Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND AGENTS FOR THE DETECTION AND MODULATION OF
CELLULAR IMMUNITY TO IMMUNE PRIVILEGED ANTIGENS
GOVERNMENTAL SUPPORT
The research leading to the present invention was supported, at least in part,
by grants from
the Department of Defense, Breast Cancer Research Award No. DAMD017-94-J-
4.277, the
National Institutes of Health Award No. MO1 RR00102, and the National Multiple
Sclerosis Society. Accordingly, the Government may have certain rights in the
invention.
FIELD OF THE INVENTION
This invention relates to diagnostic and therapeutic methods based upon the
development of
cellular immunity to immune privileged antigens and its role in the etiology
of
paraneoplastic neuronal disorders and tumor immunity, among other conditions.
BACKGROUND OF THE INVENTION
Constant surveillance of epitopes throughout those structures in the body
accessible to the -
immune system provides a very effective means for recognizing and maintaining
"self" and
destroying epitopes and their carriers which invade the body or arise
pathologically, such as
infectious microorganisms. One important role of immune surveillance is the
recognition and
destruction of neoplastic cells that are believed to arise continuously in the
body and for the
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most part are eliminated by the immune system before becoming detectable. HoW
eves,
examples of naturally-occurring tumor immunity have been elusive. Cytotoxic T
lymphocytes,
key participants in effective immune surveillance, are not expanded in
patients with active
tumors, even when these tumors express what are believed to be tumor-specific
antigens such
as the MAGE/MART antigens of melanoma.
Effective tumor immunity has been documented, however, in individuals with
paraneoplastic
neuronal disorders (PNDs). These syndromes are poorly understood diseases in
which serious
effects of cancer in the body occur in the nervous system without any direct
involvement of the
tumor. PND patients typically present to physicians with neurologic
dysfunction unaware that
they harbor a tumor. For example, patients with ovarian or breast cancer who
develop
paraneoplastic cerebellar degeneration (PCD) have an effective tumor immune
response (3,4,5;
reviewed in 1,2,6), and moreover, the tumor expresses neuron-specific proteins
{antigens).
These patients have in circulation and in the cerebrospinal fluid (CSF)
antibodies against these
tumor cell antigens, which also cross-react with the same proteins expressed
in neurons,
termed onconeural antigens. A high titer antibody recognizes the intracellular
antigen cdr2
expressed in the ovarian or breast tumor present in PCD patients (10); and
also recognizes the
antigen in Purkinje neurons of the cerebellum {10). However, as will be
elaborated below, the
existence of this antibody does not account for the etiology of the PND nor
for effective tumor
killing.
?0 Certain regions of the body, such as the brain, eye, and testis, are
protected from immune
surveillance, these sites are referred to as immune privileged. Based on the
above
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observations, the immune system is proposed to initiate PCD by recognizing the
normally
immune-privileged antigen cdr2 (10) when it is ectopically expressed in
gynecologic tumors.
This immune response is associated clinically with effective tumor immunity,
and is believed
to lead to the recognition and destruction of Purkinje neurons expressing
cdr2. cDNAs
S encoding several of the target antigens have been cloned, for example, cdr2
which has been
shown to be the correct tumor antigen (9,54). However, because the target
neuronal antigen
is cytoplasmic, the role of circulating and cerebrospinal fluid (CSF)
antibodies against these
antigens in the pathogenesis of PCD is questionable. Moreover, attempts to
reproduce the
disorder by passive or active transfer of antibodies have failed ( 11,12,13).
As the target organ,
the brain, is immune privileged, and furthermore the target antigen is
cytoplasmic, the etiology
of the paraneoplastic syndrome is difficult to reconcile. This is further
confounded by the
apparent absence of a cellular immune response against tumor antigens in
general and the
apparent absence of a cellular immune response in PCD. No cytotoxic T
lymphocytes were
found against the cdr2 protein using autologous dendritic cells in a patient
with PCD (47). The
etiology of tumor immunity in PND is enigmatic.
As described above, the paraneoplastic syndromes are serious conditions
associated with
tumors and frequently affect the central nervous system; these disorders are
collectively
referred to as paraneoplastic neuronal disorders (PND). For example, one
common
paraneoplastic disorder which is seen in patients with breast or ovarian
cancer is paraneoplastic
cerebellar degeneration, or PCD, in which a progressive and severe
neurological dysfunction
occurs involving the cerebellum, leading to dyscoordination of the legs and
arms, dizziness and
double vision. Frequently, these symptoms appear before the diagnosis of
cancer. In another
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example of neurological degeneration, Hu syndrome is associated with small
cell lung cancer
and antibodies to the onconeural antigen Hu. In other examples, opsoclonus, or
spontaneous,
chaotic eye movements, and myoclonus, jerky body movements, may accompany
breast
cancer, fallopian tube cancer, or small cell lung cancer, and are associated
with antibodies to
the onconeural antigen Nova.
The target onconeural antigens have yet to be identified for some disorders
believed to be
paraneoplastic. Patients with Hodgkin's disease and other lymphomas may
develop subacute
cerebellar degeneration that is believed to be immune mediated (22,42). Eaton-
Lambert
syndrome, a condition causing weakness in the limbs, may also accompany
intrathoracic
tumors such as lung cancer and is believed to be immune mediated (2). Some
patients who
develop spinal cord dysfunction (e.g., myelopathy), motor neuron diseases,
blindness and other
neurologic symptoms are found to have specific sets of underlying tumors and
are believed to
have immunity to unknown or partially-characterized onconeural antigens
(2,37). Less well
understood, the incidence of the muscle diseases dermatomyositis and
polymyositis is increased
in cancer patients. The dermatologic condition vitiligo, in which melanocytes
producing skin
pigment are destroyed, appears associated with a decrease in incidence of
melanoma. It is thus
apparent that an association exists between tumors, and in some cases tumor
immunity, and-
the sites of the paraneoplastic disorder symptoms, perhaps through the
existence of some
common antigens.
Several lines of evidence suggest the existence of naturally-occurring tumor
immunity in PND
patients. PND-associated tumors are typically occult (24,25); in several cases
they have been
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identified only by microscopic analysis of suspect organs following
exploratory surgery or at
autopsy. Patients with PND-associated tumors have significantly-limited
disease and an
improved tumor prognosis relative to patients with histologically-identical
tumors unassociated
with PND (20,24,26-28). In some cases PND-associated tumors have been
documented to
S regress with the onset of autoimmune neurologic disease (7).
Specific clinical data regarding anti-tumor immunity is available for several
of the PNDs.
Patients with paraneoplastic encephalomyelitis harbor high titers of an
antibody termed Hu
and small cell lung cancers (SCLCa); their tumors are typically limited to
single nodules
(S3/SS [96%J patients in the most complete study published [3J ). This is a
remarkable
i 0 finding given that most SCLCa patients from unselected series (over 60 % )
have widely
metastatic disease at the time of diagnosis (and no detectable titers of Hu
antibody). In
addition, fifteen percent of SCLCa patients without PND nonetheless have
detectable titers
of the Hu antibody (20). These patients have statistically significant
increases in the frequency
of limited stage disease, complete response to chemotherapy and longer
survival (3, S). These
1 S results suggest that anti-PND antibodies may be associated with
suppression of tumor
growth independently from their association with neurologic disease.
There are also firm associations between the presence of the Nova (Ri) (28)
and Yo (10)
antibodies in PND patients and clinically-limited malignancy. Both antibodies
are found in
women with gynecologic cancer. Of S2 Yo-antibody-positive patients with breast
or
20 ovarian cancer (4), two-thirds (34/S2) presented with neurologic symptoms
prior to the
diagnosis of cancer, and 87 % (4S/S2) had limited oncologic disease when
diagnosed;
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similarly, 4/7 Nova-positive patients presented with neurologic symptoms, 6/7
had limited
stage disease, and no tumor could be found in one patient (28). By comparison,
only 50-60%
of unselected breast cancer patients, and 25 % of ovarian cancer present with
limited stage
disease (8).
Experimental observations support the clinical evidence that there is
immunologic recognition
of tumor cells in PND. High titer anti-PND antibodies are found in the serum
and
cerebrospinal fluid of PND patients. In vitro, these antibodies react
specifically with
tumor specimens obtained from PND patients cells, as well as neurons from
clinically
affected areas of the nervous system (24,25, 29). For example, 10/10 breast or
ovarian
tumors from Yo-positive patients were immunoreactive with biotinylated Yo
antisera (4), and
3/4 breast or fallopian tumors from Nova-positive patients were immunoreactive
with
biotinylated Nova antisera (28). ~ Taken together, these observations suggest
that PND
antibodies are more than markers for neurologic disease or even the presence
of tumor
cells, but are markers, and perhaps in part reflective of effective anti-tumor
immune
responses.
The immunologic basis of the anti-tumor and antineuronal immune response in
PND is
unknown. The finding of autoantibodies with neuronal binding specificity, and
observations
on autoimmune neurologic disorders of the peripheral nervous system, have
focused attention
on the role of B cells in the pathogenesis of PND. In myasthenia gravis (MG)
and
Lambent-Eaton myasthenic syndrome (LEMS), antineuronal antibodies have been
found to
passively transfer autoimmune disease in animals (30, 31). In PND, there are
relatively higher
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titers of antibody in the CSF than serum (IgG index > 1) (32) suggestive of an
active B cell
inflammatory response within the CNS compartment. Furthermore, although the
data is not
fully compelling, there have been numerous reports that PND antibodies may be
neurotoxic
in vitro and that antibodies may be able to be taken up by neurons (33, 34).
These
observations have led clinicians to focus therapy for the PNDs on the
elimination of PhID
antibodies. Unfortunately these attempts have been uniformly unsuccessful (24,
32, 35).
Several features of the PNDs distinguish them from MG and LEMS and suggest
that B cells
might not be sufficient or even necessary for the development of PND. PND
antigens have
been found to be cytoplasmic (Yo, ~3-NAP) or nuclear (Nova, Hu) proteins,
unlike the target
antigens in MG (the acetylcholine receptor) or LEMS (the presynaptic calcium
channel) (2).
It is difficult to reconcile these observations with the premise that PND
antibodies play a
primary role in PND autoimmunity. Moreover, attempts to produce animal models
of PND,
including infusion of antibody into the CSF and immunization with cloned
fusion protein, have
failed {11, 12).
