U.S. patent application number 12/583271 was filed with the patent office on 2010-01-28 for processes for treating subjects carrying viral infectious agent.
This patent application is currently assigned to ENZO THERAPEUTICS., C/O ENZO BIOCHEM, INC.. Invention is credited to James J. Donegan, Dean L. Engelhardt, Israel Gotsman, Yaron Ilan, Elazar Rabbani.
Application Number | 20100021500 12/583271 |
Document ID | / |
Family ID | 24242629 |
Filed Date | 2010-01-28 |
United States Patent
Application |
20100021500 |
Kind Code |
A1 |
Ilan; Yaron ; et
al. |
January 28, 2010 |
Processes for treating subjects carrying viral infectious agent
Abstract
This invention provides novel processes for therapeutic
applications, including the treatment of subjects carrying
infectious agents or having impaired autoimmunity or impaired
immune condition. The therapeutic applications disclosed herein are
also directed at the treatment of cancerous subjects with malignant
tumors containing cancerous cells or malignant or cancerous cells.
Vaccination processes for preventing infections in subjects are
also provided. The novel processes comprise introducing into or
adminstering to a subject one or more antigens, or trained or
adopted immune cells. These antigens or immune cells are capable of
establishing or increasing at least one first specific immune
response and decreasing at least one second specific immune
response. Such responses include components, such as cellular
immune reaction elements, humoral immune reaction elements and
cytokines, the latter also encompassing interferons and
lymphokines. Useful compositions are also provided by this
invention.
Inventors: |
Ilan; Yaron; (Jerusalem,
IL) ; Rabbani; Elazar; (New York, NY) ;
Engelhardt; Dean L.; (New York, NY) ; Gotsman;
Israel; (Jerusalem, IL) ; Donegan; James J.;
(Long Beach, NY) |
Correspondence
Address: |
ENZO BIOCHEM, INC.
527 MADISON AVENUE (9TH FLOOR)
NEW YORK
NY
10022
US
|
Assignee: |
ENZO THERAPEUTICS., C/O ENZO
BIOCHEM, INC.
New York
NY
|
Family ID: |
24242629 |
Appl. No.: |
12/583271 |
Filed: |
August 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10470611 |
Dec 8, 2003 |
7608274 |
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12583271 |
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09561596 |
Apr 27, 2000 |
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10470611 |
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Current U.S.
Class: |
424/227.1 ;
424/184.1 |
Current CPC
Class: |
A61K 39/001 20130101;
A61K 2035/122 20130101; A61K 2039/57 20130101; A61K 39/12 20130101;
A61P 31/18 20180101; A61K 39/0005 20130101; A61P 31/20 20180101;
A61K 38/00 20130101; A61K 39/292 20130101; A61P 31/00 20180101;
A61K 2039/5152 20130101; C12N 2730/10134 20130101; A61K 2039/542
20130101; A61P 31/22 20180101; A61P 35/00 20180101; A61P 31/12
20180101 |
Class at
Publication: |
424/227.1 ;
424/184.1 |
International
Class: |
A61K 39/29 20060101
A61K039/29; A61K 39/00 20060101 A61K039/00; A61P 37/04 20060101
A61P037/04 |
Claims
1-75. (canceled)
76. A process of treating a subject having an immune system and
carrying a viral infectious agent, said process comprising the
steps of: (a) introducing into or administering to said subject one
or more antigens, wherein said one or more antigens: (1) establish
or increase at least one first immune reaction directed against:
(i) said viral infectious agent; or (ii) cells infected with said
viral infectious agent; or (iii) a combination of (1)(i) and
(1)(ii) above; and (2) decrease at least one second immune reaction
which is different from said first immune reaction (a)(1), said
second immune reaction being directed toward: (i) said viral
infectious agent; or (ii) cells infected with said viral infectious
agent; or (iii) uninfected cells; or (iv) a combination of any of
(2)(i), (2)(ii) and (2)(iii) above, wherein said first immune
reaction establishes or increases one or more of the following:
decrease in viral load by PCR; increase in specific T-cell activity
to viral surface antigen as measured by a T-cell proliferation
assay (T-cell assay); increase in number of specific T-cell clones
secreting IFN .gamma. when exposed to viral surface antigen as
measured by an ELISA Spot Assay; increase in IFN .gamma. and/or IL
10 as measured by RT PCR; increase in cytotoxic lymphocyte
response; or increase in specific serum cytokines; and wherein said
second immune reaction decreases in one or both enzyme (ALT and/or
AST) levels; decrease (improvement) in liver pathology as measured
by standard hemotoxylin & eosin (H&E) staining; decrease in
viral surface antigen staining by immunohistochemistry; or decrease
in viral core antigen staining by immunohistochemistry.
77. The process according to claim 76, wherein said first immune
reaction and said second immune reaction comprise cellular
responses, humoral responses or cytokine responses, and
combinations thereof.
78. The process according to claim 77, wherein said first immune
reaction comprises cellular responses and said second immune
reaction comprises humoral responses or cytokine responses.
79. The process according to claim 76, wherein said one or more
antigens are introduced or administered in multiple doses.
80. The process according to claim 76, wherein said one or more
antigens are introduced or administered in different dosages.
81. The process according to claim 76, wherein said one or more
antigens comprise different epitopes.
82. The process according to claim 76, wherein said one or more
antigens are introduced or administered intermittently or
periodically.
83. The process according to claim 76, wherein said one or more
antigens are introduced or administered by a single route.
84. The process according to claim 76, wherein said one or more
antigens are introduced or administered by a route comprising oral,
intravenous, parenteral, transdermal, subcutaneous, intravaginal,
intranasal, mucosal, sublingual, aural, intrabronchial, topical,
intramuscular, intraperitoneal, or rectal, and combinations of any
of the foregoing.
85. The process according to claim 76, wherein said one or more
antigens are introduced or administered orally.
86. The process according to claim 76, further comprising
introducing or administering one or more carriers.
87. The process according to claim 76, wherein said virus comprises
HBV, HCV or HIV.
88. A process of treating a subject having an immune system and
carrying a viral infectious agent, said process comprising the step
of: (a) introducing into or administering to said subject at least
two different antigens, wherein said at least two different
antigens: (1) establish or increase at least one first immune
reaction directed against: (i) said viral infectious agent; or (ii)
cells infected with said viral infectious agent; or (iii) a
combination of (1)(i) and (1)(ii) above; and (2) decrease at least
one second immune reaction which is different from said first
immune reaction (a)(1), said second immune reaction being directed
toward: (i) said viral infectious agent; or (ii) cells infected
with said viral infectious agent; or (iii) uninfected cells; or
(iv) a combination of any of (2)(i), (2)(ii) and (2)(iii) above;
wherein said first immune reaction establishes or increases one or
more of the following: decrease in viral load by PCR; increase in
specific T-cell activity to viral surface antigen as measured by a
T-cell proliferation assay (T-cell assay); increase in number of
specific T-cell clones secreting IFN .gamma. when exposed to viral
surface antigen as measured by an ELISA Spot Assay; increase in IFN
.gamma. and/or IL 10 as measured by RT PCR; increase in cytotoxic
lymphocyte response; or increase in specific serum cytokines; and
wherein said second immune reaction decreases in one or both enzyme
(ALT and/or AST) levels; decrease (improvement) in liver pathology
as measured by standard hemotoxylin & eosin (H&E) staining;
decrease in viral surface antigen staining by immunohistochemistry;
or decrease in viral core antigen staining by
immunohistochemistry.
89. The process according to claim 88, wherein said first immune
reaction and said second immune reaction are directed to at least
two different epitopes on said at least two different antigens.
90. The process according to claim 89, wherein said first immune
reaction is directed to a first epitope or epitopes on said at
least two different antigens, and said second immune reaction is
directed to a second epitope or epitopes which are different from
said first epitope or epitopes, and wherein said first immune
reaction and said second immune reaction are mediated by different
responses of said subject's immune system.
91. The process according to claim 90, wherein said response or
responses of the first immune reaction comprise cellular responses
and said response or responses of the second immune reaction
comprise humoral responses or cytokine responses.
92. The process according to claim 90, wherein said response or
responses of the first immune reaction comprise humoral responses
and said response or responses of the second immune reaction
comprise cellular responses or cytokine responses.
93. The process according to claim 90, wherein said response or
responses of the first immune reaction comprise cytokine responses
and said response or responses of the second immune reaction
comprises cellular responses or humoral responses.
94. The process according to claim 88, wherein said at least two
different antigens are introduced or administered in a single dose
or in multiple doses.
