Note: Descriptions are shown in the official language in which they were submitted.
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ABLATIVE IMMUNOTHERAPY
FIELD
[0001] The present disclosure relates generally to immunotherapy and, more
specifically, to therapeutic methods and compositions for treating tumors and
pathogen infected tissues.
BACKGROUND
[0002] Harnessing the power of the immune system to treat chronic infectious
diseases or cancer is a major goal of immunotherapy. Active immunotherapy
treatments are methods designed to activate the immune system to specifically
recognize and destroy tumor or pathogen-infected cells. For over 200 years
active immunotherapy approaches have been used to prevent numerous
infectious diseases, including small pox, rabies, typhoid, cholera, plague,
measles, varicella, mumps, poliomyelitis, hepatitis B and the tetanus and
diphtheria toxins.
[0003] Active immunotherapy concepts are now being applied to develop
therapeutic cancer vaccines with the intention of treating existing tumors or
preventing tumor recurrence as well as for treatment and prevention of chronic
viral infection. Many of these techniques have proven to successfully develop
increased frequencies of immune cells in circulation that have the ability to
specifically kill tumors or pathogen infected cells. However, despite the
ability
to generate immune cells reactive against tumor antigens, tumor escape
mechanisms can overpower this immune response resulting in eventual tumor
progression.
[0004] Active immunotherapy of cancer has been shown to be very effective in
numerous rodent models. However, the clinically disappointing results of
decades of immunotherapy trials of various types in humans have shown the
immune system in humans does not perceive the threat/danger of human cancer
cells as well as the immune system of rodent models of the same diseases.
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[0005] The same is true of chronic viral infection. The innate immune response
is able to slow down viral replication and activate cytokines which trigger
the
synthesis of antiviral proteins. The adaptive immune system neutralizes virus
particles and destroys infected cells. However, viruses have developed a
number of countermeasures to avoid immune attack and stay moving targets for
the immune system.
[0006] There is a need to provide an active immunotherapy that is capable of
overcoming tumor and viral immunoavoidance mechanisms and to train the
human immune system to perceive the threat/danger of human cancer cells and
viral infected cells resulting in an immune response which can eradicate
tumors
or pathogen-infected cells wherever they might be located in the body.
SUMMARY
[0007] The present disclosure relates to methods and compositions for inducing
a systemic, adaptive immune response against a tumor or pathogen. The method
uses a combination of an allogeneic cell therapy and a method for subjecting
the
tumor or pathogen infected tissue to cellular distress, resulting in the
liberation
of tumor specific antigen(s) or pathogen specific antigen(s).
[0008] In one aspect, the present disclosure is a method for inducing an
adaptive
immune response against a tumor or a pathogen in a subject. The method
includes the steps of: (1) administering to a subject with cancer or an
infectious
disease an aliquot of allogeneic cells that are designed to be rejected by the
subject immune system in a manner that induces anti-allogeneic Thl immunity;
(2) in the same subject, after allowing time for an anti-allogeneic Thl immune
memory to form (about 7 to 14 days), performing an in situ ablation of an
accessible tumor lesion or pathogen-infected tissue with an ablation method
which causes at least a portion of the tumor or infected tissue to die,
preferably
by necrosis, (e.g., by methods such as but not limited to electroporation,
cryoablation, chemotherapy, radiation therapy, ultrasound therapy, ethanol
chemoablation, microwave thermal ablation, radiofrequency energy or a
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combination thereof); then; (3) injecting a second aliquot of the same
allogeneic
cells intralesionally (same cells as used to prime), preferably 2-24hrs after
the
ablation step, creating an immune response that serves as an adjuvant to the
uptake of antigen(s) and the subsequent maturation of host antigen presenting
cells (i.e., dendritic cells) responding to the necrotic or apoptotic tissue.
Mature
antigen presenting cells from the lesion then migrate to the lymph nodes and
stimulate systemic anti-tumor or anti-pathogen immunity. In another aspect,
the
priming step is omitted. The tissue from the tumor or the pathogen infected
tissue is ablated in situ and an aliquot of the allogeneic cells are injected
after
the ablation to create the desired immune response.
[0009] In another aspect, the present disclosure includes a vaccine for a
patient
against a tumor or a pathogen. The vaccine includes an antigenic composition
comprising antigenic material from the tumor or pathogen and an aliquot of
allogeneic cells wherein administration of the antigenic composition to the
patient creates a rejection response and stimulates a delayed-type
hypersensitivity response to the antigens thereby acting as an adjuvant to the
stimulation of systemic anti-tumor or anti-pathogen immunity in the patient.
The vaccine can also include a priming composition wherein the priming
composition includes an aliquot of allogeneic cells.