1 S Thus, the etiology of the paraneoplastic syndromes appears to have an
immunological basis,
heretofore undefined. It is towards a better understanding of the etiology of
the paraneoplastic
neuronal disorders and the establishment of a link between effective tumor
immunity and these-
serious, remote complications of neoplasia in immune privileged sites that the
present invention
is directed, with objectives of improving the detection of tumors and
paraneoplastic disorders
in individuals in general and offering improved therapies for both tumors
expressing immune
privileged antigens and the associated syndromes.
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SUMMARY OF THE INVENTION
The inventors herein have made the surprising and remarkable finding of the
presence of tumor
antigen-specific T lymphocytes (CTLs) in patients with paraneoplastic neuronal
disorders.
This finding provides a basis for understanding the desirable and often
effective cell-based
immunologic attack on the tumors, and the effective but undesirable attack on
remote target
organs) of the paraneoplastic disorders by CTLs. Expression of the same immune-
privileged
antigen by these remote tissues as that which is expressed in the tumor cells,
and to which T
lymphocytes are targeted, explains for the first time the etiology of the
PNDs. Both activated
CTLs and memory T lymphocytes specific for the tumor and for the remote
antigen have been
detected. This finding provides an appreciation that immune privileged
antigens offer a unique
set of targets for the immune system. If expressed in tumors, they provide
targets for effective
anti-tumor immunity. If immune-privilege or tolerance to these antigens is
broken, for
example in the setting of effective anti-tumor immunity, autoimmune disease
may result. The
identification of a cellular immune response to immune-privileged antigens
that can be readily
and specifically detected, amplified, or inhibited, provides the basis for
diagnostic and
therapeutic utilities disclosed herein. Based upon this discovery, diagnostic
utilities are
disclosed for the detection and monitoring of cellular immunity to privileged
antigens, and
therapeutic methods are described for increasing the effectiveness of anti-
tumor immunity and
also for ,protecting the immune privileged site from immune-mediated
pathology. Known
diagnostic and therapeutic procedures and manipulations of the immune system
are modified
based on the discoveries herein in order to detect and modulate the immune
response to
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immune-privileged antigens.
As will be described in more detail below, only a fraction of patients with a
specific T
lymphocyte response to immune privileged antigens, especially those with
tumors, exhibit an
overt paraneoplastic disorder, yet such patients are at risk for the
development of, or may have
as-yet undetected autoimmune disease or another subclinical disorders. In
accordance with the
present invention, methods for determining in an individual the presence and
extent of a
cellular immune response to an immune-privileged antigen are provided, the
cellular immune
response associated directly or indirectly with a pathological state. Examples
of pathological
states include but are not limited to dysproliferative diseases,
paraneoplastic syndromes, and
autoimmune disorders. The method comprises quantitating in a sample of bodily
fluid from
an individual the presence and extent of T lymphocytes specific for the immune-
privileged
antigen or its fragments. The preferred method involves the detection of T
lymphocytes which
recognize paraneoplastic antigens, and most preferably, onconeural antigens
such as cdr2 and
Hu antigen. One example of a means for detection comprises determining the
extent of
activation of T lymphocytes upon exposure to the antigen by measuring cytokine
production;
another method comprises detecting the extent of recognition by the cytotoxic
T cells of target
cells expressing the antigen. Methods for detecting T lymphocytes bearing
receptors for
immune-privileged antigen are also provided.
In the instance where the T lymphocytes to be detected are memory T cells, the
methods
comprises detecting the extent of activation of memory T cells after exposure
to antigen-
presenting cells (APCs) presenting the immune-privileged antigen. In another
embodiment,
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the extent of recognition of target cells expressing the antigen is determined
after exposure of
the memory T lymphocytes to APCs presenting the immune-privileged antigen.
The present invention further provides a method for screening individuals for
the presence of
tumors expressing immune-privileged antigens as well as detecting the early
onset or
propensity to develop a pathological state caused by a cellular immune
response to an immune-
privileged antigen. This method comprises measuring the presence and extent of
T
lymphocytes specific for immune privileged antigens. Furthermore, a method is
provided for
determining whether a neoplasm expresses an immune-privileged ~ antigen by
quantitating T
lymphocytes that are specific for the antigen or its fragment. In another
embodiment, a method
is disclosed for determining whether a patient with a immune-privileged
antigen-expressing
tumor has a sufficient population of antigen-specific T lymphocytes to control
the tumor or is
a candidate for anti-cancer therapy. This method comprises quantitating T
lymphocytes
specific for the antigen or a fragment. In a still further embodiment, a
method for monitoring
the effectiveness of therapies directed to modulate the population of immune-
privileged
antigen-specific T lymphocytes in a patient is described wherein the numbers
of antigen-
specific T lymphocytes are quantitated.
The cDNAs encoding the target immune-privileged as welt as their expressed
proteins and
fragments thereof may be used in the present invention to provide reagents for
carrying out the
diagnostic and therapeutic methods as described herein, as well as being part
of a diagnostic
?0 kit. As described above, the sequence and cDNA of cdr2 is known (9,54); its
fragments that
complexes with HLA are described below.
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In a further example of a screening method for identifying the number of
immune-privileged
antigen-specific T cells in a patient sample, the following steps may be
carried out:
i) maturing dendritic cells in the blood sample;
ii) exposing the matured dendritic cells to apoptotic debris from unrelated
cells expressing an immune-privileged antigen;
iii) co-incubating the immune-privileged antigen-exposed dendritic cells
with the peripheral blood lymphocytes from the patient; and
iv) correlating the amount of interferon-y released from the lymphocytes
with the number of immune privileged antigen-specific T cells in the
sample.
By way of non-limiting example, the immune-privileged antigen may be cdr2. The
unrelated
cells expressing an immune-privileged antigen may be cells stably transfected
to express an
immune-privileged antigen, such as cdr2. The interferon-y release may be
measured in an
ELISPOT assay.
Diagnostic kits are also provided with componentry capable of measuring the
above-described
T lymphocytes and antigens comprising, for example, one or more of the
following reagents:
an isolated, immune-privileged antigen or preferably a fragment of the immune-
privileged_
antigen; a target cell expressing the immune-privileged antigen or its
fragment; a fragment of
the immune-privileged antigen in a tetrameric complex with HLA; and a reagent
such as an
antibody or labeled antibody which recognizes a fragment of the immune-
privileged antigen
in a complex with HLA. When the immune-privileged antigen is cdr2, useful
isolated
polypeptide sequences identified include cdr2 peptides referred to as Yol
through YoB, or
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cdr2-1 through cdr2-8, and identified herein as SEQ ID NO: l, SEQ ID N0:2, SEQ
ID N0:3,
SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7 and SEQ ID N0:8. The kit
may
include target cells prepared from a cell line or, for example, Drosophila,
which expressed the
immune-privileged antigen, and further may express HLA molecules and co-
stimulatory
molecules. A kit may further include components for detecting cytokine
production, suchas
y-IFN, as a means for detecting immune cell activation. To broadly screen
samples from a
variety of patients with different HLA hapiotypes, a variety of target cells
expressing the same
immune-privileged antigen, but different HLA haplotypes, may be employed in
order to detect
immune-privileged antigen specific T lymphocytes regardless of the patient's
HLA haplotype.
It is another object of the present invention to provide methods for treating
a neoplasm in a
patient in which the neoplasm expresses an immune-privileged antigen. One
preferred
embodiment is accomplished by increasing the number of immune-privileged,
antigen-specific
cytotoxic T lymphocytes present in the patient. in one, non-limiting example,
the method is
carried out by first isolating a quantity of APCs from a sample of the
patient's blood, then
exposing the APCs in vitro to the immune-privileged antigen or its fragment,
followed by
reintroducing the antigen-exposed APCs to the patient. In another related
embodiment, the
same method is followed with an additional step of exposing the antigen-
exposed APCs in vitro_
to a quantity of T lymphocytes isolated from the patient, and reintroducing
the T lymphocytes
to the patient. These examples are illustrative of methods of providing the
patient with immune
?0 privileged antigen-specific T lymphocytes and/or immune-privileged antigen-
presenting APCs
in order to develop or enhance immunity to the tumor.
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Methods for achieving presentation of the immune-privileged antigen or its
fragment on the
APCs in the aforementioned methods is achieved using any one of several
methods. For
example, APCs are provided with apoptotic cells expressing the immune-
privileged antigen or
a fragment. These can be commonly available cell lines expressing the immune
privileged
antigen, such as HeLa cells, which express the cdr2 antigen (9), or
transfected cells such_as
Drosophila cells expressing the gene encoding the immune-privileged antigen.
These cells may
also be further engineered to additionally express the gene encoding the MHC
molecule
haplotype of the patient, and even further engineered to express co-
stimulatory molecules, such
that the Drosophila cells function as an antigen-presenting cell, thus forming
a useful APC for
l0 in-vivo or ex-vivo stimulation of T iymphocytes as described above. These
cells also have
diagnostic utility, as described herein. The preferred antigen is a
paraneoplastic antigen, and
most preferred, an onconeural antigen such as cdr2 and Hu antigen. In a
further embodiment,
the immune-privileged antigen-specific T lymphocytes are derived from a donor
individual of
the same HLA haplotype as the patient.
In a further embodiment of the present invention, a method for treating a
pathological state m
a mammal is provided, wherein the pathological state is caused by the presence
in the mammal
of T lymphocytes specific for an immune-privileged antigen. The method
consists of-
administration of an effective amount of an agent which decreases the
population of activated
T lymphocytes specific for cells expressing the immune-privileged antigen. Non-
limiting
examples of such agents include tacrolimus, cyclosporin, immunosuppressive
cytokines,
corticosteroids, and combinations. The preferred agent is tacrolimus. The
immune-privileged
antigen is preferably a paraneoplastic antigen, most preferably, and
onconeural antigen such
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as cdr2 and Hu antigen and their fragments. The preferred route of
administration of the
agents is to the central nervous system. Other effective routes of
administration are also
disclosed.
In a further embodiment of the present invention, a method is provided for
decreasing the
ability of non-tumor cells expressing privileged antigens to be killed by
cytotoxic T
lymphocytes as well as decreasing the expression of paraneoplastic antigens on
non-tumor
cells. These may be achieved by several methods, for example, by reducing the
cytokine level
in contact with the affected cells; increasing the expression of Nef or Nef
like proteins,
inhibiting perform-mediated CTL killing of neurons, and inhibiting apoptosis
of the target
cells.
In another embodiment, methods and agents are provided for enhancing the
killing of tumors
expressing immune privileged antigens by T lymphocytes. These methods include
administering cytokines, inhibiting Fas-ligand expression in the tumor, and
inducing the
expression of MHC I molecules on the tumor. Other methods may be used in
combination
with increasing the immune-privileged T lymphocyte activity in the patient.
