initiate in the mammal
an immune response against the tumor cell, and (c) administering to
the mammal the isolated stress protein-peptide complex in an amount
sufficient to elicit in the mammal an immune response against the
tumor cell thereby to inhibit proliferation of any tumor cell
remaining in the mammal.
11. The method of claim 10, wherein the stress protein in the
complex is a Hsp70, a Hsp90 or a gp96.
12. The method of claim 10, wherein a peptide in the complex is non
covalently associated with the stress protein.
13. The method of claim 10, wherein administering the complex
initiates an immune response mediated by a T cell.
14. The method of claim 13, wherein administering the complex
initiates an immune response mediated by a cytotoxic T cell.
15. The method of claim 10, wherein the complex is administered to
the mammal in an amount in the range of about 1 to about 1000
micrograms of complex/kg body weight of mammal/administration.
16. The method of claim 15, wherein said amount is in the range of
about 100 to about 250 micrograms of complex/kg body weight of
mammal/administration.
17. The method of claim 10, wherein the complex is administered
repeatedly to the mammal.
18. The method of claim 10, wherein said complex is administered to
the mammal in combination with a cytokine.
Description
FIELD OF THE INVENTION
[0001] The application relates generally to the field of cancer
therapy, in particular, to the immunotherapy of human cancer.
BACKGROUND OF THE INVENTION
[0002] It has been found that inbred mice and rats can be immunized
phrophylactically against tumors derived from mice and rats of the
same genetic background (Gross (1943) Cancer Res. 3:323-326; Prehn
et al. (1957) J. Natl. Cancer Inst. 18:769-778; Klein et al. (1960)
Cancer Res. 20:1561-1572; Old et al. (1962) Ann NY Acad. Sci.
101:80-106; for review, see Srivastava et al. (1988) Immunology
Today 9:78-83). These studies not only showed that mice vaccinated
with inactivated cancer cells become immunized against subsequent
challenges of live cancer cells but also demonstrated the existence
of tumor-specific antigens.
[0003] Further studies revealed that the phenomenon of
prophylactically induced immunity is tumor-specific. Although mice
can be specifically immunized against the tumor cells that were
used to immunize them they still remain sensitive to challenges
with other unrelated tumors (Basombrio (1970) Cancer Res.
30:2458-2462, Globerson et al. (1964) J. Natl. Cancer Inst.
32:1229-1243). The demonstration of immunogenicity of cancer cells
led to a search for the cancer-derived molecules which elicit
resistance to tumor challenges. The general approach was to
fractionate cancer cell-derived proteins and test them individually
for their ability to immunize mice against the cancers from which
the fractions were prepared (see Srivastava et al. (1988) supra;
Old (1981) Cancer Res. 41:361-375). A number of proteins have been
identified by this method, however, a large proportion of these
proteins are related to a class of proteins known as stress-induced
proteins or stress proteins (Lindquist et al. (1988) Annual Rev.
Genet. 22:631-677). Because the stress proteins are among the most
highly conserved and abundant proteins in nature, they are unlikely
candidates for tumor specific antigens. Stress proteins have
subsequently been shown to non covalently associate with a variety
of peptides thereby to form stress protein-peptide complexes
(Gething et al. (1992) Nature 355:33-45; Lindquist et al. (1988)
supra; Young (1990) Annu. Rev. Immunol. 8:401-420; Flynn et al.
1991) Nature 353:726-730).
[0004] Studies have also shown that stress protein-peptide
complexes lose their immunogenicity upon treatment with ATP (Udono
et al. (1993) J. Exp. Med. 178:1391-1396). This treatment is known
to dissociate the stress-protein peptide complex into its stress
protein and peptide components. Considering that there are no
differences in the structure of stress proteins derived from normal
and tumor cells, and that stress proteins bind a wide spectrum of
peptides in an ATP dependent manner it appears that the
antigenicity of the stress protein-peptide complex results not from
the stress protein per se, but from the peptide associated with the
stress protein.
[0005] One of the major conceptual difficulties in cancer
immunotherapy has been the possibility that human cancers, like
cancers of experimental animals, are antigenically distinct.
Clearly, there is some recent evidence for existence of common
tumor antigens (Kawakami et al. (1992) J. Immunol. 148:638-643;
Darrow et al. (1989) J. Immunol. 142:3329-3334), and this augurs
well for prospects of cancer immunotherapy. Nonetheless, in light
of the overwhelming evidence from experimental and human systems,
it is reasonable to assume that at the very least, human tumors
would show tremendous antigenic diversity and heterogeneity.
[0006] The prospect of identification of the immunogenic antigens
of individual tumors from cancer patients (or even of `only`
several different types of immunogenic antigens in case the
antigens are shared), is daunting to the extent of being
impractical. Conventional cancer therapies typically are based on
the isolation and characterization of specific antigenic
determinants which then may become the target for subsequent
immunotherapies. In addition, although studies have demonstrated
that mammals can be immunized prophylactically against tumors
derived from mammals of the same genetic background, heretofore it
has not been appreciated that a mammal harboring a tumor can be
therapeutically immunized with a composition derived from its own
tumor as a means of treating a cancer preexisting in the
mammal.
