U.S. patent application number 10/265505 was filed with the patent office on 2003-02-20 for therapeutic and prophylactic methods using heat shock proteins.
This patent application is currently assigned to Fordham University. Invention is credited to Srivastava, Pramod K..
Application Number | 20030035808 10/265505 |
Document ID | / |
Family ID | 24101904 |
Filed Date | 2003-02-20 |
United States Patent
Application |
20030035808 |
Kind Code |
A1 |
Srivastava, Pramod K. |
February 20, 2003 |
Therapeutic and prophylactic methods using heat shock proteins
Abstract
The present invention relates to immunogenic complexes of heat
shock proteins (hsp) noncovalently bound to exogenous antigenic
molecules which when administered to an individual elicit specific
immunological responses in the host. Methods of prevention and
treatment of cancer and infectious disease are provided.
Inventors: |
Srivastava, Pramod K.;
(Riverdale, NY) |
Correspondence
Address: |
PENNIE AND EDMONDS
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Assignee: |
Fordham University
Bronx
NY
|
Family ID: |
24101904 |
Appl. No.: |
10/265505 |
Filed: |
October 7, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10265505 |
Oct 7, 2002 |
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09545351 |
Apr 7, 2000 |
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6461615 |
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09545351 |
Apr 7, 2000 |
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09372022 |
Aug 9, 1999 |
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6410028 |
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09372022 |
Aug 9, 1999 |
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08527547 |
Sep 13, 1995 |
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5935576 |
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Current U.S.
Class: |
424/185.1 ;
424/277.1 |
Current CPC
Class: |
A61P 37/04 20180101;
Y10S 530/822 20130101; Y10S 530/824 20130101; A61K 2039/6043
20130101; Y10S 530/826 20130101; Y10S 530/823 20130101; Y10S
530/825 20130101; A61K 2039/622 20130101; Y10S 530/82 20130101;
Y10S 530/828 20130101; A61K 39/0011 20130101; Y10S 530/827
20130101; Y02A 50/30 20180101; A61K 38/00 20130101; C07K 14/47
20130101 |
Class at
Publication: |
424/185.1 ;
424/277.1 |
International
Class: |
A61K 039/00 |
Goverment Interests
[0001] This invention was made with government support under grant
number CA44786 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
What is claimed is:
1. A composition comprising an immunogenic amount of a complex of a
heat shock protein noncovalently bound to an exogenous antigenic
molecule.
2. The composition of claim 1 wherein the exogenous antigenic
molecule is a cancer antigen or antigenic fragment or antigenic
derivative thereof.
3. The composition of claim 1 wherein the exogenous antigenic
molecule is an antigen of an infectious agent.
4. The composition of claim 1 wherein the exogenous antigenic
molecule is an antigen of a virus or antigenic fragment or
antigenic derivative thereof.
5. The composition of claim 1 wherein the exogenous antigenic
molecule is an antigen of a bacterium or antigenic fragment or
antigenic derivative thereof.
6. The composition of claim 1 wherein the exogenous antigenic
molecule is an antigen of a fungus or antigenic fragment or
antigenic derivative thereof.
7. The composition of claim 1 wherein the exogenous antigenic
molecule is an antigen of a parasite or antigenic fragment or
antigenic derivative thereof.
8. The composition of claim 1, 2, or 3 wherein the heat shock
protein is selected from the group consisting of hsp70, hsp90, gp96
and a combination thereof.
9. The composition of claim 1 wherein the heat shock protein is
hsp70.
10. The composition of claim 1 wherein the heat shock protein is
hsp 90.
11. The composition of claim 1 wherein the heat shock protein is
gp96.
12. The composition of claim 4 wherein the virus is selected from
the group consisting of 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 synctial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsachie virus, mumps virus, measles virus, rubella virus, polio
virus, human immunodeficiency virus type I (HIV-I), and human
immunodeficiency virus type II (HIV-II).
13. The composition of claim 5 wherein the bacterium is selected
from the group consisting of mycobacterium, rickettsia, mycoplasma,
neisseria and legionella.
14. The composition of claim 1 wherein the exogenous antigenic
molecule is an antigen of a protozoa or antigenic fragment or
antigenic derivative thereof.
15. The composition of claim 14 wherein the protozoa is selected
from the group consisting of leishmania, kokzidioa, and
trypanosoma.
16. The composition of claim 7 wherein the parasite is selected
from the group consisting of chlamydia and rickettsia.
17. The composition of claim 2 wherein the cancer is selected from
the group consisting of fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, 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 S carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
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, leukemia, lymphoma, multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease.
18. The composition of claim 1 further comprising a
pharmaceutically acceptable carrier.
19. The composition of claim 1 further comprising a cytokine.
20. The composition of claim 19 wherein the cytokine is selected
from the group consisting of interferon-.alpha.,
interferon-.gamma., interleukin-2, interleukin-4, interleukin-6,
and tumor necrosis factor.
21. A method of eliciting an immune response in an individual
comprising administering to the individual a complex of a heat
shock protein noncovalently bound to an exogenous antigenic
molecule.
22. The method according to claim 21 wherein the exogenous
antigenic molecule is selected from the group consisting of a
cancer antigen, an antigen of an infectious agent, and an antigenic
fragment or antigenic derivative of a cancer antigen or antigen of
an infectious agent.
23. A method of treating an individual having cancer comprising
administering to the individual a complex of a heat shock protein
noncovalently bound to an exogenous antigenic molecule.
24. The method according to claim 23 wherein the exogenous
antigenic molecule is a cancer antigen or antigenic fragment or
antigenic derivative thereof.
25. The method according to claim 23 wherein the exogenous
antigenic molecule is a tumor-specific antigen or antigenic
fragment or antigenic derivative thereof.
26. The method according to claim 24 wherein the cancer is selected
from the group consisting of fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
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, Wilius' 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, leukemia, lymphoma, multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease.
27. The method according to claim 21 or 23 wherein the heat shock
protein is selected from the group consisting of hsp70, hsp90, gp96
and a combination thereof.
28. The method according to claim 21 or 23 wherein the heat shock
protein is hsp70.
29. The method according to claim 21 or 23 wherein the heat shock
protein is hsp90.
30. The method according to claim 21 or 23 wherein the heat shock
protein is gp96.
31. A method of preventing cancer in an individual in whom
prevention of cancer is desired comprising administering to the
individual a complex of a heat shock protein noncovalently bound to
an exogenous antigenic molecule.
32. The method according to claim 31 wherein the exogenous
antigenic molecule is a cancer antigen or antigenic fragment or
antigenic derivative thereof.
33. The method according to claim 31 wherein the heat shock protein
is selected from the group consisting of hsp70, hsp90, gp96 and a
combination thereof.
34. A method of treating or preventing infectious disease in an
individual in whom such treatment or prevention is desired
comprising administering to the individual a complex of a heat
shock protein noncovalently bound to an exogenous antigenic
molecule.
35. The method according to claim 34 wherein the exogenous
antigenic molecule is an antigen of an infectious agent.
36. The method according to claim 35 wherein the infectious agent
is a virus, bacterium, fungus, parasite, or protozoa.
37. The method according to claim 35 wherein the heat shock protein
is selected from the group consisting of hsp70, hsp90, gp96, and a
combination thereof.
38. The method according to claim 21, 23, 31, or 34 wherein the
individual is a human.
39. A kit comprising in a container a purified complex of a heat
shock protein noncovalently bound to an exogenous antigenic
molecule, in a pharmaceutically acceptable carrier.
40. The kit of claim 39 wherein the exogenous antigenic molecule is
a cancer antigen.
41. The kit of claim 39 wherein the exogenous antigenic molecule is
an antigen of an infectious agent.
42. The kit of claim 41 wherein the infectious agent is a virus,
bacterium, fungus, parasite, or protozoa.
Description
1. INTRODUCTION
[0002] The present invention relates to compositions for the
prevention and treatment of primary and metastatic cancers and/or
infectious diseases. In the practice of the preventive and
therapeutic methods of the invention, compositions of noncovalent
complexes of heat shock/stress proteins (hsp) including, but not
limited to, hsp70, hsp90, gp96 alone or in combination with each
other, and antigenic molecules are used to augment the immune
responses to genotoxic and nongenotoxic factors, tumors, pathogens
and infectious agents.
2. BACKGROUND OF THE INVENTION
[0003] Studies on the cellular response to heat shock and other
physiological stresses have identified important families of
proteins that are involved not only in cellular protection against
these aggressions, but also in essential biochemical and
immunological processes in unstressed cells. The heat shock
proteins include, but are not limited to hsp70, hsp90, gp96, and
hsp100; these hsp families accomplish different kinds of
chaperoning functions. For example, hsp70, located in the cell
cytoplasm, nucleus, mitochondria, or endoplasmic reticulum,
(Lindquist, S., et al., 1988, Ann. Rev. Genetics 22:631-677) are
involved in the presentation of antigens to the cells of the immune
system, and are also involved in the transfer, folding and assembly
of proteins in normal cells. Similarly, Hsp90 located in the
cytosol are involved in chaperoning and gp96 present in the
endoplasmic reticulum are involved in antigen presentation
(Srivastava, P. K., et al., 1991, Curr. Topics in Microbiology
& Immun. 167:109-123).