95. The process according to claim 88, wherein said at least two
different antigens are introduced or administered in different
dosages.
96. The process according to claim 88, wherein said at least two
different antigens are introduced or administered in a manner
comprising continuous administration and intermittent or periodic
administration.
97. The process according to claim 88, wherein said at least two
different antigens are introduced or administered by a single route
or by at least two different routes.
98. The process according to claim 88, wherein said at least two
different antigens are introduced or administered by a route
comprising oral, intravenous, parenteral, transdermal,
subcutaneous, intravaginal, intranasal, mucosal, sublingual, aural,
intrabronchial, topical, intramuscular, intraperitoneal, rectal, or
combinations of any of the foregoing.
99. The process according to claim 88, wherein said one or more
antigens are introduced or administered orally.
100. The process according to claim 88, further comprising
introducing or administering one or more carriers.
101. The process according to claim 88, wherein said virus
comprises HBV, HCV, HIV and a combination of any of the foregoing.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of therapeutic processes
and therapeutic compositions, including treatments and compositions
directed against infectious agents, cancerous conditions and
immunity disorders. This invention also relates to therapeutic
processes and compositions in vaccination and immunization.
[0002] All patents, patent applications, patent publications,
scientific articles and the like, cited or identified in this
application are hereby incorporated by reference in their entirety
in order to describe more fully the state of the art to which the
present invention pertains.
BACKGROUND OF THE INVENTION
[0003] Antigenic stimulation of the immune system induces a series
of reactions which can be mediated by immunological components such
as the humoral, cellular or cytokine responses. The directionality
of these reactions can be considered to be of a reactive or
suppressive nature. For instance, in the context of the present
invention, an immune reaction is defined as a response that
specifically neutralizes, reduces or eliminates the presence of a
specific antigen or set of antigens in a subject. In the context of
the present invention, immune suppression is defined as a response
that specifically diminishes or reduces an immune reaction or has
the capability of blocking an immune reaction from being initiated.
Examples of humoral responses that may contribute to an immune
reaction can comprise or not be limited to the production of
antibodies or proteins involved in complement fixation. Examples of
cellular responses that may contribute to an immune reaction can
comprise but not be limited to expansion of helper T cells, Natural
killer (NK) cells, cytopathic T-lymphocytes (CTLs) and B
lymphocytes. Examples of cytokine responses that may contribute to
an immune reaction can comprise but not be limited to induction of
IFN .gamma. and IL-2. Examples of humoral responses that may
contribute to an immune suppression reaction can comprise or not be
limited to the production of anti-idiotypic antibodies. Examples of
cellular responses that may contribute to an immune suppression
reaction can comprise but not be limited to expansion of supressor
T-cells. Examples of cytokine responses that may contribute to an
immune suppression reaction can comprise but not be limited to
induction of TGF .beta., IL-4 and IL-10
[0004] The stimulation or manipulation of the immune system can be
achieved by the introduction of an antigen or antigens that are
foreign to the subject. This reaction is a major source of the
body's resistance to colonialization by viral, bacterial or
parasitic organisms. The absence of this defense in
immuno-compromised individuals has allowed what are called
opportunistic infections i.e. infections by organisms that are
normally non-pathogenic. Examples of such individuals are patients
undergoing chemotherapy or transplantation, AIDS patients and
individuals with severe combined immune deficiency. Reactivity to
foreign antigen sources is also the source of allergy immune
reactions, i.e. immunostimulation caused by exposure to antigenic
substances present in the environment including dust, pollen, hair
and other materials.
[0005] Immune stimulation can also be induced by substances that
are native to the subject or are immunologically related to native
antigens. An illustrative example of this are antigens that provoke
autoimmune responses. Since reactivity to the cells, tissues and
organs that make up an organism would be self-destructive, there is
a system of control over the induction of this form of immune
reactions. The mechanism that is most widely regarded as
responsible for this self-limitation has been called clonal
deletion. In this model, cells that are stimulated by self-antigens
are selectively eliminated in a process that begins shortly after
birth. After a certain amount of time, the repertoire of
immunogenic responses that remains is devoid of cells capable of
responding to these native stimuli. Since clonal deletion is an
irreversible process, the existence of auto-immunity has been
ascribed to a limited number of cells that were unable to achieve a
"threshold" level of stimulation by native antigens. Then at some
later point in life when clonal elimination processes were absent,
an event or events have occurred that induced a heightened immune
response to native antigens
[0006] Other example of an immune response to a native antigen is
recognition of tumor antigens. The "immune surveillance" theory
proposes that during the course of a lifetime, potentially
tumorogenic cells are constantly arising, but they are recognized
and purged by immune processes. Although proteins expressed by
these cells are derived from the genetic information of the
subject, recognition as antigens may still take place when they are
mutated or inappropriately expressed in a subject. Growth of a
tumor may then take place when there is somehow a breakdown in this
surveillance process.
[0007] Varying degrees of immune response to antigens are seen both
in terms of the intrinsic nature of the particular antigens and
also in terms of the individual response of a subject to their
presence. A given antigen may comprise a single immunostimulatory
epitope or it may comprise a number of epitopes, each of which has
its own potential level of immunostimulatory effect. Stimulatory
activity of an antigen may also be increased by the use of a
supplementary treatment called an adjuvant.
[0008] The series of events created by the presence of a particular
antigen in a subject is typically described in reviews and
textbooks on Immunology as leading to generation of a singular
immune state. For example, in immunization a specific humoral
and/or cellular response against the immunogen is induced. This
"mono-static" view predicts mutually exclusive results of either a
state of immune responsiveness or a state of immune suppression. In
prior art, attempts at alteration of a pre-existing immune state
are still of a unidirectional nature. These have been used either
for the purpose of extending or boosting a particular immune
response or leading to the reversal or suppression of the immune
response. With reference to a particular immune target, either case
is a change from one particular singular state to a different
singular state. Thus, it would be predicted that treatments that
lead to reduction or elimination of any aspect of immune reactivity
towards a pathogen should result in allowance of further
progression in either expression or growth of the pathogen by
releasing the pathogen from immune control. This point has been
discussed previously in a pending patent application, U.S. Ser. No.
08/808/629 filed Feb. 28, 1997 which is incorporated by reference
in its entirety, where it was suggested that drug treatments
suitable for the pathogen would have to be used in conjunction with
an immune therapy treatment. However, the drawback of a need for
such dual therapeutic or pathogen management procedures was
considered to be outweighed by benefits that would be provided by
the reduction of immune responses that contribute to aspects of the
disease state. Examples of such undesirable immune derived aspects
are the inflammation and tissue destruction that are the hallmarks
of chronic HBV and HCV infection. Thus, according to previous views
a decrease in undesirable immune reactivity should also induce a
decrease in other immune responses that may be beneficial for the
continued health of the subject.
SUMMARY OF THE INVENTION
[0009] The present invention provides a treatment process for
subjects, i.e., a human subject, carrying an infectious agent. The
process comprises introducing into or administering to the subject
one or more antigens. Such antigens are characterized in being
capable of (1) establishing or increasing at least one first
specific immune reaction directed against (i) the infectious agent,
or (ii) cells infected with the infectious agent, or (iii) a
combination of (1)(i) and (1)(ii). These antigens are further
characterized in being capable of (2) decreasing at least one
second specific immune reaction which is different from the first
specific immune reaction (a)(1), the second specific immune
reaction itself being directed toward (i) the infectious agent, or
(ii) cells infected with the infectious agent; or (iii) uninfected
cells; or (iv) a combination of any of (2)(i), (2)(ii) and (2)(iii)
just mentioned.
[0010] The present invention also provides a process of treating a
subject carrying an infectious agent. In this aspect of the
invention, the process comprises the steps of (a) introducing into
or administering to the subject at least two different antigens,
each of these antigens being capable of (1) establishing or
increasing at least one first specific immune reaction directed
against:(i) the infectious agent; or (ii) cells infected with the
infectious agent; or (iii) a combination of (1)(i) and (1)(ii) just
described. The antigens are further capable of (2) decreasing at
least one second specific immune reaction which is different from
the first specific immune reaction (a)(1). The second specific
immune reaction is itself directed toward (i) the infectious agent;
or (ii) cells infected with the infectious agent; or (iii)
uninfected cells; or (iv) a combination of any of (2)(i), (2)(ii)
and (2)(iii) just described.
