Note: Descriptions are shown in the official language in which they were submitted.
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A METHOD OF INDUCING AN ANTI-TUMOR RESPONSE
AGAINST A LUNG METASTASIS IN A MELANOMA PATIENT
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority pursuant to 35 U.S.C. ~119 based upon
Provisional Patent Application Serial No. 60/081,256 filed April 9,1998, the
entire disclosure
of which is hereby incorporated by reference.
REFERENCE TO GOVERNMENT GRANTS
The invention described herein was made in the course of work under a grant
or award from the US Public Health Services IVIH grant no. CA29348. The United
States
Government may have certain rights in this invention.
BACKGROUND OF THE INVENTION
In the 1960's, certain theories that tumor cells bear specific antigens (TSA)
not
present on normal cells, and that the immune response to these antigens might
help reject a
tumor, were advanced. 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 determinants, although not known, were termed "helper
determinants." At the time, speculations were made that compounds such as, for
example,
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 to act as helper
determinants. Clinically,
the hope was that an immunologic reaction would occur against the helper
determinant, as a
consequence of which the reaction to the accompanying TSA would be increased,
and tumor
cells, which would otherwise be tolerated, destroyed.
Fujiwara et al., J. Immunol.,1984,131, 1571 showed that mouse tumor cells
conjugated with the hapten trinitrophenyl (TNP), could induce systemic
immunity against
unmodified tumor cells in a murine system, provided that the mice were first
sensitized to the
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hapten in the absence of hapten-specific suppressor T cells. Spleen cells from
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 (10 mm) tumors by in situ haptenization of tumor cells;
subsequently, these
animals were specifically resistant to challenge with unconjugated tumor
cells. However,
these results could not show whether haptenized cells isolated from a human
spontaneous
tumor would be effective in immunotherapy and be capable of inducing tumor
regression.
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 ofmice 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 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
speculated 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 mg/M2) or low dose (300 mg/MZ), 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 ofperipheral blood lymphocyte non-specific
T suppressor
function (Berd et al., CancerRes.,1984, 44, 5439; CancerRes.,1987, 47, 3317),
possibly by
depleting CD4+, CD45R+ suppressor inducer T cells (Berd et al., Cancer Res.,
1988, 48,
1671). The anti-tumor effects of this immunotherapy regimen appear to be
limited by the
excessively long interval between the initiation ofvaccine administration and
the 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 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.
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.
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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 acute
myelogenous leukemia (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
autologous, leukemia cells were used; 2) the dose of leukemia cells in the
vaccine was
excessive (up to 1 O9 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).
Use of conventional therapies to treat human cancer has been generally
unsuccessful. Administration of vaccine compositions also failed to reliably
induce the
development of cell-mediated immunity until the work of the present inventors.
Hence, there
is a continued need in the art for novel methods of treating cancer, and
particularly cancer
metastasis. Applicants have now discovered an effective method for inducing an
anti-tumor
response, and particularly tumor regression, in patients suffering from
metastatic melanoma.
SUMMARY OF THE INVENTION
The present invention is directed to a method of inducing an anti-tumor
response against a melanoma by administering an effective amount of a
composition
comprising at least one of the following: (i) a hapten-modified syngeneic
mammalian
melanoma cell substantially in a no growth phase, (ii) a hapten-modified
melanoma cell
membrane, (iii) a peptide isolated from said hapten-modified melanoma cell or
membrane and
(iv) a T cell capable of mediating an anti-tumor response such as for example
regression of
a melanoma. The melanoma treated according to the present invention includes
metastatic
melanoma which may be lung, lymph node or subcutaneous metastasis, and is
preferably a
lung metastasis. The lung metastasis to be treated according to the invention
is preferably a
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small lung metastasis. In one preferred embodiment of the invention, a
melanoma metastasis
is localized to the lung of the treated mammal. The invention is further
directed to a
melanoma cell, an isolated melanoma cell membrane, a peptide isolated from
such cell or
membrane, a T cell having a property of inducing an anti-tumor response, a
composition
5 containing such cell, membrane, peptide, T cell or combinations thereof, as
well as the
methods for their isolation and preparation. The melanoma cell or membrane,
which may be
hapten modified, may be syngeneic or allogeneic. The syngeneic melanoma cell
or membrane
may be autologous. The melanoma cell membrane is preferably a tumor cell
plasma
membrane.