In a preferred embodiment, an individual with a tumor expressing an immune-
privileged
antigen and also suffering from a paraneoplastic disease or other syndrome in
which the
immune system is recognizing and attacking the same antigen at a non-tumor
site within the
body is treated by increasing the immune recognition of the immune-privileged
antigen of the
tumor exemplified by the non-limiting examples of methods disclosed herein,
while
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concurrently protecting the non-tumor site from immune attack by the
corresponding methods
disclosed herein.
It is thus a principal object of the present invention to take advantage of
the presence of
immune-privileged antigen-specific T lymphocytes to detect the existence of a
pathological
state in a patient and to monitor the efficacy of treatments based upon the
enhancement of
tumor immunity by T lymphocytes as well as their suppression in the treatment
of the
associated syndrome in the non-tumor site. It is a further object of the
present invention to
provide both diagnostic and therapeutic purposes for the detection of tumors
and paraneoplastic
syndromes, to increase the immune destruction of such tumors as well as to
protect the non-
tumor organs susceptible to disease caused by the same T lymphocytes.
These and other aspects of the present invention will be better appreciated by
reference to the
following drawings and Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a Western blot analysis of patient's serum and CSF against
the cloned cdr2
fusion protein (9). Serum (1:10,000 dilution) and CSF (1:500 dilution) from
patient 1 (lanes
1, 4), patient 2 (lanes 2, 5), or serum from a patient with an irrelevant PND
(Hu syndrome;
1:500 dilution; lane 3) was blotted. Serum and CSF from patient 3 gave similar
results.
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Figure 2 demonstrates that Cdr2-specific killer cells are present in the
peripheral blood of
PCD patients. T lymphocytes were isolated from the peripheral blood of acute
(A) and chronic
(B) HLA-A2.1+ PCD patients. (A) Isolated T lymphocytes were used directly in a
chromium
release assay using peptide pulsed T2 cells (a TAP-f- HLA-A2.1 + cell line) as
targets. Peptides
were predicted based on known anchor residues for the HLA-A2.1 binding groove,
and
designated cdr2-1 and cdr2-2. (B) Blood derived DCs were generated from PCD
and
HLA-A2.1+ matched control individuals, pulsed with peptides and co-cultured
with T cells.
After seven days, the responding T cells were tested for cytolytic activity
specific for cdr2 as
determined by 5'Cr release assay. The HLA-A2.1 immunodominant epitope derived
from the
influenza matrix protein served as a positive control for the generation of a
CTL recall
response (data not shown). Effector : target ratio = 20:1. In (A) and (B),
percent cytotoxicity
is measured as a function of spontaneous and total release. Background killing
of target cells
was 0 - 3 % in all groups. Results are representative of 6 experiments and the
values shown
represent the mean from triplicate wells.
Figure 3 depicts cytofluorography of cells isolated from the CSF of a patient
with acute PCD
indicate a Thl-type cellular immune response. (A) Cells present in the CSF
were assayed for
various phenotypic markers by FACScan~ (Becton Dickinson) using the indicated
monoclonal_
antibodies; CD56 is a marker for natural killer cells; CD19 is specific for B
cells; CD3 is
present on all T cells; CD4 and CD8 indicate helper and cytotoxic T cell
subsets, respectively;
CD25 is the IL2 receptor and is a marker for activated T cells. A second
analysis was
performed after the patient received tacrolimus. * p < 0.005. (B) Cells from
the CSF were
assayed for their intracellular cytokine profile using a dual laser
fluorocytometer (Becton
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Dickinson). Cells were treated with brefeldin A (BFA), and stained for the
presence of
accumulated cytokines using the indicated monoclonal antibodies. T-blasts were
selected based
on forward scatter and these cells consisted of approximately 10% of the CD3+
CD4+ T cell
population. (C) As a control, PBMCs were isolated at the same time and assayed
as described
in (B). In addition, the PBMCs were stimulated using phorbol 12-myristate 13-
acetate and
ionomycin allowing cytokine production to be detected as a positive control
(23).
Figure 4 shows a Western blot analysis of cdr2 expression in human ovarian
tumors. (A)
Protein extracts from 9 human ovarian tumors were run on Western blots and
probed with
biotinylated affinity purified PCD antibody. Strong cdr2 reactivity was
evident in tumors 1,
2, 6, 7 and 9, as well as in extracts of human Purkinje neurons; there was no
reactivity with
a protein extract of normal human ovary. Probing a duplicate blot with an anti-
tubulin
antibody showed immunoreactive protein in each lane. A non-specific (NS) band
was present
in Purkinje extracts and ovarian tumors that reacted with avidin-horseradish
peroxidase (HRP)
secondary alone (data not shown). (B) IEF/SDS-PAGE analysis of cdr2
expression. An
immunoreactive band of identical Mr and pI is present in extracts of mouse
brain or a human
ovarian tumor when probed with PCD antiserum and HRP conjugated secondary
antibody.
The mouse cdr2 cDNA encodes a protein that is 87%a identical with human cdr2
(10), and this
protein migrates identically with cdr2 detected in human Purkinje extracts
(data not shown).
Extraneous cross reactive bands seen in standard 1-D SDS gels (A) do not
resolve on IEF.
Figure 5 shows that apoptotic cells expressing cdr2 may be used to present
antigen to T
lymphocytes. MC (97-09) DCs were co-cultured with apoptotic uninfected HeLa
cells and
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syngeneic T cells. After 7 days, responding T cells were tested using T2 cells
pulsed with
various Yo peptides, Yol through Yo8 (corresponding to SEQ ID NO:1 through SEQ
ID
N0:8, and also referred to as cdr2-1 through cdr2-8). Negative control
included testing Matrix
peptide (M) (SEQ ID N0:9) pulsed T2 cells (HeLa cells were uninfected).
Figure 6 A-C show that apoptotic PC7 cells are capable of cross-priming cdr2-
specific CD8+
T cells in vivo. Mice were immunized with apoptotic tumor cells which
expresses cdr2, and
potent cdr2-specific killer T cells were demonstrated, in the absence of any
neurologic
dysfunction.
Figure 7 shows the results of a simplified assay of cdr2-specific T cells
present in the blood
of a patient with PCD. Dendritic cells from a blood sample are matured, then
exposed to
apoptotic cells expressing cdr2. Subsequently, exposure of the cells to
peripheral blood
lymphocytes results in gamma-interferon production by T lymphocytes as an
indicator of the
number of cdr2-specific T cells in the individual.
DETAILED DESCRIPTION OF THE INVENTION
The terms "tumor," "cancer," "neoplasm," "neoplasia" and their etymological
relatives are
used interchangeably herein to refer generally to dysproliferative diseases
and the attendant
affected cells or cell masses. Preferably, the dysproliferative cells referred
to herein express
an immune- privileged antigen.
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Cytotoxic T lymphocytes (CTLs) are effector T cells, usually CD8+, that can
mediate the lysis
of target cells bearing antigenic peptides associated with a MHC molecule.
Other cytotoxic
cells include 'y/8 and CD4+ NK 1.1 + cells.
Immune privilege and immune-privileged antigen refer to the isolation of
certain sites and
S antigens within the body from the immune system and thus relate to antigens
to which an
immune response is not normally developed. Immune-privileged antigens
expressed
ectopically (i.e. , outside of their normally immune-privileged sites) may
result in autoimmunity
or tumor immunity. Immune-privileged antigens are expressed by some tumors
resulting in
an immune response to both the tumor and to non-tumor sites expressing the
same immune-
privileged antigens. Subsequent access of the immune effector cells to the
immune privileged
sites results in immune attack of non-tumor cells. One type of such immune-
privileged
antigens are neuronal antigens, a subset of which are the onconeural or
paraneoplastic antigens,
against which an immune response will cause neurologic disease. A more
detailed description
of the onconeural antigens may be found in reference (2), herein incorporated
by reference.
I 5 Antigen presenting cells (APCs) are cells including dendritic cells,
macrophages, and B cells,
that can process and present antigenic peptides in association with class I or
class II MHG
molecules and deliver a co-stimulatory signal necessary for T cell activation.
It has been discovered by the inventors herein that the heretofore enigmatic
etiology of the
paraneoplastic syndromes, wherein individuals with a tumor experience disease
at remote
locations within the body leading to severe neurological impairment, is
explained by the
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existence of cytotoxic T lymphocytes (CTLs) and memory T lymphocytes targeted
against
tumor antigens which also recognize identical antigens expressed on neurons.
Such immune-
privileged antigens expressed by tumor cells induce a cellular immune response
which in some
cases provides effective and desirable tumor immunity, but as an undesirable
side effect
mediates immune attack on normal tissues in immune privileged sites which also
express~he
same antigen. Less well understood is access of CTLs to the immune privileged
sites as well
as the expression of the normally cytoplasmic immune-privileged antigens on
non-tumor cells.
As mentioned above, patients often experience the remote, adverse effects
before detection of
the tumor. Because the antigens recognized by specific CTLs are located in
immune privileged
sites within the body, for example, in the brain in paraneoplastic neuronal
degeneration, the
discovery herein provides an explanation for the poorly understood phenomenon
of immune
system attack on immune privileged, non-tumor antigens. In addition to the
brain, other
immune privileged sites in the body include the testis and parts of the eye.
In accordance with the present invention and as will be elucidated in the
examples and
description below, both diagnostic and therapeutic utilities are provided in
which the presence
or activity of T lymphocytes specific for immune-privileged antigens are
usefully employed.
Detection of T lymphocytes specific for immune-privileged antigens provides an
opportunity
for screening patients for the early detection of tumors which express such
antigens, and
facilitates the monitoring of patients undergoing anti-cancer therapies.
Effective tumor
immunity by such T lymphocytes may eradicate the tumor at an early stage but
leave behind
a paraneoplastic syndrome or autoimmune disease; detection of the cellular
immune response
and identification of the particular antigen to which it is directed may allow
therapeutic
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intervention before the development of the neurological disease and may
facilitate treatment
of the persisting condition.
New methods of treatment of neoplasms as well as of paraneoplastic syndromes
and
autoimmune diseases is also provided by the appreciation of the role of T
lymphocytes in-the
pathophysiology of the paraneoplastic syndromes and immunologic recognition of
privileged
antigens in general. The effectiveness of tumor immunity mediated by specific
CTLs which
recognize the immune-privileged antigen expressed by tumors is stimulated or
enhanced by
creating or expanding the population of specific CTLs or memory T lymphocytes.
