Note: Descriptions are shown in the official language in which they were submitted.
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HAPTEN-MODIFIED TUMOR CELL MEMBRANES,
AND METHODS OF MAKING AND USING
HAPTEN-MODIFIED TUMOR CELL MEMBRANES
Reference to Government Grants
The invention described herein was made in the course of work under a grant
or award from the National Institutes of Health-National Cancer Institute,
grant no.
CA-39248. The United States Government may have certain rights in this
invention.
Background of the Invention
It was theorized in the 1960's that tumor cells bear specific antigens (TSA)
which are not present on normal cells and that the immune response to these
antigens
might enable an individual to reject a tumor. It was later suggested that the
immune
response to TSA could be increased by introducing new immunological
determinants
on cells. Mitchison, Transplant. Proc., 1970, 2, 92. Such a "helper
determinant",
which can be a hapten, a protein, a viral coat antigen, a transplantation
antigen, or a
xenogenous cell antigen, could be introduced into a population of tumor cells.
The
cells would then be injected into an individual who would be expected to be
tolerant
to the growth of unmodified tumor cells. Clinically, the hope was that an
immunologic
reaction would occur against the helper determinants, as a consequence of
which the
reaction to the accompanying TSA is increased, and tumor cells which would
otherwise
be tolerated are destroyed. Mitchison, supra, also suggests several modes of
action
of the helper determinants including 1) that the unmodified cells are merely
attenuated,
in the sense that their growth rate is slowed down or their susceptibility to
immunologic attack increased; 2) that helper determinants merely provide
points of
attack and so enable the modified cells to be killed by an immune response not
directed
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2
against TSA; 3) that the helper determinants have an adjuvant action such as
binding
to an antibody or promoting localization of the cells in the right part of the
body for
immunization, in particular, in lymph nodes.
Fujiwara et al., J. Immunol., 1984, 132, 1571 showed that certain haptenized
tumor cells, i.e., tumor cells conjugated with the hapten trinitrophenyl
(TNP), could
induce systemic immunity against unmodified tumor cells in a marine system,
provided
that the mice were first sensitized to the hapten in the absence of hapten-
specific
suppressor T cells. Spleen cells from the treated mice completely and
specifically
prevented the growth of tumors in untreated recipient animals. Flood et al.,
J.
Immunol. , 1987, 138, 3573 showed that mice immunized with a TNP-conjugated,
ultraviolet light-induced "regressor" tumor were able to reject a TNP-
conjugated
"progressor" tumor that was otherwise non-immunologic. Moreover, these mice
were
subsequently resistant to challenge with unconjugated "progressor" tumor. In
another
experimental system, Fujiwara et al. , J. Immunol. , 1984, 133, 510
demonstrated that
mice sensitized to trinitrochlorobenzene (TNCB) after cyclophosphamide
pretreatment
could be cured of large (lOmm) tumors by in situ haptenization of tumor cells;
subsequently, these animals were specifically resistant to challenge with
unconjugated
tumor cells.
The teachings of Fuj iwara et al. differ from the present invention for
several
reasons including the following: A. The cells used in Fujiwara's composition
are
derived from induced transplantable marine tumors - not from spontaneous human
tumors; B. Fujiwara's composition is used in immunoprophylaxis - the present
invention uses immunotherapy; C. Fujiwara's composition is administered as a
local
therapy - the composition of the present invention is administered by systemic
inoculation; D. Fujiwara's composition did not result in tumor regression -
the
composition of the present invention results in regression and/or prolonged
survival for
at least a substantial portion of the patients treated; and E. Fujiwara
administers tumor
cells - the present invention teaches administration of tumor cell membranes.
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The existence of T cells which cross-react with unmodified tissues has
recently
been demonstrated. Weltzien and coworkers have shown that class I MHC-
restricted
T cell clones generated from mice immunized with TNP-modified syngeneic
lymphocytes respond to MHC-associated, TNP-modified "self" peptides. Ortmann,
B. , et al. , J. Immunol. , 1992, 148, 1445. In addition, it has been
established that
immunization of mice with TNP-modified lymphocytes results in the development
of
splenic T cells that exhibit secondary proliferative and cytotoxic responses
to TNP-
modified cells in vitro. Shearer, G. M. Eur. J. Immunol. ,1974, 4, 527. The
potential
of lymphocytes elicited by immunization with DNP- or TNP-modified autologous
cells
to respond to unmodified autologous cells is of considerable interest because
it may be
relevant to two clinical problems: 1) drug-induced autoimmune disease, and 2)
cancer
immunotherapy. In regard to the former, it has been suggested that ingested
drugs act
as haptens, which combine with normal tissue protein forming immunogenic
complexes
that are recognized by T cells. Tsutsui, H. , et al. , J. Immunol. , 1992,
149, 706.
Subsequently, autoimmune disease, e.g., systemic lupus erythematosus, can
develop
and continue even after withdrawal of absence of the offending drug. This
would
imply the eventual generation of T lymphocytes that cross-react with
unmodified
tissues.
The common denominator of these experiments is sensitization with hapten in
a milieu in which suppressor cells are not induced. Spleen cells from
cyclophosphamide pretreated, TNCB-sensitized mice exhibited radioresistant
"amplified helper function" i.e., they specifically augmented the in vitro
generation of
anti-TNP cytotoxicity. Moreover, once these amplified helpers had been
activated by
in vitro exposure to TNP-conjugated autologous lymphocytes, they were able to
augment cytotoxicity to unrelated antigens as well, including tumor antigens
(Fujiwara
et al., 1984). Flood et al., (1987), supra, showed that this amplified helper
activity
was mediated by T cells with the phenotype Lyt'1+, Lyt-2-, L3T4+, I-J+ and
suggests
that these cells were contrasuppressor cells, a new class of immunoregulatory
T cell.
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Immunotherapy of patients with melanoma had shown that administration of
cyclophosphamide, at high dose (1000 mgIM2) or low dose (300 mg/M2), three
days
before sensitization with the primary antigen keyhole limpet hemocyanin
markedly
augments the acquisition of delayed type hypersensitivity to-that antigen
(Berd et al.,
Cancer Res. , 1982, 42, 4862; Cancer Res. , 1984, 44, 1275). Low dose
cyclophosphamide pretreatment allows patients with metastatic melanoma to
develop
delayed type hypersensitivity to autologous melanoma cells in response to
injection
with autologous melanoma vaccine (Berd et al. , Cancer Res. , 1986, 46, 2572;
Cancer
Invest. , 1988, 6, 335). Cyclophosphamide administration results in reduction
of
peripheral blood lymphocyte non-specific T suppressor function (Berd et al. ,
Cancer
Res. , 1984, 44, 5439; Cancer Res. , 1987, 47, 3317); possibly by depleting
CD4 +,
CD45R+ suppressor inducer T cells (Berd et al., CancerRes., 1988, 48, 1671).
The
anti-tumor effects of this immunotherapy regimen appear to be limited by the
excessively long interval between the initiation of vaccine administration and
the
IS development of delayed type hypersensitivity to the tumor cells (Berd et
al., Proc.
Amer. Assoc. Cancer Res. , 1988, 29, 408 {#1626)). Therefore, there remains a
need
to increase the therapeutic efficiency of such a vaccine to make it more
immunogenic.
Most tumor immunologists now agree that infiltration of T lymphocytes, white
cells responsible for tumor immunity, into the tumor mass is a prerequisite
for tumor
destruction by the immune system. Consequently, a good deal of attention has
been
focused on what has become known as "TIL" therapy, as pioneered by Dr. Stephen
Rosenberg at NCI. Dr. Rosenberg and others have extracted from human cancer
metastases the few T lymphocytes that are naturally present and greatly
expanded their
numbers by culturing them in vitro with Interleukin 2 (IL2), a growth factor
for T
lymphocytes. Topalian et al., J. Clin. Oncol., 1988, 6, 839. However this
therapy
has not been very effective because the injected T cells are limited in their
ability to
"home" to the tumor site.
The ability of high concentrations of IL2 to induce lymphocytes to become non-
specifically cytotoxic killer cells has been exploited therapeutically in a
number of
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studies (Lotze et al. , J. Biol. Response, 1982, 3, 475; West et al. , New
Engl. J. Med. ,
1987, 316, 898). However, this approach has limitations due to the severe
toxicity of
high dose intravenous IL2. Less attention has been given to the observation
that much
lower concentrations of IL2 can act as an immunological adjuvant by inducing
the
5 expansion of antigen-activated T cells (Talmadge et al. , Cancer Res. ,
1987, 47, 5725;
Meuer et al., Lancet, 1989, l, 15). Therefore, there remains a need to
understand and
attempt to exploit the use of IL2 as an immunological adjuvant.