[0011] Also provided by the present invention is a process of
treating a subject carrying an infectious agent in which immune
cells are usefully trained or adopted. Here, the steps involve (a)
removing immune cells from said subject, (b) training or adopting
said removed cells, (c) introducing into or administering to the
subject the immune cells which have been trained or adopted, e.g.,
in vivo or in vitro. Such immune cells are capable of (1)
establishing or increasing at least one first specific immune
reaction directed against:(i) the infectious agent; or (ii) cells
infected with the infectious agent; or (iii) a combination of
(1)(i) and (1)(ii) just described. The immune cells are also
capable of (2) decreasing at least one second specific immune
reaction which is different from the first specific immune reaction
(a)(1). The second specific immune reaction is directed toward:(i)
the infectious agent; or (ii) cells infected with the infectious
agent; or both of the foregoing.
[0012] Still provided by this invention is a process of treating a
subject carrying an infectious agent, the process utilizing immune
cells and multiple steps. First, immune cells are removed from a
trained donor, or from a naive donor wherein the immune cells have
been trained in a surrogate or in vitro. Second, the removed immune
cells are introduced into or administered to the subject. These
immune cells are characterized in being capable of (1) establishing
or increasing at least one first specific immune reaction directed
against (i) the infectious agent; or (ii) cells infected with the
infectious agent; or (iii) a combination of (1)(i) and (1)(ii) just
described. The immune cells are also capable of (2) decreasing at
least one second specific immune response which is different from
the first specific immune reaction (a)(1). Here, the second
specific immune response is directed toward (i) the infectious
agent; or (ii) cells infected with the infectious agent; or (iii)
uninfected cells; or (iv) a combination of any of (2)(i), (2)(ii)
and (2)(iii) as just described. Finally, the subject is managed,
monitored or treated for graft-versus-host complications.
[0013] Another process provided herein is a process for treating a
cancerous subject who could have such cancer in the form of a tumor
containing cancerous cells, or in the form of cancerous cells. This
process comprises the step or steps of (a) introducing into or
administering to the subject one or more specific antigens which
are capable of two significant functions. First, these specific
antigens are capable of (1) establishing or increasing at least one
first specific immune reaction directed against (i) cancer
associated antigens; or (ii) cancerous cells; or (iii) a
combination of (1)(i) and (1)(ii) just described. These specific
antigens are also capable of (2)decreasing at least one second
specific immune reaction which is different from the first specific
immune reaction, in that the second specific immune reaction is
directed toward (i) any cancer associated antigens; or (ii) any
cancerous cells; or (iii) any non-cancerous cells; or (iii) a
combination of these last three elements.
[0014] Another useful process provided by this invention involves
treating a cancerous subject who has a tumor containing cancerous
cells, or who has cancerous cells. Here, the process comprising the
steps of (a) removing immune cells from the cancerous subject, (b)
training or adopting said removed cells, (c) introducing into or
administering to said subject said immune cells which have been
rendered capable of (1) establishing or increasing at least one
first specific immune reaction directed against (i) cancer
associated antigens; or (ii) cancerous cells; or (iii) a
combination of (1)(i) and (1)(ii) just described. The immune cells
are further capable of (2) decreasing at least one second specific
immune reaction which is different from the first specific immune
reaction (a)(1). This second specific immune reaction is directed
toward (i) the cancer associated antigens; or (ii) the cancerous
cells; or (iii) non-cancerous cells; or (iii) a combination of
(2)(i), (2)(ii) and (2)(iii) just described.
[0015] Another process provided herein is useful for treating a
cancerous subject who has a tumor containing cancerous cells, or
who has cancerous cells. This process comprises the first step of
(a) removing immune cells from a trained donor, or from a naive
donor wherein the immune cells have been trained in a surrogate or
in vitro. The next step involves (b) introducing into or
administering to the subject the immune cells which were removed.
The immune cells have been rendered capable of (1) establishing or
increasing at least one first specific immune reaction directed
against (i) cancer associated antigens; or (ii) cancerous cells; or
(iii) a combination of (1)(i) and (1)(ii) as just described. The
immune cells are further capable of (2) decreasing at least one
second specific immune response which is different from the first
specific immune reaction (a)(1). The second specific immune
response is directed toward (i) cancer associated antigens; or (ii)
cancerous cells; or (iii) non-cancerous cells; or (iii) a
combination of (2)(i), (2)(ii) and (2)(iii) as just described. The
next step of the process calls for (c) managing or treating the
subject for graft-versus-host complications.
[0016] Another process is provided for enhancing the immunized
state of a subject vaccinated against an infectious agent. This
process comprises the step or steps of (a) introducing into or
administering to the subject one or more specific antigens, such
antigen or antigens being capable of (1) establishing or increasing
at least one first specific immune reaction directed against the
infectious agent; and (2) decreasing at least one second specific
immune reaction which is different from the first specific immune
reaction (a)(1). The second specific immune reaction is directed
toward (i) the infectious agent; or (ii) uninfected cells; or (iii)
a combination of (2)(i) and (2)(ii) just described.
[0017] Another process is useful for enhancing the immunized state
of a subject vaccinated against an infectious agent. Here, the
process comprises the steps of: (a) removing immune cells from the
subject, (b) training or adopting the cells so removed, and (c)
introducing into or administering to the subject these immune cells
which have been rendered capable of two significant biological
functions. First, the immune cells are capable of (1) establishing
or increasing at least one first specific immune reaction directed
against the infectious agent; and (2) decreasing at least one
second specific immune reaction which is different from the first
specific immune reaction (a)(1). The second specific immune
reaction is directed toward (i) the infectious agent; or (ii)
uninfected cells; or (iii) a combination of (2)(i) and (2)(ii) as
just described.
[0018] Still yet another process is useful for enhancing the
immunized state of a subject vaccinated against an infectious
agent. This process comprises the steps of (a) removing immune
cells from a trained donor, or from a naive donor wherein the
immune cells have been trained in a surrogate or in vitro, and (b)
introducing into or administering to the subject the removed immune
cells which have been rendered capable of two significant
biological or immunological functions. First, these immune cells
are capable of (1) establishing or increasing at least one first
specific immune reaction directed against the infectious agent; and
(2) decreasing at least one second specific immune reaction which
is different from said the specific immune reaction (a)(1). This
second specific immune reaction is directed toward (i) the
infectious agent; or (ii) uninfected cells; or (iii) a combination
of the last-described elements, (2)(i) and (2)(ii). Another step in
this process involves (c)managing or treating said subject for
graft-versus-host complications.
[0019] Another process herein is useful for vaccinating a subject
against an infectious agent, this process comprising the steps of
(a) introducing into or administering to the subject one or more
first antigens capable of establishing an immune response against
the infectious agent; and (b) introducing into or administering to
the subject one or more second specific antigens capable of: (1)
establishing or increasing at least one first specific immune
reaction directed against the infectious agent; and (2) decreasing
at least one second specific immune reaction which is different
from the first specific immune reaction (a)(1), the second specific
immune reaction being directed toward (i) the infectious agent; or
(ii) uninfected cells; or both.
[0020] Yet another useful process is directed toward vaccinating a
subject against an infectious agent, the process comprising the
steps of (a) introducing into or administering to the subject one
or more first antigens capable of establishing an immune response
against the infectious agent; and (b) introducing into or
administering to the subject immune cells capable of (1)
establishing or increasing at least one first specific immune
reaction directed against the infectious agent; and (2) decreasing
at least one second specific immune reaction which is different
from the first specific immune reaction (a)(1), this second
specific immune reaction being directed toward (i) the infectious
agent; (ii) uninfected cells, or both. In this process, the immune
cells have been removed from the subject and otherwise trained or
adopted prior to the aforementioned introducing or administering
step (b).
[0021] Another process for vaccinating a subject against an
infectious agent comprises the steps of (a) introducing into or
administering to the subject one or more first antigens capable of
establishing an immune response against the infectious agent, (b)
introducing into or administering to the subject immune cells
capable of (1) establishing or increasing at least one first
specific immune reaction directed against the infectious agent; and
(2) decreasing at least one second specific immune reaction which
is different from the first specific immune reaction (a)(1), the
second specific immune reaction being directed toward (i) the
infectious agent; or (ii) uninfected cells, or both. Notably, prior
to the introducing or administering step (b), the immune cells have
been removed from a trained donor, or from a naive donor wherein
the immune cells were trained in a surrogate or in vitro. Another
step of this process calls for (c) managing or treating the subject
for graft-versus-host complications.