APCs
exposed to the immune-privileged antigen are employed to enhance the immune
response.
Methods for the protection of non-tumor cells from immune attack are also
provided, in order
to protect the non-tumor target sites from pathology, especially when an
immune response
against the tumor is enhanced.
Among the various paraneoplastic syndromes, paraneoplastic cerebellar
degeneration (PCD)
is associated with gynecological tumors such as those of the ovary and breast.
As will be seen
in the examples below, CTLs present in PCD patients specifically lyse target
cells presenting
peptides derived from the PCD antigen called cdr2. Thus, cdr2 antigen,
normally an immune-
privileged antigen of neuronal cells, is expressed in gynecological tumors,
enabling the
induction of a cellular immune response to the antigen. T lymphocytes assayed
directly from
the serum of an acute PCD patient, as well as dendritic cell-stimulated memory
T cells present
in patients with chronic PCD, demonstrated cdr2-specific cytotoxicity.
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The PCD antigen has been identified as cdr2, a protein expressed in neuronal
cells and in
gynecological tumors (9, 54). An investigation by the present inventors into
the processing
of the cdr2 antigen for presentation by APCs to T lymphocytes has led to the
identification of
polypeptide fragments of cdr2 which are targets for naturally-occurring CTLs
in PCD patients.
These peptides are believed to be those presented by dendritic cells in the
development of
cellular immunity. Eight peptides, namely cdr2-1 or Yol (SEQ ID NO:1), cdr2-2
or Yo2
(SEQ ID N0:2), cdr2-3 or Yo3 (SEQ ID N0:3), cdr2-4 or Yo4 (SEQ ID N0:4), cdr2-
5 or
Yo5 (SEQ ID NO:S), and cdr2-6 or Yo6 (SEQ ID N0:6), cdr2-7 or Yo7 (SEQ ID
N0:7)
and cdr2-8 or Yo8 (SEQ ID N0:8) have been synthetically prepared, and are used
diagnostically and therapeutically in the practice of the present invention.
Furthermore, and
as will be described below in detail, engineering of cells to express these
peptides has
additional diagnostic and therapeutic utilities in the detection and treatment
of cancer and
paraneoplastic diseases.
In several other diseases, cellular immunity against antigens expressed by a
tumor is
responsible or presumed responsible for the attack of non-tumor cells
expressing the same
antigens. As described above, these syndromes may be subacute or acute,
causing serious
complications. Hu -antigen is expressed by small cell lung tumors; Hu syndrome
is another
example of a neurological disease brought about by a cellular immune response
to an immune-
privileged antigen. The identification by the inventors herein of the role of
cellular immunity
in the etiology of these diseases provides the link between expression of non-
tumor antigens
at the affected site of the paraneoplastic syndrome and the expression of the
antigen in the
tumor.
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As described above, the presence of cdr2-specific CTLs on a wide range of
gynecological
tumors suggests that breast or ovarian tumor cells expressing cdr2 are
responsible for initiating
PCD. However, the detection of cdr2 in a high percentage of non-PCD-associated
tumors
indicates that there are additional factors responsible for successful tumor
immunity. Relevant
factors include tumor cell expression of MHC-I (demonstrated in PND-associated
tumors;_ref
18) and the proximity of dendritic cells (DCs) to apoptotic tumors that may be
necessary for
cross-priming (19). It has been reported that 15 % of patients with non-PND
associated small
cell lung cancer harbor low titers of the PND Hu antibody, and that this
antibody response
predicts limited tumor spread and a complete response to chemotherapy (5,20).
The invention
described herein is extended to the presence of Hu-specific CTLs in such
patients. Similarly,
it is expected that a significant percentage of patients with non-PND-
associated gynecological
tumors expressing cdr2 may be amenable to diagnosis and therapy by particular
embodiments
of this invention whereby the presence of cdr2-specific cytotoxic T
lymphocytes are detected
and their activity modulated in these patients. The present invention includes
immune
privileged antigens generally, not limited to those described herein in
addition to Nova, ~3-
NAP, etc.) and are taught to be relevant by this invention.
Thus, it is towards the detection and the modulation of the T lymphocyte
response to immune
privileged antigens that the present invention is directed. Enhancement of the
immune
response increases the effectiveness of antitumor activity. Suppression of the
immune response
alleviates the paraneoplastic or autoimmune disease. The diseases and
syndromes that arise
as a result of a cellular immune response to privileged antigens include, but
are not limited to,
paraneoplastic neuronal degeneration, paraneoplastic cerebellar degeneration,
Hu syndrome,
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the Ri syndrome (opsoclonus-myoclonus ataxia associated with breast, fallopian
tube and small
cell lung cancer, and the Ri or Nova antigen), opsoclonus and myoclonus
associated with
neuroblastoma, vitiligo, myasthenia gravis, subacute motor neuropathy,
subacute necrotic
myelopathy, polyneuropathy, Eaton-Lambert syndrome, dermatomyositis and
polymyositis.
These conditions appear to be directly or indirectly related to neoplasia in
the patient, either
undetectable, overt, or as a result of a tumor which spontaneously regressed.
In some cases,
the identity of the antigen is not yet elucidated, but the course of the
disease and its relationship
with neoplasia and the other, better-studied diseases indicates a role for an
as-yet identified
immune-privileged antigen in the etiology of the disease. Furthermore, it is
suspected that the
above-mentioned diseases as well as various autoimmune disease may in fact
arise from a
cellular immune response to a neoplasm which was effectively eradicated by the
immune
response, but results in T lymphocytes attacking privileged antigen and
evoking a syndrome
far after the neoplasm is eradicated. The diagnostic and therapeutic methods
of the present
invention directed to the paraneoplastic diseases will also find utility in
the diagnosis and
therapy of the other immune-privileged antigen-related diseases, including
autoimmune
diseases.
In one embodiment of the present invention, quantitation of immune-privileged
antigen-specifiE
cytotoxic and memory T lymphocytes is performed in order to identify the
presence and extent
of a tumor or to confirm the diagnosis of a paraneoplastic or other syndrome,
such as an
autoimmune disorder, e.g. vitiligo. This test is performed as a routine assay
such as a
screening test or for individuals undergoing physical examination. It may also
be performed
in individuals suspected of having a neoplasm or a disease related to a
cellular immune
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response to an immune-privileged antigen. In order to carry out the test, a
sample of blood or
other appropriate bodily fluid containing lymphocytes is obtained, such as
cerebrospinal fluid
(CSF).
In order to determine the presence of cytotoxic T lymphocytes that are
specific for a particular
immune-privileged antigen, any one of several types of assays is performed,
all of which are
known to one or ordinary skill in the art. By way of non-limiting examples, an
assay is
performed which identifies the presence of T lymphocyte receptors that
recognize the immune-
privileged antigen, i.e., peptide fragments of the antigen complexed in an HLA
tetramer (51).
In this assay, a specific antibody reagent is prepared that recognizes
peptides of the immune-
privileged antigen complexed with HLA; this reagent is used to detect T
lymphocytes
expressing the particular receptor. The reagent must be specific for the
particular HLA
haplotype of the patient. In a routine screening test, various combinations of
immune-
privileged antigens and HLA haplotypes is sought. Detection is achieved using
any of a
number of means, for example, with a fluorescent labeled reagent using
fluorescence-activated
cell sorting (FACS) techniques, or by using a detectable label such as a
radioactive or
enzymatic tag and quantitating the binding of the reagent to T lymphocytes in
the sample by
standard techniques. These various methods are provided by way of non-limiting
examples
to illustrate the practice of the invention, based upon the detection of CTLs
that recognize
immune-privileged antigens.
In another example of the means for detecting immune-privileged antigen-
specific CTLs, the
ability of such CTLs to lyse target cells expressing the immune-privileged
antigen or a peptide
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thereof in the context of HLA is employed. Lysis of such cells by CTLs is
detected by
methods known to the skilled artisan. For preparation of the target cells
expressing the
appropriate HLA haplotype HLA-antigen peptide, any one of several methods is
used. For
example, the target cell may be one that expresses HLA, such as the cell line
T2 (TAP'- HLA-
A2.1 +), and will incorporate peptides into the HLA complex. The cells are
pulsed with
immune-privileged antigen peptides such as the cdr2 peptides described herein,
and
subsequently used as targets to detect specific CTLs. To detect specific
recognition of the
target cells by CTLs, lysis of the target cells is determined. For example,
the target cells is
preloaded with a marker such as Nas'Cr04; lysis of the target cells results in
the release of the
label. Alternatively, lysis is assayed by the release of other intracellular
markers such as
intracellular enzymes, e.g., lactate dehydrogenase. These various methods are
known to one
of ordinary skill in the art. In a particular embodiment of the aforementioned
method, the
following steps are performed:
i) obtaining a sample of a bodily fluid;
ii) isolating T lymphocytes from the sample;
iii) preparing a sample of target cells bearing on the cell surface the immune-
privileged antigen or a fragment thereof in the context of HLA;
iv) incubating the isolated T lymphocytes with the target cells; -
v) quantitating the viability of the target cells; and
iv) correlating the viability of said target cells with the presence of immune-
privileged antigen-specific cytotoxic T lymphocytes in the sample.
In a further and preferred embodiment, the identification of CTLs specific for
an immune-
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privileged antigen is readily determined by incubating T lymphocytes with the
above-described
target cells, and subsequently detecting the release of specific mediators
from the CTLs
indicative of the specific recognition and subsequent activation. CTLs
encountering the antigen
to which they are targeted are known to release y-interferon and other
cytokines including
TNF-a, RANTES, MIP-la and other chemokines, as well as to lyse target cells
bearing the
antigen. As an example of the practice of this preferred method, the following
steps are
carried out:
i} obtaining a sample of bodily fluid from an individual which contains T
lymphocytes;
ii) optionally isolating T lymphocytes from the sample;
iii) exposing the body fluid sample or the isolated T lymphocytes to the
immune-
privileged antigen or fragment in the context of HLA;
iv) quantitating the level of a mediator produced by the T lymphocytes.