[0010] In yet another aspect, the present disclosure includes a method of
vaccinating a patient. The method includes the steps of: (1) administering to
the
subject with cancer or an infectious disease a priming composition that
includes
an aliquot of allogeneic cells that are designed to be rejected by the subject
immune system in a manner that induces anti-allogeneic Thl immunity; (2) in
the same subject, after allowing time for an anti-allogeneic Thl immune
memory to develop (about 7 to 14 days), injecting, preferably intradermally,
an
antigenic composition containing a source of tumor antigen or pathogen
antigens
(e.g., attenuated virus, tumor lysates, heat shock proteins), preferably
containing
from the same individual autologous lysates of the infected or cancerous
tissue,
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the lysates preferably containing chaperone proteins, and such lysates
formulated with an aliquot of allogeneic cells (same cells used to prime the
patient) to create a rejection response and stimulate a delayed-type
hypersensitivity response to the alloantigens which serve to adjuvant the
stimulation of systemic anti-tumor or anti-pathogen immunity. In a further
aspect, this method may be practiced without the priming step.
[0011] In another aspect, the present disclosure includes a therapeutic
composition for treating a tumor or a pathogen in a patient comprising tumor
antigens or pathogen antigens generated in situ and allogeneic cells, wherein
the
allogeneic cells and the in situ generated antigens elicit an immune response
whereby subsequent maturation of the patient's antigen presenting cells
systemically stimulate anti-tumor or anti pathogen immunity. The tumor
antigens or pathogen antigens are generated in situ from necrosis of the tumor
or
the pathogen infected tissue. The therapeutic composition may also include a
priming composition containing allogeneic cells. The allogeneic cells in the
priming composition and in the antigenic composition may include between
about 1 x 108 and about 1 x 1010 cells.
[0012] In yet a further aspect, the present description includes a vaccine
composition for treating a tumor or a pathogen in a patient comprising
antigens
generated in situ that include tumor antigens or pathogen antigens and
allogeneic cells wherein injecting the patient with the allogeneic cells in
the
presence of in situ generated antigens creates an immune response whereby
subsequent maturation of the patient's antigen presenting cells systemically
stimulate anti-tumor or anti pathogen immunity. The tumor antigens in the
composition are derived from in situ necrosis of the tumor. The pathogen
antigens in the composition are derived from in situ necrosis of the pathogen
infected tissue. The composition may also include a priming composition
containing allogeneic cells. The allogeneic cells in the priming composition
and
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in the vaccine composition may include between about 1 x 108 and about 1 x
1010 cells.
[0013] In a further aspect, the present disclosure includes a method of
vaccinating a patient. The method includes the steps of: (1) administering to
the
subject with cancer or an infectious disease a priming composition that
includes
an aliquot of allogeneic cells that are designed to be rejected by the subject
immune system in a manner that induces anti-allogeneic Thl immunity; (2) in
the same subject, after allowing time for an anti-allogeneic Thl immune
memory to develop (about 7 to 14 days), ablating the tumor or pathogen
infected
tissue in situ to release tumor antigens or pathogen antigens (3)
administering a
composition of allogeneic cells (same cells used to prime the patient) to
create a
rejection response and stimulate a delayed-type hypersensitivity response to
the
alloantigens which serve to adjuvant the stimulation of systemic anti-tumor or
anti-pathogen immunity. In a further aspect, this method may be practiced
without the priming step.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0014] The present disclosure includes a method for stimulating anti-tumor or
anti-pathogen immunity in patients. The method involves first "priming" of the
patient to develop Thl anti-alloantigen immune memory by infusion of an
aliquot of allogeneic cells. It is desired that the infusion of allogeneic
cells
stimulates the patient's immune system to react against the allogeneic cells.
A
time period is allowed to elapse until the patient's immune system is allowed
to
form an anti-allogeneic memory. In some embodiments, a patient may need a
booster of allogeneic cells to develop the appropriate Thl immune memory.
[0015] Patient as used herein includes not only mice but also humans. Thl
response as used herein refers to production of a cytokine profile that
activates
T-cells and macrophages. Thl response is to be distinguished from Th2
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response which activates mainly an immune response that depends upon
antibodies and is antagonistic to the Thl response.
[0016] The next step includes injury and/or death of cells within a tumor bed
or
pathogen-infected tissue after the patient develops sufficient anti-allogeneic
Thl
immune memory. Tissue injury or death releases cellular components and
recruits scavenger cells to the injury site. The release of cellular
components
during tissue injury or tissue death by necrosis is critical for the in situ
generation of tumor or pathogen antigens. A variety of methods are known in
the art to cause tissue injury or death within a tumor bed or pathogen
infected
tissue. Preferably death is by necrosis, which causes recruitment of scavenger
cells to the injury site. In some preferred embodiments, tissue death or
injury is
by cryoablation or by irreversible electroporation. Alternatively, tissue is
ablated ex-vivo and the released components injected into the patient.