[0004] 2.1. Immunotherapy
[0005] In modern medicine, immunotherapy or vaccination has
virtually eradicated diseases such as polio, tetanus, tuberculosis,
chicken pox, measles, hepatitis, etc. The approach using
vaccinations has exploited the ability of the immune system to
prevent infectious diseases. Such vaccination with non-live
materials such as proteins generally leads to an antibody response
or CD4+ helper T cell response. Raychaudhuri, S. and Morrow, W. J.
W., 1993, Immunology Today, 14:344-348. On the other hand,
vaccination or infection with live materials such as live cells or
infectious viruses generally leads to a CD8+ cytotoxic T-lymphocyte
(CTL) response. A CTL response is crucial for protection against
cancers, infectious viruses and bacteria. This poses a practical
problem, for, the only way to achieve a CTL response is to use live
agents which are themselves pathogenic. The problem is generally
circumvented by using attenuated viral and bacterial strains or by
killing whole cells which can be used for vaccination. These
strategies have worked well but the use of attenuated strains
always carries the risk that the attenuated agent may recombine
genetically with host DNA and turn into a virulent strain. Thus,
there is need for methods which can lead to CD8+ CTL response by
vaccination with non-live materials such as proteins in a specific
manner.
[0006] The era of tumor immunology began with experiments by Prehn
and Main, who showed that antigens on the methylcholanthrene
(MCA)-induced sarcomas were tumor specific in that transplantation
assays could not detect these antigens in normal tissue of the mice
(Prehn, R. T., et al., 1957, J. Natl. Cancer Inst. 18:769-778).
This notion was confirmed by further experiments demonstrating that
tumor specific resistance against MCA-induced tumors can be
elicited in the autochthonous host, that is, the mouse in which the
tumor originated (Klein, G., et al., 1960, Cancer Res.
20:1561-1572).
[0007] In subsequent studies, tumor specific antigens were also
found on tumors induced with other chemical or physical carcinogens
or on spontaneous tumors (Kripke, M. L., 1974, J. Natl. Cancer
Inst. 53:1333-1336; Vaage, J., 1968, Cancer Res. 28:2477-2483;
Carswell, E. A., et al., 1970, J. Natl. Cancer Inst. 44:1281-1288).
Since these studies used protective immunity against the growth of
transplanted tumors as the criterion for tumor specific antigens,
these antigens are also commonly referred to as "tumor specific
transplantation antigens" or "tumor specific rejection antigens."
Several factors can greatly influence the immunogenicity of the
tumor induced, including, for example, the specific type of
carcinogen involved, immunocompetence of the host and latency
period (Old, L. J., et al., 1962, Ann. N.Y. Acad. Sci. 101:8015
106; Bartlett, G. L., 1972, J. Natl. Cancer Inst. 49:493-504).
[0008] Most, if not all, carcinogens are mutagens which may cause
mutation, leading to the expression of tumor specific antigens
(Ames, B. N., 1979, Science 204:587-593; Weisburger, J. H., et al.,
1981, Science 214:401-407). Some carcinogens are immunosuppressive
(Malmgren, R. A., et al., 1952, Proc. Soc. Exp. Biol. Med.
79:484-488). Experimental evidence suggests that there is a
constant inverse correlation between immunogenicity of a tumor and
latency period (time between exposure to carcinogen and tumor
appearance) (Old, L. J., et al., 1962, Ann. N.Y. Acad. Sci.
101:80-106; and Bartlett, G. L., 1972, J. Natl. Cancer Inst.
49:493-504). Other studies have revealed the existence of tumor
specific antigens that do not lead to rejection, but, nevertheless,
can potentially stimulate specific immune responses (Roitt, I.,
Brostoff, J and Male, D., 1993, Immunology, 3rd ed., Mosby, St.
Louis, pp. 17.1-17.12).
[0009] 2.2. Tumor-Specific Immunogenicities of Heat Shock/Stress
Proteins hsp70, hsp90 and gp96
[0010] Srivastava et al. demonstrated immune response to
methylcholanthrene-induced sarcomas of inbred mice (1988, Immunol.
Today 9:78-83). In these studies it was found that the molecules
responsible for the individually distinct immunogenicity of these
tumors were identified as cell-surface glycoproteins of 96 kDa
(gp96) and intracellular proteins of 84 to 86 kDa (Srivastava, P.
K., et al., 1986, Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich,
S. J., et al., 1986, Proc. Natl. Acad. Sci. USA 83:3121-3125.
Immunization of mice with gp96 or p84/86 isolated from a particular
tumor rendered the mice immune to that particular tumor, but not to
antigenically distinct tumors. Isolation and characterization of
genes encoding gp96 and p84/86 revealed significant homology
between them, and showed that gp96 and p84/86 were, respectively,
the endoplasmic reticular and cytosolic counterparts of the same
heat shock proteins (Srivastava, P. K., et al., 1988,
Immunogenetics 28:205-207; Srivastava, P. K., et al., 1991, Curr.
Top. Microbiol. Immunol. 167:109-123). Further, hsp70 was shown to
elicit immunity to the tumor from which it was isolated but not to
antigenically distinct tumors. However, hsp70 depleted of peptides
was found to lose its immunogenic activity (Udono, M., and
Srivastava, P. K., 1993, J. Exp. Med. 178:1391-1396). These
observations suggested that the heat shock proteins are not
immunogenic per se, but are carriers of antigenic peptides that
elicit specific immunity to cancers (Srivastava, P. K., 1993, Adv.
Cancer Res. 62:153-177).
[0011] 2.3. Pathobiology of Cancer
[0012] Cancer is characterized primarily by an increase in the
number of abnormal cells derived from a given normal tissue,
invasion of adjacent tissues by these abnormal cells, and lymphatic
or blood-borne spread of malignant cells to regional lymph nodes
and to distant sites (metastasis). Clinical data and molecular
biologic studies indicate that cancer is a multistep process that
begins with minor preneoplastic changes, which may under certain
conditions progress to neoplasia.
[0013] Pre-malignant abnormal cell growth is exemplified by
hyperplasia, metaplasia, or most particularly, 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.) Hyperplasia is a form of controlled cell proliferation
involving an increase in-cell number in a tissue or organ, without
significant alteration in structure or function. As but one
example, endometrial hyperplasia often precedes endometrial cancer.
Metaplasia is a form of controlled cell growth in which one type of
adult or fully differentiated cell substitutes for another type of
adult cell. Metaplasia can occur in epithelial or connective tissue
cells. Atypical metaplasia involves a somewhat disorderly
metaplastic epithelium. Dysplasia is frequently a forerunner of
cancer, and is found mainly in the epithelia; it is the most
disorderly form of non-neoplastic cell growth, involving a loss in
individual cell uniformity and in the architectural orientation of
cells. Dysplastic cells often have abnormally large, deeply stained
nuclei, and exhibit pleomorphism. Dysplasia characteristically
occurs where there exists chronic irritation or inflammation, and
is often found in the cervix, respiratory passages, oral cavity,
and gall bladder.
[0014] The neoplastic lesion may evolve clonally and develop an
increasing capacity for invasion, growth, metastasis, and
heterogeneity, especially under conditions in which the neoplastic
cells escape the host's immune surveillance (Roitt, I., Brostoff, J
and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps.
17.1-17.12).
3. SUMMARY OF THE INVENTION
[0015] The present invention provides pharmaceutical compositions,
methods, and kits for prevention and treatment of cancer and/or
infectious diseases by enhancing the host's immunocompetence and
activity of immune effector cells. The pharmaceutical compositions
of the invention comprise complexes of hsps noncovalently bound to
exogenous antigenic molecules. The exogenous antigenic molecules
differ from the peptides endogenously complexed with hsps in vivo
and which copurify with the hsps. The exogenous antigenic molecules
are antigens/immunogens or antigenic/immunogenic fragments or
derivatives thereof. Such antigenic molecules can be selected from
among those known in the art or assayed by the ability to bind to
antibody or MHC molecule (antigenicity) or generate immune response
(immunogenicity) by standard immunoassays known in the art. The
antigenic molecules are noncovalently complexed with hsps in vitro
prior to administration to a patient. For treatment or prevention
of cancer, the antigenic molecules are molecules that will induce
an immune response against the cancer, e.g., tumor-specific
antigens, or tumor-associated antigens, preferably of human tumors.