[0007] Accordingly, it is an object of the instant invention to
provide a novel method for therapeutically inhibiting proliferation
of tumors in a mammal. The method described herein does not require
the isolation and characterization of specific antigenic
determinants, and accordingly provides a more rapid approach for
making and using immunogenic compositions effective in inhibiting
the proliferation of specific predetermined tumors in mammals.
[0008] This and other objects and features of the invention will be
apparent from the description and claims which follow.
SUMMARY OF THE INVENTION
[0009] The observation that stress proteins chaperone the antigenic
peptides of the cells from which they are derived provides an
approach for readily isolating antigenic peptides for a preselected
tumor. Once isolated, the stress protein-peptide complexes are
administered back to the animal from which they were derived in
order to elicit an immune response against a preexisting tumor.
Accordingly, this approach circumvents the necessity of isolating
and characterizing specific tumor antigens and enables the artisan
to readily prepare immunogenic compositions effective against a
preselected tumor.
[0010] In its broadest aspect, the invention provides a method for
inhibiting proliferation of a preselected tumor in a mammal. The
method comprises administering to the mammal undergoing therapy a
composition comprising a pharmaceutically acceptable carrier in
combination with a stress protein-peptide complex. The complex
having been isolated from a tumor cell previously excised from the
mammal and characterized in that it is operative to initiate in the
mammal an immune response against the tumor cells from which it was
derived. The complex subsequently is administered back to the
mammal in an amount sufficient to elicit in the mammal an immune
response against the tumor cells thereby to inhibit proliferation
of any tumor cells still remaining in the mammal.
[0011] It is contemplated that this approach may be used in
combination with other conventional cancer therapies which include,
for example, surgery, radiation therapy and chemotherapy. For
example, following surgical excision of cancerous tissue the
artisan, using the principles described herein, may isolate stress
protein-peptide complexes from the excised tissue and administer
the complex back to the mammal. The complex subsequently induces a
specific immune response against any remaining tumor cells that
were not excised during surgery. The approach is amenable to cancer
therapy when the primary tumor has metastasized to different
locations with the body.
[0012] The term "tumor" as used herein, is understood to mean any
abnormal or uncontrolled growth of cells which may result in the
invasion of normal tissues. It is contemplated also that the term
embraces abnormal or uncontrolled cell growths that have
metastasized, i.e., abnormal cells that have spread from a primary
location in the body (i.e., primary tumor) to a secondary location
spatially removed from the primary tumor.
[0013] The term "stress protein" as used herein, is understood to
mean any cellular protein which satisfies the following criteria.
It is a protein whose intracellular concentration increases when a
cell is exposed to stressful stimuli, is capable of binding other
proteins or peptides, and is capable of releasing the bound
proteins or peptides in the presence of adenosine triphosphate
(ATP) and/or low pH. Stressful stimuli include, but are not limited
to, heat shock, nutrient deprivation, metabolic disruption, oxygen
radicals, and infection with intracellular pathogens.
[0014] The first stress proteins to be identified were the heat
shock proteins (Hsp's). As their name suggests, Hsp's typically are
induced by a cell in response to heat shock. Three major families
of mammalian Hsp's have been identified to date and include Hsp60,
Hsp70 and Hsp90. The numbers reflect the approximate molecular
weight of the stress proteins in kilodaltons. The members of each
of the families are highly conserved, see for example, Bardwell et
al. (1984) Proc. Natl. Acad. Sci. 81:848-852; Hickey et al. (1989)
Mol. Cell Biol. 9:2615-2626; Jindal (1989) Mol. Cell. Biol.
9:2279-2283, the disclosures of which are incorporated herein by
reference. Members of the mammalian Hsp90 family identified to date
include cytosolic Hsp90 (also known as Hsp83) and the endoplasmic
reticulum counterparts Hsp90 (also known as Hsp83), Hsp87, Grp94
(also known as ERp99) and gp96. See for example, Gething et al.
(1992) Nature 355:33-45 the disclosure of which is incorporated
herein by reference. Members of the Hsp70 family identified to date
include: cytosolic Hsp70 (also known as p73) and Hsc70 (also known
as p72); the endoplasmic reticulum counterpart BiP (also known as
Grp78); and the mitochondrial counterpart Hsp 70 (also known as
Grp75), Gething et al. (1992) supra. To date, members of the
mammalian Hsp60 family have only been identified in the
mitochondria, Gething et al. (1992) supra.
[0015] In addition, it has been discovered that the Hsp-60, Hsp-70
and Hsp-90 families are composed of proteins related to the stress
proteins in amino acid sequence, for example, having greater than
35% amino acid identity, but whose expression levels are not
altered by stressful stimuli. Accordingly, it is contemplated that
the definition of stress protein, as used herein, embraces other
proteins, muteins, analogs, and variants thereof having at least
35% to 55%, preferably 55% to 75%, and most preferably 75% to 85%
amino acid identity with members of the three families whose
expression levels in a cell are stimulated in response to stressful
stimuli.
[0016] The term "peptide", as used herein, is understood to mean
any amino acid sequence isolated from a mammalian tumor cell in the
form of a stress protein-peptide complex.