[0017] The scavenger cells, including immature dendritic cells can pick up
antigens released from the damaged or dead tissues. A second aliquot of
allogeneic cells are injected intralesionally in order to cause the maturation
of
dendritic cells (DC) for the priming of Thl immunity to the antigens. By
intralesionally is meant administration of the composition of this invention
through injection or otherwise directly into a cancerous area or tumor or a
pathogen infected tissue. In preferred embodiments, all of the allogeneic
cells
administered to the patient are from the same source. Preferably, the
allogeneic
cells are administered between about 2 and about 24 hours after ablation of
the
tissue. This method is especially useful in the treatment of solid or
metastatic
tumors, particularly in patients with tumor lesions resident in the prostate,
breast, bone, liver, lung, or kidney.
[0018] It is desirable for the patient to develop a strong delayed-type
hypersensitivity (DTH) reaction upon introduction of the second aliquot of
allogeneic cells resulting in rejection of the allogeneic cells due to the
fact that
the patient has been primed to Thl immunity against the allogeneic cells
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introduced by the first aliquot. The by-stander effect of the anti-alloantigen
DTH reaction can produce "danger signals" which serve to cause the DC,
collecting and processing antigens from the damaged tissue, to mature and
migrate to the draining lymph nodes. The combination of the induction of
tissue
injury and the DTH rejection response can create an inflammatory environment
which leads to Thl immunity against the antigens released from the damaged
tissue.
[0019] The general state of inflammation caused by the treatment process can
serve to cause the DC to program T-cells to Thl immunity against antigens in
the damaged tissue resulting in a systemic adaptive immune response to the
tumor or pathogen-infected cells and the disabling of tumor and pathogen-
mediated immune avoidance mechanisms. By adaptive immunity is meant that
the patient's defenses are mediated by B and T cells following exposure to
antigen and that such defenses exhibit specificity, diversity, memory, and
self/nonself recognition. Such adaptive immunity is systemic within the
patient.
Adaptive immunity is to be distinguished from innate immunity which is non-
specific and exists prior to exposure to the antigen.
[0020] In some embodiments, ablation followed by administration of the
allogeneic cells may be sufficient to generate the desired response. In other
words, the priming of the patient by a first aliquot of allogeneic cells may
be
omitted. In these embodiments, tissue from the tumor or infected by a pathogen
is ablated followed by injection of an aliquot of allogeneic cells.
[0021] The present invention also includes a method of vaccinating a patient
having cancerous cells or an infected tissue. This method can be used to
vaccinate a patient using in situ generated antigens from the cancerous cells
or
infected tissue in conjunction with administration of allogeneic cells. This
method can also be used for patients with hematological malignancies (e.g.,
Chronic Lymphocytic Leukemia, Multiple Myeloma, and non-Hodgkin's
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lymphomas) or viral infectious diseases (e.g., hepatitis B or C, herpes, HIV)
and
other disorders where the affected lesions are not easily assessable for
ablation.
[0022] The method involves first "priming" of the patient to develop Thl anti-
alloantigen immune memory by infusion of a first aliquot of allogeneic cells.
It
is desired that the infusion of allogeneic cells stimulates the patient's
immune
system to react against the allogeneic cells. A time period is allowed to
elapse
until the patient's immune system is allowed to form an anti-allogeneic
memory.
In some embodiments, a patient may need a booster of allogeneic cells to
develop the appropriate Thl immune memory.
[0023] The next step includes exposing the patient's immune system to antigens
from the cancerous cells or the pathogen infected tissue in addition to the
activated allogeneic cells described herein. In preferred embodiments, the
antigens are generated in situ by ablation of the cancerous cells/tumors or
the
pathogen infected tissue. In situ ablation of the tumors or the pathogen
infected
tissue results in the release of tumor antigens or pathogen antigens in the
patient.
The subsequent administration of the allogeneic cells at site of the lesion
results
in the desired immune response by creating a rejection response in the patient
and stimulating a delayed-type hypersensitivity response to the antigens. The
ablation can be by cryoablation, electroporation or other means that result in
necrotic death of tumors or pathogen infected tissue
[0024] In other embodiments, after the priming step, the method can include
administering into the patient an antigenic composition that includes an
autologous lysate containing antigens from the cancerous cells or the infected
tissue. This composition also includes an aliquot of the allogeneic cells,
i.e.
allogeneic cells that are from the same source as the allogeneic cells used in
the
priming step. The injection of the antigenic composition can create a
rejection
response in the patient and can stimulate a delayed-type hypersensitivity
response to the antigens.