[0022] Also provided by the present invention are useful
compositions of matter. These include the following a therapeutic
composition of matter comprising specific antigens capable of (1)
establishing or increasing at least one first specific immune
reaction directed against an infectious agent of interest, cells
infected with the infectious agent, or both, and (2) decreasing at
least one second specific immune reaction which is different from
the first specific immune reaction, the second specific immune
reaction being directed toward the infectious agent, cells infected
with the infectious agent, uninfected cells, or a combination of
any of the infectious agent, the infected cells and the uninfected
cells.
[0023] Another therapeutic composition of matter comprises trained
or adopted immune cells capable of (1) establishing or increasing
at least one first specific immune reaction directed against an
infectious agent of interest, cells infected with the infectious
agent, or both, and (2) decreasing at least one second specific
immune reaction which is different from the first specific immune
reaction, the second specific immune reaction being directed toward
the infectious agent, cells infected with the infectious agent,
uninfected cells, or a combination of any of the infectious agent,
infected cells and uninfected cells.
[0024] Another therapeutic composition of matter comprises trained
or adopted immune cells capable of (1) establishing or increasing
at least one first specific immune reaction directed against cancer
associated antigens, cancerous cells, or a combination of the
cancer associated antigens and the cancerous cells; and (2)
decreasing at least one second specific immune reaction which is
different from the first specific immune reaction, the second
specific immune reaction being directed toward the cancer
associated antigens; cancerous cells; non-cancerous cells; or a
combination of cancer associated antigens, cancerous cells and
non-cancerous cells.
[0025] Further yet is a therapeutic composition of matter
comprising trained or adopted immune cells capable of (1)
establishing or increasing at least one first specific immune
reaction directed against cancer associated antigens; cancerous
cells; or a combination of such cancer associated antigens and
cancerous cells; and (2) decreasing at least one second specific
immune reaction which is different from the first specific immune
reaction (1), the second specific immune reaction being directed
toward the cancer associated antigens; cancerous cells,
non-cancerous cells, and a combination of cancer associated
antigens, cancerous cells and non-cancerous cells.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 shows the average levels of antibody production for
each group of donors.
[0027] FIG. 2 shows the average tumor size at varying times for the
different experimental groups of mice described in the
examples.
[0028] FIG. 3 provides the average level of AFP at varying times
for the different experimental groups of mice described in the
examples.
[0029] FIG. 4 presents the average weight at varying times for the
different experimental groups of mice described in the
examples.
[0030] FIG. 5 depicts the survival rate for the different
experimental groups of mice described in the examples.
[0031] FIG. 6 is a gel showing RT-PCR results for varying cytokines
for the different experimental groups of mice described in the
examples.
[0032] FIG. 7 shows ELISA results for the average levels of IFN
.gamma. synthesis for the different experimental groups of mice
described in the examples.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention provides for novel methods and
compositions that when introduced into a subject having a
particular immune state towards a given immune target, can achieve
a new state which exhibits not only more than one change in said
state, but these changes are in more than one direction. Such a
dual or multi-faceted alteration in a given immune state may lead
to an overall enhancement of immune response towards immunological
targets such as infectious agents or cancer cells. Furthermore,
these methods and compositions may provide reduction or elimination
of undesirable consequences in the initial immune state towards the
immune target.
[0034] A novel and unanticipated result of the present invention is
that introduction of a viral antigen to an infected subject can
achieve an alteration of the immune state that comprises both a
decrease in one or more immune reactions towards antigens carried
by the pathogen or related cellular targets and simultaneously a
display of one or more enhanced or increased specific immune
reactions towards said immunological target. Prior art is incapable
of either predicting or explaining such a dual response. As
described previously, prior art predicts that the introduction of a
viral antigen into an infected subject should lead to a single
change in the immunological state towards the infectious agent,
either enhancement of the immune reaction or loss of immune
reactivity.
[0035] The prior view that immune reactive state towards a
particular immune target is not only monostatic but a given
manipulation of immunological systems that can change over state
would only lead to a new immunological state that again is
monostatic. To put this in other words, in immunological processes
that change, a given specific immune are perceived or intended to
be unidirectional in character; thus, they could only lead to a
single new immunological state (new response or no response).
[0036] The present invention provides novel methods and
compositions that when introduced into a subject carrying an
infectious agent having an immune state directed towards the
infectious agent, the said novel methods and compositions are
capable of producing a dual effect of a decrease or inversion of at
least one component of the immune response towards an epitope or
antigen carried by the infectious agent and simultaneously and in
the opposite direction and enhancement or increase in the immune
response to an epitope or antigen of the same epitope. The
decrease, inversion, enhancement or increase may be directed
towards different epitopes or antignes or they may be the same
antigen. When they are the same epitope or antigen the simultaneous
presence is carried out by different components of the immune
reaction.
[0037] In contrast to this prediction, it has now been demonstrated
that oral introduction of HBV antigens into infected subjects
simultaneously gave indications of both a decrease in specific
immune reactivity towards HBV antigens and related immunological
targets such as hepatocytes and an increase in other specific
immune reactions towards HBV antigens. The simultaneous presence of
these apparently antagonistic effects was independently measured by
various parameters and components of the immune system. For
instance, evidence for a loss or diminishment of immune reactivity
towards viral antigens in the subjects could be observed by a
decrease in enzyme activities (ALT and AST) and histology markers
associated with liver inflammation and tissue destruction. In
contrast to previous views that would have predicted a
proliferation of viral activity when immune reactivity towards the
immunological target was decreased, the subjects unexpectedly also
showed evidence of enhanced immune reactivity towards virus
antigens. Markers that demonstrated the simultaneous presence of
this surprising increase in the specific immune response towards
the virus included induction of antigen-specific T cell
proliferation responses, antiviral cytokine synthesis (as measured
by ELISA and RT-PCR assays of IFN .gamma.) and antigen-specific CTL
responses. Lastly and most notably virus copy number measurements
showed that instead of an increase in viral load, in some subjects
there were decreases as large as three orders of magnitude lower
than initial levels. This drop in viral loads indicates that even
after a decrease in some elements of immune reactivity towards HBV
antigens, there are other components of the immune system that are
capable of providing an increased immune response that has either
inhibited viral production or enhanced virus clearance. Thus the
present invention provides a binary immune response that can
provide decreased immune reactivity that should ameliorate the
chronic inflammation that is responsible for liver damage in
chronic HBV infection and at the same time the present invention
provides for an increased immune reaction towards the virus that
can decrease the viral load. The present invention can find utility
in other infections where a complex change in immune reactions is
desired rather than a unitary effect of either a gain or loss in
immune reactivity. In addition to HBV, other pathogens that may
benefit from application of the present invention can comprise but
not be limited to: HCV, HIV, HTLV, CMV, herpes and herpes zoaster,
varicella, EBV, chronic fatigue syndrome, (with and without EBV
infections), STD, bacterial infections (with immune mediated
phenomena such as endocarditis or sepsis), mycobacteria,
rickettsia, fungi and parasites.
[0038] The unexpected and unanticipated result of a duality in the
immune response in an infected subject with a decrease in at least
one immune reaction while simultaneously demonstrating an enhanced
immune reaction to the pathogen could be explained further. In this
view, immunological manipulations do not lead to a unidirectional
change in immune reactivity or immune response but rather a
bi-directional effect that can simultaneously increase or decrease
the effects of various elements or components of immune response to
different extents or directions. Thus, in immunogical systems there
can be an effector that can act as an inversion factor with regard
to immune reactivity, immune suppression or both that can lead to
induction of a dual response. These different responses can be
manifested through different elements or components of the immune
system such as the humoral, cellularor cytokine responses or
through two different epitopes of the same immunological
target.
[0039] Another aspect of the present invention is directed towards
immune manipulation prior to infection by a pathogen for
vaccination purposes. For some infective agents, prevention by
immunological means has been a failure. Notable examples of this
have been attempts at vaccination against HCV and HIV. In contrast
to prior art where only induction or enhancement of immune
reactivity was undertaken, the present invention recognizes and
uses the binary effect of immune manipulation to provide a more
effective immune response towards these potential pathogens. The
present invention carries this out by providing a reduction of
specific immune reactivity towards one or more antigens of a
pathogen while also providing an induction or increase in the
immune reactivity towards one or more antigens of the pathogen. In
other words, the present invention teaches that in order to achieve
an overall stronger immune response towards a pathogen or
immunological target, one has to decrease at least one aspect of
the undesirable immune response in a subject. For example, a
subject could be immunized against a target virus by injection of
an antigen with or without an adjuvant. After a specific immune
state has been established, the same or different antigens are
orally introduced into the subject such that a decrease of at least
one immune reaction towards the immunological target takes place
while achieving an increase in the immune reactivity towards the
pathogen. This seemingly antagonistic effect could take place
either simultaneously or sequentially. The subject may be further
treated with other immunological manipulations that may increase
the overall immune responsiveness to the pathogen. It can be seen
that this example is actually a parallel to the previously
described therapy for HBV infection that resulted in a heightened
immune response after oral administration of HBV antigens to HBV
infected subjects.