Human melanomas are believed to express unique surface antigens recognizable
by T lymphocytes. Old, L. J., Cancer Res., 1981, 41, 361; Van der Bruggen, P.,
et
al., Science,1991, 254, 1643; Mukherji, B., et al., J. Immunol.,1986,136,
1888; and
Anichini, A. , et al. , J. Immunol. , 1989, 142, 3692. However,
immunotherapeutic
approaches prior to work done by the present inventor had been limited by the
difficulty of inducing an effective T cell-mediated response to such antigens
in vivo.
There are several models proposed to explain what appears to be tolerance to
human tumor-associated antigens. They include:
1) Tumor antigen-specific suppressor cells that down-regulated incipient anti-
tumor responses. Mukherji, et al., supra; Berendt, M. J. and R. J. North., J.
Exp.
Med. , 1980, 151, 69.
2) Failure of human tumor cells to elicit T helper cells or to provide
costimulatory signals to those T cells. Fearon, E. R., et al., Cell, 1990, 60,
397;
Townsend, S. E. and J. F. Allison, Science, 1993, ?,59, 368; and
3) Reduced surface expression of major histocompatibility products on tumor
cells which limits their recognition by T cells. Ruiter, D. J., Seminars in
Cancer
Biology,1991, 2, 35. None of these hypotheses has yet been corroborated in a
clinical
system.
Regardless of whether such explanations are true or not, there is a continuing
need for more effective treatment of various malignancies.
In regard to acute myelogenous leukemia (AML), the treatment for AML is
divided into one or two initial induction phases and several courses of post-
remission,
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also known as consolidation, chemotherapy. Initial induction chemotherapy may
induce
a complete response in 55 to 88% of the patients, depending on the protocol
used.
However, the vast majority of these patients relapse, and the long-term (5
year +)
survival of AML patients is only 20-30% . The addition of-high-dose
chemotherapy
and bone marrow transplantation {BMT) to this therapeutic regime during the
first
remission can bring about modest improvements in result. For example, patients
undergoing allogeneic BMT are afforded a 5 to 10% increase in the 5 year
survival.
However, the strict eligibility criteria for BMT (e. g. , age, availability of
an HLA-
matched donor) severely limit the number of patients who can be treated. Once
AML
patients relapse, there is only a 30 % chance of achieving a second remission,
and very
few of these patients remain disease-free in the long run. Treatment
modalities on
relapse include similar protocols to those used in achieving the first
remission
(induction therapy followed by several courses of consolidation chemotherapy),
although high dose of a single agent and BMT can also be used (Keating et al).
Experience with bone marrow transplantation has suggested that immunological
rejection may play a role in the control of the disease. Graft-versus-host
disease
(GVHD) and relapse are the two main causes of death of patients treated with
BMT.
The risk of relapse decreases if mild GVHD occurs (Horowitz et al). Therefore
it has
been hypothesized that grafted lymphocytes are able to imrnunologically reject
host
leukemia cells (graft-versus-leukemia reaction, GVL). This GVL reaction could
be
mediated by a T-cell response against specific leukemia cell antigens,
although
immunogenic human leukemia antigens have not yet been demonstrated (the same
is
true for melanoma). It is known that human AML cells strongly express both
class I
and class II major histocompatibility complex (MHC) antigens (Ashman et al;
Andreasen et al) which are prerequisites for the induction of CD8- and CD4-
mediated
T cell responses, respectively. However, induction of a T cell response
targeted to
leukemia cells has not been successful.
Several immunological approaches have been used for the treatment of acute
leukemia (Foon et al; Caron and Scheinberg). These approaches are divided into
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non-specific, such as Bacillus Calmette Guerin (BCG), interleukin-2,
levamisole,
methanol-extraction residue of tubercle bacillus, and specific, such as
monoclonal
antibodies and vaccines (harvested leukemia cells, cell free extracts and
cultured cells).
The majority of these studies have been performed in patients already in
remission, in
which immunotherapy would have to be successful in controlling residual
disease.
In the late 1960's and early 1970's, the research group of R. Powles at St.
Barthlomew's Hospital in England conducted a series of studies of vaccine
treatment
of AML patients after chemotherapy-induced remission (Powles, 1974; Powles et
al,
1977). They used allogeneic AML cells with BCG as an adjuvant. Several trials
were
performed, all with small sample sizes (N=10-15). There was some prolongation
of
survival using a combination of chemotherapy and immunotherapy compared to
chemotherapy alone, but no prolongation of relapse-free survival. No serious
toxicity
was observed; autoimmunity (e. g. , toxicity to normal bone marrow) was not
seen. In
retrospect, there were a number of technical problems with these trials: 1)
allogeneic,
rather than autoiogous, leukemia cells were used; 2) the dose of leukemia
cells in the
vaccine was excessive (up to 109 cells/dose); 3) the BCG dose was very high
and BCG
administration was separated by time and location from the leukemia cell
vaccine; and
4) the vaccine was administered while the patients were receiving cytotoxic
drugs
(maintenance or consolidation chemotherapy).
The immunochemical basis of the limited success of the above-mentioned
treatrnents remains speculative, but several hypotheses are being tested. Kim
and Jang
(1992) have suggested that the lack of T cell response to a particular epitope
may not
be due to absence of a T cell repertoire, but rather to difficulty in
generating the
particular epitope. Martin et al (1993) have explained their results by
hypothesizing
the existence of autoreactive T cells that escape thymic selection because of
low affinity
for "self" peptides.
Conventional attempts to treat human cancer have been unsuccessful.
Administration of compositions, exemplified by those set forth above, failed
to reliably
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induce the development of cell-mediated immunity as indicated by delayed-type
hypersensitivity (DTH), T cell infiltration, and inflammatory immune response.
Accordingly, despite the number of attempts based on various theories proposed
for the immunological effects reported in the treatments of cancer, there
remains a need
S for a composition which, upon administration to a mammal, is capable of
eliciting T
lymphocytes that infiltrate a tumor, eliciting an inflanunatory immune
response to a
tumor, and eliciting a delayed-type hypersensitivity response to a tumor.
Applicants
have now surprisingly discovered that using membranes isolated from either
syngeneic
or allogeneic tumor cells have these desired properties.
Summary of the Invention
The present invention is directed to an isolated tumor cell membrane, a
composition containing such membrane, methods for isolating and preparing the
tumor
cell membrane and compositions containing such membrane, and their use in
vitro and
for treating cancer. The tumor cell membrane, which may be hapten modified, is
IS preferably a tumor cell plasma membrane that may be syngeneic or
allogeneic. The
syngeneic tumor cell membrane may be autologous. The cancer to be treated
includes
carcinomas and non-solid tumors, including leukemia (such as acute myelogenous
leukemia), lymphoma, multiple myeloma, ovarian, colon, rectal, colorectal,
melanoma,
breast, lung, kidney, and prostate cancer.
In one aspect, the present invention relates to an isolated mammalian,
preferably human, tumor cell membrane modified with a hapten. The hapten may
be
selected from the group consisting of dinitrophenyl, trinitrophenyl, N-
iodoacetyl-N'-(S-
sulfonic 1-naphthyl) ethylene diamine, trinitrobenzenesulfonic acid,
fluorescein
isothiocyanate, arsenic acid benzene isothiocyanate, trinitrobenzenesulfonic
acid,
2S phosphorylcholine, sulfanilic acid, arsanilic acid and dinitrobenzene-S-
mustard and
combinations thereof.
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In another aspect, the present invention is directed to a composition
comprising
a hapten modified mammalian tumor cell membrane, alone or in combination with
a
hapten modified mammalian tumor cell.
In yet another aspect, the invention provides for. a vaccine composition
comprising a therapeutically effective amount of a mammalian, preferably
human,
tumor cell membrane for administration to a mammal which suffers from a
malignant
tumor of the same type as the tumor cell membrane.
In another aspect of the invention, the composition contains an adjuvant, such
as, for example, Bacille Calmette-Guerin, QS-21, detoxified endotoxin and
cytokines
such as interleukin-2, interleukin-4, gamma interferon (IFN-y), interleukin-
12,
interleukin-15 and GM-CSF.