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[0025] The scavenger cells, including immature dendritic cells can pick up the
antigens resulting from the in situ ablation or from the autologous lysate.
The
allogeneic cells can cause the maturation of dendritic cells for the priming
of
Thl immunity to the antigens. It is desirable for the patient to develop a
strong
DTH reaction upon introduction of the allogeneic cells at the site of the
necrotic
lesion due to the fact that the patient has been primed to Thl immunity
against
the allogeneic cells introduced by the first aliquot of allogeneic cells
during the
priming step.
[0026] The general state of inflammation caused by the treatment process can
serve to cause the DC to program T-cells to Thl immunity against the antigens
at the ablation site resulting in a systemic adaptive immune response to the
tumor or pathogen-infected cells and the disabling of tumor and pathogen-
mediated immune avoidance mechanisms.
[0027] The present disclosure also provides a method for enhancing the
immunogenicity of weakly immunogenic or non-immunogenic tumors and a
method to deviate an immune response from a non-protective immune response
(e.g., Th2 response) to a protective immune response (e.g., Th1). Such
diseases
include, for example, all types of cancers and diseases caused by infections
with
a variety of pathogens (e.g., Hepatitis viruses, fungal infections such as
aspergillus, HIV, malaria, typhoid, cholera, herpes viruses, Chlamydia, and
HPV).
[0028] The present disclosure also includes a therapeutic composition for
treating a tumor or a pathogen in a patient. The therapeutic composition
preferably includes allogeneic cells and antigens, generated in situ,
comprising
the products of tumor necrosis or pathogen infected tissue necrosis. The
therapeutic composition may also include a priming composition. The priming
composition generally contains allogeneic cells that are injected into the
patient
to generate a rejection response by the patient's immune system in a manner
that
induces an allogeneic Thl immunity.
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[0029] The therapeutic composition includes antigenic material from the tumor
or pathogen-infected tissue and an aliquot of allogeneic cells. In preferred
embodiments, the antigenic material is in situ released antigenic material
containing antigens from the cancerous cells or from infected tissue. The
antigenic material can be derived in situ from tissue necrosis of the tumor or
the
pathogen-infected tissue. Preferably, the antigenic material is derived from
ablation of the tumor or pathogen-infected tissue.
[0030] The ablation may be done in vivo, in situ or ex vivo. In some
embodiments, the antigenic material includes heat shock proteins released upon
ablation of the tissue from a tumor or pathogen-infected tissue.
[0031] The therapeutic composition also includes allogeneic cells. In
embodiments with in situ generated antigenic material, the therapeutic
composition includes allogeneic cells that are administered directly into the
lesion site containing the in situ generated antigens. The allogeneic cells,
when
injected into the patient, can create a rejection response and stimulate a
delayed-
type hypersensitivity response to the antigens thereby acting as an adjuvant
to
the stimulation of systemic anti-tumor or anti-pathogen immunity in the
patient.
[0032] The therapeutic compositions may include other components that act as
adjuvants to the response generated by the priming composition and the
allogeneic cells. The priming composition and allogeneic cells may include
other components generally found in therapeutic composition, for example,
preservatives. The addition of these components are within the scope of this
invention.
[0033] In some embodiments, the therapeutic composition may only include the
allogeneic cells and in situ generated antigens and not the priming
composition.
The priming composition may not be needed to obtain the desired immune
response.
[0034] The present invention also includes a vaccine composition for a patient
against a tumor or a pathogen. The vaccine preferably includes a priming
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composition and an antigenic composition. The priming composition generally
contains allogeneic cells which are injected into the patient to generate a
rejection response by the patient's immune system in a manner that induces an
allogeneic Thl immunity.
[0035] The antigenic composition includes antigenic material from the tumor or
pathogen-infected tissue and an aliquot of allogeneic cells. In some
embodiments, the antigenic material is an autologous lysate containing
antigens
from the cancerous cells or from infected tissue. The antigenic material can
be
derived from tissue necrosis of the tumor or the pathogen-infected tissue.
Preferably, the antigenic material is derived from ablation of the tumor or
pathogen-infected tissue. The ablation may be done in vivo or ex vivo. In some
embodiments, the antigenic material includes heat shock proteins released upon
ablation of the tissue from a tumor or pathogen-infected tissue. The antigenic
composition also includes allogeneic cells. The antigenic material and the
allogeneic cells may be combined together or packaged separately.
[0036] The present disclosure also includes an in situ generated vaccine
composition. The antigenic material in this vaccine is generated in situ and
the
allogeneic cells are administered to the patient as an adjuvant, preferably
intralesionally. The in situ generation of antigenic material can be
accomplished
by ablation of the tumor or pathogen infected tissue within the patient
releasing
the antigenic material. The ablation can be by cryoablation, electroporation
or
other means that result in necrotic death of tumors or pathogen infected
tissue.