[0040] Another aspect of the present invention is directed towards
manipulation of the immune response towards tumors for management
of cancer. As described previously, recognition by the immune
system of cancer cells as being "foreign" is believed to be one of
the mechanisms of prevention of tumor growth. Thereby, the
continued presence and growth of cancerous cells in a subject
represents a lapse, defect or suppression of the immune
surveillance program. One factor that may be involved in this
"escape" process is the induction of cytokines or other cellular
factors that inhibit the expansion or immune reactivity of T-cells
towards the malignant cells. Previous attempts have had a
limitation that their efforts to heighten immune reactivity has
been negated by an increased induction of these factors. For some
tumors, there is the paradoxical effect that the higher the degree
of immune reactivity, the faster the tumor is able to grow (L. H.
Sigal and Y. Ron in Immunology and Inflammation: Basic Mechanisms
and Clinical Consequences, page 528 McGrawHill, Inc, NY, N.Y.,
1994).
[0041] Chronic infection by HBV has been discussed earlier in the
context of viral infection. One of the reasons that this is a
matter of concern is due to the increased likelihood of development
of hepatocellular carcinoma (HCC). Hepatocellular carcinoma rate is
increasing worldwide, especially in-patients with chronic viral
hepatitis. Currently there is no effective treatment for this
malignant neoplasm, and the prognosis is limited. The mechanism of
HCC development and the exact role of Hepatitis B virus (HBV) in
tumor induction are not well understood. Approximately one third of
patients with HBV associated HCC express the HBV envelope antigen
(HBsAg) on their cell surface which in this particular situation,
may serve as a tumor associated antigen. Patients with persistent
HBV infection have a defective or deviant_immune response against
the virus that not only fails to clear it, but there is a
pathological immune response such as induction of severe liver
injury and a potential role in enablement of neoplasm growth.
[0042] It has previously been shown that oral tolerance towards
adenoviral antigens effectively can prevent an anti-viral immune
response (U.S. patent application Ser. No. 08/808,629, supra). In
addition, adoptive transfer of tolerance by transplantation of
immune cells from orally tolerized donors to sublethally irradiated
recipients, supports the existence of suppresser cells in this
setting. Previously oral tolerance was shown to induce
antigen-specific immune suppression of HBsAg by feeding HBV
antigens (U.S. patent application Ser. No. 08/808/629 supra).
Therefore, adoptive transfer of this immune suppression should
cause immune hyporesponsiveness to HBsAg via suppressor cells. In
the case of HCC expressing HBsAg, the HBV antigen may be considered
a tumor associated antigen. Based on prior art that has been cited
previously, it would have been predicted that a decrease in a
specific immune response to tumor cells or tumor associated
antigens would allow unbridled growth of tumors.
[0043] Contrary to this expectation, the present invention
demonstrates that it is possible to manipulate the immune system
such that an effective immune response is achieved or enhanced
towards malignant cells while exhibiting a decrease in other
aspects of the immune response towards cancerous cells. This binary
effect was evident in experiments where donor immune cells were
implanted into recipient mice carrying human cancerous cells.
Without donor cell implantation these mice showed numerous
malignant growths and early death (group D in Example 2). In
contrast, when the donor immune cells were trained by inoculation
of HBV antigens prior to implantation, no evidence of tumor growth
was seen (Group C in Example 2). Moreover, if the donor cells were
given a dual treatment of oral administration of antigens as well
as the inoculation, a binary immune response was observed (Groups A
and B in Example 2). Evidence of a decrease in immune reactivity in
these last two groups was demonstrated by reduced levels of
anti-HBs antibodies as compared to the control. The presence of an
immune reaction to the HBV and/or cancer antigens was demonstrated
by the eventual disappearance of a marker for the tumor (AFP) and
lack of any macroscopic evidence for the presence of tumor growth.
Evidence for an enhanced immune reaction in Groups A and B was seen
by the increase in the levels of IFN.gamma. compared to Group C
which was treated with only inoculation. These results demonstrate
that immunological manipulation can lead to a reduction of a
specific immune reaction towards tumor specific antigen or antigens
(lowering antibody levels) while achieving an enhancement of an
antigen specific immune reaction (increase in IFN.gamma. levels).
Even in the presence of reduced levels of antibodies, there was
prevention of tumor growth thereby demonstrating the ability of the
enhanced immune response to manage cancer cells. This treatment may
thereby reduce undesirable immune components or-elements such as
suppressor cells or cytokines that promote tumor growth and allow
an enhanced immune reactivity towards the tumor
[0044] Malignancies that may find utility in the present invention
can comprise but not be limited to Hematological malignancies
(including leukemia, lymphoma and myeloproliferative disorders),
Hypoplastic and aplastic anemia (both virally induced and
idiopathic), myelodysplastic syndromes, all types of
_paraneoplastic syndromes (both immune mediated and idiopathic) and
solid tumors (including lung, liver, breast, colon, prostate GI
tract, pancreas and Karposi)
[0045] Induction of the extent and nature of an immune response can
be determined by a number of factors. Illustratively, these can
comprise the nature of an antigen, modifications of an antigen, the
amount of an antigen, the method of introduction of an antigen into
the subject, application of secondary treatments and other methods
that are well known in the art. Antigens can be prepared from
biological sources or they can be obtained synthetically or from
recombinant DNA technology. Antigens from biological sources can be
derived from cells, cell extracts, cell membranes and biological
matrixes.
[0046] The form of the antigen may present opportunities for
manipulations of immune response. For example bovine .gamma.
globulin (BGG) in saline solution results in an immune state
characteristic of an immunoreactive response. However, if the same
solution of BGG is separated out into monomeric and polymeric
forms, the monomeric form can actually be seen to induce tolerance
while the polymeric forms maintains properties that result in an
immune reactivity response (pg. 304 in Immunology: a Short Course,
E. Benjamini and S. Leskowitz, Eds. Wiley-Liss , NY, N.Y., USA). It
should also be pointed out that the unfractionated solution should
be viewed as a balance of immune reactive and immune suppressive
factors where the immune reactive potential is stronger in this
experimental system. Therefore, polymerization and degradation,
fractionation and chemical modification, are all capable of
altering the properties of a particular antigen in terms of
potential immune responses.
[0047] Antigens have been discussed as if each was a singular
homogeneous entity, but although an antigen may comprise a single
epitope it may also comprise a number of different epitopes. The
particular properties of each epitope of an antigen may be
dissimilar; this is reflected in the immune response to an antigen
where there may be particularly strong responses to some epitopes
and little or no response to others. In addition the nature of the
immune response can be variable as well. For instance, different
fragments of myelin basic protein may have completely opposite
effects with some epitopes inducing immune reactivity and other
fragments inducing immune suppression (page 107, D. P. Stites and
A. I Terr in Basic and Clinical Immunology, Appleton & Lange,
Norwalk, Conn., 1991). Therefore, smaller fragments could provide a
subset of epitopes compared to the complete antigen. The particular
choice and modifications of these fragments can provide more
flexibility in the elicitation or alteration of immune responses in
a subject. These smaller segments, fragments or epitopes can either
be isolated or synthesized.
[0048] Antigen dosage can serve as a way of manipulating
immunological responses. For example, it has been noted that
extremes in dosage of some antigens induce immune suppression
whereas a range of dosages in between induces immune reactivity.
Thus the same set of antigenic epitopes are capable of invoking
either of two opposite results. Furthermore, even when the same
response is evoked it can be by two different pathways. For
instance, with regard to oral tolerance, high dosages have been
linked to a clonal deletion mode of induction whereas low dosages
have been identified with the induction of suppressor cells. (Oral
Tolerance: Mechanisms and Applications, H. L. Weiner and L. F.
Mayer, eds. Annals of the New York Academy of Sciences Volume
778).