For treatment or prevention of infectious diseases, the antigenic
molecules are molecules that will induce an immune response against
the infectious agent, e.g., antigens of viruses, bacteria, fungi,
parasites etc., preferably agents that infect humans. In a specific
embodiment, the pharmaceutical compositions of the present
invention include hsps complexed not only to a single antigen but
also more than one antigen or an entire cocktail of (e.g., tumor
specific) antigens. Preferably, the patient is a human, and the
hsps are human hsps. The hsps in the complexes can be autologous or
allogeneic to the patient.
[0016] Particular compositions of the invention and their
properties are described in the sections and subsections which
follow. Preferably, the complex of hsp and antigenic molecules
comprises hsp70, hsp90, gp96, or a combination thereof.
[0017] In another embodiment, the pharmaceutical compositions
further comprise effective amounts of a biological response
modifier, including but not limited to the cytokines
interferon-.alpha. (IFN-.alpha.), IFN-.gamma., interleukin-2
(IL-2), IL-4, IL-6, tumor necrosis factor (TNF), or other cytokine
growth factor.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Effect of administration of hsp70 complexed with
ovalbumin or cytotoxicity of T cells against the EG7 cell line
(expresses ovalbumin antigen) or the EL4 cell line (negative for
ovalbumin antigen).
[0019] FIG. 1A: Two mice in each group were immunized with: a) a
control vehicle (squares); b) ovalbumin alone (plus sign); c) hsp70
alone (triangles); or d) hsp70-ovalbumin complex. T cells taken
from the immunized mice were tested for cytotoxicity against the
EG7 cells (FIG. 1A) or EL4 cells (FIG. 1B). The results demonstrate
that the hsp70-ovalbumin complex is a far better reagent at
inducing a cytotoxic T-lymphocyte response than ovalbumin alone or
hsp70 alone (FIG. 1A). The T cells did not respond in the presence
of the EL4 cells which lack the ovalbumin antigen (FIG. 1B).
5. DETAILED DESCRIPTION OF THE INVENTION
[0020] Compositions and methods for the prevention and treatment of
primary and metastatic cancers and/or infectious diseases are
described. The invention provides pharmaceutical compositions of
hsp noncovalently bound to exogenous antigenic molecules.
[0021] The exogenous antigenic molecules differ from the peptides
endogenously complexed with hsps in vivo and which copurify with
the hsps. The exogenous antigenic molecules are antigens/immunogens
or antigenic/immunogenic fragments or derivatives thereof. Such
antigenic molecules can be selected from among those known in the
art or assayed by the ability to bind to antibody or MHC molecule
(antigenicity) or generate immune response (immunogenicity) by
standard immunoassays known in the art. The antigenic molecules are
noncovalently complexed with hsps in vitro prior to administration
to a patient. For treatment or prevention of cancer, the antigenic
molecules are molecules that will induce an immune response against
the cancer, e.g., tumor-specific antigens, or tumor-associated
antigens, preferably of human tumors. For treatment or prevention
of infectious diseases, the antigenic molecules are molecules that
will induce an immune response against the infectious agent, e.g.,
antigens of viruses, bacteria, fungi, parasites etc., preferably
agents that infect humans. In a specific embodiment, the
pharmaceutical compositions of the present invention include hsps
complexed not only to a single antigen but also more than one
antigen or an entire cocktail of (e.g., tumor specific) antigens.
Preferably, the patient is a human, and the hsps are human hsps.
The hsps in the complexes can be autologous or allogeneic to the
patient.
[0022] The methods of the invention comprise methods of eliciting
an immune response in an individual in whom the treatment or
prevention of infectious diseases or cancer is desired by
administering a composition comprising an effective amount of a
complex, in which the complex consists essentially of a hsp
noncovalently bound to an exogenous antigenic molecule. The hsp
and/or the antigenic molecule can be isolated from the individual
or from others or by recombinant production methods using a cloned
hsp originally derived from the individual or from others.
Exogenous antigens and fragments and derivatives (both peptide and
non-peptide) thereof for use in complexing with hsps, can be
selected from among those known in the art, as well as those
readily identified by standard immunoassays know in the art by the
ability to bind antibody or MHC molecules (antigenicity) or
generate immune responses (immunogenicity).
[0023] The hsps of the present invention that can be used include
but are not limited to, hsp70, hsp90, gp96 alone or in combination.
Preferably, the hsps are human hsps.
[0024] Heat shock proteins, which are also referred to
interchangeably herein as stress proteins, useful in the practice
of the instant invention can be selected from among any cellular
protein that satisfies any one of the following criteria. It is a
protein whose intracellular concentration increases when a cell is
exposed to a stressful stimuli, it is capable of binding other
proteins or peptides, and it is capable of releasing the bound
proteins or peptides in the presence of adenosine triphosphate
(ATP) or low pH; or it is a protein showing at least 35% homology
with any cellular protein having any of the above properties.
[0025] The first stress proteins to be identified were the heat
shock proteins (hsps). As their name implies, hsps are synthesized
by a cell in response to heat shock. To date, three major families
of hsp have been identified based on molecular weight. The families
have been called 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 Welch,
May 1993, Scientific American 56-64; Young, 1990, Annu. Rev.
Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething, et
al., 1992, Nature 355:33-45; and Lindquist, et al., 1988, Annu.
Rev. Genetics 22:631-677), the disclosures of which are
incorporated herein by reference. It is contemplated that
hsps/stress proteins belonging to all of these three families can
be used in the practice of the instant invention.
[0026] The major hsps can accumulate to very high levels in
stressed cells, but they occur at low to moderate levels in cells
that-have 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).
[0027] Heat-shock 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
excoriates (Bardwell, et al., 1984, Proc. Natl. Acad. Sci.
81:848-852). The hsp60 and hsp90 families also show similarly high
levels of intra families conservation (Hickey, et al., 1989, Hol.
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. The
purification of stress proteins belonging to these three families
is described below.
[0028] The immunogenic complexes of hsp and exogenous antigenic
molecules of the invention include any complex containing an hsp
and an exogenous antigenic molecule that is capable of inducing an
immune response in a mammal. The antigenic molecules are
noncovalently associated with the hsps. Preferred complexes
comprise hsp60, hsp70, or hsp90, noncovalently bound to a protein
antigen. In a specific embodiment, the complex comprises an hsp
called gp96 which is present in the endoplasmic reticulum of
eukaryotic cells and is related to the cytoplasmic hsp90s.
[0029] Although the hsps can be allogeneic to the patient, in a
preferred embodiment, the hsps are autologous to (derived from) the
patient to whom they are administered. The hsps and/or antigenic
molecules can be purified from natural sources, chemically
synthesized, or recombinantly produced. The invention provides
methods for determining doses for human cancer immunotherapy by
evaluating the optimal dose of hsp noncovalently bound to peptide
complexes in experimental tumor models and extrapolating the data.
Specifically, a scaling factor not exceeding a fifty fold increase
over the effective dose estimated in animals, is used as the
optimal prescription method for cancer immunotherapy or vaccination
in human subjects.
[0030] The invention provides compositions comprising the
hsp-antigenic molecule complexes which enhance the immunocompetence
of the host individual and elicit specific immunity against
infectious agents or specific immunity against preneoplastic and
neoplastic cells. The therapeutic regimens and pharmaceutical
compositions of the invention are described below. These
compositions are believed to have the capacity to prevent the onset
and progression of infectious diseases and prevent the development
of tumor cells and to inhibit the growth and progression of tumor
cells indicating that such compositions can induce specific
immunity in infectious diseases and cancer immunotherapy.
[0031] The complexes of the invention can be used to induce an
inflammatory reaction at the tumor site and ultimately cause a
regression of the tumor burden in the cancer patients treated.
Cancers which can be treated with complexes of hsps noncovalently
bound to exogenous antigenic molecules include, but are not limited
to, human sarcomas and carcinomas.
[0032] Accordingly, the invention provides methods of preventing
and treating cancer in an individual comprising administering a
composition which stimulates the immunocompetence of the host
individual and elicits specific immunity against the preneoplastic
and/or neoplastic cells. As used herein, "preneoplastic" cell
refers to a cell which is in transition from a normal to a
neoplastic form; and morphological evidence, increasingly supported
by molecular biologic studies, indicates that preneoplasia
progresses through multiple steps. Non-neoplastic cell growth
commonly consists of hyperplasia, metaplasia, or most particularly,
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).
[0033] The therapeutic regimens and pharmaceutical compositions of
the invention may be used with additional immune response enhancers
or cytokines including, but not limited to, the cytokines
IFN-.alpha., IFN-.gamma., IL-2, IL-4, IL-6,, TNF, or other cytokine
affecting immune cells. In accordance with this aspect of the
invention, the complexes of the hsp and antigenic molecule are
administered in combination therapy with one or more of these
cytokines.