Accordingly, in one aspect, the present invention is directed to a method of
inducing an anti-tumor response against a melanoma metastasis by administering
to a
mammal, preferably a human, a composition comprising a therapeutically
effective amount
of at least one of the following: (i) a hapten-modified syngeneic mammalian
melanoma cell
substantially in a no growth phase, (ii) a hapten-modified melanoma cell
membrane, (iii) a
peptide isolated from said hapten-modified melanoma cell or membrane and (iv)
a T cell
capable of mediating an anti-tumor response.
The present invention also relates to inducing at least one of the following
anti-
tumor responses: tumor necrosis, tumor regression, tumor inflammation, tumor
infiltration by
activated T lymphocytes, stable disease and prolongation of patient survival.
In another aspect, the invention relates to a method of inducing an anti-tumor
response against a lung metastasis, which is preferably a complete or a
partial regression of
the metastasis, and/or prolongation of survival.
In yet another aspect, the present invention relates to an isolated mammalian,
preferably human, melanoma cell or membrane modified with a hapten, the
peptides isolated
from such cells and membranes, and T cells capable of mediating an anti-tumor
response, as
well as compositions thereof.
In yet another aspect, the invention provides for a vaccine composition and
dosage forms containing a therapeutically effective amount of a mammalian,
preferably
human, melanoma cell or membrane modif ed with a hapten, the peptides isolated
from such
cells and membranes, T cells, or combinations thereof adapted for
administration to a
mammal which suffers from a metastatic melanoma, preferably lung metastasis.
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In another aspect of the invention, the composition contains an adjuvant, such
as, for example, Bacillus Calmette-Guerin (BCG), QS-21, detoxified endotoxin
and cytokines
such as interleukin-2, interleukin-4, gammainterferon (IFN-~y), interleukin-
12, interleukin-15
and GM-CSF.
Brief Description of the Drawings
Figure 1 A represents Patient # 1 - chest x-ray, July, 1991, pre-vaccine:
shows multiple small
lung nodules, best seen in left lower lobe
Figure 1B represents Patient #1- chest x-ray, September, 1991, pre-vaccine:
shows increased
size of multiple lung nodules, best seen in left lower lobe
Figure 1 C represents Patient # 1 - chest x-ray, January, 1992, post-vaccine:
shows regression
of multiple lung nodules
Figure 1D represents Patient # 2 - CT, January, 1997, pre-vaccine: 1 cm
diameter lung
metastasis left lung adjacent to aorta
Figure lE represents Patient #2 - CT, November, 1997, post-vaccine: regression
of lung
metastasis noted on D
Figure 1 F represents Patient #3 - CT, April, 1996, pre-vaccine: approximately
2 cm diameter
metastasis in right lower lobe adjacent to hear boarder.
Figure 1 G represents Patient #3 - CT, April, 1998, post-vaccine: regression
of approximately
2 cm diameter metastasis noted on F
Figure 1H represents Patient # 4 - CT, January, 1997, pre-vaccine:
approximately 1 cm
diameter metastasis in periphery of right lower lobe
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Figure lI represents Patient #4 - CT, January, 1998, post-vaccine: regression
of metastasis
noted in H
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.
The present invention is directed to a method of inducing an anti-tumor
response against a melanoma, preferably a lung metastasis, by administering an
effective
amount of a composition comprising at least one of the following: (i) a hapten-
modified
syngeneic mammalian melanoma cell substantially in a no growth phase, (ii) a
hapten-
modified melanoma cell membrane, (iii) a peptide, isolated from said hapten-
modified
melanoma cell or membrane and (iv) a T cell capable of mediating an anti-tumor
response
such as, for example, regression of a metastasis.
The cells, membranes, peptides and T cells of the invention have the property
of inducing at least one of the following anti-tumor responses: tumor
necrosis, tumor
regression, tumor inflammation, tumor infiltration by activated T lymphocytes,
delayed-type
hypersensitivity response, and prolongation of patient survival. The cells,
membranes,
peptides, and compositions thereof are capable of 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.
The melanoma cells, membranes, peptides and T cells of the invention and
compositions thereof may be used for treating melanoma in a mammal, preferably
a human,
including treating metastatic and primary melanoma. Metastatic melanomas may
include
lymph node, lung and subcutaneous metastasis. Stage I, II, III, or IV cancer
may be treated
with the preparations, compositions and methods of the present invention,
preferably stages
III and IV. The lung metastasis to be treated according to the invention is
preferably a small
lung metastasis. For purposes of the present invention, a "small" lung
metastasis is less than
about 2 cm in diameter, preferably less than about 1.5 cm in diameter and most
preferably less
than about 1 cm in diameter. In one preferred embodiment of the invention, a
melanoma
metastasis in a mammal is localized to the lung of said mammal. Lung
metastases to be
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treated according to the present invention may be single or multiple nodules.