[0017] The term "immunogenic stress protein-peptide complex", as
used herein, is understood to mean any complex which can be
isolated from a mammalian tumor cell and comprises a stress protein
non covalently associated with a peptide. The complex is further
characterized in that it is operative to induce in the mammal an
immune response against the tumor cells from which the complex was
derived.
[0018] The term "immune response" is understood to mean any
cellular process that is produced in the mammal following
stimulation with an antigen and is directed toward the elimination
of the antigen from the mammal. The immune response typically is
mediated by one or more populations of cells characterized as being
lymphocytic and/or phagocytic in nature.
[0019] In a more specific aspect of the invention, the stress
protein in the stress protein-peptide complex is selected from the
group consisting of Hsp70, Hsp90 and gp96. Stress protein-peptide
complexes which include Hsp70-peptide, Hsp90-peptide and
gp96-peptide complexes may be isolated simultaneously from a batch
of tumor cells excised from a mammal. During immunotherapy it is
contemplated that one or more of the aforementioned complexes may
be administered to the mammal in order to stimulate the optimal
immune response against the tumor.
[0020] It is contemplated that the method described herein is
particularly useful in the treatment of human cancer. However, it
is contemplated that the methods described herein likewise will be
useful in immunotherapy of cancers in other mammals, for example,
farm animals (i.e., cattle, horses, goats, sheep and pigs) and
household pets (i.e., cats and dogs).
[0021] In another aspect of the invention, it is contemplated that
the immune response is effected by means of a T cell cascade, and
more specifically by means of a cytotoxic T cell cascade. The term
"cytotoxic T cell", as used herein, is understood to mean any T
lymphocyte expressing the cell surface glycoprotein marker CD8 that
is capable of targeting and lysing a target cell which bears a
class I histocompatibility complex on its cell surface and is
infected with an intracellular pathogen.
[0022] In another aspect of the invention, the stress
protein-peptide complexes may be administered to the mammal in
combination with a therapeutically active amount of a cytokine. As
used herein, the term "cytokine" is meant to mean any secreted
polypeptide that influences the function of other cells mediating
an immune response. Accordingly, it is contemplated that the
complex can be coadministered with a cytokine to enhance the immune
response directed against the tumor. Preferred cytokines include,
but are not limited to, interleukin-1.alpha. (IL-1.alpha.),
interleukin-1.beta. (IL-1.beta.), 1.beta.), interleukin-2 (IL-2),
interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5),
interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8),
interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11
(IL-11), interleukin-12 (IL-12), interferon .alpha. (IFN.alpha.),
interferon .beta. (IFN.beta.), interferon .gamma. (IFN.gamma.),
tumor necrosis factor .alpha. (TNF.varies.), tumor necrosis factor
.beta. (TNF.beta.), granulocyte colony stimulating factor (G-CSF),
granulocyte/macrophage colony stimulating factor (GM-CSF), and
transforming growth factor .beta. (TGF-.beta.).
[0023] The complex may be administered to a mammal when combined
with a conventional pharmaceutically acceptable carrier, adjuvant,
or excipient using techniques well known in the art. The dosage and
means of administration of the family of stress protein-peptide
complexes necessarily will depend upon a variety of factors such as
the stability of the complex under physiological conditions, the
effectiveness of the complex at eliciting an immune response, the
size and distribution of the tumor, and the age, sex and weight of
the mammal undergoing therapy.
[0024] Typically, the complex should be administered in an amount
sufficient to initiate in the mammal an immune response against the
tumor from which the complex was derived and in an amount
sufficient to inhibit proliferation of the tumor cells in the
mammal. The amount of stress protein-peptide complex administered
preferably is in the range of about 1-1000 micrograms of complex/kg
body weight of the mammal/administration, and most preferably about
100-250 micrograms of complex/kg body weight of the
mammal/administration. It is contemplated that typical dose will be
in the range of about 5 to about 20 mg for a human subject weighing
about 75 kg. In addition, it is contemplated that the strength of
the immune response may be enhanced by repeatedly administering the
complex to the individual. The mammal preferably receives at least
two doses of the stress protein-peptide complex at weekly
intervals. If necessary, the immune response may be boosted at a
later date by subsequent administration of the complex. It is
contemplated, however, that the optimal dosage and immunization
regimen may be found by routine experimentation by one skilled in
the art.
DETAILED DESCRIPTION
[0025] The invention is based on the observation that stress
protein-peptide complexes chaperone antigenic peptides of the cells
from which they are derived. Conventional cancer therapies are
based upon the isolation an characterization of tumor specific
antigens which then become the target for a specific therapeutic
regime. Because of the antigenic diversity of mammalian-cancers the
isolation and characterization of specific tumor antigens for each
specific tumor can be impractical. The instant invention thus
provides an alternative approach to cancer immunotherapy by
obviating the necessity of isolating and characterizing tumor
specific antigens for each tumor being treated.