[0037] In embodiments with in situ generated antigens, the antigenic
composition administered to the patient includes allogeneic cells and these
cells
are administered at the necrotic lesion site containing the released antigens.
The
antigenic composition, when injected into the patient, can create a rejection
response and stimulate a delayed-type hypersensitivity response to the
antigens
thereby acting as an adjuvant to the stimulation of systemic anti-tumor or
anti-
pathogen immunity in the patient.
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[0038] The vaccine may include other components that act as adjuvants to the
response generated by the priming composition and the antigenic composition.
The priming composition and antigenic composition may include other
components generally found in vaccines, for example, preservatives. The
addition of these components are all within the scope of this invention.
[0039] In some embodiments, the vaccine may only include the antigenic
composition and not the priming composition. The antigenic composition may
be sufficient to obtain the desired immune response.
[0040] The therapeutic vaccines of the present invention are useful for the
prevention and treatment of diseases such as cancer or chronic viral diseases
that
develop and/or persist by suppressing or escaping the immune response.
Priming Step
[0041] The purpose of the priming step is to create anti-allogeneic Thl
immunity in a patient that can be recalled upon subsequent exposure to the
alloantigens. Priming occurs by exposing a patient to an aliquot of allogeneic
cells and the subsequent rejection of these allogeneic cells when a second
aliquot is administered to the patient by the patient's immune system
resulting
from immune memory. Preferably, the patients are not immunosuppressed prior
to priming, as this will inhibit the ability of the patient to reject the
infused
allogeneic cells and will also inhibit the development of anti-alloantigen Thl
immunity.
[0042] In one embodiment of the present invention, the patient's immune
system is skewed to generate Thl immunity. It is preferable to manipulate the
allogeneic cells such that Thl and not Th2 immunity develops in response to
the
rejection of the allogeneic cells. In one embodiment, the patient's immune
system can be skewed to produce Th-1 response by administering allogeneic
cells that are producing Thl cytokines (e.g., IFN-gamma and TNF-alpha) when
infused. Thl cytokines can assist in skewing the immune response to the
alloantigens to Thl type immunity. Other methods of skewing a patient's
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immune system to produce Th-1 immunity are also within the scope of this
invention.
[0043] The allogeneic cells used to first prime the patients and then later
used
for either intralesional administration (after induction of cell death) or as
an
adjuvant to a source of pathogenic or tumor material, are preferably
allogeneic
activated T-cells, more preferably allogeneic activated CD4+ Thl cells, more
preferably allogeneic CD4+ T-cells that have differentiated into effector or
memory cells and produce high levels of Type 1 cytokines, such as IL-2, IL-15,
IFN-gamma, TNF-alpha and also express, preferably at high density, effector
molecules such as CD40L, TRAIL and FasL on the cell surface but do not
produce IL-4 or other Type 2 cytokines. CD40 ligation of innate immune cells
(e.g., dendritic cells, macrophages and NK cells) has the capacity to induce
high
levels of the cytokine IL-12, which polarizes CD4+ T cells toward the Th 1
type
immunity, enhances proliferation of CD8+ T cells, and activates NK cells.
These pro-inflammatory events can enable the consistent development of Thl
immunity to the alloantigens on the allogeneic cells upon rejection by the
patient's immune system.
[0044] In the priming step, the activated allogeneic T-cells are administered
to
the patient, preferably intravenously, but can also be administered
intradermally.
The allogeneic cells are preferably derived from a deliberately HLA-
mismatched donor. Preferred dosage in an aliquot of allogeneic cells for
intravenous infusion is at least about 1 x 107 cells and more preferred is
between
about 1 x 108 to 1 x 1010 cells. Dosages of allogeneic cells outside this
range
that can primarily generate an immune response are also within the scope of
this
invention.
[0045] It is desirable to test the patients for development of Thl anti-
alloantigen
immunity prior to the ablation of affected tissue or administration of the
antigenic composition. The development of Thl anti-alloantigen immunity may
take at least about 7 days. Preferably, the patient is allowed between about 7
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days to about 14 days to develop Thl anti-alloantigen immunity. The
development of Thi anti-alloantigen immunity can be measured by, for example,
ELISPOT assay. Other methods of testing patients for development of Thl anti-
alloantigen immunity are also within the scope of this invention. If the Thl
anti-
alloantigen immunity is weak, additional booster injections of allogeneic
cells
can be administered. Booster injections are preferably made intradermally to
generate a delayed type hypersensitivity (DTH) reaction in the skin.