[0049] Methods that can be used to introduce an antigen or antigens
into a subject may comprise but are not limited to intramuscular,
intravenous, and intrathymic injection, nasal inhalation, oral
feeding and gastral intubation. In addition to administration of
antigen to a subject to induce a desired immune response in a
subject, the desired immune response or responses themselves may be
introduced into the subject. This can be carried out by a
process_that has been termed adoptive transfer. The particular
immune cells used for the transfer may have originated from the
subject (autologous transfer) or they may be from a syngeneic or
non-syngeneic donor (non-autologous transfer). The storage, growth
or expansion of the transferred immune cells may have taken place
in vivo or in vitro.
[0050] Methods for in vivo storage, growth or expansion of cells of
a subject in a surrogate host prior to reimplantation have been
described in U.S. patent application Ser. No. 08/876,635 filed on
Jun. 16, 1997). Methods for in vitro storage, growth or expansion
of cells prior to transfer are well known to practitioners of the
art. When the immune cells intended for use in a transfer are
derived from a donor, these cells may also undergo storage, growth
or expansion in vivo or in vitro as described above. Immune cells
that are to be transferred may be naive or they may have been
exposed to an immunological reagent such that they are immune
reactive, immune suppressive or as described previously, a mixture
of both. In vivo methods can be used to introduce an immunological
reagent to a surrogate host or a donor in order to render immune
cells immune reactive and/or immune suppressive toward a specific
antigen or antigens.
[0051] In addition, prior to implantation immune cells can be
rendered immune reactive and/or immune suppressive by exposure of
the immune cells to at least one specific antigen during in vitro
conditions. Such conditional or adoptive immune training would
provide immune cells with immune responsiveness towards at least
one specific antigen. In addition the immune cells may be
genetically modified by any of a number means known to those
skilled in the art. These modifications can include but not be
limited to genetic editing (Wetmur et al., U.S. Pat. No. 5,958,681)
and capability of anti-sense (Inouye et al., U.S. Pat. No.
5,272,065) or gene expression. Antisense expression can include but
not be limited to resistance to virus infection and elimination of
native gene expression. An example of anti-sense to native gene
expression would include but not be limited to major
histocompatibility (MHC) genes. Gene expression that is conferred
by genetic manipulation can include expression of native or
non-native gene products. These may include but not be limited to
antibodies, growth factors, cytokines, hormones, and drug
resistance.
[0052] The immune cells may be used as a mixture or sub-populations
may be segregated or isolated for use. For instance, it may be
desirable to separate out immune reactive cells such as CD4.sup.+,
CD8.sup.+ or CD34.sup.+ or other cells. In another example, in a
population of immune reactive cells it may be desirable to isolate
immune cells that synthesize one particular form of antibody from
immune cells that synthesize other forms or immune cells that are
cytotoxic to cells expressing one or more specific cell surface
markers. When the source of the cells used for adoptive transfer
are not native to the subject but are from a donor (non-autologous
transfer), additional steps may be required for successful
implantation. Such treatments can comprise partial or total
ablation of the subjects immune system prior to transfer or the use
of immune suppressive drugs. Alternatively or in combination, the
subject can further be treated to manage Graft versus Host
complications as described in U.S. patent application Ser. No.
08/808/629 supra.
[0053] In the present invention, auxiliary treatments may also in
conjunction with introduction of an antigen or antigens to the
subject. For example, provision of adjuvants, immunosuppressive
reagents, anti-inflammatory reagents and cytokines can all be used
in conjunction with the present invention by shifting various
components of the immune response.
[0054] The present invention has been described in terms of
accomplishment of a binary response by means of a single mode of
treatment. In another aspect of the present invention, more than
one therapeutic treatment is carried out either sequentially or
simultaneously. Thus, one can use one or more treatments that are
anticipated increase immune reactivity towards one or more epitopes
or antigens and one or more treatments that are anticipated to
decrease immune reactivity towards one or more epitopes or
antigens. Thereby a new immune state can be achieved where the
various elements and components that comprise the sets of immune
reactions and immune suppressions have been enhanced or
diminished.
[0055] Understanding the duality of the immune response allows the
prediction that after cessation of treatment there may be a
reversion to a state that is closer to the pre-treatment immune
states. To manage such a potential reversion, the subject may be
maintained continuously under treatment or alternatively the
treatment can be carried out periodically. The timing of periodic
treatments can be carried out at set intervals or may be determined
by observations of the onset of immune reversion. During continuous
or periodic treatment, the mode of the treatment, the nature of the
antigen or the dosage may stay the same or they may be varied as
needed.
[0056] Thus, contrary to prior art, the present invention predicts
that a change in immunological state (through manipulation) does
not have to be unidirectional but may lead to a dual or multi
changes in opposite directions (inxcrease and decrease in one or
more components in the immune response toward an antigen or
antigens. By this manipulation, it is possible to reduce the
undesirable aspects or components of the immune response that may
be the underlying cause or a contributory factor to disease
development such as destruction of the liver.
Description of the Preferred Embodiments
Example 1
Materials and Methods
[0057] Fifteen subjects were enrolled in the clinical study. The
subjects were men or women with a diagnosis of active HBV infection
(acute or chronic) based on liver biopsy (active inflammatory
response), and positive for HBsAg with liver enzymes at least twice
above normal. The subjects were required to meet one or more of the
following criteria: (1) failed treatment with interferon or were
unable to receive interferon; (2) hepatocellular carcinoma and
active inflammatory response; (3) fulminant liver failure or severe
deteriorating synthetic liver functions; (4) liver transplant
recipient with evidence of reinfection of the graft and active
inflammatory reaction in the liver, who failed or were unable to
receive interferon or lamivudine; and (5) had HBV immune mediated
disease (i.e., cryo, PAN, neuropathy, kidney involvement).
[0058] The subjects were fed with recombinant HbsAg preS1+preS2
twice a day for 20 weeks. The HBV antigen was given in liquid form,
diluted in calf serum. The subjects were given 1 tablet of
Omeprazole (20 mg/day/orally) 4 hours before the HBV antigen to
prevent the effect of gastric acidity on the ingested antigen.
[0059] The subjects were followed for 20 weeks of feeding and 20
weeks after completion of feeding. The subjects were tested every
other week for the 20 weeks of feeding and continued monthly for 20
weeks after feeding. A liver biopsy was performed before the study
began and again after completion of the 20-week feeding period. The
biopsies were stained using the standard hematoxylin and eosin
(H&E) stain. HB surface antigen and HB core antigen were
determined using immunohistochemical staining techniques. Liver
enzymes, ALT and AST levels were followed bimonthly. HBV DNA (viral
load) was quantified bimonthly using PCR.
[0060] Cytotoxic lymphocyte response and specific T-cell activity
to HB surface antigen determined by a T-cell proliferation assay
were assayed as described (Chisari et al., "Hepatitis B virus
immunopathogenesis"; Ann. Rev. Immunol. 13:29-45 (1995); Rehermann
et al., "Cytotoxic T lymphocyte responsiveness after resolution of
chronic hepatitis B virus infection;" J. Clin. Invest. 7:1655-1665
(1996); Guidotti et al., "Viral clearance without destruction of
infected cells during acute HBV infection;" Science 284:825-829
(1999); Ishikawa et al., "Polyclonality and multispecificity of the
CTL response to a single viral epitope;" J. Immunol. 161:5842-5850
(1998)).
[0061] The number of specific T-cell clones secreting IFN .gamma.
when exposed to HB surface antigen was measured by an ELISA Spot
Assay (Hauer et al., "An analysis of interferon gamma, IL-4, IL-5
and IL-10 production by ELISPOT and quantitative reverse
transcriptase-PCR in human Peyer's patches;" Cytokine 10:627-634
(1998); Larsson et al., "A recombinant vaccinia virus based ELISPOT
assay detects high frequencies of Pol-specific CD8 T cells in
HIV-1-positive Individuals;" AIDS 13:767-777 (1999)).
[0062] IFN .gamma. and IL 10 were quantified using RT PCR (Ilan et
al., "Insertion of the Adenoviral E3 region into a recombinant
viral vector prevents antiviral humoral and cellular immune
responses and permits long term gene expression;" Proc. Nat. Acad.
Sci. (USA) 94:2587-2592. (1997); Ilan et al., "Oral tolerization to
adenoviral antigens permits long term gene expression using
recombinant adenoviral vectors;" J. Clin. Invest. 99:1098-1106
(1997)). Specific serum cytokines were measured as described by
Ilan et al. (Ilan et al., "Treatment of experimental colitis by
oral tolerance induction: a central role for suppressor
lymphocytes;" Am. J. Gastroenterol. 95:966-973 (2000)).