[0026] The invention described herein provides a method for
inhibiting proliferation of a preselected tumor in a mammal. The
method comprises isolating or obtaining tumor cells from the mammal
undergoing therapy. This is accomplished readily using conventional
surgical procedures well known in the art. Typically, tumor cells
are excised from the mammal during routine surgical recision of the
tumor. The method then involves isolating stress protein-peptide
complexes from the excised tumor cells. This is accomplished using
any one of the isolation procedures described in detail herein
below. The stress protein-peptide complexes are characterized in
that when they are administered back to the mammal they are capable
of initiating a specific immune response against the same type of
tumor cells that they were derived from. Finally, the method
comprises the step of administering back to the mammal the isolated
stress protein-peptide complex in an amount sufficient to elicit in
the mammal an immune response against the tumor cells thereby
inhibiting proliferation of any tumor cells remaining in the
mammal.
[0027] It is contemplated that this approach may be used in
combination with one or more conventional cancer therapies which
include, for example, surgery, radiation therapy and chemotherapy.
For example, following surgical excision of cancerous tissue the
artisan, using the principles described herein, may isolate stress
protein-peptide complexes from the excised tissue and administer
the complex back to the mammal. The complex then induces in the
mammal a specific immune response against any tumor cells that were
not removed during surgery. Alternatively, the method described
herein provides a novel approach for treating cancer when the
primary tumor has metastasized to multiple locations with the body.
For example, when the cancer has metastasized, making surgical
intervention impractical, a stress protein-peptide complex may be
used either alone or in combination with another standard
chemotherapeutic agent in the treatment of the cancer.
[0028] It is contemplated that the invention has particular utility
in the immunotherapy of human cancer, however, it is appreciated
that the methodologies described herein may be applied to the
treatment of cancers occurring in, for example, farm animals (i.e.,
cattle, horses, sheep, goats and pigs) and household pets (i.e.,
cats and dogs).
[0029] The main advantage this approach has over conventional
methodologies is that it is not necessary to isolate and
characterize the tumor specific antigen for each tumor. Once the
stress protein-peptide complex has been isolated it is simply
administered back the mammal without further characterization.
Since the procedures for isolating the immunogenic complexes are
routine and well known in the art, the artisan may rapidly and
routinely prepare a specific immunogenic composition "tailor-made"
for each individual being treated.
[0030] Another advantage of the instant method over previous
methodologies is that the administration of purified stress
protein-peptide complexes back to the individual from which they
were derived eliminates the risk of inoculating the mammal
undergoing therapy with potentially transforming agents (i.e.,
transforming DNA) and/or immunosuppressive agents which can be an
issue when the complex is present in a biochemically undefined
tumor or tumor extract. In addition, stress protein-peptide
complexes can induce significant tumor immunity in the absence of
adjuvants. Accordingly, while adjuvants may further enhance the
immunotherapeutic properties of the complex, their availability is
not a pre-condition for inducing a significant immune response.
[0031] It is contemplated that this method can be used in the
treatment of a variety of tumors, for example, tumors that are
mesenchymal in origin (sarcomas) i.e., fibrosarcomas; myxosarcomas;
liposarcomas; chondrosarcomas; osteogenic sarcomas; angiosarcomas;
endotheliosarcomas; lymphangiosarcomas; synoviosarcomas;
mesotheliosarcomas; Ewing's tumors; myelogenous leukemias;
monocytic leukemias; malignant lymphomas; lymphocytic leukemias;
plasmacytomas; leiomyosarcomas and rhabdomyosarcoma.
[0032] In addition, it is contemplated that this method can be used
in the treatment of tumors that are epithelial in origin
(carcinomas) i.e., squamous cell or epidermal carcinomas; basal
cell carcinomas; sweat gland carcinomas; sebaceous gland
carcinomas; adenocarcinomas; papillary carcinomas; papillary
adenocarcinomas; cystadenocarcinomas; medullary carcinomas;
undifferentiated carcinomas (simplex carcinomas); bronchogenic
carcinomas; bronchial carcinomas; melanocarcinomas; renal cell
carcinomas; hepatocellular carcinomas; bile duct carcinomas;
papillary carcinomas; transitional cell carcinomas; squamous cell
carcinomas; choriocarcinomas; seminomas; embryonal carcinomas
malignant teratomas and teratocarcinomas. Generic methodologies
useful in the preparation of compositions effective at inducing an
immune response against these tumors are discussed in detail herein
below.
[0033] Although not wishing to be bound by theory, it is
contemplated that the stress protein-peptide complexes stimulate an
immune response against the tumor cells from which they are derived
by means of a T cell cascade. Previous experiments have
demonstrated that mice immunized prophylactically with stress
protein-peptide preparations derived from a tumor originating in
the same strain of mouse or rat develop immunological resistance to
the tumor from which it was isolated. The mice, however, fail to
develop immunity against antigenically distinct tumors.
Furthermore, stress protein-peptide complexes derived from normal
tissues do not elicit resistance to any tumors tested. See for
example, Srivastava et al. (1984) Int. J. Cancer 33:417; Srivastava
et al. (1986) Proc. Natl. Acad. Sci. USA 83:3407; Palladino et al.
(1987) Cancer Res. 47:5074; Feldweg et al. (1993) J. Cell Biochem.
Suppl. 17D:108 (Abst.); Udono et al. (1993) J. Cell. Biochem.
Suppl. 17D:113 and Udono (1993) J. Exp. Med.178:1391-1396, the
disclosures of which are incorporated herein by reference.