In one
embodiment, the present invention is used to treat domestic animals such as,
for example,
members of feline, canine, equine and bovine families.
It will be understood that any disclosure in this specification with respect
to
use of isolated melanoma cells equally applies to use of melanoma cells,
membranes,
peptides, T cells or to a combination thereof.
Preparations of the Invention and Compositions thereof
Melanoma cells, membranes, peptides and T cells are herein collectively
referred to as the preparations of the invention. Furthermore, a term
"extract" may be used
to collectively refer to disrupted melanoma cells, isolated melanoma cell
membranes and
melanoma cell peptides.
The isolated melanoma cell, membrane or peptide of the present invention are
prepared from mammalian, preferably human, melanoma cells. The source of
melanoma cells
may be lung, lymph node or subcutaneous tumor masses including metastatic
masses. In one
embodiment of the invention, these materials are isolated from a melanoma of
an animal from
a feline, canine, equine or bovine family. T cells of the invention are
isolated from tumor
masses, such as for example metastatic lung and lymph node masses, and may be
expanded
in vitro.
For the purposes of the present invention, a melanoma cell is intended to
include both whole and disrupted melanoma cells. The melanoma cells for use in
the vaccine
composition of the invention, as well as those melanoma cells from which
membranes and
peptides are isolated, may be live, attenuated, or killed cells. Melanoma
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. As used herein, the phrase "cells in a state of
no growth" means
live, attenuated or killed cells, cells in GO phase, whole or disrupted (or
both whole and
disrupted), i.e., 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.
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Melanoma cells may be irradiated, for example at 2500 R. Melanoma cell
membranes and
peptides may be isolated from either melanoma cells in a no growth state or
melanoma cells
that are capable of growing and dividing in vivo. Preferably, in the latter
case, the melanoma
cell membrane or peptide preparation is not contaminated with melanoma cells
that are
capable of dividing in vivo.
Melanoma cells, membranes and peptides are isolated from the melanoma cells
which may be of lymph node, lung and subcutaneous origin. Preferably, the
melanoma cells
originate from the same subject who is to be treated. The melanoma cells are
preferably
syngeneic (e.g. autologous). To be defined as "syngeneic," the melanoma 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 and the patient is generally sufficient. Additionally, there may be
genetic identity
between a particular antigen on the melanoma cell and an antigen present on
the patient's
tumor cells. Genetic identity may be determined according to the methods known
in the art.
For purposes of the present invention, a melanoma 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 is also within the meaning of the term "syngeneic"
melanoma cell.
However, such cells may also be referred to as "MHC-identical" or "MHC-
compatible."
Melanoma cells from mammals of the same species that differ genetically, such
as allogeneic
cells, may also be used for the preparation of melanoma cell membranes and
peptides of the
invention. The melanoma 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.
Melanoma cell membranes may include all cellular membranes, such as outer
membrane, nuclear membranes, mitochondrial membranes, vacuole membranes,
endoplasmic
reticular membranes, golgi complex membranes, and lysosome membranes. In one
embodiment of the invention greater than about SO% of the membranes are
melanoma cell
plasma membranes. Preferably, greater than about 60% of the membranes consist
of
melanoma cell plasma membranes, with greater than about 70% being more
preferred, 80%
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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
5 than about 100 cells and/or nuclei in about 2 x 108 cell equivalents (c.e.)
of membrane
material. A cell equivalent is that amount of membrane isolated from the
indicated number
of cells. An isolated melanoma cell membrane which is substantially free of
cells and/or
nuclei may contain lymphocytes and/or lymphocyte membranes.
Preferably, the isolated melanoma cell membranes are the outer cell
10 membranes, i.e., melanoma 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.
The isolated melanoma cells as well as melanoma cell membranes may be
modified, for example, with a hapten. Such modified melanoma cells and
membranes 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.
The peptides of the present invention may be isolated from a hapten modified
melanoma cell or membrane. The peptides of the present invention may be hapten-
modified.
For the purpose of the present invention, peptides are compounds of two or
more amino acids.