The membrane and the composition of the present invention, have (when
administered to a mammal, preferably a human, suffering from a malignant tumor
of
the same type as the tumor cell from which the membrane was isolated) at least
one of
the following properties: (i) eliciting T lymphocytes that infiltrate the
tumor of a
treated mammal, (ii) eliciting an inflammatory immune response against the
tumor of
the mammal, and (iii) eliciting a delayed-type hypersensitivity response to
the tumor
of the mammal. The membrane and the composition of the invention also have the
property of stimulating T cells in vitro.
In yet another aspect, the present invention is directed to a method of
treating
cancer comprising administering to a mammal, preferably a human, a composition
comprising a therapeutically effective amount of a hapten modified human tumor
cell
membrane wherein said mammal suffers from a malignant tumor of the same type
as
said tumor cell membrane.
Yet further, the present invention relates to a method of eliciting T
lymphocytes
that have a property of infiltrating said tumor of said mammal, preferably a
human,
and, optionally, measuring said T lymphocytes that infiltrate said tumor of
said
mammal. The invention further relates to a method of eliciting an inflammatory
immune response to said tumor of said mammal and, optionally, measuring said
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inflammatory immune response, or to a method of eliciting a delayed-type
hypersensitivity response to said tumor of said mammal and, optionally,
measuring said
delayed-type hypersensitivity response. The invention also relates to a method
of
stimulating T cell in vitro.
5 In yet further aspect of the invention, the invention relates to a method of
making a hapten-modified tumor cell membrane.
Detailed Description of the Invention
All patents, patent applications and references cited herein are hereby
incorporated by reference. In case of inconsistencies, the present disclosure
governs.
10 The present invention is directed to an isolated modified and un-modified
tumor
cell membrane, a novel composition containing such membrane, methods of
isolating
and making the membrane and compositions containing such membrane, and methods
for using the membrane and compositions of the invention.
The membranes and compositions of the present invention may be used for
treating cancer in a mammal, preferably a human, including metastatic and
primary
cancers, solid and non-solid cancers such as, for example, rectal, colorectal,
melanoma, breast, lung, kidney, and prostate cancers. Stage I, II, III, or IV
cancer
may be treated with the isolated modified membranes, the compositions and
methods
of the present invention, preferably stages III and IV, even more preferably
stage III.
Mammals, particularly humans, having metastatic cancer of the foregoing type
may be
treated with the membranes, the compositions and methods of the present
invention.
In one embodiment, the present invention is used to treat domestic animals
such as, for
example, members of feline, canine, equine and bovine families.
The membranes and compositions of the invention may also be used for
eliciting T lymphocytes that have a property of infiltrating a mammalian
tumor,
eliciting an inflammatory immune response to a mammalian tumor, eliciting a
delayed
type hypersensitivity response to a mammalian tumor and/or stimulating T
lymphocytes
in vitro.
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It will be understood that any disclosure in this specif cation with respect
to use of isolated tumor cells equally applies to use of tumor cells
membranes, or to
a combination of tumor cells and tumor cell membranes.
TUMOR CELLS MEMBRANES AND COMPOSITIONS THEREOF
The isolated, modified tumor cell membranes of the present invention are
prepared from mammalian, preferably human, tumor cells. In one embodiment of
the
invention, tumor cell membrane are isolated from a tumor of an animal from a
feline,
canine, equine or bovine family.
Included within the definition of a tumor cell for purposes of the present
invention are whole and disrupted tumor cells. The tumor cells from which
membranes are isolated may be live, attenuated, or killed cells. Tumor cells
which do
not grow and divide after administration into the subject such that they are
substantially
in a state of no growth can be used in the present invention. Such cells are
preferred
if they are administered to the patient alone or in combination with isolated
tumor cell
membranes. It is to be understood that "cells in a state of no growth" means
live,
attenuated or killed, whole or disrupted (or both whole and disrupted) cells
that do not
divide in vivo. Conventional methods of suspending cells in a state of no
growth are
known to skilled artisans and may be useful in the present invention. For
example,
cells may be irradiated prior to use such that they do not grow and divide.
Tumor cells
may be irradiated, for example at 2500 R, to prevent the cells from growing
after
administration. Alternatively, tumor cell membranes may also be isolated from
tumor
cells that can grow and divide in vivo. Preferably, in such a case, the tumor
cell
membrane preparation is not contaminated with tumor cells that are capable of
dividing
in vivo.
Tumor cell membranes are isolated from the tumor cells of the same type as the
cancer to be treated. For example, membranes to be used for treating ovarian
cancer
are isolated from ovarian cancer cells. Preferably, the tumor cells originate
from the
same subject who is to be treated. The tumor cells are preferably syngeneic
(e.g.
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autologous), but may also be allogeneic to that subject. To be defined as
"syngeneic,"
the tumor cell need not be completely (i. e. , 100 % ) genetically identical
to either the
tumor cell or the non-tumor, somatic cell of the treated patient. Genetic
identity of the
MHC molecules between the tumor cell (from which membranes are isolated) and
the
patient is generally sufficient. Additionally, there may be genetic identity
between a
particular antigen on the tumor cell used as a membrane source and an antigen
present
on patient's tumor cells. Genetic identity may be determined according to the
methods
known in the art. A syngeneic tumor cell also means a cell that has been
genetically
altered (using for example recombinant DNA technology) to become genetically
identical with respect to, for example, the particular MHC molecules of the
patient
and/or the particular antigen on the patient's cancer cells. Tumor cells from
animals
of the same species that differ genetically, such as allogeneic cells, may
also be used
for the preparation of tumor cell membranes of the invention. The tumor cells
may be,
and are not limited to, cells dissociated from biopsy specimens or from tissue
culture.
Membranes isolated from allogeneic cells and stem cells are also within the
scope of
the present invention.
Tumor cell membranes may include all cellular membranes, such as outer
membrane, nuclear membranes, mitochondria) membranes, vacuole membranes,
endoplasmic reticular membranes, golgi complex membranes, and lysosome
membranes. In one embodiment of the invention greater than about 50% of the
membranes are tumor cell plasma membranes. Preferably, greater than about b0%
of the
membranes consist of tumor cell plasma membranes, with greater than about 70%
being
more preferred, 80% being even more preferred, 90% being even more preferred,
95%
being even more preferred, and 99% being most preferred.
Preferably, the isolated membranes are substantially free of nuclei and cells.
For
example, a membrane preparation is substantially free of nuclei or cells if it
contains less
than about 100 cells and/or nuclei in about 2 x 10$ cell equivalents (c.e.) of
membrane
material. A cell equivalent is that amount of membrane isolated from the
indicated
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number of cells. An isolated tumor cell membrane which is substantially free
of cells
and/or nuclei may contain lymphocytes and/or lymphocyte membranes.
Preferably, the isolated tumor cell membranes are the outer cell membranes, i.
e.,
tumor cell plasma membranes. The membrane preparation of the invention may
contain
the entire outer membrane or a fraction thereof. An isolated membrane of the
invention
containing a fraction of the outer membrane contains at least an MHC molecule
fraction
and/or a heat shock protein fraction of the outer membrane. The size of
membrane
fragments is not critical.
Allogeneic tumor cell membranes may also be used in the methods of the present
invention with syngeneic (e.g. autologous) antigen presenting cells. This
approach
permits immunization of a patient with tumor cell membranes originating from a
source
other than the patient's own tumor. Syngeneic antigen-presenting cells process
allogeneic membranes such that the patient's cell-mediated immune system may
respond
to them.
The isolated tumor cell membranes as well as tumor cells may be modified, for
example, with a hapten. Such modified tumor cell membranes (and tumor cells)
have at
least one of the following properties: (i) eliciting T lymphocytes that
infiltrate the tumor
of a treated mammal, (ii) eliciting an inflammatory immune response against
the tumor
of the mammal, and (iii) eliciting a delayed-type hypersensitivity response to
the tumor
of the mammal. Modified tumor cell membranes and cells also have the property
of
stimulating T cells in vitro.
A tumor cell membrane (modified or un-modified) as referred to in this
specification includes any form in which such membrane preparation rnay be
stored or
administered such as, for example, a membrane resuspended in a diluent, a
membrane
pellet, or a frozen or a lyophilized membrane.