[0034] The invention further relates to administration of complexes
of hsp-antigenic molecules to individuals at enhanced risk of
cancer due to familial history or environmental risk factors.
[0035] The compositions comprising hsp noncovalently bound to
exogenous antigenic molecules are administered to elicit an
effective specific immune response to the complexed antigenic
molecules (and not to the hsp).
[0036] In a preferred embodiment, hsp70, hsp90 and/or gp96 are
noncovalently complexed with exogenous antigenic molecules.
[0037] In accordance with the methods described herein, the
exogenous antigenic molecules are immunogenic or antigenic proteins
or other molecules or immunogenic/antigenic fragments or
derivatives thereof. For example, exogenous antigenic molecules
include but are not limited to different tumor specific
translatable antigens (e.g., tyrosinase, gp100, melan-A, gp75,
mucins, etc.) and viral antigens including, but not limited to,
proteins of immunodeficiency virus type I (HIV-I), human
immunodeficiency virus type II (HIV-II), 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 and polio virus.
[0038] In a specific embodiment, antigens of cancers (e.g., tumors)
or infectious agents (e.g., viral antigen, bacterial antigens,
etc.) can be obtained by purification from natural sources, by
chemical synthesis, or recombinantly, and, through in vitro
procedures such as that described below, noncovalently complexed to
hsps.
[0039] 5.1. Purification of Hsps
[0040] In an embodiment wherein the hsp portion of the
hsp-antigenic molecule complex is desired to be purified from
cells, exemplary purification procedures such as described in
Sections 5.1.1-5.1.3 below can be employed to purify hsp-peptide
complexes, after which the hsps can be purified from the endogenous
hsp-peptide complexes in the presence of ATP or low pH, for
subsequent in vitro complexing to exogenous antigenic molecules.
Although described for tumor cells, the protocols described
hereinbelow may be used to isolate hsps from any eukaryotic cells,
for example, tissues, isolated cells, or immortalized eukaryote
cell lines infected with a preselected intracellular pathogen,
tumor cells or tumor cell lines.
[0041] Alternatively to isolation of native hsps from cells as
described in Sections 5.1.1-5.1.3, hsps can be chemically
synthesized or recombinantly produced.
[0042] 5.1.1. Preparation and Purification of Hsp70-Peptide
Complexes
[0043] The purification of hsp70-peptide complexes has been
described previously, see, for example, Udono et al., 1993, J. Exp.
Med. 178:1391-1396. A procedure that may be used, presented by way
of example but not limitation, is as follows:
[0044] Initially, tumor cells are suspended in 3 volumes of
1.times. Lysis buffer consisting of 5 mM sodium phosphate buffer
(pH 7), 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
<nicated, 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 minutes and then homogenized in a
dounce homogenizer until >95% cells are lysed.
[0045] Then the lysate is centrifuged at 1,000 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 2.times. 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 minutes. Then the resulting supernatant is harvested
and applied to a Mono Q FPLC column equilibrated in 20 mM
TrisAcetate 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 then 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).
[0046] 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). If necessary the hsp70
preparation thus obtained can be repurified through the Mono Q FPCL
Column as described above.
[0047] 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.
[0048] The present invention further describes a new and rapid
method for purification of hsp70. This improved method uses column
chromatography with ATP affixed to a solid substratum (e.g.,
ATP-agarose). The hsp70 yields are believed to be increased
significantly and have high purity. By way of example but not
limitation, purification of hsp70 by ATP-agarose chromatography was
carried out as follows: Meth A sarcoma cells (500 million cells)
were homogenized in hypotonic buffer and the lysate was centrifuged
at 100,000 g for 90 minutes at 4.degree. C. The supernatant was
divided into two and was applied to an ADP-agarose or an
ATP-agarose column. The columns were washed in buffer and were
eluted with 3 mM ADP or 3 mM ATP, respectively. The eluted
fractions were analyzed by SDS-PAGE: in both cases, apparently
homogeneous preparations of hsp70 were obtained. However, when each
of the preparations was tested for presence of peptides, the
ADP-bound/eluted hsp70 preparation was found to be associated with
peptides, while the ATP-bound/eluted hsp70 preparation was not.
[0049] 5.1.2. Preparation and Purification of Hsp90-Peptide
Complexes
[0050] A procedure that can be used, presented by way of example
and not limitation, is as follows:
[0051] Initially, tumor cells are suspended in 3 volumes of
1.times. Lysis buffer consisting of 5 mM sodium phosphate buffer
(pH 7), 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 minutes and then homogenized in
a dounce homogenizer until >95% cells are lysed.
[0052] Then the lysate is centrifuged at 1,000 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 2.times. 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 minutes. Then
the resulting supernatant is harvested and applied to a Mono Q FPLC
column equilibrated with lysis buffer. The proteins are then eluted
with a salt gradient of 200 mM to 600 mM NaCl.
[0053] The eluted fractions are fractionated by SDS-PAGE and
fractions containing the hsp90-peptide complexes identified by
immunoblotting using an 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.
[0054] 5.1.3. Preparation and Purification of gp96-Peptide
Complexes
[0055] A procedure that can be used, presented by way of example
and not limitation, is as follows:
[0056] A pellet of tumors is resuspended in 3 volumes of buffer
consisting of 30 mM sodium bicarbonate buffer (pH 7.5) and 1 mM
PMSF and the cells allowed to swell on ice 20 minutes. The cell
pellet then is homogenized in a Dounce homogenizer (the appropriate
clearance of the homogenizer will vary according to each cells
type) on ice until >95% cells are lysed.
[0057] The lysate is centrifuged at 1,000 g for 10 minutes to
remove unbroken cells, nuclei and other debris. The supernatant
from this centrifugation step then is recentrifuged at 100,000 g
for 90 minutes. The gp96-peptide complex can be purified either
from the 100,000 pellet or from the supernatant.
[0058] When purified from the supernatant, the supernatant is
diluted with equal volume of 2.times. lysis buffer and the
supernatant mixed for 2-3 hours at 4.degree. C. with Con A
Sepharose equilibrated with PBS containing 2 mM Ca.sup.2+ and 2 mM
Mg.sup.2+. Then, the slurry is packed into a column and washed with
1.times. lysis buffer until the OD.sub.280 drops to baseline. Then,
the column is washed with 1/3 column bed volume of 10% a-methyl
mannoside (.alpha.-MM) dissolved in PBS containing 2 mM Ca.sup.2+
and 2 mM Mg.sup.2+, the column sealed with a piece of parafilm, and
incubated at 37.degree. C. for 15 minutes. Then the column is
cooled to room temperature and the parafilm removed from the bottom
of the column. Five column volumes of the a-MM buffer are applied
to the column and the eluate analyzed by SDS-PAGE. Typically the
resulting material is about 60-95% pure, however this depends upon
the cell type and the tissue-to-lysis buffer ratio used. Then the
sample is applied to a Mono Q FPLC column (Pharmacia) equilibrated
with a buffer containing 5 mM sodium phosphate, pH 7. The proteins
then are eluted from the column with a 0-1 M NaCl gradient and the
gp96 fraction elutes between 400 mM and 550 mM NaCl.
[0059] The procedure, however, may be modified by two additional
steps, used either alone or in combination, to consistently produce
apparently homogeneous gp96-peptide complexes. One optional step
involves an ammonium sulfate precipitation prior to the Con A
purification step and the other optional step involves
DEAE-Sepharose purification after the Con A purification step but
before the Mono Q FPLC step.
[0060] In the first optional step, the supernatant resulting from
the 100,000 g centrifugation step is brought to a final
concentration of 50% ammonium sulfate by the addition of ammonium
sulfate. The ammonium sulfate is added slowly while gently stirring
the solution in a beaker placed in a tray of ice water. The
solution is stirred from about 1/2 to 12 hours at 4.degree. C. and
the resulting solution centrifuged at 6,000 rpm (Sorvall SS34
rotor). The supernatant resulting from this step is removed,
brought to 70% ammonium sulfate saturation by the addition of
ammonium sulfate solution, and centrifuged at 6,000 rpm (Sorvall
SS34 rotor). The resulting pellet from this step is harvested and
suspended in PBS containing 70% ammonium sulfate in order to rinse
the pellet. This mixture is centrifuged at 6,000 rpm (Sorvall SS34
rotor) and the pellet dissolved in PBS containing 2 mM Ca.sup.2+
and Mg.sup.2+. Undissolved material is removed by a brief
centrifugation at 15,000 rpm (Sorvall SS34 rotor). Then, the
solution is mixed with Con A Sepharose and the procedure followed
as before.