Cells expressing particular immune privileged antigens useful for the practice
of these
embodiment of the present invention include but is not limited to cells which
naturally express
immune privileged antigens, such as cdr2 expressed by HeLa cells; cells
transfected with a
gene which results in expression of the desired antigen, such as Drosophila
cells; othet_
examples are known to one of ordinary skill in the art. As described above,
such transfected
calls may additionally be engineered to express molecules of a particular HLA
haplotype, and
in addition may express co-stimulatory molecules. These cells may thus
function as target cells
which express the antigen in the context of HLA molecules, useful for the
identification of
immune-privileged antigen-specific T lymphocytes. A series of such cells may
be prepared,
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each expressing a different HLA hapiotype, for use in screening.
The level of y-interferon or other mediators produced is directly related to
the numbers of T
lymphocytes specific for the immune-privileged antigen or fragment thereof
present in the
sample. This value is then used to identify a patient in which a tumor is or
had been present,
S and the possibility of the development of a paraneoplastic syndrome or other
disorder
characterized by the presence of immune-privileged antigen-specific CTLs.
Other methods
for detecting antigen-specific CTLs are applicable to the practice of the
present invention as
adapted for measuring CTLs specific immune-privileged antigens. Examples
provided to
illustrate the invention are not intended to be limiting. For example, methods
as described
above to directly identify CTLs against immune-privileged antigens include
detecting on the
surface of T lymphocytes receptors capable of detecting tetramers comprising
HLA molecule
and immune-privileged antigen peptides. The selection of the immune-privileged
antigen or
its fragments for the assays of the present invention may be general or
specific to the particular
syndrome to be detected. For general screening, for example for cancer, a test
comprises a
mixture of the various known paraneoplastic antigens or fragments. After
identifying a patient
as having specific CTLs against a mixture of antigens, further screening is
carried out to
pinpoint the particular antigen. Such screening and further identification may
then be used to_
direct the future course of therapy for the patient, for example, therapies to
increase the CTLs
against the particular tumor, and to reduce the severity of the paraneoplastic
syndrome by
suppressing the CTLs in non-tumor sites within the body; these therapeutic
utilities are
described in further detail below.
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It has also been found by the inventors herein that memory T cells specific
for the
paraneoplastic antigen are present in individuals with paraneoplastic
syndrome. In order to
screen for or detect the presence and extent of memory T cells in a patient
sample, suspected
memory T cells must be exposed to APCs .presenting the immune-privileged
antigen.
Detection of the resulting activated T lymphocytes is quantitated in a similar
fashion to the
detection of CTLs directly in a patient sample as described above. As a
general example of
the method, the assay is carried out by following sups:
I) obtaining a sample of bodily fluid containing T lymphocytes;
ii) optionally isolating T lymphocytes from the sample of bodily fluid;
iii) preparing differentiated APCs that have processed and are presenting the
immune-privileged antigen;
iv) co-incubating the immune-privileged antigen-presenting APCs with the
sample
or the isolated T lymphocytes;
v) ~ measuring immune-privileged antigen-specific T lymphocytes.
Measuring the immune-privileged antigen-specific T lymphocytes is accomplished
by any one
of a number of methods known in the art. For example, expression of receptors
recognizing
the HLA-peptide tetramer may be measured, or the extent of secretion of
mediators from the
T lymphocytes may be determined; alternatively, the cytolytic activity of the
T lymphocytes
towards target cells expressing the immune-privileged antigen in the context
of HLA may be
detected.
By way of non-limiting examples, the APCs may be dendritic cells, macrophages,
B cells,
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microglial cells, fibrocytes, engineered cells containing MHC and secreting co-
stimulatory
molecules, among others. Various known methods are used to prepare the target
cells
expressing the desired peptide; for example, it may be achieved by delivering
antigen through
apoptotic cells which express the antigen or a peptide fragment, by use of
heat shock proteins
which direct proteins to the MHC, and the direct pulsing of the cells with
protein or peptides.
These examples are merely illustrative of examples of the practice of the
present invention and
are not intended to be limiting.
In the specific example wherein mediators such as y-interferon production is
used as the read-
out of the assay, its level will be directly related to the numbers of memory
T lymphocytes in
the patient sample, and thus is correlated with the presence and extent of the
neoplasm in said
individual or the prior presence of a neoplasm. Cytolysis and quantitation of
specific receptors
also provides similar data.
In another aspect of the invention, an assay is provided for the detection of
cdr2-specific T
cells. This assay is rapid and offers the ability to screen large numbers of
patient samples for
I 5 the presence of cdr2-specific T cells in the form of a kit. The steps of
ahis method are as
follows:
i) obtaining a sample of blood;
ii) maturing dendritic cells in the blood sample;
iii) exposing the matured dendritic cells to apoptotic debris from unrelated
cells
expressing an immune-privileged antigen;
iv) co-incubating the immune-privileged antigen-exposed dendritic cells with
the
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patient's peripheral blood lymphocytes; and
v) measuring interferon-y released from the lymphocytes as a measure of
stimulation.
The number of T cells stimulated by the immune privileged antigen-fed
dendritic cells is an
indication of the number of immune privileged antigen-specific T cells in the
patient's
peripheral blood. The apoptotic debris to which the dendritic cells are
exposed may be, by
way of non-limiting example, apoptotic, transfected cells expressing an immune
privileged
antigen such as cdr2. A negative. control may be used in the assay, for
example, the same cell
line but not expressing the antigen.
As an example of the practice of the above procedure, peripheral blood is
obtained from a
patient and dendritic cells were matured as described in Example 1. These
cells were then fed
with apoptotic debris from unrelated (mouse) cells that do or do not express
the cdr2 protein.
Such cells expressing cdr2 protein may be prepared for example, as described
in Example 6
below, such as PC7 or EC2 cells, by stably transfecting EL4 cells with pcDNA-
cdr2, and
determination of protein expression made by Western blot analysis. These fed
DCs are then
incubated with patient's peripheral blood lymphocytes, and interferon gamma
(IFN-y) release -
measured as an index of stimulation. Although IFN-y release may be measured in
any one of
a number of ways, the standard ELISPOT assay provides a rapid method. For
example, 105
T cells are placed in a 96-well plate, previously coated with a monoclonal
antibody specific for
IFN-y, and incubated with 104 -irradiated stimulator cells (such as EC2 or
EL4). After 20
hours, the cells are washed out and IFN-y spot forming cells (SFCs) are
detected using a
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biotinylated anti-IFN-y antibody, and an HRP-AEC (3-amino-9-ethyl-carbazole}
staining
procedure. SFCs reported per million cells.
In this example, nitrocellulose-bottom wells are plated with antibody to IFN-
y, release allowed
to occur for 20 hours, plates are washed, and a second anti-IFN-y antibody
is.added which was
conjugated to biotin to allow colorimetric detection. The number of spots
secreting IFN-y
directly correspond to the number of T cells in the assay that are stimulated
by the DCs. By
comparing the number of T cells stimulated by EL4-fed DCs (negative control)
with the
number stimulated by EC2-fed DCs, the number of cdr2-specific T cells in a
patient's
peripheral blood can be determined.
There are several advantages of this assay. First, there is no HLA restriction
required as there
is for CTL assays in which T2 (HLA A2.1) targets are killed, so that patients
of any MHC
haplotype can be assayed by this method. Second, the assay is relatively
simple, can be
performed from a peripheral blood draw, and can be performed by automated
ELISPOT robots
and readers. Third, although current methods allow DCs to be grown from a
single 50 ml
1 S peripheral blood draw, this assay can ultimately be done without DCs as
antigen presenting
cells (APCs), using peripheral blood monocytes, which as a mixed cell
population, have -
sufficient APCs to allow T cell stimulation.
Furthermore, the above-described assays are useful to determine whether a
patient with a
immune-privileged antigen-expressing tumor has a sufficient population of
antigen-specific T
lymphocytes to control the tumor or is a candidate for anti-cancer therapy, by
quantitating T
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lymphocytes specific for the particular antigen. In addition, the method can
be used for
monitoring the effectiveness of therapies intended to modulate the population
of antigen-
specific T lymphocytes in a patient, i.e., increase the population for anti-
cancer therapy, and
decrease the population for protection of non-tumor sites and alleviation of
for example the
paraneoplastic syndrome, by measuring the numbers of cytotoxic and memory T
lymphocytes
in accordance with the methods described above. The assay readout can be
compared to pre-
established standards and ranges to enable the health care professional to
direct the appropriate
course of therapy based on assay results.
As a consequence of the discovery of the role of CTLs in paraneoplastic
syndromes, the
inventors herein have identified a further diagnostic modality to monitor the
progression or
predict the potential success of immune-privileged antigen CTL-based therapies
as described
hereinabove, and to determine the propensity for the development of the
syndrome as a
consequence of the participation of cytokines. Cytokines are known to promote
the expression
of cytoplasmic antigen via MHC-I molecules (21,22), and thus increased levels
of cytokines
may enhance the effectiveness of the killing of tumor cells by the therapeutic
methods of the
present invention. Conversely, cytokines present in pathological states, such
as neoplasia or
a paraneoplastic syndrome, can induce, accelerate or exacerbate the disease
process by
promoting the expression of antigens on non-tumor cells. This may also help
explain the still
poorly understood phenomenon of how immune-privileged antigen-specific CTLs
which arise
from the expression of the antigen on tumor cells, are able to first gain
access to normally
immune privileged body sites such as the central nervous system, perhaps
through a cytokine-
mediated weakening of the endothelium barrier, and secondly, to recognize and
attack neuronal
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and other cells in which the antigen is normally cytoplasmic and not expressed
on the surface.
Measurement of cytokine levels in contact with the paraneoplastic syndrome-
affected tissue is
thus useful in assessing the severity or potential severity of the disease.
Diagnostic kits are embodied by the present invention which provide particular
immune-
privileged antigens or fragments for the use in detecting antigen-specific T
lymphocytes in a
sample of bodily fluid, in accordance with the various methods described
above. For instance,
immune-privileged antigen peptides, such as by way of non-limiting example,
the several
previously-described peptides of cdr2 predicted to bind to the binding domain
of HLA, may
be included in a kit for the preparation of target cells bearing antigenic
peptides in the context
of HLA. Another component of a kit comprises a labeled or tetrameric complex
containing
the HLA molecule and peptides, for direct detection of CTLs with receptors
specific for the
complex. Kit componentry will be specific to the type of assay to be performed
and the type
or types of immune privileged antigens to be detected.
As a result of the discovery by the inventors herein of the role of T
lymphocytes in both the
progression of paraneoplastic syndromes and in tumor inununity, several
therapeutic modalities
are provided that are directed to the enhancement of the immune response to
the tumor, and
the suppression of the immune response at the site of the affected non-tumor
tissues. These
therapeutic modalities are generally directed to either the enhancement or the
diminution of the
cellular immune response to privileged antigens, and has utility in the
treatment of cancer,
paraneoplastic syndromes and in other diseases in which an inappropriate
immune response to
immune-privileged antigens results in pathology, for example, autoimmune
diseases.