The membranes of the invention may be employed in the methods of the
invention singly or in combination with other compounds, including and not
limited to
other compositions of the invention. Accordingly, tumor cells and tumor cell
membranes
may be used alone or co-administered. For purposes of the present invention,
co-
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14
administration includes administration together and consecutively. Further,
the tumor
cells and tumor cell membranes may be co-administered with other compounds
including and not limited to cytokines such as interleukin-2, interleukin-4,
gamma
interferon (IFN-y), interleukin-12, interleukin-15 and GM-GSF. The tumor cells
and
tumor cell membranes of the invention may also be used in conjunction with
other cancer
treatments including and not limited to chemotherapy, radiation, antibodies,
and antisense
oligonucleotides. However, it is the advantage of the present invention that
it can be
useful alone as a cancer treatment, such that the need for additional
therapies is
unnecessary.
A composition of the present invention may contain the isolated tumor cell
membrane of the invention (modified or unmodified) and a pharmaceutically
acceptable
carrier or diluent, such as and not limited to Hanks solution, saline,
phosphate-buffered
saline, sucrose solution, and water. In general, the pharmaceutically-
acceptable carrier
is selected with regard to the intended route of administration and the
standard
pharmaceutical practice. The proportional ratio of active ingredient to
carrier naturally
depends on the chemical nature, solubility, and stability of the compositions,
as well as
the dosage contemplated and can be optimized using common knowledge in the
art.
In one preferred embodiment of the invention, a composition of the invention
is
a vaccine composition containing an effective amount of isolated modified
tumor cell
membrane. For purposes of this disclosure, "an effective amount" is the amount
necessary to achieve a desired result. For example, in a method for treating
cancer, "an
effective amount" means that amount of isolated modified tumor cell membranes
that has
the property of causing at least one of the following: (i) eliciting T
lymphocytes that
infiltrate tumor, (ii) eliciting an inflammatory response against the tumor,
(iii) eliciting
a delayed-type hypersensitivity response to a tumor, and (iv) tumor
regression. Similarly,
in a method for stimulating T cells in vitro, "an effective amount" is that
amount of
membranes that results in T cell stimulation.
The vaccine composition may contain, for example, at least 104 c.e. of
isolated
membranes per dose, preferably at least l Os c.e., and most preferably at
least 106 c.e. A
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dose is that amount of the vaccine composition that is administered in a
single
administration. In one embodiment, the vaccine composition contains from about
1 OS to
about 2.5 x 10' c.e. membranes per dose, more preferably about 5 x 106 c.e.
The amount
of the tumor cells and tumor cell membranes of the invention to be used
generally
5 depends on such factors as the affinity of the compound for cancerous cells,
the amount
of cancerous cells present and the solubility ofthe composition. Dosages may
be set with
regard to weight, and clinical condition of the patient.
A vaccine composition of the invention may be packaged in a dosage form
suitable for intradermal, intravenous, intraperitoneal, intramuscular, and
subcutaneous
10 administration. Alternatively, the dosage fonm may contain isolated tumor
cell
membranes to be reconstituted at the time of the administration with, for
example, a
suitable diluent.
HAPTEN
The tumor cells and tumor cell membranes of the present invention may be used
15 as modified, unmodified, or a combination of modified and unmodified tumor
cells and
tumor cell membranes. For purposes of the present invention, modified includes
and is
not limited to modification with a hapten. Any small molecule that does not
alone induce
an immune response (but that enhances immune response against another molecule
to
which it is conjugated or otherwise attached) may function as a hapten.
Generally, the
molecule used should have less than about 1,000 mw.
A variety of haptens are known in the art such as for example: TNP (Kempkes et
al., J. Immunol.1991 147:2467); phosphorylcholine (Jang et al., Eur. J.
Immunol. 1991
21:1303); nickel (Pistoor et al., J. Invest. Dermatol.1995145:92); arsenate
(Nalefski and
Rao, J. Immunol. 1993150: 3806).
Generally, haptens suitable for use in the present invention have the property
of
binding to a hydrophilic amino acid (such as for example lysine). Hapten can
be
conjugated to a cell via s-amino groups of lysine or -COON groups.
Additionally, hapten
that can bind to hydrophobic amino acids such as tyrosine and histidine via
diaza
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16
coupling can also be used. Examples of haptens suitable for use in the present
invention
are: dinitrophenyl, trinitrophenyl, N-iodoacetyl-N'-{5-sulfonic 1-naphthyl)
ethylene
diamine, trinitrobenzenesulfonic acid, fluorescein isothiocyanate, arsenic
acid benzene
isothiocyanate, trinitrobenzenesulfonic acid, phosphorylcholine, sulfanilic
acid,
arsanilic acid, dinitrobenzene-S-mustard (Nahas and Leskowitz, Cellular
Immunol.
1980 54:241) and combinations thereof. Once armed with the present disclosure,
skilled artisans, would be able to choose haptens for use in the present
invention. For
example, haptens can be routinely tested using a delayed type hypersensitivity
(DTH)
test.
ADJi7VANT
In one preferred embodiment, the tumor cell or tumor cell membrane is
administered with an immunological adjuvant. The adjuvant has the property of
augmenting an immune response to hapten modified tumor cells and membranes.
Representative examples of adjuvants are Bacille Calmette-Guerin, BCG, or the
synthetic adjuvant, QS-21 comprising a homogeneous saponin purified from the
bark
of Quillaja saponaria, Corynebacterium parvum, McCune et al. , Cancer 1979
43:1619, saponins in general, detoxified endotoxin and cytokines such as
interleukin-2,
interleukin-4, gamma interferon {IFN-'y), interleukin-12, interleukin-15, GM-
CSF and
combinations thereof.
It will be understood that the adjuvant may be subject to optimization. In
other
words, the skilled artisan may use routine experimentation to determine the
most
optimal adjuvant to use.
METHODS OF MAKING TUMOR CELL MEMBRANES OF THE INVENTION
The tumor cells for use in the present invention may be prepared as follows.
Tumors are processed as described by Berd et al. (1986), supra, Sato, et al.
(1997),
U.S. Patent No. 5,290,551, and applications U.S. Serial Nos. 081203,004,
081479,016, 081899,905, 08/942,794, or corresponding PCT application
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17
PCT/US96/09511, each of which is incorporated herein by reference in its
entirety.
Briefly, the cells are extracted by dissociation, such as by enzymatic
dissociation with
collagenase and DNase, by mechanical dissociation in a blender, by teasing
with
tweezers, using mortar and pestle, cutting into small pieces using a scalpel
blade, and
the like. With respect to liquid tumors, blood or bone marrow samples may be
collected and tumor cells isolated by density gradient centrifugation.
Tumor cell membranes are prepared from tumor cells by disrupting the cells
using, for example, hypotonic shock, mechanical dissociation and enzymatic
dissociation, and separating various cell components by centrifugation.
Briefly, the
following steps may be used: lysing tumor cells, removing nuclei from the
lysed tumor
cells to obtain nuclei-free tumor cells, obtaining substantially pure
membranes free
from cells and nuclei, and subjecting the tumor cell membranes to a hapten to
obtain
hapten-modified tumor cell membranes. Membrane isolation may be conducted in
accordance with the methods of Heike et al.
In one embodiment of the invention, intact cells and nuclei may be removed by
consecutive centrifugation until membranes are substantially free of nuclei
and cells,
as determined microscopically. For example, lysed cells may be centrifuged at
low
speed, such as for example, at about 500-2,000 g for about five minutes. The
separation procedure is such that less than about 100 cells andlor nuclei
remain in
about 2 x 10g cell equivalents (c.e.) of membrane material. The postnuclear
supernatant containing membranes may be pelleted by ultracentr~fugation at
about
100,000 g for about 90 minutes, for example. The pellet contains total
membranes.
Membranes may be resuspended, for example, in about 8 % sucrose, 5 mM Tris, pH
7.6 and frozen at about -80°C until use. Any diluent may be used,
preferably one that
acts as a stabilizer. To determine the quality of membrane preparation (about
6 x 10'
c.e. membranes) may be regularly cultured. Cell colonies should not develop
and cells
or nuclei should not be detected by light microscopy.
Modification of the prepared cells or membranes with DNP or another hapten
may be performed by known methods, e.g. by the method of Miller and Claman, J.
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18
Immunol. , 1976, 117, 1519, incorporated herein by reference in its entirety,
which
involves a 30 minute incubation of tumor cells or membranes with a hapten
under
sterile conditions, followed by washing with sterile saline. The hapten-
modification
may be confirmed by flow cytometry using a monoclonal anti-hapten antibody.