Recently, it has been established prophylactic immunity typically
is mediated by means of a T cell cascade, more specifically by
means of a cytotoxic T cell cascade. See for example, Blachere et
al. (1993) J. Immunother. 14:352-356, the disclosure of which is
incorporated by reference herein. Accordingly, it is contemplated
that the stress-protein complexes may also mediate their effect
therapeutically by a similar mechanism; specifically, via a
cytotoxic T cell cascade.
[0034] It is contemplated that the stress protein-peptide complexes
typically will be isolated directly from tumor tissue excised from
the mammal being treated. Under certain conditions, however, the
amount of tumor tissue available for isolation of the complex may
be limiting. Accordingly, it is contemplated that the excised tumor
tissue may be proliferated using techniques well known in the art
prior to the isolation of the stress protein-peptide complexes. For
example, the excised tumor tissue may be proliferated either in
vivo, for example, by transfecting a nude mouse with a sample of
the tumor tissue, or in vitro, for example, by serially passaging
the tumor cells in culture. The proliferated tumor tissue
subsequently can be harvested and used as a starting material for
the isolation of the stress protein-peptide complex.
[0035] Stress proteins useful in the practice of the instant
invention may be defined as any cellular protein that satisfies the
following criteria. It is a protein whose intracellular
concentration increases when a cell is exposed to a stressful
stimuli, is capable of binding other proteins or peptides, and is
capable of releasing the bound proteins or peptides in the presence
of adenosine triphosphate (ATP) or low pH.
[0036] The first stress proteins to be identified were the Hsp's
which are synthesized in a cell in response to heat shock. To date,
three major families of mammalian Hsp's have been identified and
include Hsp60, Hsp70 and Hsp90 where the numbers reflect the
approximate molecular weight of the stress proteins in kilodaltons.
Many members of these families were found subsequently to be
induced in response to other stressful stimuli including, but not
limited to, nutrient deprivation, metabolic disruption, oxygen
radicals, and infection with intracellular pathogens. See for
example: Welch (May 1993) Scientific American 56-64; Young (1990)
supra; Craig (1993) Science 260:1902-1903; Gething et al (1992)
supra; and Lindquist et al. (1988) supra, the disclosures of which
are incorporated herein by reference. It is contemplated that
mammalian stress proteins belonging to all three families may be
useful in the practice of the instant invention.
[0037] The major stress proteins accumulate to very high levels in
stressed cells but occur at low to moderate levels in cells that
have not been stressed. For example, the highly inducible mammalian
Hsp70 is hardly detectable at normal temperatures but becomes one
of the most actively synthesized proteins in the cell upon heat
shock (Welch et al. (1985), J. Cell. Biol. 101:1198-1211). In
contrast, Hsp90 and Hsp60 proteins are abundant at normal
temperatures in most, but not all, mammalian cells and are further
induced by heat (Lai et al. (1984), Mol. Cell. Biol. 4:2802-10; van
Bergen en Henegouwen et al. (1987), Genes Dev., 1:525-31).
[0038] Members of the mammalian Hsp90 family identified to date
include cytosolic Hsp90 (also known as Hsp83).and the endoplasmic
reticulum counterparts Hsp90 (also known as Hsp83), Hsp87, Grp94
(also known as ERp99) and gp96 (Gething et al. (1992) supra).
Members of the Hsp70 family identified to date include: cytosolic
Hsp70 (also known as p73) and Hsc70 (also known as p72), the
endoplasmic reticulum counterpart BiP (also known as Grp78) and the
mitochondrial counterpart Hsp 70 (also known as Grp75), Gething et
al. (1992) supra. To date, members of the mammalian Hsp60 family
have only been identified in the mitochondria, Gething et al.
(1992) supra.
[0039] Stress proteins are among the most highly conserved proteins
in existence. For example, DnaK, the Hsp70 from E. coli has about
50% amino acid sequence identity with Hsp70 proteins from
eukaryotes (Bardwell et al. (1984) Proc. Natl. Acad. Sci.
81:848-852). The Hsp60 and Hsp90 families similarly exhibit high
levels of intrafamilial conservation (Hickey et al. (1989) Mol.
Cell Biol. 9:2615-2626; Jindal (1989) Mol. Cell. Biol.
9:2279-2283). In addition, it has been discovered that the Hsp60,
Hsp70 and Hsp90 families are composed of proteins that are related
to the stress proteins in sequence, for example, having greater
than 35% amino acid identity, but whose expression levels are not
altered by stress. Therefore it is contemplated that the definition
of stress protein, as used herein, embraces other proteins,
muteins, analogs, and variants thereof having at least 35% to 55%,
preferably 55% to 75%, and most preferably 75% to 85% amino acid
identity with members of the three families whose expression levels
in a cell are enhanced in response to a stressful stimulus.
[0040] The immunogenic stress protein-peptide complexes of the
invention may include any complex containing a stress protein non
covalently associated with a peptide that is capable of inducing an
immune response in a mammal. Preferred complexes include, but are
not limited to, Hsp70-peptide, Hsp90-peptide and gp96- peptide
complexes. For example, the mammalian stress protein gp96 which is
the endoplasmic reticulum counterpart of the cytosolic Hsp90 may be
used in the practice of the instant invention.