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Depending on the patient's condition, the extent of application of either of
these therapies may
be appropriate; ideally, these may be applied simultaneously for optimizing
the anti-cancer
benefits of cellular immunity while protecting non-tumor cells from immune
destruction. The
effectiveness of these therapies disclosed as a consequence of the present
invention is enhanced
by concurrently providing other known means to increase the effectiveness of
cellular
immunity .
The therapeutic regimen of enhancing killing of tumor cells in a patient by
increasing the
number or activation state of immune privileged antigen-specific cytotoxic T
cells in a patient
is supported experimentally by the ability to develop an animal model wherein
such cytotoxic
T cells are elevated, yet the animal is not neurologically compromised. As
will be shown in
more detail in Example 5 below, this has been achieved using mice that were
immunized using
an apoptotic tumor cell, which results in the generation of potent cdr2-
specific killer T cells.
The apoptotic tumor cell was a cell line (PC7) generated by stably
transfecting EL4 cells (a T
cell lymphoma) with pcDNA-cdr2, and determination of protein expression made
by Western
blot analysis. Mice were immunized with irradiated PC7 cells, followed by
harvesting the
spleens of primed and naive littermates. Mixed lymphocyte / tumor cell
cultures were
established using MHC-matched target cells, EC2 cells, or TIB84 cells for
purposes of
restimulating primed T cells. After 5 days, responding T cells were collected
and tested at
various ratios in a standard chromium release assay. Alternatively, CD8+ T
cells were purified
from the spleens of primed and naive mice using the MACS cell isolation
system. Briefly, anti-
CD8 antibody coupled to iron conjugated microbeads are incubated with
splenocytes and CD8+
T cells are positively selected by passing the cells through a magnetic
column. The positively
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selected CD8+ T cells were used directly in an ELISPOT assay.
The results of the experiment showed specificity as demonstrated by the
ability of T
lymphocytes to lyse MHC-matched target cells expressing cdr2, EC2 cells, but
not MHC-
matched cells that lack cdr2 expression. As the PC7 cells are MHC-mismatched
with respect
to the C57/B6 mouse that was immunized, it is believed that this model system
is analogous
to the cross-priming of tumor cells evident in patients with PCD.
In addition to the killing assay, IFN-'y release was also demonstrated from T
cells purified
from PC7 immunized mice. This short term assay confirmed that high levels of
CTL
precursors exist in the immunized mice. As no immunized mice exhibited signs
of neurologic
dysfunction, these data indicate the ability to separate tumor immunity from
the autoimmune
neurodegeneration. As described above, cdr2-specific killer T cells have been
identified in
patients with effective tumor suppression and PCD. In addition, significant
numbers of breast
and ovarian tumors present in neurologically normal patients express the cdr2
target antigen.
Therefore, the present study demonstrates in this model that stimulation of T
cells able to kill
cdr2-expressing tumor cells is possible without inducing autoimmune neurologic
disease.
In a first embodiment, enhanced anti-tumor therapy is provided to a patient
with a neoplasm
expressing an immune-privileged antigen by increasing the number of immune-
privileged
antigen-specific cytotoxic T lymphocytes in the patient. The lymphocytes to be
stimulated may
either be the patient's own cells, stimulated in vivo or ex vivo, or they may
be HLA-matched
cells from another source, as will be described below. This increase can be
effected by
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exposing APCs such as dendritic cells isolated from the patient ex vivo to one
or more peptides
of the particular antigen, or by other known means, whereby the antigen will
be available for
presentation to T lymphocytes. The antigen-exposed APCs are then reintroduced
into the
patient to stimulate the activation of specific T lymphocytes in vivo, or, the
APCs, for
example, dendritic cells, may be further exposed in vitro to T lymphocytes
.isolated from the
patient, whereby presentation to T lymphocytes will induce the activation of
antigen-specific
CTLs. T lymphocytes or dendritic cells are reintroduced into the patient,
wherein the CTLs
will promote anti-tumor activity, and dendritic cells will stimulate
additional CTLs in vivo.
For example, to practice the first method, the following steps are followed:
i) isolating a quantity of dendritic cells from a sample of patient's blood;
ii) exposing the dendritic cells in vitro to the immune-privileged antigen
fragment;
iii) reintroducing the antigen-exposed dendritic cells to the patient.
The aforementioned ex-vivo therapies may be achieved by any one of several,
non-limiting
methods, known to the skilled artisan. For example, memory T lymphocytes may
be activated
1 S ex vivo by exposure to dendritic cells presenting the desired immune
privileged antigen. In
another non-limiting example, immune-privileged antigen-specific T lymphocytes
may be
isolated using the HLA-peptide tetramer as described above, and then expanded
with cytokines, -
e.g., IL-2, or in the presence of dendritic cells, before reintroduction to
the patient. Bulk T
cells isolated from the patient may be exposed to dendritic cells presenting
the antigen; then
the activated, antigen-specific cytotoxic T lymphocytes may then be
reintroduced to the patient.
As mentioned above, these methods may be enhanced by concurrent therapies
which increase
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the effectiveness of T lymphocyte killing. Non-limiting examples include
increasing cytokine
levels, inhibiting the expression of Fas-ligand expression (44) on tumor cells
to block
apoptosis, inducing the expression of MHC I molecules on the tumor using, for
example, Nef
inhibitor or Nef-like protein(41), radiation therapy, tumor chemotherapy, Bax
induction in the
tumor (52) and inducing apoptosis of tumor cells using FLIP inhibitors (49).
In a further embodiment of the above example, to achieve an object of the
present invention
in enhancing cellular immune-based therapy to cancer patients in those at risk
for the
development of, or exhibiting, a PND, the above-described T lymphocytes and/or
dendritic
cells may be engineered to be sensitive to a drug such as gancyclovir by
methods known to the
I0 skilled artisan (53). After introduction of the immune cells to the
patient, any induction or
exacerbation of a PND may be controlled by suppressing the introduced immune
cell
population by administration of the drug to which the cells are sensitive.
In the practice of the above method, certain immune-privileged antigens may
not be adequately
taken up by dendritic cells for presentation on the cell surface, nor will
exposure of the
dendritic cells to the intact antigen or its peptides allow for processing and
presentation. In a
further embodiment, the antigen is provided in a form which can be readily
processed and
presented. Among various known means for increasing antigen presentation by
poorly
immunogenic or poorly processed antigens, use of apoptotic cells expressing
the desired
antigen to deliver antigen to dendritic cells (I7), in addition to other known
means such as the
use of viral vectors, naked and plasmid DNA, RNA, liposomes with nucleic acid
to thereby
transfect dendritic cells (50) have been described. In a preferred embodiment,
delivery is
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achieved using cells which already express the desired antigen; for example,
HeLa cells, which
are useful in the above-described methods for the treatment of PND because
they express the
cdr2 antigen. These immune-privileged antigen-expressing cells are induced to
become
apoptotic before exposure to the APCs.
In another embodiment of the invention, a method of treatment of a patient
with a
paraneopiastic syndrome or other inappropriate cellular immune response to an
immune
privileged antigen is provided wherein suppression of cellular immunity is
desired to intervene
in the attack of non-tumor cells by antigen-specific CTLs. Such methods of
treatment are
targeted at decreasing or suppressing the cellular immune response against the
specific
immune-privileged antigen. Agents useful for this method of treatment include,
but are not
limited to, immunosuppressive agents such as tacrolimus, cyclosporin,
corticosteroids, and
azathioprine, which have been shown to eliminate CTLs. These agents are useful
for the
treatment of paraneoplastic syndromes as described herein, whereby CTLs
targeted against
immune-privileged antigens are eliminated. In a further embodiment,
suppression of the attack
of CTLs on immune privileged sites such as the brain is achieved by sealing
the blood-brain
barrier. This may be accomplished by the use of various agents known in the
art, for example,
corticosteroids. Protection of the brain to maintain immune privilege also may
be achieved by_
upregulating Fas ligand expression (40).
Administration of an agent to suppress the cellular immune response against an
tmmune-
privileged antigen is directed to the body in general or to specific locations
for increased
effectiveness, for example, in the case of paraneoplastic syndromes, to the
central nervous
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system, by intracranial or intrathecal administration. Such agents may be
administered by
parenteral injection, or for oral, pulmonary, nasal or other forms of
administration.
Appropriate dosage levels for treatment of the various conditions in various
patients will be
ascertainable by the ordinary skilled worker, considering the therapeutic
context, age and
general health of the recipient.
As a consequence of the discovery of the role of CTLs in paraneoplastic
syndromes, the
inventors herein have identified a further therapeutic modality to intervene
in the development
or progression of the syndrome by limiting the expression of the immune-
privileged antigen
by non-tumor cells. This is achieved by reducing the level of cytokines in
contact with the
non-tumor cells, as it is known that cytokines will promote the expression of
endogenous
antigen via MHC-I molecules (21,22). Treatment with inhibitors of cytokine
production, such
as corticosteroids, or anti-cytokine agents such as anti-cytokine antibodies,
are provided as
non-limiting examples. In the example of PND wherein CTLs attack neuronal
cells in the
central nervous system, anti-cytokine therapy delivered to the CNS is provided
by means such
as intrathecal administration as described above.
In a further embodiment, a method is provided for decreasing the recognition
of non-tumor
cells expressing paraneoplastic antigens by paraneoplastic antigen-specific T
lymphocytes by
contacting non-tumor cells with an agent which interferes with the recognition
of
paraneoplastic antigen by lymphocytes. Such agents include those which
suppress the
expression of MHC, and other agents which suppress antigen presentation, such
as those which
may switch a Thl response to a Th2 response.
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In a still further embodiment, means to decrease the sensitivity of non-tumor
cells to CTLs are
known and may be used in conjunction with the methods and agents of the
present invention.
By way of non-limiting examples; perforin-mediated CTL killing may be
inhibited (43);
apoptosis may be inhibited for example, by inhibitors of FLIP (39); reducing
Bax expression
in neurons (45); cytokines that promote the immune-privileged state may be
administered,
such as IL-10 and TCF-~3 (46); and MHC I expression may be decreased (41).