The dissociated cells or isolated membranes may be used fresh or stored
frozen,
such as in a controlled rate freezer or in liquid nitrogen until needed. The
cells and
membranes are ready for use upon thawing. Preferably, the cells or membranes
are
thawed shortly before they are to be administered to a patient. For example,
on the
day that a patient is to be skin tested or treated, the cells or membranes may
be thawed.
Optionally, the cells or membranes may be washed, and optionally irradiated to
2500
R. They may be washed again and then suspended in Hanks balanced salt solution
without phenol red.
Allogeneic tumor cell membranes may be prepared as described above.
However, prior to administaration to a subject they are co-incubated with
syngeneic
(e.g. autologous) antigen presenting cells. Syngeneic antigen-presenting cells
process
allogeneic membranes such that the patient's cell-mediated immune system may
respond to them. This approach permits immunization of a patient with tumor
cell
membranes originating from a source other than the patient's own tumor.
Allogeneic
tumor cell membranes are incubated with antigen-presenting cells for a time
period
varying from about several hours to about several days. The membrane-pulsed
antigen
presenting cells are then wsshed and injected into the patient.
Antigen-presenting cells may be prepared in a number of ways including for
example the methods of Grabbe et al. , 1995 and Sierra et al. , 1995 .
Briefly, blood is
obtained, for example by venipuncture, from the patient to be immunized.
Alternatively, bone marrow may be obtained. Alternatively, blood leukocytes
may be
obtained by leukapheresis. From any of these sources, mononuclear leukocytes
are
isolated by gradient centrifugation. The leukocytes may be further purified by
positive
selection with a monoclonal antibody to the antigen, CD34.
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The purified leukocytes are cultured and expanded in tissue culture medium
(for
example, RPMI-164.0 supplemented with serum, such as fetal calf serum, pooled
human serum, or autologous serum). Alternatively, serum-free medium may be
used.
To stimulate the growth of antigen-presenting cells, cytokines may be added to
the
culture medium. Cytokines include and are not limited to granulocyte
macrophage-
colony stimulating factor (GM-CSF), interleukin 4 (IL4), TNF (tumor necrosis
factor),
interleukin 3 (IL3), FLT3 ligand and granulocyte colony stimulating factor (G-
CSF).
The antigen-presenting cells isolated and expanded in culture may be
characterized as dendritic cells, monocytes, macrophages, and Langerhans
cells, for
example.
METHODS OF USING TUMOR CELL MEMBRANES AND COMPOSITIONS
Method for TreatinE Cancer The present invention relates to a method of
treating a mammal, preferably a human, diagnosed with or suspected of having
cancer
by administering a pharmaceutically acceptable amount of a hapten modified
tumor cell
membrane, hapten modified tumor cell, or a combination thereof. The membranes
and/or cells may be mixed with an immunological adjuvant and/or a
pharmaceutically
acceptable carrier. A pharmaceutically acceptable amount of a low-dose
cyclophosphamide or another low-dose chemotherapy may be administered
preceding
the administration of the composition. The haptenized composition may
optionally be
followed by administration of a pharmaceutically acceptable amount of a non-
haptenized tumor cell or tumor cell membrane. A non-haptenized composition may
also be administered in accordance with the methods of the present invention.
Any malignant tumor may be treated according to the present invention
including metastatic and primary cancers and solid and non-solid tumors. Solid
tumor
include carcinomas, and non-solid tumors include hematologic malignancies.
Carcinomas include and are not limited to adenocarcinomas and epithelial
carcinomas.
Hematologic malignancies include leukemias, lymphomas, and multiple myelomas.
The
following are non-limiting examples of the cancers treatable with isolated
modified
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tumor cell membranes according to the methods of the present invention:
ovarian,
including advanced ovarian, leukemia, including and not limited to acute
myelogenous
leukemia, colon, including colon metastasized to liver, rectal, colorectal,
melanoma,
breast, lung, kidney, and prostate cancers. The ovarian cancers may be
adenocarcinomas
5 or epithelial carcinomas. Colon and prostate cancers are adenocarcinomas.
Leukemias
may originate from myeloid bone marrow or lymph nodes. Leukemias may be acute,
exhibited by maturation arrest at a primitive stage of development, and
chronic, exhibited
by excess accrual of mature lymphoid or myeloid cells. Stage I, II, III, or IV
cancer may
be treated according to the present invention, preferably stages III and IV,
even more
10 preferably stage III.
Mammals, particularly humans, having metastatic cancer of the foregoing type
may be treated with the membranes, the compositions and methods of the present
invention. In one embodiment of the invention, domestic animals rnay be
treated.
Prior to administration of the vaccine composition of the invention, the
subject
15 may be immunized to the hapten which is to be used to modify tumor cells
and
membranes by applying it to the skin. For example, dinitrofluorobenzene (DNFB)
may
be used. Subsequently (about two weeks later, for example), the subject may be
injected
with a tumor cell membrane composition. The composition may be administered
(such
as by reinjection) for a total of at least three and preferably at least six
treatments. In one
20 embodiment, the total number of administrations (including the initial
administration)
may be eight, and in another embodiment may be ten. The vaccination schedule
may be
designed by the attending physician to suit the particular subject's
condition. The vaccine
injections may be administered, for example, every 2 weeks, and preferably
every week.
A booster vaccine may be administered. Preferably, one or two booster vaccines
are
administered. The booster vaccine may be administered, for example, after
about six
months or about one year after the initial administration.
The immune response of the subj ect may be augmented with drugs. For example,
cyclophosphamide (C~ may be administered prior to each administration.
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21
The present invention may be used following conventional treatment for cancer,
such as surgery. In case of solid tumors, such as ovarian cancer, the tumor
may be
optimally or sub-optimally debulked. Optimally debulked refers to excising the
tumor
so that only small tumor pieces remain in the treated subject. _ Sub-optimally
debulked
refers to excising the tumor while large pieces remain in the subject. In the
case of non-
solid tumors, an appropriate blood or bone marrow sample may be collected, and
cancer
cells isolated by known techniques any reference. Excised tumors or collected
tumor
cells may be used to prepare tumor cell membranes as described above.
Tumor cell membranes may be administered by any suitable route, including
inoculation and injection, for example, intradermal, intravenous,
intraperitoneal,
intramuscular, and subcutaneous. There may be multiple cites of administration
per each
vaccine treatment. For example, the vaccine composition may be administered by
intradermal injection into at least two, and preferably three, contiguous
sites per
administration. In one embodiment of the invention, the vaccine composition is
administered on the upper arms or legs.
The effectiveness of the vaccine may be improved by administering various
biological response modifiers. These agents work by directly or indirectly.
stimulating
the immune response. Biological response modifiers of the present invention
include and
are not limited to interleukin-I2, interleukin-15 and gamma interferon. In one
embodiment, IL 12 is given following each vaccine injection. Administration of
IL 12 to
patients with inflammatory responses may cause the T lymphocytes within the
tumor
mass to proliferate and become more active. The increased T cell numbers and
functional
capacity leads to immunological destruction and regression of the tumors.
Human cancer vaccines have been developed and tested by a number of workers.
Although they can sometimes induce weak immunity to a patient's cancer, they
rarely
cause tumor regression or prolong survival. Evidence of an inflammatory
response was
surprisingly found with the vaccine of the present invention. Microscopically,
infiltration
of T lymphocytes is observed. Therefore, this approach, which increases the
inflammatory response and.the number of lymphocytes, is a significant advance
in the
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22
art. Therefore, the present invention also provided for methods of eliciting T
cells that
have at least one of the following properties: (i) eliciting T lymphocytes
that infiltrate the
tumor of a treated mammal, (ii) eliciting an inflammatory immune response
against the
tumor of the mammal, and (iii) eliciting a delayed-type hypersensitivity
response to the
tumor of the mammal when administered.
Method for Stimulating T Cells Isolated tumor cell membranes may be used
to stimulate T cells in vitro. This assay can be used to assess, for example,
whether a
therapy using a particular tumor membrane is likely to be successful. T cell
for use in
this assay may be obtained according to the following method which is
described in
humans but may be applied to any mammalian subject.
T cells are generated by administering to a patient diagnosed with cancer of a
certain type, a pharmaceutically acceptable amount of a composition comprising
hapten-modified tumor cells, tumor cell membranes, or a combination thereof.