[0061] In the second optional step, the gp96 containing fractions
eluted from the Con A column are pooled and the buffer exchanged
for 5 mM sodium phosphate buffer, pH 7,300 mM NaCl by dialysis, or
preferably by buffer exchange on a Sephadex G25 column. After
buffer exchange, the solution is mixed with DEAE-Sepharose
previously equilibrated with 5 mM sodium phosphate buffer, pH 7,
300 mM NaCl. The protein solution and the beads are mixed gently
for 1 hour and poured into a column. Then, the column is washed
with 5 mM sodium phosphate buffer, pH 7, 300 mM NaCl, until the
absorbance at 280 nM drops to baseline. Then, the bound protein is
eluted from the column with five volumes of 5 mM sodium phosphate
buffer, pH 7, 700 mM NaCl. Protein containing fractions are pooled
and diluted with 5 mM sodium phosphate buffer, pH 7 in order to
lower the salt concentration to 175 mM. The resulting material then
is applied to the Mono Q FPLC column (Pharmacia) equilibrated with
5 mM sodium phosphate buffer, pH 7 and the protein that binds to
the Mono Q FPLC column (Pharmacia) is eluted as described
before.
[0062] It is appreciated, however, that one skilled in the art may
assess, by routine experimentation, the benefit of incorporating
the second optional step into the purification protocol. In
addition, it is appreciated also that the benefit of adding each of
the optional steps will depend upon the source of the starting
material.
[0063] When the gp96 fraction is isolated from the 100,000 g
pellet, the pellet is suspended in 5 volumes of PBS containing
either 1% sodium deoxycholate or 1% oxtyl glucopyranoside (but
without the Mg.sup.2+ and Ca.sup.2+) and incubated on ice for 1
hour. The suspension is centrifuged at 20,000 g for 30 minutes and
the resulting supernatant dialyzed against several changes of PBS
(also without the Mg.sup.2+ and Ca.sup.2+) to remove the detergent.
The dialysate is centrifuged at 100,000 g for 90 minutes, the
supernatant harvested, and calcium and magnesium are added to the
supernatant to give final concentrations of 2 mM, respectively.
Then the sample is purified by either the unmodified or the
modified method for isolating gp96-peptide complex from the 100,000
g supernatant, see above.
[0064] The gp96-peptide complexes can be purified to apparent
homogeneity using this procedure. About 10-20 g of gp96 can be
isolated from 1 g cells/tissue.
[0065] 5.2 Exogenous Antigenic Molecules
[0066] 5.2.1. Peptides from MHC Complexes
[0067] It has been found that potentially immunogenic peptides may
be eluted from MHC-peptide complexes using techniques well known in
the art (Falk, K. et al., 1990 Nature 348:248251; Elliott, T., et
al., 1990, Nature 348:195-197; Falk, K., et al., 1991, Nature
351:290-296). Once isolated, the amino acid sequence of each
antigenic peptide may be determined using conventional amino acid
sequencing methodologies. Such antigenic molecules can then be
produced by chemical synthesis or recombinant methods, purified,
and complexed to hsps in vitro.
[0068] Thus, potentially immunogenic or antigenic peptides may be
isolated from endogenous MHC-peptide complexes for use subsequently
as exogenous antigenic molecules, by complexing in vitro to hsps.
Exemplary protocols for isolating peptides and/or antigenic
components from MHC complexes are set forth below in Section
5.2.1.1.
[0069] 5.2.1.1. Peptides from MHC-Peptide Complexes.
[0070] The isolation of potentially immunogenic peptides from MHC
molecules is well known in the art and so is not described in
detail herein (See, Falk, et al., 1990, Nature 348:248-251;
Rotzsche, at al., 1990, Nature 348:252-254; Elliott, et al., 1990,
Nature 348:191-197; Falk, et al., 1991, Nature 351:290-296; Demotz,
et al., 1989, Nature 0.343:682-684; Rotzsche, et al., 1990, Science
249:283-287), the disclosures of which are incorporated herein by
reference.
[0071] Briefly, MHC-peptide complexes may be isolated by a
conventional immunoaffinity procedure. The peptides then may be
eluted from the MHC-peptide complex by incubating the complexes in
the presence of about 0.1% TFA in acetonitrile. The eluted peptides
may be fractionated and purified by reverse phase HPLC, as
before.
[0072] The amino acid sequences of the eluted peptides may be
determined either by manual or automated amino acid sequencing
techniques well known in the art. Once the amino acid sequence of a
potentially protective peptide has been determined the peptide may
be synthesized in any desired amount using conventional peptide
synthesis or other protocols well known in the art.
[0073] Peptides having the same amino acid sequence as those
isolated above 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-.alpha.-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-.alpha.-deprotected amino acid to an .alpha.-carboxy group of an
N-.alpha.-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-.alpha.-protecting groups
include Boc which is acid labile and Fmoc which is base labile.
[0074] Briefly, the C-terminal N-.alpha.-protected amino acid is
first attached to the polystyrene beads. The N-.alpha.-protecting
group is then removed. The deprotected .alpha.-amino group is
coupled to the activated .alpha.-carboxylate group of the next
N-.alpha.-protected amino acid. The process is repeated until the
desired peptide is synthesized. The resulting peptides are then
cleaved from the insoluble polymer support and the amino acid side
chains deprotected. Longer peptides can be derived by condensation
of protected peptide fragments. Details of appropriate chemistries,
resins, protecting groups, protected amino acids and reagents are
well known in the art and so are not discussed in detail herein
(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).
[0075] Purification of the resulting peptides is 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 and so are not described in detail herein.
[0076] 5.2.2. Other Exogenous Antigenic Molecules
[0077] Antigens or antigenic portions thereof can be selected for
use as antigenic molecules, for complexing to hsps, from among
those known in the art or determined by immunoassay to be able to
bind to antibody or MHC molecules (antigenicity) or generate immune
responses (immunogenicity). To determine immunogenicity or
antigenicity by detecting binding to antibody, various immunoassays
known in the art can be used, including but not limited to
competitive and non-competitive assay systems using techniques such
as radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in vivo immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
western blots, immunoprecipitation reactions, agglutination assays
(e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody is detected
by detecting binding of a secondary antibody or reagent to the
primary antibody. In a further embodiment, the secondary antibody
is labelled. Many means are known in the art for detecting binding
in an immunoassay and are envisioned for use. In one embodiment for
detecting immunogenicity, T cell-mediated responses can be assayed
by standard methods, e.g., in vitro cytoxicity assays or in vivo
delayed-type hypersensitivity assays.
[0078] Potentially useful antigens or derivatives thereof for use
as antigenic molecules can also be identified by various criteria,
such as the antigen's involvement in neutralization of a pathogen's
infectivity (wherein it is desired to treat or prevent infection by
such a pathogen) (Norrby, 1985, Summary, in Vaccines 85, Lerner, et
al. (eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., pp. 388-389), type or group specificity, recognition by
patients' antisera or immune cells, and/or the demonstration of
protective effects of antisera or immune cells specific for the
antigen. In addition, where it is desired to treat or prevent a
disease caused by pathogen, the antigen's encoded epitope should
preferably display a small or no degree of antigenic variation in
time or amongst different isolates of the same pathogen.
[0079] Preferably, where it is desired to treat or prevent cancer,
known tumor-specific antigens or fragments or derivatives thereof
are used. For example, such tumor specific or tumor-associated
antigens include but are not limited to KS 1/4 pan-carcinoma
antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal,
1988, Hybridoma 7(4):407-415); ovarian carcinoma antigen (CA125)
(Yu, et al., 1991, Cancer Res. 51(2):468-475); prostatic acid
phosphate (Tailer, et al., 1990, Nucl. Acids Res. 18(16):4928);
prostate specific antigen (Henttu and Vihko, 1989, Biochem.
Biophys. Res. Comm. 160(2):903-910; Israeli, et al., 1993, Cancer
Res. 53:227-230); melanoma-associated antigen p97 (Estin, et al.,
1989, J. Natl. Cancer Inst. 81(6):445-446); melanoma antigen gp75
(Vijayasardahl, et al., 1990, J. Exp. Med. 171(4):1375-1380); high
molecular weight melanoma antigen (Natali, et al., 1987, Cancer
59:55-63) and prostate specific membrane antigen.
[0080] In a specific embodiment, an antigen or fragment or
derivative thereof specific to a certain tumor is selected for
complexing to hsp and subsequent administration to a patient having
that tumor.
[0081] Preferably, where it is desired to treat or prevent viral
diseases, molecules comprising epitopes of known viruses are used.
For example, such molecules comprise epitopes from proteins of
viruses including, but not limited to, hepatitis type A hepatitis
type B, hepatitis type C, influenza, varicella, adenovirus, herpes
simplex type I (HSVI), herpes simplex type II (HSV-II), rinderpest,
rhinovirus, echovirus, rotavirus, respiratory syncytial virus,
papilloma virus, papova virus, cytomegalovirus, echinovirus,
arbovirus, huntavirus, coxsachie virus, mumps virus, measles virus,
rubella virus, polio virus, human immunodeficiency virus type I
(HIV-I), and human immunodeficiency virus type II (HIV-II).