Generation of Allogeneic T-cells
[0046] It is desirable that allogeneic T-cells can be generated such that,
upon,
activation and infusion into a patient, a Th-1 immunity can be generated by
the
patient. A preferred method for producing allogeneic cells with the properties
necessary for stimulation of anti-allogeneic Thl immunity involves: (1) the
collection of mononuclear cell source material by leukapheresis from normal
screened donors; (2) the isolation of CD4 T-cells from the source material;
(3)
the activation of the CD4+ cells with immobilized anti-CD3 and anti-CD28
monoclonal antibodies (mAbs) on days 0, 3 and 6; (4) the activation of the
cells
again on day 9 with immobilized anti-CD3 and anti-CD28 mAbs and the
infusion of the cells within 24h of activation.
Cell Death Step
[0047] In situ cell death or cell injury can result in recruitment of DC to
the
lesion and provide a source of antigen for uptake by DC. It is preferable that
target tissues be destroyed by a process which causes death by necrosis. By
necrosis it is meant the death of individual cells or groups of cells such
that
amounts of intracellular components are released to the environment. For
purposes of this application, necrosis includes a cell death by a variety of
methods including cryoablation, irreversible electroporation, chemotherapy,
radiation therapy, ultrasound therapy, ethanol chemoablation, microwave
thermal ablation, radio frequency energy or a combination thereof.
Necrotically
killed cells activate endogenous signals of distress responsible for the
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recruitment and maturation of DC, stimuli that would not be generated by
healthy or apoptotically dying cells. Further, exposure of immature DC to
these
stimuli provides maturation signals, critical for the initiation of local and
systemic Thl immunity.
[0048] In one preferred embodiment, in order to cause death by necrosis, it is
preferred that the target tissue is frozen in situ. Cryosurgery is a well-
aimed and
controlled procedure capable of inducing tissular necrosis by the application
of
liquid N2 or argon gas. The biologic changes that occur during and after
cryosurgery have been studied in vitro and in vivo. Tissue injury and necrosis
is
induced by cell freezing and by the vascular stasis that develops after
thawing.
Cryosurgery (in situ freezing) has been known to elicit an antigenic stimulus
(comparable to that obtained through the parenteral administration of antigen)
capable of generating a specific immunologic response against autologous
antigens of the frozen tissue.
[0049] Cryoablation can cause peptides to be released from lysed tumor or
pathogen-infected cells for antigen processing by DC and creates a pro-
inflammatory cytokine environment. Cytokines released after cryoablation such
as IL-1, IL-2, TNF-a, IFN-y, and GM-CSF can activate the T, NK, and
Langerhans cells essential to an immune response capable of destroying cancer
or pathogen infected cells.
[0050] In another preferred embodiment, in order to cause death by necrosis,
it
is preferred that the target tissue is subject to irreversible
electroporation.
Irreversible electroporation is a tissue ablation technique in which micro to
milli-second electrical pulses are delivered to the tissue to produce cell
necrosis
through irreversible cell membrane permeabilization. In
irreversible
electroporation, the cellular membranes of the cells between the electrodes
are
disrupted causing cellular necrosis. Irreversible electroporation can cause
antigens to be released from lysed tumor or pathogen¨infected cells for
antigen
processing by DC and creates a pro-inflammatory cytokine environment.
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[0051] Another preferred method for generating a source of antigen is to
isolate
autologous chaperone proteins, also known as heat shock proteins (HSP), from
dead infected tissue or tumors. HSPs are among the major targets of the
immune response to bacterial, fungal and parasitic pathogens. Certain
chaperones in extracellular milieu may also modulate innate and adaptive
immunity due to their ability to chaperone polypeptides and to interact with
the
host's immune system, particularly professional antigen-presenting cells.
Vaccination with heat shock proteins from tumor have been shown to elicit an
anti-tumor response. Current studies indicate that the immunogenicity of HSPs
is derived from the antigenic peptides with which they associate.
[0052] A preferred method for isolation of chaperone proteins for use as an
antigen source is described by Katsantis in US Pat No. 6,875,849. Additional
methods are described by Srivastava in US Pat Nos. 6,797,480; 6,187,312,
6,162,436; 6,139,841; 6,136,315; and 5,837,251.
Adjuvant Step
[0053] The purpose of the adjuvant step is to cause the maturation of DC to
stimulate Thl immunity against antigens taken up in the lesions containing
dead
target tissue. This can be accomplished by the injection of the same
allogeneic
cells, i.e. allogeneic cells of the same origin as those used to prime the
patient.