The
composition may optionally contain an immunological adjuvant and/or a
pharmaceutically acceptable carrier. A pharmaceutically acceptable amount of a
low-
dose cyclophosphamide or another low-dose chemotherapy, such as and not
limited to
melphalan, about 5 to about lOmg/Mz, may optionally be administered preceding
the
administration of the first tumor cell composition. The haptenized composition
may
optionally be followed by administration of a pharmaceutically acceptable
amount of a
non-haptenized vaccine composition containing non-haptenized membranes, tumor
cells
or a combination thereof. A non-haptenized composition may be administered in
accordance with the methods of the present invention.
Peripheral blood lymphocytes (PBL) rnay be obtained from patients who develop
a strong delayed type hypersensitivity (DTH) reaction to hapten-modified
autologous
cells or membranes following administration thereof. The DTH reaction
preferably has
a diameter of about 10 mm, even more preferably of about greater than 10 mm. A
T cell
line may be established from PBL by repeated stimulation with hapten-modified
cancer
cells. The T cells can be isolated by known techniques, such as preparation of
single cell
suspension, filtration, depletion of monocytes and isolation of a subset
expressing a
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23
particular T cell receptor (TCR) type by causing that subset to expand in the
presence of
TCR-subtype specific antibody and/or in the presence of IL-2 and/or in the
presence of
a superantigen. The T cells of interest may be expanded in vivo since they are
collected
from infiltrates from or within the tumor which are already enriched in the T
cells of
interest.
The modified tumor cells and tumor cell membranes each have the property of
stimulating T cells. "Stimulation" for purposes of the present invention
refers to inducing
proliferation Qf T cells as well as production of cytokines by T cells in
vitro. Membranes
and tumor cells each independently have the ability to stimulate T cells.
Proliferation of
IO T cells may be detected and measured by the uptake by T cells of modified
nucleotides,
such as and not limited to 3H thymidine, 'ZSIUDR (iododeoxyuridine); and dyes
such as
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) which
stains live
cells. In addition, production of cytokines such as and not limited to IFNY,
tumor
necrosis factor (TNF), and IL-2 rnay be useful in exhibiting T cell
proliferation.
Production of cytokines may be detected and measured using tests well known in
the art.
Cytokine production should be above the background level, which is generally
above 25
picograms/ml, and is preferably above 100 picograms/ml.
T cells are lymphocytes which mediate two types of immunologic functions,
effector and regulatory, secrete proteins (lymphokines), and kill other cells
(cytotoxicity).
Effector functions include reactivity such as delayed type hypersensitivity,
allograft
rejection, tumor immunity, and graft-versus-host reactivity. Lymphokine
production and
cytotoxicity are demonstrated by T cell effector functions. Regulatory
functions of T
cells are represented by their ability to amplify cell-mediated cytotoxicity
by other T cells
and immunoglobulin production by B cells. The regulatory functions also
require
production of lymphokines. T cells produce gamma interferon (IFNY) in response
to an
inducing stimulus including and not limited to mitogens, antigens, or lectins.
In one embodiment of the invention, a T cell line may be developed as follows.
PBL (1 x 106) are mixed with autologous DNP-conjugated B lymphoblastoid cells
(1 x
1 OS) in 24 well flat bottom plates in lymphocyte culture medium. After 7 days
of culture,
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IL2100 U/ml (fetus Oncology, Emeryville, CA) is added. Expanding T cell
cultures are
maintained in medium containing IL2 and are split as needed to maintain a
concentration
of about 2 x 106 cells in a 22 mm diameter well. Every 14 days, the cultures
are
restimulated by adding autologous DNP-conj ugated B lymphoblastoid cells.
Phenotypes
may be determined by flow cytometry with a panel of monoclonal antibodies
(Becton-
Dickinson, San Jose, CA). Separation of CD8+ and CD4+ T cells is accomplished
by
indirect panning in which T cells coated with anti-CD8 or anti-CD4 monoclonal
antibodies are adhered to anti-immunoglobulin-coated dishes using standard
techniques
according to the methods of Wysocki, L. J. and V. L. Sato, Proc. Natl. AcaaL
Sci. USA,
197$, 75, 2844, incorporated herein by reference in its entirety; the adherent
cells are
isolated and expanded with DNP-modified stimulators, including and not limited
to those
set forth below, melanoma cells and (3 lymphoblastoid cells; and IL2.
Phenotypically homogeneous subpopulations of T cells are obtained, for
example,
by culturing at limiting dilution in round-bottom rnicrotiter wells in
lymphocyte culture
medium containing 2 x 105 irradiated allogeneic feeder cells, 200 U/ml IL2,
and
phytohemagglutinin. Wells with growing lymphocyte colonies are screened for
ability
to proliferate in response to DNP-modified B lymphoblastoid cells. Positive
wells are
expanded in IL2 and restimulated with autologous DNP-conjugated B
lymphoblastoid
cells every 14 days.
Peripheral blood lymphocytes (PBL) rnay be tested as responder cells. They are
suspended in lymphocyte culture medium (RPMI-1640,10% pooled human AB'' serum,
insulin-transfen~in-selenite media supplement (Sigma Chemical Co.) 2 mM L-
glutamine,
1% non-essential amino acids, 25 mM HEPES buffer, penicillin + streptomycin)
and
added to 96-well, round bottom microtiter plates at 1 x 105 cells/well.
Stimulator cells,
including: 1 ) autologous or allogeneic PBL, 2) autologous or allogeneic B
lymphoblastoid lines made by transfection with Epstein-Barr virus, 3)
autologous
cultured melanoma cells; inactivated by irradiation (5000 R), are also added.
In most
experiments, the responderatimulator ratio is preferably 1:1. The plates are
incubated
in a C02 incubator at 37°C for S days; then the wells pulsed with'Z5I-
labeled IUDR (ICN
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Radiochemical, Costa Mesa, CA) for 6 hours, harvested with an automatic
harvesting
device, and counted in a gamma counter. The mean of triplicate wells is
calculated.
Cultured T cells are also tested for a lymphoproliferative response in
accordance with the
above methods.
S PBL, obtained and cryopreserved from patients at the time of maximum DTH
reactivity to DNP-modified autologous cells, are thawed and tested for in
vitro
proliferative responses. DNFB application alone does not result in detectable
numbers
of circulating responding cells. Reactive PBL are expected to be detected
after two
injections of DNP-vaccine (day 63) and will continue to be detected throughout
the
10 period of vaccine treatment, based on prior experience with DNP-modified
melanoma
cells.
To test for cytokine production, T cells may be added to round bottom
microtiter
plates at about 1 x lOs cells/well. An equal number of stimulators (DNP-
modified
autologous B lymphoblastoid cells) is added, and supernatants are collected
after 18
15 hours incubation. Commercially available ELISA kits are used to measure
gamma
interferon (Endogen, Boston, MA; sensitivity = 5 pg/ml).
To determine the MHC-dependence of the response, stimulator cells may be pre-
incubated with monoclonal antibodies to MHC class I (W6/32) or MHC class II
(L243)
at a concentration of 10 ~,g/ml for one hour before adding responder cells.
Non-specific
20 mouse immunoglobulin at the same concentration may be tested as a negative
control.
DNP-reactive CD8+ T cells obtained by panning of the bulk population are able
to be maintained in long-term (> 3 months) culture in IL2-containing medium by
repeated stimulation with DNP-modified autologous B lymphoblastoid cells; they
retained the stable phenotype, CD3+, CDS+. Two lines of evidence suggest that
their
25 response will be MHC class I restricted: 1 ) Gamma interferon production
will be blocked
by pre-incubation of stimulator cells with anti-class I framework antibody,
but not by
anti-class II antibody, 2) The T cells will be able to respond to aIlogeneic
DNP-modified
stimulators that are matched at one or both HLA-A loci, but not to stimulators
that are
HLA-A mismatched.
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To test for gamma interferon production by T cells, lymphocytes from a
patient's
blood may be obtained. About 1,000,000 lymphocytes are mixed with DNP modified
autologous melanoma cell membranes to stimulate T cells. Every seven days, 100
U/ml
of interleukin-2 may be added. The T cells are expanded by passage. The T
cells are
then restimulated by the DNP modified autologous melanoma cell membranes. An
enriched population of T cells results which are responsive to the DNP
modified
autologous melanoma cells. Stimulation is determined by the amount of gamma
interferon production by the T cells. Generally the production of gamma
interferon at
greater than 15 picograms/ml is considered significant.