[0041] Typical procedures for isolating stress protein-peptide
complexes useful in the practice of the instant invention are set
forth in detail below.
Purification of Hsp70-peptide Complexes
[0042] The purification of Hsp70-peptide complexes has been
described previously, see for example, Udono et al. (1993)
supra.
[0043] Initially, tumor cells are suspended in 3 volumes of 1X
Lysis buffer consisting of 5 mM sodium phosphate buffer (pH7), 150
mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 1 mM phenyl methyl
sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice,
until >99% cells are lysed as determined by microscopic
examination. As an alternative to sonication, the cells may be
lysed by mechanical shearing and in this approach the cells
typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM
PMSF, incubated on ice for 20 min and then homogenized in a dounce
homogenizer until >95% cells are lysed.
[0044] Then the lysate is centrifuged at 1000 g for 10 minutes to
remove unbroken cells, nuclei and other cellular debris. The
resulting supernatant is recentrifuged at 100,000 g for 90 minutes,
the supernatant harvested and then mixed with Con A Sepharose
equilibrated with phosphate buffered saline (PBS) containing 2 mM
Ca.sup.2+ and 2 mM Mg.sup.2+. When the cells are lysed by
mechanical shearing the supernatant is diluted with an equal volume
of 2X Lysis buffer prior to mixing with Con A Sepharose. The
supernatant is then allowed to bind to the Con A Sepharose for 2-3
hours at 4.degree. C. The material that fails to bind is harvested
and dialyzed for 36 hours (three times, 100 volumes each time)
against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM
PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34
rotor) for 20 min. Then the resulting supernatant is harvested and
applied to a Mono Q FPLC column equilibrated in 20 mM Tris-Acetate
pH 7.5, 20 mM NaCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. The
column is then developed with a 20 mM to 500 mM NaCl gradient and
the eluted fractions fractionated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
characterized by immunoblotting using an appropriate anti-Hsp70
antibody (such as from clone N27F3-4, from StressGen).
[0045] Fractions strongly immunoreactive with the anti-Hsp70
antibody are pooled and the Hsp70-peptide complexes precipitated
with ammonium sulfate; specifically with a 50%-70% ammonium sulfate
cut. The resulting precipitate is then harvested by centrifugation
at 17,000 rpm (SS34 Sorvall rotor) and washed with 70% ammonium
sulfate. The washed precipitate is then solubilized and any
residual ammonium sulfate removed by gel filtration on a
Sephadex.sup.R G25 column (Pharmacia).
[0046] The Hsp70-peptide complex can be purified to apparent
homogeneity using this method. Typically 1 mg of Hsp70-peptide
complex can be purified from 1 g of cells/tissue.
Purification of Hsp90-peptide Complexes
[0047] Initially, tumor cells are suspended in 3 volumes of 1X
Lysis buffer consisting of 5 mM sodium phosphate buffer (pH7), 150
mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 1 mM phenyl methyl
sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice,
until >99% cells are lysed as determined by microscopic
examination. As an alternative to sonication, the cells may be
lysed by mechanical shearing and in this approach the cells
typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM
PMSF, incubated on ice for 20 min and then homogenized in a dounce
homogenizer until >95% cells are lysed.
[0048] Then the lysate is centrifuged at 1000 g for 10 minutes to
remove unbroken cells, nuclei and other cellular debris. The
resulting supernatant is recentrifuged at 100,000 g for 90 minutes,
the supernatant harvested and then mixed with Con A Sepharose
equilibrated with PBS containing 2 mM Ca.sup.2+ and 2 mM Mg.sup.2+.
When the cells are lysed by mechanical shearing the supernatant is
diluted with an equal volume of 2X Lysis buffer prior to mixing
with Con A Sepharose. The supernatant is then allowed to bind to
the Con A Sepharose for 2-3 hours at 4.degree. C. The material that
fails to bind is harvested and dialyzed for 36 hours (three times,
100 volumes each time) against 10 mM Tris-Acetate pH 7.5, 0.1 mM
EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is centrifuged at
17,000 rpm (Sorvall SS34 rotor) for 20 min. Then the resulting
supernatant is harvested and applied to a Mono Q FPLC column
equilibrated equilibrated with lysis buffer. The proteins are then
eluted with a a salt gradient of 200 mM to 600 mM NaCl.
[0049] The eluted fractions are fractionated by SDS-PAGE and
fractions containing the Hsp90-peptide complexes identified by
immunoblotting using a anti-Hsp90 antibody such as 3G3 (Affinity
Bioreagents). Hsp90-peptide complexes can be purified to apparent
homogeneity using this procedure. Typically, 150-200 .mu.g of
Hsp90-peptide complex can be purified from 1 g of cells/tissue.
Purification of gp96-peptide Complexes
[0050] Initially, tumor cells are suspended in 3 volumes of 1X
Lysis buffer consisting of 5 mM sodium phosphate buffer (pH7), 150
mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2 and 1 mM phenyl methyl
sulfonyl fluoride (PMSF). Then, the pellet is sonicated, on ice,
until >99% cells are lysed as determined by microscopic
examination. As an alternative to sonication, the cells may be
lysed by mechanical shearing and in this approach the cells
typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mM
PMSF, incubated on ice for 20 min and then homogenized in a dounce
homogenizer until >95% cells are lysed.