[0082] Preferably, where it is desired to treat or prevent
bacterial infections, molecules comprising epitopes of known
bacteria are used. For example, such antigenic epitopes may be from
bacteria including, but not limited to, mycobacteria rickettsia,
mycoplasma, neisseria and legionella.
[0083] Preferably, where it is desired to treat or prevent
protozoal infectious, molecules comprising epitopes of known
protozoa are used. For example, such protozoa include, but are not
limited to, leishmania, kokzidioa, and trypanosoma.
[0084] Preferably, where it is desired to treat or prevent
parasitic infectious, molecules comprising epitopes of known
parasites are used. For example, such antigenic epitopes may be
from parasites including, but not limited to, chlamydia and
rickettsia.
[0085] 5.3. In Vitro Production of Stress Protein-Antigenic
Molecule Complexes
[0086] As will be appreciated by those skilled in the art,
exogenous antigenic molecules, either purified from natural sources
or chemically synthesized or recombinantly produced, may be
reconstituted with a variety of naturally purified or chemically
synthesized or recombinantly produced stress proteins in vitro to
generate immunogenic noncovalent stress protein-antigenic molecule
complexes. A preferred, exemplary protocol for noncovalently
complexing a stress protein and an exogenous antigenic molecule in
vitro is described below.
[0087] Prior to complexing, the hsps are pretreated with ATP or low
pH to remove any peptides that may be associated with the hsp of
interest. When the ATP procedure is used, excess ATP is removed
from the preparation by the addition of apyranase as described by
Levy, et al., 1991, Cell 67:265-274. When the low pH procedure is
used, the buffer is readjusted to neutral pH by the addition of pH
modifying reagents.
[0088] The antigenic molecules (1 .mu.g) and the pretreated hsp (9
.mu.g) are admixed to give an approximately 5 antigenic molecule:1
stress protein molar ratio. Then, the mixture is incubated for 15
minutes to 3 hours at room temperature in a suitable binding buffer
such as one containing 20 mM sodium phosphate, pH 7.2, 350 mM NaCl,
3 mM MgCl.sub.2 and 1 mM phenyl methyl sulfonyl fluoride (PMSF).
The preparations are centrifuged through Centricon 10 assembly
(Millipore) to remove any unbound peptide. The association of the
peptides with the stress proteins can be assayed by SDS-PAGE. This
is the preferred method for in vitro complexing of peptides
isolated from NHC-peptide complexes of peptides disassociated from
endogenous hsp-peptide complexes.
[0089] In an alternative embodiment of the invention, preferred for
producing complexes of hsps70 to exogenous antigenic molecules that
are proteins, 5-10 micrograms of purified hsp is incubated with
equimolar quantities of the antigenic molecule in 20 mM sodium
phosphate buffer pH 7.5, 0.5 M NaCl, 3 mM MgCl.sub.2 and 1 mM ADP
in a volume of 100 microliter at 37.degree. C. for 1 hr. This
incubation mixture is further diluted to 1 ml in phosphate-buffered
saline.
[0090] In an alternate embodiment of the invention, preferred for
producing complexes of gp96 or hsp90 to peptides 5-10 micrograms of
purified gp96 or hsp90 is incubated with equimolar or excess
quantities of the antigenic peptide in a suitable buffer such as
one containing 20 mM sodium phosphate buffer 7.5, 0.5 m NaCl, 3 mM
MgCl.sub.2 at 60-65.degree. C. for 5-20 minutes. This incubation
mixture is allowed to cool to room temperature and centrifuged more
than once if necessary through Centrican 10 assembly (Millipore to
remove any unbound peptide).
[0091] Following complexing, the immunogenic stress
protein-antigenic molecule complexes can optionally be assayed in
vitro using for example the mixed lymphocyte target cell assay
(MLTC) described below. Once immunogenic complexes have been
isolated they can be optionally characterized further in animal
models using the preferred administration protocols and excipients
discussed below.
[0092] 5.4. Determination of Immunogenicity of Stress
Protein-Peptide Complexes
[0093] In an optional procedure, the purified stress
proteinantigenic molecule complexes can be assayed for
immunogenicity using the mixed lymphocyte target culture assay
(MLTC) well known in the art.
[0094] By way of example but not limitation, the following
procedure can be used. Briefly, mice are injected subcutaneously
with the candidate stress protein-antigenic molecule complexes.
Other mice are injected with either other stress protein peptide
complexes or whole infected cells which act as positive controls
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 restimulated
subsequently in vitro by the addition of dead cells that expressed
the complex of interest.
[0095] For example, 8.times.10.sup.6 immune spleen cells may be
stimulated with 4.times.10.sup.4 mitomycin C treated or
.gamma.-irradiated (5-10,000 rads) infected cells (or cells
transfected with an appropriate gene, as the case may be) in 3 ml
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 restimulated with
antigenically distinct cells, to determine the specificity of the
cytotoxic T cell response.
[0096] Six days later the cultures are tested for cytotoxicity in a
4 hour .sup.51Cr-release assay (See, Palladino, et al., 1987,
Cancer Res. 47:5074-5079 and Blachere, at al., 1993, J.
Immunotherapy 14:352-356). In this assay, the mixed lymphocyte
culture is added to a target cell suspension to give different
effector:target (E:T) ratios (usually 1:1 to 40:1). The target
cells are prelabelled by incubating 1.times.10.sup.6 target cells
in culture medium containing 200 mCi .sup.51Cr/ml for one hour at
37.degree. C. The cells are washed three times following labeling.
Each assay point (E:T ratio) is performed in triplicate and the
appropriate controls incorporated to measure spontaneous .sup.51Cr
release (no lymphocytes added to assay) and 100% release (cells
lysed with detergent). After incubating the cell mixtures for 4
hours, the cells are peletted by centrifugation at 200 g for 5
minutes. The amount of .sup.51Cr 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.
[0097] 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%.
[0098] 5.5. Formulation
[0099] Complexes of the invention (of hsps noncovalently bound to
exogenous antigenic molecules) may be formulated into
pharmaceutical preparations for administration to mammals for
treatment of tumors and infectious diseases. Preferred dosages,
routes of administration and therapeutic regimens are described in
copending application by P. Srivastava entitled "Compositions and
Methods for the Prevention and Treatment of Primary and Metastatic
Neoplastic Diseases and Infectious Diseases with Heat Shock/Stress
Proteins, filed on even date herewith, which is incorporated by
reference herein in its entirety. For example, pharmaceutical
formulations are provided, based on a newly-discovered
extrapolation and scaling of animal dosage to human, comprising
compositions of complexes of antigenic molecules and heat
shock/stress proteins, including but not limited to hsp70, hsp90,
gp96 either alone or in combination. Specifically, interspecies
dose-response equivalence for hsp noncovalently bound to antigenic
molecules for a human dose is estimated as the product of the
therapeutic dosage observed in mice and a single scaling ratio, not
exceeding a 50-fold increase. In preferred aspects, an amount of
hsp70- and/or gp96-antigenic molecule complex is administered to a
human that is in the range of about 10-600 .mu.g, preferably 10-100
.mu.g, most preferably about 25 .mu.g, given once weekly for about
4-6 weeks, subcutaneously with the site of administration varied
sequentially. Preferred amounts for hsp90-antigenic molecule
complexes are in the range of 50-5,000 .mu.g, preferably 100
.mu.g.
[0100] Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may be prepared,
packaged, and labelled for treatment of the indicated tumor, such
as human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
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, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytqma, 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. Alternatively, it can
be formulated and labeled for treatment of the appropriate
infectious disease.
[0101] If the complex 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 compounds 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 or, in the case
of tumors, directly injected into a solid tumor.
[0102] 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.
[0103] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0104] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0105] For administration by inhalation, the compounds for use
according to the present invention are 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 compound and a
suitable powder base such as lactose or starch.
[0106] The compounds 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 multi-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.
[0107] The compounds 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.
[0108] In addition to the formulations described previously, the
compounds 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 compounds 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.
[0109] 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 for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0110] 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 the hsp-antigenic molecule complexes in pharmaceutically
acceptable form. The hsp-antigenic molecule complex 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 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 to form a solution for
injection purposes.
[0111] In another embodiment, a kit of the invention further
comprises a needle or syringe, preferably packaged in sterile form,
for injecting the complex, and/or a packaged alcohol pad.
Instructions are optionally included for administration of
hsp-antigenic molecule complexes by a clinician or by the
patient.
[0112] 5.6. Target Infectious Diseases
[0113] Infectious diseases that can be treated or prevented by the
methods of the present invention are caused by infectious agents
including, but not limited to viruses, bacteria, fungi protozoa and
parasites.