The invention is further illustrated by means of the following Examples which
is
meant to be an illustration only and is not intended to limit the present
invention to these
specific embodiments.
Example 1 In vitro Stimulation of T Cells by Isolated Melanoma Membranes
Establishment of T Cell Line Peripheral blood lymphocytes (PBL) were
obtained from a patient who developed a strong delayed type hypersensitivity
(DTH)
reaction to DNP-modified autologous melanoma cells following DNP-vaccine
administration according to the present method. PBL were separated from blood
by
density gradient centrifugation, suspended in freezing medium, such as RPMI-
1640 with
2.5% human albumin and DMSO, frozen in a control-rate freezer, and stored in
liquid
nitrogen until use (Sato et al.,1995).
A T cell line was established from these PBL by repeated stimulation with
DNP-modified autologous melanoma cells (DNP-Mel) and maintained with
recombinant
interleukin 2 (IL-2) (Sato et al.,1995).
Melanoma Cells Melanoma cells were enzymatically extracted as described
above from metastatic masses surgically removed from the same patient and
cryopreserved by a previously described method (Sato et al., 1997). An
autologous
melanoma cell line was established from the melanoma cell suspension. Briefly,
melanoma cells were enzymatically dissociated from metastatic masses and
suspended
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in tissue culture medium (RPMI-1640 with fetal calf serum or human serum) and
added
to tissue culture plates. After several days, non-adherent tumor cells were
removed and
fresh medium was added. After several weeks, adherent melanoma cells began to
rapidly
proliferate. When the cells grew to confluence on the culture plate, they were
split by
removing the cells with EDTA and adding to fresh tissue culture plates.
The melanoma cell line cells were modified with DNP by the method of Miller
and Claman. This involves a 30 minute incubation of tumor cells with
dinitrnfluorobenzene (DNFB, Sigma Chemical Co.) under sterile conditions,
followed
by washing out of excess DNFB with Hanks solution. The DNP-modification was
confirmed by flow cytometry with a mouse monoclonal anti-DNP antibody (SPE-7;
Sigma Immunochemicals, St. Louis, MO) ( 100 % of the cells were shown to be
modified
with DNP).
As an alternative procedure, cryopreserved melanoma cells were modified
with DNP as described above without the intervening step of establishing a
cell line.
Cell Membrane Extraction Cell membranes were extracted from DNP-
modified melanoma cells (DNP-Mel) by the methods of Heike et al. Briefly,
DNP-modified cells were lysed by hypotonic shock in 5 volumes of 30 mM sodium
bicarbonate buffer, 1 mM phenyl methyl sulfonyl fluoride (PMSF), and by Dounce
homogenization (10-20 strokes). Residual intact cells and nuclei were removed
by
consecutive centrifugation at 1,000 g for 5 minutes, until supernatant was
free of nuclei
and cells. Then, the membranes were pelleted by centrifugation at 100,000 g
for 90
minutes. The total membranes in the pellet were resuspended in 8 % sucrose, 5
mM Tris,
pH 7.6 at 10' cell equivalent units (i.e., membranes extracted from 10'
cells)/ml and
frozen at -80° C until use.
As an alternate procedure, cell membranes were isolated from unmodified
melanoma cells in an identical manner as described above. The membranes were
suspended in Hanks solution without albumin at various cell equivalent
concentrations
(from 105 -109 cell equivalents/ml). Then DNFB was added as described above.
Then,
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28
the membranes were pelleted by centrifugation at 100,000 g for 90 minutes and
washed
twice with saline.
Cvtokine Production in Response to Membrane Preparation T cell responses
induced by DNP-modified melanoma membranes were measured by IFN-gamma
production. The T cells obtained from patients PBL were plated into a 96 round-
bottom
well plate at 105 cells/well in 100 ~l culture medium (RAMI 1640 supplemented
with 10
human AB serum, 2 mM L-glutamate, 100 mg/m1/100 U/ml streptomycin/penicillin,
mM HEPES, 1 % non-essential amino acids). Various amounts (about 105 to about
1 O8 cell equivalents) of cell membranes were added into each well and
additional culture
10 medium was added to make the total volume of each well to 250 ~tl.
Supernatants were collected for IFN-gamma assay after 18 hour incubation. The
concentration of IFN-gamma in supernatants was measured by a commercially
available
ELISA kit (Endogen, Boston, MA; sensitivity = 5 pg/ml).
Significant IFN-gamma production by T cells (750 pg/ml) was detected after
incubation with autologous DNP-Mel membranes. The production of IFN-gamma by T
cells was related to the amount of coincubated DNP-Mel membranes. No
significant
response to unmodified Mel membranes was elicited. Two T cell sublines were
developed by enriching for CD4+ and CD8+ T cells by the positive panning
technique.
Each subline responded to DNP-Mel membranes by IFN-gamma production. The
response of the CD4+ T subline to DNP-Mel membranes was blocked by antibody to
MHC class II, and the response of the CD8+ subline was blocked by antibody to
MHC
class I (73% and 80% blocking, respectively).
These results show that hapten-modified tumor cell membranes may be
successfi~lly used to vaccinate patients in need of tumor treatment.
Example 2: Treating Ovarian Stage III Cancer with Modified Tumor Cell
Membranes
Patients may be initially treated according to standard medical practice
(debulking
surgery followed by chemotherapy). After the completion of chemotherapy, a six
week
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29
course of treatment with a vaccine containing ovarian cancer cell membranes
modified
with the hapten, dinitrophenyl (DNP) may be administered. Low dose
cyclophosphamide
may be administered prior to the first injection. After the completion of the
course of
treatment, patients may be tested for delayed type hypersensitivity to
carcinoma cell
membranes, both DNP-modified and unmodified. In vitro studies may be performed
with cryopreserved lymphocytes extracted from metastatic tumors and/or
separated from
peripheral blood.
Patients receiving surgical debulking or patients exhibiting tumor reduction
by
chemotherapy, for example, may be selected for treatment. The mass of tumor
excised
from each patient may be sufficient to obtain at least 100x106 viable tumor
cells. Such
patient may receive chemotherapy, such as carboplatin and taxol, and are
preferably
clinically tumor-free following completion of chemotherapy (i.e., normal
physical
examination and CT studies and serum CA-125 <35 IU/L).
Patients may be excluded from receiving the treatment of the present invention
based upon: insufficient quantity of tumor cells for preparing a vaccine and
skin-testing
(< 100 x 106 cells), Karnovsky performance status less than 80, major field
radiation
therapy within the preceding 6 months, current administration of systemic
corticosteroids, hematocrit <30% or WBC <3000, age <18, active autoimmune
disease,
active, serious infection, another active maligmancy, evidence of infection
with hepatitis
B virus (circulating antigen) or with HIV (circulating antibody), or inability
to provide
informed consent.
Patients undergo surgical resection of the tumor and debulking of metastases.
Patients who undergo either optimal or suboptimal debulking may be eligible.
Tumor
tissue may be delivered to the laboratory and processed to obtain membranes.
The
membranes may be cryapreserved and stored in liquid nitrogen.
Both syngeneic and allogeneic tumor cell membranes may be prepared and used
as described in this specification.
Beginning within six (6) weeks after surgery, patients may begin chemotherapy,
such as with carboplatin or cisplatin + taxol, according to the following
dosage-schedule:
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carboplatin AUC 7.5 or cisplatin 75 mg/M2 - every 3 weeks, taxol 175 mg/M2
i.v. over
3 hours - every 3 weeks. Six cycles of chemotherapy may be administered. Any
other
chemotherapy may be administered.
Approximately, four weeks after completion of chemotherapy, patients may
5 undergo a metastatic evaluation to include computer tomography (CT) chest-
abdomen-
pelvis. Only patients with no evidence of recurrent carcinoma may be eligible
for
vaccine treatment. Patients with elevated serum level of-CA125 may be eligible
providing that CT studies are negative for recurrence. The tumor cell membrane
therapy
may be started at least 4 weeks after, and no more than 12 weeks after, the
last
10 administration of chemotherapy.
On day -7, patients may be skin-tested with: 1) autologous ovarian cancer
cells
or membranes modified with DNP, 2) diluent (Hanks balanced salt solution with
0.1
human albumen, and 3) PPD intermediate. DTH reactions may be measured on day -
5.