[0051] Then the lysate is centrifuged at 1000 g for 10 minutes to
remove unbroken cells, nuclei and other cellular debris. The
resulting supernatant is recentrifuged at 100,000 g for 90 minutes,
the supernatant harvested and mixed with Con A Sepharose slurry
equilibrated with PBS containing 2 mM Ca.sup.2+ and 2 mM Mg.sup.2+.
When the cells are lysed by mechanical shearing the supernatant is
diluted with an equal volume of 2X Lysis buffer prior to mixing
with Con A Sepharose. The supernatant is then allowed to bind to
the Con A Sepharose for 2-3 hours at 4.degree. C. The slurry is
then packed into a column and washed with 1X lysis buffer until the
OD.sub.280 drops to baseline. Then the column is washed with 1/2
column bed volume of 10% a-methyl mannoside (.alpha.-MM), the
column sealed with parafilm and incubated at 37.degree. C. for 15
min. The column is then cooled to room temperature, the parafilm
removed from the bottom of the column, and five column volumes of a
.alpha.-MM is applied to the column. The eluate is then
fractionated and characterized by SDS-PAGE. Typically, the
resulting gp96-peptide complex is about 60 to 95% pure depending
upon the cell type and the tissue to lysis buffer ratio used.
[0052] If further purification is required, the sample can be
applied to a Mono Q FPLC column equilibrated with a buffer
containing 5 mM sodium phosphate, pH7. The proteins are then eluted
from the column with a 0-1M NaCl gradient. The gp96 fraction elutes
between 400 mM and 550 mM NaCl.
[0053] As an alternative procedure, the gp96 fraction isolated from
the 100,000 g pellet can be resuspended in 5 volumes of PBS
containing 1% sodium deoxycholate (without Ca.sup.2+ and Mg.sup.2+)
and incubated on ice for 1 h. The resulting suspension is
centrifuged for 30 min at 20,000 g and the resulting supernatant
harvested and dialyzed against several changes of PBS (without
Ca.sup.2+ and Mg.sup.2+) to remove the detergent. The resulting
dialysate is centrifuged for 90 min at 100,000 g and the
supernatant purified further. Then calcium and magnesium are both
added to the supernatant to give final concentrations of 2 mM. Then
the sample is applied to a Mono Q HPLC column equilibrated with a
buffer containing 5 mM sodium phosphate, pH7 and the proteins
eluted with a 0-1M NaCl gradient. The gp96 fraction elutes between
400 mM and 550 mM NaCl.
[0054] The gp96-peptide complexes can be purified to apparent
homogeneity using this procedure. Typically about 10-20 .mu.g of
gp96 can be isolated from 1 g cells/tissue using this method.
Formulation and Administration of the Complexes
[0055] Once stress protein-peptide complexes have been purified
from the excised tumor they are administered back to the mammal
undergoing therapy in order to stimulate in the mammal an immune
response against tumor cells from which the complex was derived.
The stress protein-peptide complexes of the invention may either be
stored or prepared for administration by mixing with
physiologically acceptable carriers, excipients, or stabilizers.
These materials should be non-toxic to the intended recipient at
dosages and concentrations employed.
[0056] When the complex is water soluble it may be formulated in an
appropriate buffer, for example PBS (5 mM sodium phosphate, 150 mM
NaCl, pH7.1) 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.
[0057] Useful solutions for oral or parenteral administration may
be prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences, (Gennaro, A., ed.), Mack Pub., 1990. Formulations may
include, for example, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like. Formulations for direct administration, in particular,
may include glycerol and other compositions of high viscosity.
Biocompatible, preferably bioresorbable polymers, including, for
example, hyaluronic acid, collagen, tricalcium phosphate,
polybutyrate, polylactide, polyglycolide and lactide/glycolide
copolymers, may be useful excipients to control the release of the
stress protein-peptide complexes in vivo.
[0058] Formulations for inhalation may contain as excipients, for
example, lactose. Aqueous solutions may contain, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Oily
solutions may be useful administration in the form of nasal drops.
Gels may be applied topically intranasally.
[0059] The compounds provided herein can be formulated into
pharmaceutical compositions by admixture with pharmaceutically
acceptable nontoxic excipients and carriers. In addition the
formulations may optionally contain one or more adjuvants.
Preferred adjuvants include, but are not limited to, pluronic
tri-block copolymers, muramyl dipeptide and its derivatives,
detoxified endotoxin, saponin and its derivatives such as QS-21 and
liposomes. The present invention further envisages sustained
release formulations in which the complex is released over an
extended period of time.
[0060] The mode of administration of the family of stress
protein-peptide complexes prepared in accordance with the invention
will necessarily depend upon the stability of the complex under
physiological conditions, and the size and distribution of the
tumor within the mammal being treated. The preferred dosage of
complex to be administered also is likely to depend on such
variables as the size and distribution of the tumor, the age, sex
and weight of the intended recipient, the overall health status of
the particular recipient, the relative biological efficacy of the
complex, the formulation for the complex, the presence and types of
excipients in the formulation, and the route of administration.