Analysis of Results
[0063] Patients were considered to have reacted positively to the
hepatitis B virus antigens if they demonstrated one or more
indications of a decrease in a specific immune response and one or
more indications of an increase in a specific immune reaction.
[0064] Indications of a decrease in a specific immune response can
be one or more of the following: [0065] 1) Decrease in one or both
enzyme (ALT and/or AST) levels; [0066] 2) Decrease (improvement) in
liver pathology as measured by standard hemotoxylin & eosin
(H&E) staining; [0067] 3) Decrease in HB surface antigen
staining by immunohistochemistry; and [0068] 4) Decrease in HB core
antigen staining by immunohistochemistry.
[0069] Indications of an increase in a specific immune reaction can
be one or more of the following: [0070] 1) Decrease in viral load
by PCR; [0071] 2) Increase in specific T-cell activity to HB
surface antigen as measured by a T-cell proliferation assay (T-cell
assay); [0072] 3) Increase in number of specific T-cell clones
secreting IFN .gamma. when exposed to HB surface antigen as
measured by an ELISA Spot Assay; [0073] 4) Increase in IFN .gamma.
and/or IL 10 as measured by RT PCR; [0074] 5) Increase in cytotoxic
lymphocyte response; and
[0075] 6) Increase in specific serum cytokines.
TABLE-US-00001 TABLE 1 Summary of Immune Reactions Decrease in
Specific Increase in Specific Subject Immune Response Immune
Reaction 502 LA ALT levels decreased Viral load decreased T-cell
proliferation increased Specific T-cell clones secreting IFN
.gamma. (ELISA spot assay) increased 503 RM Not fully responding
yet Viral load decreased Cytotoxic lymphocyte response increased
506 ASA Liver pathology (H&E) decreased HB T-cell proliferation
increased core antigen staining decreased 509 II ALT levels
decreased T-cell proliferation increased Specific T-cell clones
secreting IFN .gamma. (ELISA spot assay) increased 511 EBH ALT
levels decreased Viral load decreased AST levels decreased T-cell
proliferation increased Liver pathology (H&E) decreased
Specific T-cell clones secreting HB surface antigen decreased IFN
.gamma. (ELISA spot assay) increased HB core antigen staining IFN
and IL 10 positive by RT PCR decreased Cytotoxic lymphocyte
response increased 517 FA HB surface antigen decreased Viral load
decreased HB core antigen staining T-cell proliferation increased
decreased Specific T-cell clones secreting IFN .gamma. (ELISA spot
assay) increased 518 IZ HB core antigen staining Viral load
decreased decreased T-cell proliferation increased Specific T-cell
clones secreting IFN .gamma. (ELISA spot assay) increased IFN and
IL 10 positive by RT PCR 520 YB AST levels decreased T-cell
proliferation increased Specific T-cell clones secreting IFN
.gamma. (ELISA spot assay) increased IFN and IL 10 positive by RT
PCR 505 NS ALT levels decreased Viral load decreased AST levels
decreased T-cell proliferation increased HB core antigen staining
Specific T-cell clones secreting decreased IFN .gamma. (ELISA spot
assay) increased 519 KS ALT levels decreased T-cell proliferation
increased AST levels decreased Specific T-cell clones secreting HB
surface antigen staining IFN .gamma. (ELISA spot assay) increased
decreased IFN and IL 10 positive by RT PCR 513 PW Liver pathology
(H&E) decreased T-cell proliferation increased HB surface
antigen staining Specific T-cell clones secreting decreased IFN
.gamma. (ELISA spot assay) increased HB core antigen staining IFN
and IL 10 positive by RT PCR decreased 514 TY ALT levels decreased
Viral load decreased AST levels decreased T-cell proliferation
increased Liver pathology (H&E) decreased IFN and IL 10
positive by RT PCR HB surface antigen staining decreased HB core
antigen staining decreased 515 JH ALT levels normal T-cell
proliferation increased AST levels normal Specific T-cell clones
secreting IFN .gamma. (ELISA spot assay) increased IFN .gamma. in
serum increased 504 GE Liver pathology (H&E) decreased T-cell
proliferation increased HB surface antigen staining Specific T-cell
clones secreting decreased IFN .gamma. (ELISA spot assay) increased
HB core antigen staining IFN and IL 10 positive by RT PCR decreased
Cytotoxic lymphocyte response increased 521 MH Liver pathology
(H&E) decreased T-cell proliferation increased Specific T-cell
clones secreting IFN .gamma. (ELISA spot assay) increased IFN and
IL 10 positive by RT PCR
[0076] In some subjects the specific response was reversed after
treatment. This may indicate that the effect of treatment may be
transient and/or reversible and continued or repeated treatment may
be recommended.
[0077] In the subjects introduction of hepatitis B surface antigen
achieved a dual effect, exhibiting an increase in at least one
aspect of the immune reaction towards HBV while exhibiting a
decrease in at least one aspect of the immune reaction towards HBV
or hepatocytes.
Example 2
Materials & Methods:
[0078] Mice: Female immunocompetent (heterozygous) and athymic
Balb/c mice were purchased from Jackson Laboratories, Bar Harbor,
Me. All animals were kept in laminar flow hoods in sterilized
cages, receiving irradiated food and sterile acidified water as
described (Shouval et al., "Comparative morphology and
tumorigenicity of human hepatocellular cell carcinoma lines in
athymic rats and mice;" Vichow's Archives A. Path. His.
412:595-606, (1988)).
[0079] Cell cultures: The human hepatoma cell line Hep-3B which
secretes HBsAg was grown in culture as a monolayer, in medium
supplemented with non essential amino acids and 10% heat
inactivated fetal bovine serum as described (American Type Culture
Collection, ATCC, HB-8064, HB-8065; Shouval et al., Vichow's
Archives A. Path. His., supra;).
[0080] Induction of anti-HBV immune response: BioHepB recombinant
hepatitis B vaccine (BioTechnology General LTD, Israel) which
contains three surface antigens of the hepatitis B virus: HBsAg,
PreS1 and preS2, was used for induction of anti-HBV immune
response. Immunocompetent Balb/c donor mice were immunized against
HBV with 1 .mu.g HBsAg intraperitonealy (i.p.) at one month,
followed by a boost vaccine one week before splenocyte
transplantation.
[0081] Preparation of HCC antigens: HCC cells were used as tumor
associated antigens. After growth in cell cultures, the cells were
filtered through a 40 .quadrature.m nylon cell strainer. The intact
cells were spun down and removed. Proteins were quantified using a
protein assay kit (BioRad Laboratories, Hercules, Calif.).
[0082] Oral administration of HCC cells or HBV antigens: Hep-3B
cells (50 .quadrature.g protein) expressing HBsAg or recombinantly
prepared HBsAg +PreS1+PreS2 antigens (BioHepB, BioTechnology
General LTD, Israel) or low dose HBV antigens (BioHepB, 1 mcg) were
administered orally. The antigens were administered with a feeding
atraumatic-needle, on alternate days for 10 days (a total of 5
doses) prior to HBV vaccine immune induction. A control group
received similar doses of bovine serum albumin (BSA).
[0083] Assessment of anti-HBs humoral immune response: Mice in all
groups were followed for anti-HBs antibody titers at sacrifice
(prior to splenocyte transplantation) 30 days following inoculation
of the BioHepB vaccine, 7 days following the boost vaccination. HBs
antibodies were measure by a commercial solid phase
radioimmunoassay (RIA).
[0084] Tumor and splenocyte transplantation in athymic mice:
Athymic mice were used as splenocyte recipients and conditioned
with sub-lethal radiation (600 cGy). Twenty four hours after
irradiation, the animals were injected subcutaneously. in the right
shoulder with 10.sup.7 human hepatoma Hep3B cells as described in
Shouval et al. infra (Shouval et al., "Adoptive transfer of
immunity to hepatitis B virus in mice following bone marrow
transplantation through immunization of bone marrow donors;"
Hepatology 17:955-959 (1993)). Seven days after irradiation,
athymic mice received splenocyte transplantation as follows: on
transplantation day, donor mice were sacrificed and spleens were
harvested. Splenocyte recipients were then injected I.V. with
spleen cells at 2.times.10.sup.6 cells/mouse (Shouval et al.,
Hepatology, supra).
[0085] Follow-up of tumor growth: Recipient mice were followed at
weekly intervals for 2 months with monitoring of tumor growth by
calipers, and periodic serum measurements of HBsAg and
alfa-fetoprotein (AFP) levels. Blood samples were obtained weekly
by retrobulbar puncture and serum was separated and frozen at
-20.degree. C. until assayed by a commercial solid phase
radioimmunoassay (RIA).