As mentioned above, an optimal treatment regimen for a patient in need of a
cellular immune-
based anti-cancer therapy provides both the enhancement of the anti-cancer
therapy and
protects the non-tumor tissues and cells from the therapy. Thus, treatment to
stimulate or
expand the CTL population while protecting the non-tumor cells from attack by
the CTLs is
desirable. Since CTLs normally pass very minimally into the CSF, no additional
therapeutic
intervention may be needed; if additional measures are desirable, this can be
achieved, for
example, by administering to the patient an anti-cytokine agent as described
above prior to the
introduction of CTLs, or limiting the effective period of CTL therapy by
providing a
I S population of CTLs which may be specifically inactivated as described
above. By way of non-
limiting examples, immortalized, immune-privileged antigen-specific CTLs can
be prepared
from an immortalized cell line of the same HLA type as the patient. The
antigen-specific
CTLs can be expanded in vitro and introduced into the patient. In order to
control the
population of these cells within the patient and to avert the potential attack
by these cells of
non-tumor cells, the cells can be engineered to be sensitive to a certain
drug, such as
gancyclovir (53). In patients with tumors who otherwise are neurologically
normal, such
control may be unnecessary. Alternatively, in an example of an course of
therapy using such
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cells for a patient with an existing PND, the patient can be first treated
with an anti-cytokine
agent and a blood brain sealing agent to reduce expression of the privileged
antigen on non-
tumor cells and to restrict access of the CTLs to the brain, respectively.
Other means for
protecting the brain include increasing FAS ligand expression in the brain
(40), decreasing Bax
expression in neurons (45), and decreasing cytokine levels in the brain (46).
A gancyclov_ir-
sensitive (53), immune-privileged antigen-specific, immortalized CTL line may
then be
introduced, and allowed to attack the tumor. The patient is closely monitored
for the
appearance of or the exacerbation of the paraneoplastic syndrome, which, if it
begins to occur,
the patient is administered gancyclovir to suppress the therapy partially or
completely. This
cycle may be repeated as necessary the effect the destruction of the neoplasm.
As it is expected
that most immune-privileged tumor patients do not have PND, this method is
preferred in
monitoring such patients.
As described above, the inventors herein have identified certain peptide
fragments of
paraneoplastic antigens, such as the cdr2-1 (SEQ ID NO:1) and cdr2-2 (SEQ ID
N0:2)
fragments of cdr2, which are believed to be the natively processed cdr2
peptides to which
CTLs are targeted. As such, these peptides have utility in the diagnostic
methods provided
herein for identifying antigen-specific T lymphocytes, as well as therapeutic
utilities in
producing dendritic cells and other APCs presenting specific peptides.
Based on the above-described therapeutic utilities of the present invention,
additional
embodiments comprise kits for carrying out one or more of the therapeutic
modalities described
herein. In one embodiment, a kit for stimulating the production of T
lymphocytes specific for
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immune privileged antigens comprises cells which express an immune-privileged
antigen as
well as MHC molecules which match that of the patient to be treated; the cells
may further
express co-stimulatory molecules. These cells may be derived from a cell line,
such as a
Drosophila cell line. The cells may be used for the in-vivo or ex-vivo
stimulation of T
lymphocytes. The kit may further comprise T lymphocytes from donors with the
same HLA
haplotype as the patient, in order to participate in the further stimulation
of a cellular immune
response to a tumor.
The present invention may be better understood by reference to the following
non-limiting
Examples, which are provided as exemplary of the invention. The following
examples are
presented in order to more fully illustrate the preferred embodiments of the
invention. They
should in no way be construed, however, as limiting the broad scope of the
invention.
General Methods
Peripheral blood was obtained from HLA A2.1 + PCD patients and normal donors
in
heparinized syringes or by leukapheresis. PBMCs were isolated using Ficoll-
Hypaque
(Pharmacia Biotech). T cell enriched (ER+) and T cell depleted (ER- )
populations were_
prepared by rosetting with neuraminidase-treated sheep red blood cells as
previously described
(14). T cells were further purified from ER+ cells for the CTL recall assays
by removing
monocytes, natural killer (NK) cells, and B cells as described (14). DCs were
generated from
peripheral blood precursors by culturing ER- cells for 7 days in the presence
of GM-CSF
(Immunex Corp.) and IL-4 (Schering-Plough Corp.), followed by 4 days in
monocyte
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conditioned medium (15).
Activated CTLs were detected using T-cells as effector cells in a conventional
Na5'Cr04 release
assay directly after purification. T2 cells (a TAP-/-, HLA-A2.1 +, class II-
cell line) were
pulsed for 1 hr with 1 mM of various peptides, loaded with Nas'Cr04 and used
as targets
(14). Alternatively, memory CTL responses were stimulated using DCs pulsed for
4 hours with
1 mM of various peptides. After 7 days, responding T cells were assayed for
cytolytic activity.
Again, T2 cells pulsed with peptide served as targets. Specific iysis was
determined by
subtracting the percent cytotoxicity of unpulsed T2 cells (0 - 3 %). PCD
peptides, designated
cdr2-1 (KLVPDSLYV) (SEQ ID NO:1) and cdr2-2 (SLLEEMFLT) (SEQ ID N0:2), were
predicted based on anchor residues for HLA A2.1 and synthesized (Biosysnthesis
Inc). Six
other peptides derived from cdr2 were tested (data not shown). The control for
these
experiments included the use of the immunodominant influenza matrix peptide,
GILGFVFTL
(SEQ ID N0:9)
Monoclonal antibodies (MoAbs) to the following antigens were used: CD19, CD56,
CD3,
CD4, CDB, a~iTCR, CD25, IFNy, TNFa, IL2, IL4, CD14, HLA-DR (Becton Dickinson);
CD83 (Coulter Corp.). Cell populations from the peripheral blood and spinal
fluid were _.
phenotyped with a panel of MoAbs listed above and analyzed on a FACScan
(Becton
Dickinson). Additionally, the DCs prepared from the patients were assayed for
phenotypic
markers (CD14- CD83+ HLA-DR+). Dead cells and contaminating red blood cells
were
excluded by forward and side scatter properties. Intracellular cytokine
profiles were assessed
using a dual laser fluorocytometer (Becton Dickinson). Cells were treated with
BFA, an
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inhibitor of secretion, followed by cell fixation and permeabilization, and
then intracytoplasmic
staining of accumulated cytokines (23). As a control, PBMCs were treated with
BFA and
cytokine production was stimulated using phorbol 12-myristate 13-acetate [PMA)
and
ionomycin [I] {23) . T helper cells were delineated by a CD3 + CD4 + phenotype
and levels
of IFNy, TNFa, IL2 and IL-4 were determin;.d.
Example 1 Identification of cdr2-specific Cytotoxic and Memory T Lymphocytes
in
Patients with Paraneoplastic Cerebellar Degeneration
Three PCD patients were studied to explore the nature of the immune response
in the serum
and spinal fluid. Ail patients had HLA-A2.1 + phenotypes. One (Patient 1) had
acute disease
and two (Patients 2 and 3) had chronic disease (seen 18 days, 9 months, and 6
months,
respectively, after the onset of cerebellar dysfunction). The diagnosis of PCD
was confirmed
in each patient by demonstrating the presence of high titer cdr2 antibodies
reactive with cloned
fusion protein (Figure 1), and peripheral blood lymphocytes were obtained for
cellular immune
assays. The possibility of that CD8+ CTLs are involved in tumor immunity in
PCD was
investigated using peptide epitopes derived from cdr2 in a standard chromium
release assay-
Target cells were T2 cells, a TAP-r~ HLA-A2.1 + cell line pulsed with cdr2
peptides (predicted
for HLA-A2.1 based on determined anchor residues) and loaded with Nas'CrO~.
Effector T
cells were obtained from peripheral blood (14) and incubated directly with
targets at various
effector : target ratios. In patient 1, cdr2-specific CTLs were detected
showing specificity for
the cdr2-2 and, to a lesser extent, cdr2-1 peptides (Figure 2A). This response
was titratable and
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specific for acute PCD, as no response was detected in an HLA-A2.1 + normal
control (Figure
2A) or in either patient with chronic PCD (data not shown).
In order to examine whether memory T cells were present in the peripheral
blood of PCD
patients, an in vitro recall assay was established. Patients were
leukapheresed, providing a
source of peripheral blood mononuclear cells (PBMCs). Terminally
differentiated dendritic
cells (DCs) were prepared by culturing a T cell depleted mononuclear fraction
for 7 days in
granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4
(IL 4),
followed by 4 days in monocyte conditioned medium (15). The DCs generated had
a typical
stellate morphology, were nonadherent, expressed characteristic maturation
markers (i.e.
CD83), and had potent T cell-stimulating capacity in mixed leukocyte reactions
at stimulator
to responder ratios of 300:1 or less (data not shown). These blood derived DCs
were pulsed
with eight different cdr2 peptides and co-cultured with purified syngeneic T
cells (14). After
7 days, responding T cells were tested for cytolytic activity specific for
cdr2 epitopes using
peptide pulsed T2 cells as targets. In patients 2 and 3 (with chronic PCD),
cdr2-specific CTLs
were detected (Figure 2B) using the cdr2-1 and cdr2-2 peptides. This CTL
activity was not
detected in 4 control individuals or in patient 1 (with acute PCD; Figure 2B
and data not
shown). As a control for these experiments, CTL responses specific for the
immunodominant
HLA-A2.1 epitope derived from the influenza matrix protein were determined
(data not
shown) .
Example 2 Detection of Activated Cytotoxic T Lymphocytes in the Cerebrospinal
Fluid
of a Patient with Paraneoplastic Cerebellar Degeneration; Treatment with
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Tacrolimus w
Cerebrospinal fluid (CSF) analysis revealed that Patient 1 had a CSF
pleiocytosis when first
seen (99 WBC/mm3). This enabled an evaluation of the autoimmune neurologic
component
of the patient's disorder by directly analyzing the CSF cells with
cytofluorography and
monoclonal antibodies specific for phenotypic cellular markers. Greater than
75 % of the cells
present were CD 3+ ap T cells (Figure 3A); approximately 40% of these were
activated T
blasts (CD25+CD3+). Less than S% of the cells were natural killer {NK) cells
(CD56+),
" 10 % were B cells (CD 19 +), and less than 2 % of these cells were CD 4- /
CD 8- T cells
(Figure 3A). As a result of the acute nature of this patient's disease, some
clinically evident
residual cerebellar function, and the presence of activated T cells in her CSF
(characterized by
a CD25+ CD3+ phenotype), patient 1 was treated with tacrolimus (FK506), a drug
which
inhibits activation of T cells and partitions favorably into the CSF (16). The
patient tolerated
treatment for 10 days without side effects; however, evidence of recovery of
cerebellar
function was not observed. On day 11, treatment was discontinued and CSF was
obtained for
analysis, which revealed that the CD25+ T cells had been eradicated (Figure
3A). These
results show that tacrolimus can effectively suppress activated T cells in the
CSF of a PCD
patient, and may be an effective alternative to treatments aimed at
suppressing B cells or
removing antibodies. Early intervention may be necessary to arrest clinical
disease before
there is excessive Purkinje cell death.