[0114] 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,
coxsachie virus, mumps virus, measles virus, rubella virus, polio
virus, human immunodeficiency virus type I (HIV-I), and human
immunodeficiency virus type II (HIV-II).
[0115] Bacterial diseases that can be treated or prevented by the
methods of the present invention are caused by bacteria including,
but not limited to, mycobacteria rickettsia, mydoplasma, neisseria
and legionella.
[0116] 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 trypanosoma.
[0117] Parasitic diseases that can be treated or prevented by the
methods of the present invention are caused by parasites including,
but not limited to, chlamydia and rickettsia.
[0118] 5.7. Target Cancers
[0119] Cancers that can be treated or prevented by the methods of
the present invention include, but not limited to human sarcomas
and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, 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, Wilms'
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. Specific examples of
such cancers are described in the sections below.
[0120] 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 therapy
(e.g., chemotherapy radiation) prior to administration of the
hsp-antigenic molecule complexes of the invention. In another
specific embodiment, the cancer is a tumor.
[0121] 5.7.1. Colorectal Cancer Metastatic to the Liver
[0122] In 1992, approximately 150,000 Americans were diagnosed with
colorectal cancer and more than 60,000 died as a result of
colorectal metastases. At the time of their deaths, 80 percent of
patients with colorectal cancer have metastatic disease involving
the liver, and one-half of these patients have no evidence of other
(extrahepatic) metastases. Most metastatic tumors of the liver are
from gastrointestinal primaries. Unfortunately, the natural history
of metastatic liver lesions carries a grave prognosis and systemic
chemotherapy regimens have been unable to induce significant
response rates or alter length of survival (Drebin, J. A., et al.,
in Current Therapy In Oncology, ed. J. E. Niederhuber, B. C.
Decker, Mosby, 1993, p.426).
[0123] Colorectal cancer initially spreads to regional lymph nodes
and then through the portal venous circulation to the liver, which
represents the most common visceral site of metastasis. The
symptoms that lead patients with colorectal cancer to seek medical
care vary with the anatomical location of the lesion. For example,
lesions in the ascending colon frequency ulcerate, which leads to
chronic blood loss in the stool.
[0124] Radical resection offers the greatest potential for cure in
patients with invasive colorectal cancer. Before surgery, the CEA
titer is determined. Radiation therapy and chemotherapy are used in
patients with advanced colorectal cancer. Results with
chemotherapeutic agents (e.g., 5-fluorouracil) are mixed and fewer
than 25 percent of patients experience a greater than 50 percent
reduction in tumor mass (Richards, 2d., F., et al., 1986, J. Clin.
Oncol. 4:565).
[0125] Patients with widespread metastases have limited survival
and systemic chemotherapy has little impact in this group of
patients. In addition, systemically administered chemotherapy is
often limited by the severity of toxicities associated with the
various agents, such as severe diarrhea, mucositis and/or
myelosuppression. Other techniques, including hepatic radiation,
systemic chemotherapy, hepatic arterial ligation, tumor
embolization and immunotherapy have all been explored, but, for the
most part, have proven ineffectual in prolonging patient
survival.
[0126] In a specific embodiment, the present invention provides
compositions and methods for enhancing tumor specific immunity in
individuals suffering from colorectal cancer metastasized to the
liver, in order to inhibit the progression of the neoplastic
disease.
[0127] Accordingly, as an example of the method of the invention,
gp96 is administered to a patient diagnosed with colorectal cancer,
with or without liver metastasis, via one of many different routes
of administration, the preferred routes being subcutaneous at
different anatomical sites, e.g., left arm, right arm, left belly,
right belly, left thigh, right thigh, etc. These routes of
administration are used in sequence and the site of injection is
varied for each weekly injection as described in Section 7. The
preparations and use of therapeutically effective compositions for
the prevention and treatment of primary and metastatic cancers are
described in detail in the sections which follow and by way of
example, infra.
[0128] 5.7.2. Hepatocellular Carcinoma
[0129] Hepatocellular carcinoma is generally a disease of the
elderly in the United States. Although many factors may lead to
hepatocellular carcinoma, the disease is usually limited to those
persons with preexisting liver disease. Approximately 60 to 80
percent of patients in the United States with hepatocellular
carcinoma have a cirrhotic liver and about four percent of
individuals with a cirrhotic liver eventually develop
hepatocellular carcinoma (Niederhuber, J. E., (ed.), 1993, Current
Therapy in Oncology, B. C. Decker, Mosby). The risk is highest in
patients whose liver disease is caused by inherited hemochromatosis
or hepatic B viral infection (Bradbear, R. A., et al., 1985, J.
Natl. Cancer Inst. 75:81; Beasley, R. P., et al., 1981, Lancet
2:1129). Other causes of cirrhosis that can lead to hepatocellular
carcinoma include alcohol abuse and hepatic fibrosis caused by
chronic administration of methotrexate. The most frequent symptoms
of hepatocellular carcinoma are the development of a painful mass
in the right upper quadrant or epigastrium, accompanied by weight
loss. In patients with cirrhosis, the development of hepatocellular
carcinoma is preceded by ascites, portal hypertension and
relatively abrupt clinical deterioration. In most cases,
abnormal-values in standard liver function tests such as serum
aminotransferase and alkaline phosphatase are observed.
[0130] CT scans of the liver are used to determine the anatomic
distribution of hepatocellular carcinoma and also provide
orientation for percutaneous needle biopsy. Approximately 70
percent of patients with hepatocellular carcinoma have an elevated
serum alpha-fetoprotein concentration (McIntire, K. R., et al.,
1975, Cancer Res. 35:991) and its concentration correlates with the
extent of the disease.
[0131] Radical resection offers the only hope for cure in patients
with hepatocellular carcinoma. Such operative procedures are
associated with five-year survival rates of 12 to 30 percent. Liver
transplantation may improve survival of some younger individuals.
However, most patients are not surgical candidates because of
extensive cirrhosis multifocal tumor pattern or scarcity of
compatible donor organs. Chemotherapeutic agents have been
administered either by intravenous route or through an intrahepatic
arterial catheter. Such therapy has sometimes been combined with
irradiation to the liver. Reductions in the size of measurable
tumors of 50% or more have been reported in some patients treated
with either systemic doxorubicin or 5-fluorouracil. However,
chemotherapy often induces immunosuppression and rarely causes the
tumor to disappear completely and the duration of response is
short. The prognosis for patients with hepatocellular carcinoma is
negatively correlated with cirrhosis and metastases to the lungs or
bone. Median survival for patients is only four to six months. In
another specific embodiment, the present invention provides
compositions and methods for enhancing specific immunity in
individuals suffering from hepatocellular carcinoma in order to
inhibit the progression of the neoplastic disease and ultimately
irradiate all preneoplastic an neoplastic cells.
[0132] 5.7.3. Breast Cancer
[0133] Another specific aspect of the invention relates to the
treatment of breast cancer. The American Cancer Society estimated
that in 1992 180,000 American women were diagnosed with breast
cancer and 46,000 succumbed to the disease (Niederhuber, J. E. ed.
Current Therapy in Oncology B. C. Decker, Mosby, 1993). This makes
breast cancer the second major cause of cancer death in women,
ranking just behind lung cancer. A disturbing fact is the
observation that breast cancer has been increasing at a rate of 3
percent per year since 1980 (Niederhuber, J. E., ed. Current
Therapy in Oncology, B. C. Decker, Mosby, (1993)). The treatment of
breast cancer presently involves surgery, radiation, hormonal
therapy and/or chemotherapy. Consideration of two breast cancer
characteristics, hormone receptors and disease extent, has governed
how hormonal therapies and standard-dose chemotherapy are sequenced
to improve survival and maintain or improve quality of life. A wide
range of multidrug regimens have been used as adjuvant therapy in
breast cancer patients, including, but not limited to combinations
of 2 cyclophosphamide, doxorubicin, vincristine methotrexate,
5-fluorouracil and/or leucovorin. In a specific embodiment, the
present invention provides hsp compositions and methods for
enhancing specific immunity to preneoplastic and neoplastic mammary
cells in women. The present invention also provides compositions
and methods for preventing the development of neoplastic cells in
women at enhanced risk for breast cancer, and for inhibiting cancer
cell proliferation and metastasis. These compositions can be
applied alone or in combination with each other or with biological
response modifiers.
[0134] 5.8. Prevention and Treatment of Primary and Metastatic
Neoplastic Diseases
[0135] 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 and surgery, with anesthesia,
and subsequent chemotherapy, may worsen the immunosuppression, then
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.