On day 0, patients may receive cyclophosphamide 300 mg/M2 as a rapid i.v.
infusion.
15 Three days later they may be injected intradermally with a tumor cell
membrane
composition and this may be repeated weekly for six (6) weeks. Vaccines may
consist
of DNP-modified, ovarian cancer cell membranes mixed with BCG. Vaccines may be
injected into the upper arm. If for some reason a left axillary lymph node
dissection had
been performed, the right arm may be used.
20 Two and a half weeks after the sixth vaccine, patients may undergo clinical
evaluation, consisting of CBC, SMA-12, CA125, and chest x-ray. They may be
tested
for DTH to the following materials: autologous carcinoma cells, both DNP-
modified and
unmodified; autologous peripheral blood lymphocytes, both DNP-modified and
unmodified; diluent; and PPD intermediate. Also, they may be tested for
contact
25 sensitivity to dinitrofluorobenzene (DNFB).
Patients who remain relapse-free may be given a seventh (booster) vaccine at
the
six month point (measured from beginning the vaccine program). For each
patient at
least one cryopreserved vial of tumor cell membranes may be saved for the six-
month
booster injection. If the number of cells available is anticipated to be
insufficient for 6
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31
weekly vaccines plus the six-month booster, then the initial course of weekly
injections
may be reduced to 5. Another booster vaccine may be administered in one year,
but only
if a sufficient number of cells is available. Just prior to the one-year
booster, patients
may be skin-tested with autologous tumor cells or membranes to determine
whether their
previous level of immunity has been maintained.
Example 3. Treating Melanoma Cancer with Modified Tumor Cell Membranes
Tumor masses may processed as previously described. Briefly, cells may be
extracted by enzymatic dissociation with collagenase and DNase and by
mechanical
dissociation. Cell membranes may be isolated as described in this
specification, and
frozen in a controlled rate freezer, and stored in liquid nitrogen until
needed. On the day
that a patient is to be treated, the membranes may be thawed, washed, and
resuspended
in Hanks balanced salt solution without phenol red. Modification with DNP may
be
performed by the method of Miller and Claman (1976). This involves a 30 minute
incubation of tumor cells with dinitrofluorobenzene (DNFB) under sterile
conditions,
1S followed by washing with sterile saline.
The vaccine composition may contain a minimum of 2.5x106 c.e. trypan-blue-
excluding melanoma cell membranes, and a maximum of 7.5x106 c.e. melanoma cell
membranes, suspended in 0.2 ml Hanks solution. Each vaccine treatment may
consist
of three inj ections into contiguous sites.
The freeze-dried material may be reconstituted with 1 ml sterile water or
phosphate buffered saline, pH 7.2 (PBS). Appropriate dilutions may be made in
sterile
buffered saline. Then 0.1 ml may be drawn up and mixed with the vaccine just
before
injection. The first and second vaccines may be mixed with 0.1 ml of a 1:10
dilution of
Tice BCG ("Tice-1 "). BCG is a Tice strain (substrain of the Pasteur Institute
strain)
obtained from Organon Teknika Corporation (Durham, NC). The third and fourth
vaccines may be mixed with 0.1 ml of a 1:100 dilution ("Tice-3 "). The fifth
and sixth
and booster vaccines may be mixed with 0.1 ml of a 1:1000 dilution ("Tice-5").
The
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32
ideal vaccine reaction is an inflammatory papule with no more than small (<
Smm)
central ulceration.
Skin testing may be performed by the intradermal injection of 0.1 ml of test
material on the forearm, and DTH is assessed at 48h by measuring the mean
diameter of
induration. The following materials may be tested: 1 ) 1 xl 06 autologous
melanoma cell
membranes cells unmodified and modified with DNP; both enzymatically-
dissociated
(TCE) and mechanically-dissociated (TCM) tumor cells may be used; 2) 3x106
autologous peripheral blood lymphocytes unmodified and modified with DNP; 3)
Hanks
solution; and 4) PPD-intermediate strength. Also, contact sensitivity to DNFB
may be
tested by applying 200 ~g DNFB to the skin of the ventral surface of the upper
arm and
examining the area for a circle of indwation at 48 hours. The full battery of
DTH tests
may be performed following the six week course of vaccine administration. Pre-
treatment DTH testing may be limited to DNP-modified melanoma cell membranes,
PPD, and diluent. This strategy is designed to avoid: 1 ) sensitizing patients
to DNP-
modified lymphocytes and 2) tolerizing patients by injection of unmodified
tumor cells.
All patients may have blood collected for separation and cryopreservation of
lymphocytes and serum each time skin-testing is performed. Periodically, these
may be
tested for: response to autologous cancer cells, as measwed by proliferation,
cytokine
release, and cytotoxicity.
Patients may be evaluated for metastatic disease before vaccine therapy
begins.
After the end of the first eight weeks of vaccine therapy, evaluations are
performed every
three months. Evaluations rnay continue through year two, every fow months in
year
three, and every six months thereafter. Physical examination and routine
bloodwork
(CBC, SMA-12, and CA125) may be performed with each evaluation. CT of the
chest-
abdomen-pelvis may be performed prior to the administration of vaccine, at 6
months and
12 months (before vaccine boosters), and then as clinically indicated. Relapse-
free and
total swvival may be measwed. All patients may be followed for at least five
years or
until time of death.
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33
Patients are expected to develop a local reaction to BCG, consisting of a
draining,
tender pustule that heals in 2-3 months leaving a smallpox vaccination-like
scar. As
patients develop sensitivity to BCG, the intensity of these reactions may
increase.
Anaphylaxis, other allergic phenomena, and auto-immunity have never been
observed
in haptenized-vaccine patients.
Reactions at the vaccine sites may be graded as follows: 0 - no symptoms; 1 -
itching or discomfort, but no interference with arm movement or normal
activity; 2 -
discomfort causing interference with arm movement, but not requiring
modification of
normal activity; 3 - discomfort causing major interference with arm movement
and
requiring modification of normal activity; and 4 - discomfort causing
inability to use the
extremity for normal activity.
Cyclophosphamide may be reconstituted in sterile water and the proper dosage
may be administered by rapid i.v. infusion. Typically, about one third of
patients may
experience nausea and about 10% may have vomiting after low dose
cyclophosphamide.
Leukopenia, alopecia, and cystitis do not occur at this dose. It is expected
that this
protocol be associated with a lower incidence of nausea and vomiting, since
cyclophosphamide rnay be administered only once in association with the final
vaccine
inoculation.
Patients may be observed following injection of the vaccine. Patients
experiencing unexpected symptoms or signs are instructed to contact the
physician to be
evaluated immediately. Fever that causes discomfort may be treated with
acetaminophen. Nausea caused by low dose cyclophosphamide may be treated with
oral
prochlorperazine (Compazine). If severe local reactions (> 5 mm ulceration)
occur at the
vaccine site, subsequent doses of BCG may be reduced (see above).
Patients who are relapse-free at the 1 year evaluation may receive a final
booster
injection of vaccine. Then their condition may be followed without further
treatment.
Patients who develop metastases may be taken off study and treated as
clinically
indicated (usually surgery or chemotherapy).
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34
An efficacy study to determine whether DNP-vaccine prolongs relapse-free
and/or
total survival in these patients may also be conducted. Survival parameters
(Kaplan-
Meier method} may be measured.
All Thomas Jefferson University, NIH, and FDA regulations regarding informed
consent are be followed in regard to informed consent.
Our prior studies of DNP-modified autologous vaccine for melanoma using
hapten-modified melanoma cells showed the following results:100% of patients
(N=60)
developed a positive DTH response (z Smm diameter of induration) to DNP-
modified
autologous tumor cells following treatment, and 85% developed a large positive
response
(s l Omm diameter of induration). Similar success is expected with a vaccine
of DNP-
modified autologous carcinoma membrane vaccine, i. e. , patients are expected
to develop
DTH to DNP-modified and to unmodified autologous carcinoma cell membranes.
Various modifications of the invention in addition to those shown and
described
herein will be apparent to those skilled in the art from the foregoing
description. Such
modifications are also intended to fall within the scope of the appended
claims.
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U.S. Patent No. 5,290,551
U.S. Patent application Serial No. 08/203,004
U.S. Patent application Serial No. 08/479,016
U.S. Patent application Serial No. 08/899,905
U.S. Patent application Serial No. 08/942,794
PCT US96/09S 11