Example 3 Intracellular Cytokine Staining of CSF Cells of Patient 1
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To .further define the activated cell population present in the CSF of patient
1, intracellular
cytokine staining was performed and cells were assayed by four color
cytofluorographic
analysis. This revealed helper T cells present in the CSF which produced IL-2,
IFNy and
TNFa but not IL-4 -- a cytokine profile characteristic of Thl cells (Figure
3B). Furthermore,
it was possible to demonstrate that the cells responsible for the production
of these cytokines
were activated T blasts (selected based on their increased forward scatter).
In contrast, CD3+
CD4+ cells isolated from the peripheral blood of the same patient produced no
cytokine unless
stimulated with phorbol 12-myristate (PMA) and ionomycin (I) prior to analysis
(Figure 3B).
Example 4 Demonstration of cdr2 Antigen on Gynecological Tumors
Despite the rarity of the paraneoplastic neurologic disorders, the target PND
antigen in one
such disorder (the Hu antigen) has been found to be expressed ubiquitously in
its associated
tumor type (small cell lung cancer). To evaluate whether the cdr2 antigen is
present in a larger
set of gynecologic tumors than the rarity of PCD might suggest, ovarian and
breast tumor
tissues obtained from neurologically normal individuals were examined for
expression of cdr2
by Western blot analysis. Twelve of 19 tumors of ovarian epithelial cell
origin expressed
robust amounts of protein immunoreactive with PCD antisera (Fig. 4A and data
not shown).
To confirm that this antigen corresponded to the cdr2 antigen, the migration
of immunoreactive
antigen from cerebellum were compared with that from the tumor samples by 2D
IEF/SDS-PAGE (Fig. 4B). These experiments confirm that the immunoreactive cdr2
band
co-migrates in brain and tumor tissues, and demonstrate that a high percentage
of non-PCD
ovarian tumors express the cdr2 tumor antigen. Similar results were found in
samples of
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non-PCD associated breast tumors, where at least 25 percent of tumors express
the cdr2
antigen.
Example 5 Use of Apoptotic Cells to Deliver Immune-Privileged Antigen to
Antigen-
Presenting Cells _
MC (97-09) DCs were co-cultured with apvptotic uninfected HeLa cells and
syngeneic T cells.
After 7 days, responding T cells were tested using T2 cells pulsed with
various Yo peptides,
Yol through Yo8 (corresponding to SEQ ID NO:1 through SEQ ID N0:8, and also
referred
to as cdr2-1 through cdr2-8). Negative control included testing Matrix peptide
(M) (SEQ ID
N0:9) pulsed T2 cells (HeLa cells were uninfected). These results show that
successful
induction of a cytotoxic T lymphocyte response to certain cdr2 peptides may be
achieved by
the use of apopototic cells delivering the target antigen.
Example 6 Generation of a mouse model that recapitulates aspects of the tumor
immunity in the disorder in the absence of autoimmune neurologic disease
A mouse model of PCD was generated that recapitulates aspects of the tumor
immunity in the
disorder in the absence of autoimmune neurologic disease. Mice were immunized
using an -
apoptotic tumor cell, PC7, which results in the generation of potent cdr2-
specific killer T cells.
The following cell lines were used in evaluating the model. For example, EC2
cells were
generated by stably transfecting EL4 cells with pcDNA-cdr2, and determination
of protein
expression made by Western blot analysis.
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TABLE 1
Cell Line Mouse of OriginMHC allele Antigen Description
expression
PC7 DBA/2 H-2'' cdr2 cdr2-transfected
P815 cells
P815 DBA/2 H-2'' parental Mastocytoma tumor
line
EC2 C57BL/6 H-2'' cdr2 cdr2-transfected
EL4 cells
TIB84 Balb/c H-2b Minor H of DBA/2Fibroblast line
from
congenic-strain
EL4 C57BL/6 H-2'' parental T cell lymphoma
C57BL/6 mice were immunized with 10' -irradiated PC7 cells, subcutaneously~,
at one week
intervals for a total of two injections. One to three weeks after the second
injection, the spleens
of primed and naive littermates were harvested. Mixed lymphocyte / tumor cell
cultures were
established using EC2 cells or TIB84 cells for purposes of restimulating
primed T cells. After
5 days, responding T cells were collected and tested at various ratios in a
standard chromium
release assay. Target cells included EC2 and EL4, demonstrating the generation
of
cdr2-specific killer T cells {A) and TIB84 and EL4, demonstrating the
generation of
alto-reactive T cells capable of recognizing minor histocompatibility antigens
in the context of
self MHC I (B). Average values of triplicates from experimental wells (E) are
compared to
average values of spontaneous (S) and total (T) release as follows: %
cytotoxicity = ((E-S) /
(T-S)) x 100. Naive littermates were used as negative controls. Alternatively,
CD8+ T~ cells-
were purified from the spleens of primed and naive mice using the MACS cell
isolation system.
Briefly, anti-CD8 antibody coupled to iron conjugated microbeads are incubated
with
splenocytes and CD8+ T cells are positively selected by passing the cells
through a magnetic
column. The positively selected CD8+ T cells were used directly in an ELISPOT
assay. 105
T cells were placed in a 96-well plate, previously coated with a monoclonal
antibody specific
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for IFN-, and incubated with 104 -irradiated stimulator cells (EC2 or EL4).
After 20 hours, the
cells were washed out and IFN- spot forming cells (SFCs) were detected using a
biotinylated
anti-IFN- antibody, and an HRP-AEC (3-amino-9-ethyl-carbazole) staining
procedure. SFCs
reported per million cells. (C).
_
The results of the experiment are shown in Figure 6. The specificity was
demonstrated as
measured by the ability to lyse MHC-matched target cells expressing cdr2, EC2
cells, but not
MHC-matched cells that lack cdr2 expression, EL4 (Fig. 6A). As the PC7 cells
are MHC-
mismatched with respect to the C57/B6 mouse that was immunized, it is believed
that this
model system is analogous to the cross-priming of tumor cells evident in
patients with PCD.
A control for this experiment included the use of TIB84 cells as targets in
the CTL assay (Fig.
6B), demonstrating the generation of allo-reactive T cells capable of
recognizing minor
histocompatibility antigens in the context of self-MHC I. As the PC7 cell also
harbor
allogeneic antigens with respect to the C57/B6 mouse, it was possible to
stimulate alto-specific
i 5 T cells that target the congenic line, TIB84. Congenic lines are useful as
they contain allo-
antigen presented in the context of self MHC molecules. Table 1 above provides
the MHC
haplotypes and cdr2 expression.
In addition to the killing assay, IFN-y release was also demonstrated from T
cells purified
from PC7 immunized mice (Fig. 1C). This short term assay confirmed that high
levels of CTL
precursors exist in the immunized mice. No immunized mice exhibited signs of
neurologic
dysfunction. These data indicate the ability to separate tumor immunity from
the autoimmune
neurodegeneration. As described above, cdr2-specific killer T cells have been
identified in
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patients with effective tumor suppression and PCD. In addition, significant
numbers of breast
and ovarian tumors present in neurologically normal patients express the cdr2
target antigen.
Therefore, the present study demonstrates that stimulation of T cells able to
kill cdr2-
expressing tumor cells is possible without inducing autoimmune neurologic
disease.
Example 7 ELISPOT assay for the detection of cdr2-specific T cells
A new assay for the detection of cdr2-specific T cells was developed. This
assay is faster and
offers the ability to screen large numbers of patient samples for the presence
of cdr2-specific
T cells in the form of a kit. Peripheral blood was obtained from patients and
dendritic cells
were matured as described above. These cells were then fed with apoptotic
debris from
unrelated (mouse) cells that did not (EL4) or that did (EC2) express the cdr2
protein. EC2
cells were generated by stably transfecting EL4 cells with pcDNA-cdr2, and
determination of
protein expression made by Western blot analysis; please refer to Example 6
and Table 1
regarding these cells. These fed DCs were then incubated with patient's
peripheral blood
lymphocytes, and interferon gamma (IFN-y) release measured as an index of
stimulation. The
assay for IFN-y release is a standard ELISPOT assay. In this instance, the
assay was done
manually, by plating nitrocellulose-bottom wells with antibody to IFN-y,
allowing release to
occur for 20 hours, washing plates, and adding a second anti-IFN-y antibody
which was
conjugated to biotin to allow colorimetric detection. The number of spots
secreting IFN-y
directly correspond to the number of T cells in the assay that were stimulated
by the DCs. By
comparing the number of T cells stimulated by EL4-fed DCs (negative control)
with the
number stimulated by EC2-fed DCs, the number of cdr2-specific T cells in a
patient's
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peripheral blood can be determined.
In an example of the above method, DCs were grown from an HLA A2.1 + patient
with PCD
and ovarian cancer that was in remission. These cells were pulsed with either
nothing, the
HLA A2.1 immunodominant matrix peptide (MP) as a positive control, or
apoptotic
{irradiated) EL4 or EC2 cells. These DCs were then cultured together with T
cells in varying
ratios of T cell:apoptotic cell, as indicated. T cell activation was measured
by counting spots
corresponding to IFN-y release as described. The results of the assay are
shown in Figure 7.
This patient had a significant number of cdr2+ T cells evident by the large
numbers of spots
seen with EC2 stimulation, and the differences in spot number seen with EL4
versus EC2 fed
cells. Negative controls included T cells incubated with apoptotic debris in
the absence of
DCs, and T cells incubated with neither DC nor apoptotic debris; neither
control led to T cell
stimulation.
This invention may be embodied in other forms or carried out in other ways
without departing
from the spirit or essential characteristics thereof. The present disclosure
is therefore to be
considered as in all respects illustrative and not restrictive, the scope of
the invention being
indicated by the appended Claims, and all changes which come within the
meaning and range _
of equivalency are intended to be embraced therein.
Various publications are cited herein, the disclosures of which are
incorporated by reference
in their entireties.
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SEQUENCE LISTING
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