[0061] In general terms, the compounds of this invention may be
provided in an aqueous physiological buffer solution containing
about 0.001 to 10% w/v compound for parenteral administration.
Preferred dosages range from about 1 to about 1000 micrograms of
complex/kg body weight of recipient/administration and most
preferably range from about 100 to about 250 micrograms of
complex/kg body weight of recipient/administratio- n. In
particular, it is contemplated that a typical dose will range from
about 5 mg to about 20 mg for a human subject weighing about 75 kg.
These quantities, however, may vary according to the adjuvant
coadministered with the complex.
[0062] The complex preferably comprises part of an aqueous solution
which may be administered using standard procedures, for example,
intravenously, subcutaneously, intramuscularly, intraorbitally,
ophthalmically, intraventricularly, intracranially,
intracapsularly, intraspinally, intracisternally,
intraperitoneally, buccal, rectally, vaginally, intranasally or by
aerosol administration. The aqueous solution preferably is
physiologically acceptable so that in addition to delivery of the
desired complex to the mammal, the solution does not otherwise
adversely affect the mammal's electrolyte and/or volume balance.
The aqueous medium for the complex thus may comprise normal
physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4 or other
pharmaceutically acceptable salts thereof.
[0063] Preferably the recipient should be vaccinated three times at
two week intervals. If necessary, the responses may be boosted at a
later date by subsequent administration of the complex. It is
contemplated that the optimal dosage and vaccination schedule may
be determined empirically for each stress protein-peptide complex
using techniques well known in the art.
[0064] Various cytokines, antibiotics, and other bioactive agents
also may be coadministered with the stress protein-peptide
complexes. For example, various known cytokines, i.e.,
interleukin-1.alpha. (IL-1.alpha.), interleukin-1.beta.
(IL-1.beta.), interleukin-2 (IL-2), interleukin-3 (IL-3),
interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),
interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9),
interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12
(IL-12), interferon .alpha. (IFN.alpha.), interferon .beta.
(IFN.beta.), interferon .gamma. (IFN.gamma.), tumor necrosis factor
.alpha. (TNF.varies.), tumor necrosis factor .beta. (TNF.beta.),
granulocyte colony stimulating factor (G-CSF),
granulocyte/macrophage colony stimulating factor (GM-CSF), and
transforming growth factor .beta. (TGF-.beta.) may be
coadministered with the complexes in order to maximize the
physiological response. However, it is anticipated that other but
as yet undiscovered cytokines may be effective in the invention. In
addition, conventional antibiotics may be coadministered with the
stress protein-peptide complex. The choice of suitable antibiotics
will however be dependent upon the disease in question.
EXAMPLE I
[0065] In this example, C57BL/6 and C3H mice approximately 100 g in
weight, are purchased from Jackson Laboratories, Bar Harbor, Me.
Malignant tumor cells are then injected subcutaneously into mice in
order to induce experimental tumors in the mice. Specifically,
malignant spindle cell carcinoma 6139 cells are injected
subcutaneously into the C3H mice, malignant mouse Lewis lung
carcinoma cells are injected subcutaneously into C57BL/6 mice and
malignant mouse B16 melanoma cells are injected subcutaneously into
C57BL/6 mice.
[0066] When the tumors have grown to a size such that they are both
visible and palpable, a sample of the tumor tissue is excised. As a
control, normal non malignant tissue is excised from some mice
bearing the experimental tumors.
[0067] Then gp96-peptide, Hsp90-peptide and Hsp70-peptide complexes
are isolated from both the excised normal and tumor derived tissues
using the methods described hereinabove. Once isolated, the
complexes are combined with PBS and administered back to the mice
from which the complexes were derived. Usually 6 mice are tested in
each experiment. The experiments are performed using the schedule
set forth below:
Experiment
Composition administered back to mice
[0068]
1 1 gp96-peptide 2 Hsp70-peptide 3 Hsp90-peptide 4 gp96-peptide and
Hsp70-peptide 5 gp96-peptide and Hsp90-peptide 6 Hsp70-peptide and
Hsp90-peptide 7 Hsp70-peptide, Hsp90-peptide and gp96-peptide 8
bnffer alone
[0069] In one series of experiments the complexes are isolated from
tumor cells whereas in a second series the complexes are isolated
from normal cells. The mice are inoculated three times at weekly
intervals with 20 micrograms (total weight) of the preselected
complex(es). During therapy, the size of each tumor is measured
daily. After 4 weeks the mice are sacrificed and the development of
the tumor examined histologically. In addition, the sacrificed mice
are examined for the presence or absence of metastasis.
[0070] It is expected that the tumors in mice treated with
complexes derived from normal tissue will continue to grow and
metastasize. In contrast, it is expected that the tumors in the
mice treated with the complexes derived from the tumor tissue will
be exhibit slower growth than the tumors in the control animals,
and in some cases, it is expected that the tumor mass may get
smaller and the tumor exhibit remission during therapy.
Other Embodiments
[0071] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
* * * * *