[0086] Cytokine production: To evaluate the effect of immune
reactivity on the balance of pro-inflammatory and anti-inflammatory
cytokines, TNF.quadrature.,
IFN.quadrature..quadrature..quadrature.IL2, TGF.quadrature. and
IL10 mRNA production were measured periodically in recipient mice
by RT-PCR. Serum levels of the cytokines were measured by a highly
sensitive RIA according to the manufacturers' instructions.
[0087] Radioimmunoassays for detection of serum HBsAg, anti-HBs and
alpha-feto-protein: HBsAg and antibodies to HBsAg were determined
by commercial solid phase RIA (Ausria II and Ausab, Abbott
Laboratories, North Chicago, Ill.; R&D Systems, Minneapolis,
Minn.). A World Health Organization reference serum was used for
quantitative analysis of anti-HBs by RIA, utilizing the Hollinger
formula and data expressed in mlU/ml (Hollinger et al., "Improved
double antibody radioimmunoassay (RIA-D) methodology for detecting
hepatitis B antigen and antibody" [Abstract], Am. Soc. Microbiol.
72:213 (1972)). Alpha feto protein (AFP) was measured by RIA (AFP,
Bridge Serono, Italy) and expressed in ng/ml.
[0088] Experimental Groups: Donor mice were divided into 4 groups
of 10 mice each (Table 2). Groups A to C received oral feedings
prior to HBV vaccine. Experimental group A received oral feedings
of Hep3B hepatoma cells. Experimental group B received oral
feedings of HBV antigens. Control group C received oral feedings of
BSA (Table 2). The above groups received HBV vaccination as
described. Control group D was neither vaccinated nor fed antigens.
Recipient mice consisted of 4 parallel groups A to D and received
injections of Hep3B cells as described above and then received
splenocytes from the donor mice.
TABLE-US-00002 TABLE 2 Experimental groups. Donor mice: Donor mice:
Group: Immunization to HbsAg Oral feedings A Immunized Hep3B
hepatoma cells B Immunized HBV antigens C Immunized BSA D None
None
Analysis of Results:
[0089] Evaluation of the effect of oral administration of HCC
proteins or HBV antigens on anti-viral humoral immune response: The
effect of oral feedings of HCC extracted proteins expressing HBsAg
or HBV antigens on anti-HBV peripheral immune reactivity was
evaluated by measuring serum anti-HBsAg antibody production. This
was measured at sacrifice--prior to splenocyte transplantation, 30
days following inoculation of the BioHepB vaccine and 7 days
following a boost vaccination. Administration of HCC extracted
proteins markedly decreased the anti-viral humoral immune response.
A lesser degree of decrease was evident in mice exposed to HBV
antigens. At sacrifice, 30 days following inoculation with the
vaccine, serum anti-HBs antibody levels were 157.+-.271 vs.
382.+-.561 and 664.+-.757 mlU/ml in HCC fed mice, (group A),
compared with HBV-envelope proteins fed mice (group B) and BSA-fed
controls (group C), respectively (p<0.05 between groups A and C
(FIG. 1).
[0090] Effect of adoptive transfer of HBV immunity on tumor growth
as manifested by tumor volume and serum AFP levels:
[0091] Tumor growth was suppressed completely in mice that received
splenocytes immunized to HBsAg (group C). After transplantation, no
tumor grew and there was no macroscopic evidence of tumor growth.
This correlated with AFP serum levels that were negative for the
duration of the experiment (12 weeks) (FIGS. 2 and 3).
[0092] Tumor growth was significant in mice that received naive
splenocytes (group D) and the mice had big tumors after 2 weeks of
tumor transplantation. Tumor growth was rapid and tumor size was
151.+-.78 mm.sup.2and 165.+-.24 mm.sup.2 at 2 and 4 weeks
respectively (FIG. 2a; p<0.0001 between groups C and D). This
correlated with AFP serum levels that rose in parallel to tumor
growth. AFP serum levels were 2320.+-.2123 ng/ml and 2500.+-.1431
ng/ml at 2 and 4 weeks respectively (FIG. 2b; p<0.02 between
groups C and D). Due to enormous tumor size after 4 weeks and
deterioration in general state of these mice with 25% mortality,
they were sacrificed.
[0093] Effect of oral administration of HBV or HCC proteins on
tumor growth as manifested by tumor volume and serum AFP
levels:
[0094] Mice receiving splenocytes from mice fed HCC extracted
proteins (group A) showed only transient tumor growth. While tumor
growth was not evident macroscopically, AFP serum levels were
significantly elevated after two weeks and declined thereafter and
were negative after 6 weeks. AFP serum levels were 574.4.+-.539
ng/ml and 418.+-.520 ng/ml at 2 and 4 weeks respectively (FIGS. 2
and 3). This was significant compared to mice immunized against HBV
(group C); p<0.02 between groups A and C).
[0095] Mice receiving splenocytes immunized against HBV and exposed
to oral feedings of HBV antigens (group B) had no evidence of tumor
growth. No evidence of macroscopic tumor growth or rise in serum
AFP levels was seen in these mice.
[0096] Effect of tumor growth on weight gain, mortality and general
appearance in the various groups: Mice that received HBV immunized
splenocytes and completely suppressed tumor growth with no evidence
of tumor growth showed continued weight gain throughout the 12 week
experiment (group C). This was in contrast to mice receiving naive
splenocytes (with significant tumor growth) that had in parallel a
significant reduction in body weight (group D). This body weight
loss became worse during the 4 weeks of follow-up and correlated
with tumor growth, general deterioration and mortality (FIG. 4).
Body weight in these mice was significantly reduced as compared
with mice in group C. Body weight was 17.7.+-.1.8 and 20.7.+-.1.3;
respectively at 2 weeks (p<0.003) and 17.1.+-.1.8 gr 21.4.+-.0.6
gr respectively at 3 weeks (p<0.00004). Mice in group C that did
not show tumor growth appeared well and there was no mortality
throughout the 12 week follow-up. This was in contrast to mice in
group D (that showed tumor growth) that appeared extremely sick,
performed poorly and had a mortality rate of 12.5% after 2 weeks
and 25% after 3 weeks (FIG. 5).
[0097] Mice receiving splenocytes from mice fed HCC extracted
proteins (group A) that showed transient tumor growth had in
parallel an initial reduction in body weight that was significantly
lower that group C mice that did not have tumor growth. A similar
but less significant reduction in weight was evident in mice
receiving splenocytes immunized to HBsAg and exposed orally to HBV
antigens (group B; FIG. 4). Body weights were 16.2.+-.2.0 and
17.8.+-.2.4 in groups A and B respectively at 2 weeks (p<0.0006;
P<0.01 respectively compared to group C) and 18.5.+-.51.9 gr and
18.5.+-.2.0 gr respectively at 4 weeks (p<0.002; p<0.003
compared to group C). No significant difference in body weight was
evident between groups A, B and D during the four weeks. After 4
weeks there was gradual increase in body weights in groups A and B
that correlated with tumor suppression in group A as evident by
negative AFP levels in this group. Mice in groups A and B initially
looked sick in correlation with weight loss and had an early
mortality rate at 4 weeks of 40% in both groups (FIG. 5). However,
after four weeks these mice were looking better and although they
did not look as healthy as mice in group C there was some
improvement in their general appearance and performance. After 4
weeks there was no mortality in these groups, similar to group
C.
Effect of Tumor Growth on Cytokine Profile:
[0098] Mice in group A that received splenocytes from HCC-fed mice
had elevated levels of interferon gamma production evident by
RT-PCR of splenocytes. Lesser levels were evident in group B. This
was in contrast to group C (that had no tumor growth) that had no
evidence of interferon production in splenocytes by RT-PCR (FIG.
6,7).
TABLE-US-00003 TABLE 3 Anti-HCC Immune Response Adptive Tumor
Suppression Tumor Promotion Transfer of Tumor Activated
Non-Specific Towards Non- Splenocytes Growth anti HBV Anti-Tumor
HBV specific Anti HBV - +1 + + + Immunized Anti HBV - +2 + - -
Immunized Orally fed with HBV antigens Anti HBV - +3 - - Immunized
Orally fed with HCC antigens Naive + - + +
[0099] Many obvious variations will no doubt be suggested to those
of ordinary skill in the art in light of the above detailed
description and examples of the present invention. All such
variations are fully embraced by the scope and spirit of the
invention as more particularly defined in the claims that now
follow.
* * * * *