This aliquot of the allogeneic cells are, preferably, injected
intralesionally, i.e.
directly into the necrotic lesion caused by the cryoablation, or other method
of
cell death. Alternatively, when chaperone proteins are used as the source of
antigen, the same allogeneic cells used to prime the patient are injected with
the
chaperone proteins, preferably intradermally. The dosage of the allogeneic
cells
to generate the desired immune response is generally at least about 1 x 107
cells
and more preferred is between about 1 x 108 to 1 x 1010 cells. Dosages of
allogeneic cells outside this range that can generate the desired immune
response are also within the scope of this invention. The preparation of the
allogeneic cells is the same as described above.
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[0054] To initiate an immune response and overcome the natural tolerance the
immune system has to self tissues, the antigens released after necrotic cell
death
or associated with the chaperone proteins must be taken up by DC and presented
with immune activating components that signal "danger". The memory immune
response against the allogeneic cells create this "danger".
[0055] The tissue resident DC, termed immature DC, are able to capture the in
situ generated antigens from the environment, but are deficient in stimulating
T
cells. In response to pathogen infection and the ensuing inflammatory
response,
DC undergo a differentiation process called maturation, whereby they up-
regulate the capacity to migrate to draining lymph nodes and present the
captured antigens to T cells. To activate Thl CD4+ T cells and CTL, the DC has
to integrate a number of maturation/differentiation stimuli. At the site of
pathogen or tumor encounter, exposure to pathogen or tumor-derived
determinants, proinflammatory cytokines, and/or cell debris induces the first
steps in the maturation process. This includes the up-regulation of
costimulatory
molecules and chemokine receptors, whereby the DC acquire the ability to
present antigens to T cells and migrate to the lymph node, respectively. At
the
lymph node, encounter of cognate CD4+ T cells provides additional
differentiation stimuli to the DC, which regulate the survival of the
activated T
cells and the polarization of the CD4+ T cells.
[0056] The maturation of DC occurs at the site of antigen uptake and the
recall
rejection response serves as an adjuvant to provide the appropriate
inflammatory
danger signals necessary for DC maturation, migration to the lymph nodes and
the programming for Thl immunity against the antigens uptaken in the lesion.
Examples
Animals
[0057] Balb/c mice were hosts and C57B1/6 (B6) mice were used as source of
Thl cells. All mice were 6 to 10 weeks old, were maintained in a specific
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pathogen-free facility at the Hadassah-Hebrew University Medical Center, and
were treated on an approved animal protocol.
Preparation of Allogeneic Thl Memory Cells
[0058] Spleen cells from male C57BL/6 mice were harvested and treated with
ammonium chloride-potassium (ACK) buffer for lysis of red blood cells.
Approximately 70-100 million cells were isolated per spleen. CD4+ T-cells
were then purified by positive selection (purity >98%) using CD4
immunomagnetic particles on an MS column (Miltenyi Biotec, Germany),
approximately 8-12 million CD4 cells were isolated with a yield of 50-60%.
Thl memory cells were generated by expansion with anti-CD3 and anti-CD28¨
coated paramagnetic beads (CD3/CD28 T-cell expander beads,
Dynal/Invitrogen) at an initial bead:CD4 cell ratio of 3:1. The purified CD4
cells were incubated with 20 IU/mL recombinant mouse (rm)IL-2, 20 ng/mL
rmIL-7, and 10 ng/mL rmIL-12 (Peprotech, New Jersey) and 10 lug/mL
antimurine IL-4 mAb (Becton Dickenson) in RPMI 1640 media containing 10%
FBS, penicillin-streptomycin-glutamine, nonessential amino acids (NEAA)
(Biological Industries, Israel) and 3.3 mM N-acetyl-cysteine (NAC; Sigma)
(complete media). Additional cytokine-containing complete media with rmIL-2
and rmIL-7 was added to the CD4 cultures daily from days 3 to 6 to maintain
the
cell concentration between 0.5 and 1 x 106 cells/mL. Additional CD3/CD28
beads were added daily from day 3 to day 6. The number of beads added was
calculated to maintain a 1:1 bead:cell ratio as the cells expanded. After 6
days
in culture, the CD4 cells expanded approximately 80 to100-fold and were
harvested and debeaded by physical disruption and passage over a magnet. The
phenotype of the harvested cells used in experiments were >95% CD4+,
CD45R0+, CD62L10, IFN-a+ and IL-4-.