[0136] In a specific embodiment, the preventive and therapeutic
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. 5.9. Monitoring of Effects During Cancer Prevention
and Immunotherapy with Hsp-Peptide Complexes
[0137] The effect of immunotherapy with hsp-antigenic molecule
complexes on development and progression of neoplastic diseases can
be monitored by any methods known to one skilled in the art,
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 tumor specific antigens, e.g.,
carcinoembryonic (CEA) antigens; d) changes in the morphology of
tumors using techniques such as a computed tomographic (CT) scan;
e) changes in levels of putative biomarkers of risk for a
particular cancer in individuals at high risk, and f) changes in
the morphology of tumors using a sonogram.
[0138] 5.9.1. Delayed Hypersensitivity Skin Test
[0139] Delayed hypersensitivity skin tests are 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, T., et al, 1995, Clin. Immunol. Pathol.
74:35-43).
[0140] Proper technique of skin testing requires that the antigens
be stored sterile at 4.degree. C., protected from light and
reconstituted shorted before use. A 25- or 27-gauge need ensures
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.
[0141] 5.9.2. Activity of Cytolytic T-Lymphocytes In Vitro
[0142] 8.times.10.sup.6 Peripheral blood derived T lymphocytes
isolated by the Ficoll-Hypaque centrifigation gradient technique,
are restimulated with 4.times.10.sup.4 mitomycin C treated tumor
cells in 3 ml 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.
[0143] In order to measure the primary response of cytolytic
T-lymphocytes after immunization, T cells are cultured without the
stimulator tumor cells. In other experiments, T cells are
restimulated with antigenically distinct cells. After six days, the
cultures are tested for cytotoxity in a 4 hour .sup.51Cr-release
assay. The spontaneous .sup.51Cr-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 M., et
al., J. Immunotherapy 15:165-174).
[0144] 5.9.3. Levels of Tumor Specific Antigens
[0145] Although it may not be possible to detect unique tumor
antigens on all tumors, many tumors display antigens that
distinguish them from normal cells. The monoclonal antibody
reagents have permitted the isolation and biochemical
characterization of the antigens and have been invaluable
diagnostically for distinction of transformed from nontransformed
cells and for definition of the cell lineage of transformed cells.
The best-characterized human tumor-associated antigens are the
oncofetal antigens. These antigens are expressed during
embryogenesis, but are absent or very difficult to detect in normal
adult tissue. The prototype antigen is carcinoembryonic antigen
(CEA), a glycoprotein found on fetal gut an human colon cancer
cells, but not on normal adult colon cells. Since CEA is shed from
colon carcinoma cells and found in the serum, it was originally
thought that the presence of this antigen in the serum could be
used to screen patients for colon cancer. However, patients with
other tumors, such as pancreatic and breast cancer, also have
elevated serum levels of CEA. Therefore, monitoring the fall and
rise of CEA levels in cancer patients undergoing therapy has proven
useful for predicting tumor progression and responses to
treatment.
[0146] Several other oncofetal antigens have been useful for
diagnosing and monitoring human tumors, e.g., alpha-fetoprotein, an
alpha-globulin normally secreted by fetal liver and yolk sac cells,
is found in the serum of patients with liver and germinal cell
tumors and can be used as a matter of disease status.
[0147] 5.9.4. Computed Tomographic (CT) Scan
[0148] CT remains the choice of techniques for the accurate staging
of cancers. CT has proved more sensitive and specific than any
other imaging techniques for the detection of metastases.
[0149] 5.9.5. Measurement of Putative Biomarkers
[0150] The levels of a putative biomarker for risk of a specific
cancer are measured to monitor the effect of hsp noncovalently
bound to peptide complexes. For example, in individuals at enhanced
risk for prostate cancer, serum prostate-specific antigen (PSA) is
measured by the procedure described by Brawer, M. K., et. al.,
1992, J. Urol. 147:841845, and Catalona, W. J., et al., 1993, JAMA
270:948-958; or in individuals at risk for colorectal cancer CEA is
measured as described above in Section 4.5.3; and in individuals at
enhanced risk for breast cancer, 16-.alpha.-hydroxylation of
estradiol is measured by the procedure described by Schneider, J.
et al., 1982, Proc. Natl. Acad. Sci. USA 79:3047-3051. The
references cited above are incorporated by reference herein in
their entirety.
[0151] 5.9.6. Sonogram
[0152] A sonogram remains an alternative choice for technique for
the accurate staging of cancers.
6. EXAMPLE
Administration of HSP-Exogenous Antigens in the Treatment of
Hepatocellular Carcinoma
[0153] Patients with hepatocellular carcinoma are injected with
hsp-antigenic molecule peptide complexes prepared in vitro from
purified hsp and purified antigen. The antigen used is
carcinoembryonic antigen (CEA). Treatment with hsp-antigen
complexes is started any time after surgery. However, if the
patient has received chemotherapy, hsp-antigen complexes are
usually administered after an interval of four weeks or more so as
to allow the immune system to recover. The immunocompetence of the
patient is tested by procedures described in sections 5.9
above.
[0154] The therapeutic regimen includes weekly injections of the
hsp-antigen complex, dissolved in saline or other physiologically
compatible solution.
[0155] The dosage used for hsp70 or gp96 is in the range of 10-600
micrograms, with the preferred dosage being 10-100 micrograms. The
dosage used for hsp90 is in the range of 50 to 5,000 micrograms,
with the preferred dosage being about 100 micrograms.
[0156] The route and site of injection is varied each time, for
example, the first injection is given subcutaneously on the left
arm, the second injection on the right arm, the third injection on
the left abdominal region, the fourth injection on the right
abdominal region, the fifth injection on the left thigh, the sixth
injection on the right thigh, etc. The same site is repeated after
a gap of one or more injections. In addition, injections are split
and each half of the dose is administered at a different site on
the same day.
[0157] Overall, the first four to six injections are given at
weekly intervals. Subsequently, two injections are given at
two-week intervals, followed by a regimen of injections at monthly
intervals. The effect of hsp-antigen complexes therapy is monitored
by measuring: a) delayed hypersensitivity as an assessment of
cellular immunity; b) activity of cytolytic T-lymphocytes in vitro;
c) levels of tumor specific antigens, e.g., carcinoembryonic (CEA)
antigens; d) changes in the morphology of tumors using techniques
such as a computed tomographic (CT) scan; and e) changes in
putative biomarkers of risk for a particular cancer in individuals
at high risk.
[0158] Depending on the results obtained, the therapeutic regimen
is developed to maintain and/or boost the immunological responses
of the patient, with the ultimate goal of achieving tumor
regression and complete eradication of cancer cells.
7. EXAMPLE
Administration of HSP-Exogenous Antigen Complexes in the Treatment
of Colorectal Cancer
[0159] Hsp-antigen complexes (comprising gp96, hsp70, hsp90 or a
combination thereof) are administered as adjuvant therapy and as
prophylactic adjuvant therapy in patients after complete reduction
of colorectal cancer to eliminate undetectable micrometastases and
to improve survival.
[0160] The therapeutic and prophylactic regimens used in patients
suffering from colorectal cancer are the same as those described in
Section 6 above for patients recovering with hepatocellular
carcinoma. The antigen used as the exogenous antigenic molecule is
carcinoembryonic antigen. The methods of monitoring of patients
under clinical evaluation for prevention and treatment of
colorectal cancer is done by procedures described in Section
5.9.
8. EXAMPLE
Induction of CTL-Response to HSP70-Ovalbumin complex
[0161] 8.1. Materials and Methods
[0162] Hsp70-ovalbumin complex was prepared in vitro. Briefly, 5-10
micrograms of purified hsp70 was incubated with equimolar
quantities of ovalbumin in 20 mM sodium phosphate buffer pH 7.5,
0.5 NaCl, 3 mM MgCl.sub.2 and 1 mM ADP in a volume of 100
microliter at 37.degree. C. for 1 hour. This incubation mixture was
then further diluted to 1 ml in phosphate-buffered saline and
injected sub-cutaneously into the mammal of choice, the C57BL/6
strain of mice.
[0163] The injections were repeated once a week interval. The
hsp70-ovalbumin complex was prepared fresh for each injection. A
total of two injections was administered before sacrificing the
animals. Two mice in each group were immunized with: a) control
vehicle; b) ovalbumin alone; c) hsp70 alone; or d) hsp70-ovalbumin
complex.
[0164] T cells were isolated from the spleen of each mouse using
the T cell-gradient centrifugation technique and 8.times.10.sup.5 T
cells were incubated with 4.times.10.sup.4 EG7 cells (positive for
ovalbumin antigen) or EL4 cells (negative for ovalbumin antigen).
The CTL response was measured as % .sup.51Cr release.
[0165] 8.2. Results
[0166] Hsp70-ovalbumin complex induced a far greater CTL response
than ovalbumin alone or hsp70 alone (FIG. 1A). However, the T cells
did not respond to the EL4 cells which lack the ovalbumin antigen
(FIG. 1B).
[0167] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0168] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
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