CD3/CD28 Nanobead Preparation
[0059] Biotinylated mouse anti-CD3 and anti-CD28 mAbs (BD Pharmingen)
were each diluted in 4001,t1 of PBS to a final concentration of 251Ag/m1 and
then
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mixed in a 1:1 ratio so that the final volume was 8001,t1. 201,t1 of
Strepavidin-
coated nanobeads (Miltenyi, Germany) were washed and diluted to a final
volume of 2001,t1 in PBS. The 8001,t1 of the CD3/CD28 mAb solution and the
2001,t1 of diluted nanobeads were then mixed so that the final concentration
of
each mAb was 10pg/m1 in a total volume of lml. The mixture was placed on a
rotating mixing device for 30 min at RT. The mAb conjugated nanobeads were
then passed over an MS column (Miltenyi, Germany) on a magnet and washed
thoroughly. The retained nanobeads were then released from the column and
resuspended in 2001,t1 of PBS. The nanobeads were not able to activate naïve T-
cells. Therefore, the nanobeads were tittered against harvested Thl memory
cells that had been previously activated 6 days prior with CD3/CD28 T-cell
expander beads (Dynal, Norway). While there were slight variations per batch,
generally 201,t1/107cells was found to provide optimal activation of
previously
activated Thl memory cells.
CD3/CD28 cross-linking
[0060] In experiments that required the infusion of activated Thl memory
cells,
the harvested Thl cells were incubated with a pre-tittered concentration of
CD3/CD28-conjugated nanobeads prior to infusion. For optimal activation, the
cells had to be incubated with the nanobeads for a minimum of 4h and a
maximum of 18h. Optimal activation caused production of IFN-a and
upregulation of CD4OL and FasL on the cell surface. For these experiments, all
infusions of CD3/CD28 cross-linked Thl memory cells occurred after 4-8h of
pre-incubation. Cells were thoroughly washed prior to infusion to remove any
unassociated nanobeads. Cross-linked Thl memory cells used in these
experiments expressed FasL and CD4OL on the cell surface and produced in
excess of 2000ng/m1/106 cells/6h IFN-a and less than 20pg/m1 IL-4 per 106
cells/6h. Thl memory cells without CD3/CD28 cross-linking did not produce
cytokines or express FasL or CD4OL.
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Cryotherapy
[0061] Cryotherapy was performed with a spherical nitrous oxide cryoprobe,
3 mm in diameter. The gas was maintained at a pressure of 50 bars and the
Joule-Thomson effect allowed to attain temperatures ranging from ¨30 to
¨40 C in the tissue. An incision was made in the centre of the tumor, the
cryoprobe was placed in contact with the tumor (it was inserted 1-2 mm deep):
the aim was to influence it by freezing but not to destroy it completely.
Three
cycles of rapid freezing (lasting for 20 s) followed by slow thawing were
applied. The ice ball was produced at the center of the lesion and reached
about
two thirds of the total tumor volume.
Example #1:
[0062] To test the ability of allogeneic Thl cells to stimulate systemic anti-
tumor immunity in extensive metastatic disease, the following protocol was
tested. Lethal doses of tumor cells including BCL1 leukemia, 4T1 breast cancer
and 3LL lung cancer were infused intravenously into mice on day 0 and the
tumor cells were also injected intradermally to establish a solid tumor mass.
On
day 7, the mice were given a 1 x 105 dose of allogeneic Thl cells. On day 14,
the mice were treated intratumorally by injection of either: (a) saline; (b)
saline+partial cryoablation of tumor; (c) allogeneic Thl cells at a dose of
103
cells; or (d) allogeneic Thl cells+partial cryoablation of tumor. The results
of
surviving animals at 90 days is shown below (n=10):
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Intratumoral BCL1 4T1 3LL
treatment Leukemia Breast Lung
Saline 0 (0%) 0 (0%) 0 (0%)
S aline+cryo ablation 0 (0%) 0 (0%) 0 (0%)
Thl alone 1( 10%) 1(10%) 2 (20%)
Thl+cryo ablation 4 (40%) 5 (50%) 8 (80%)
Example #2
[0063] In order to investigate whether treatment of patients with solid tumors
might benefit from the present invention, the experiment design above was
repeated in animals that only received intradermal injections of tumors
creating
solid tumor masses. The results were similar to those obtained with animals
with metastatic disease.
Intratumoral BCL1 4T1 3LL
treatment Leukemia Breast Lung
Saline 0 (0%) 0 (0%) 2 (20%)
Thl alone 0 (0%) 0 (0%) 1 (10%)
s aline+cryo ablation 0 (0%) 0 (0%) 2 (20%)
Thl+cryo ablation 6 (60%) 7 (70%) 9 (90%)
[0064] The combination of Thl cells with cryotherapy results in high cure
rates.
Cryotherapy kills tumors by necrosis, which is thought to be a more
pathological
type of cell death than death by apoptosis (the type of death caused by
chemotherapy). It is thought that the cryotherapy makes the tumors more
immunogenic and therefore the combination of allogeneic Thl cells with
necrotic tumor death creates a type of tumor vaccine leading to systemic anti-
tumor immunity.
[0065] Although the present invention has been described with reference to
preferred embodiments, workers skilled in the art will recognize that changes
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may be made in form and detail without departing from the spirit and scope of
the invention.