Peptides will preferably be of low molecular weight, of about 1,000 kD to
about 10,000 kD,
more preferably of about 1,000 to about 5,000. The peptide may preferably be
about 8 to
about 20 amino acids, in addition the peptide may be haptenized. Peptides may
be isolated
from the cell surface, cell interior, or any combination'of the two locations.
The extract may
be particular to type of cancer cell (versus normal cell}. The peptide of the
present invention
includes and is not limited to a peptide which binds to the major
histocompatibility complex
or to a cell surface-associated protein such as a heat shock protein. This
peptide may be a
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protein encoded by cancer oncogenes or mutated anti-oncogenes. Both individual
peptides
and small peptide-containing fractions of a melanoma cell or membrane are
within the scope
of the present invention. A small peptide-containing fraction is that fraction
of melanoma
peptides which has the ability to stimulate T cells and contains peptides that
are isolated
together in a particular purification step. For example, a pool of samples
eluted from an
HPLC column may represent such a small peptide-containing fraction.
The useful peptides of the invention have a property of stimulating T
lymphocytes. The ability of peptides to stimulate T cells can be identified by
using standard
assays, such as by measuring uptake of labelled nucleotides by T cells or by
measuring
production of cytokines such as and not limited to gamma interferon, tumor
necrosis factor
(TNF), and IL-2.
Allogeneic melanoma cell membranes and peptides isolated from allogeneic
melanoma cells 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
melanoma cell membranes or peptides originating from a source other than the
patient's own
melanoma. Syngeneic antigen-presenting cells process allogeneic membranes or
peptides
such that the patient's cell-mediated immune system may respond to them.
A melanoma cell, membrane or peptide (modified or un-modified) as referred
to in this specification includes any form in which such preparation may be
stored or
administered such as, for example, resuspended in a diluent, as a pellet,
frozen or lyophilized.
Mammalian T cells capable of mediating regression of a tumor or another
specific immune response directed against a tumor (as evidenced, for example,
by T cell
expansion in vitro, or T cell cytotoxicity) are also within the scope of the
present invention.
The T cell may be elicited in vivo by immunization of the patient with a
composition
comprising haptenized cells of the same tumor type or may be produced from
such T cells by
cloning in vitro. In one preferred embodiment, the isolated human T cell
expresses a Vii
receptor, which may be V~31, VAS, V(313, or V~314. The T cells to be used in
the method of
the present invention, may be cytotoxic T lymphocytes (CTL), or more
generally, tumor
infiltrating lymphocytes (TIL), i.e., a type of effector lymphocyte associated
with cell
mediated immunity directed against the tumor. The T cells isolated from a
patient may be
CD8+ T cells and MHC class I specific.
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The cells, membranes, peptides and T cells 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, cells,
membranes, peptides
or T cells may be used alone or co-administered. For purposes of the present
invention, co-
y administration includes administration together and consecutively. Further,
the preparations
of the invention 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-CSF. They 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 melanoma
cell, membrane, peptide, T cell of the invention (modified or unmodified) or a
combination
thereof and a pharmaceutically acceptable Garner or diluent, such as and not
limited to Hanks
IS solution, saline, phosphate-buffered saline, sucrose solution, and water.
In general, the
pharmaceutically-acceptable Garner 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 an isolated hapten-
modified
melanoma cell, membrane, peptide, T cell or a combination thereof. For
purposes of this
disclosure, "an effective amount" is the amount necessary to achieve a desired
result. For
example, in a method for inducing an anti-tumor response, "an effective
amount" means the
amount of melanoma cell, membrane, peptide or T cell that has the property of
causing at least
one of the following: tumor necrosis, tumor regression, tumor inflammation,
tumor infiltration
by activated T lymphocytes, and prolongation of patient survival. Similarly,
in a method for
stimulating T cells in vitro, "an effective amount" is that amount of cells,
membranes or
peptides that results in T cell stimulation.
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The vaccine composition may contain, for example, at least 104 melanoma
cells per dose, preferably at least 105 cells, and most preferably at least
106 cells. A dose is
that amount of the vaccine composition that is administered in a single
administration. With
respect to melanoma membranes, the vaccine composition may contain, for
example, at least
104 c.e. of isolated membranes per dose, preferably at least 105 c.e., and
most preferably at
least 106 c.e. In one embodiment, the vaccine composition contains from about
105 to about
2.5 x 10' cells, c.e. membranes, or a combination thereof per dose, more
preferably about 5
x 106 cells/c.e. In another embodiment, the vaccine composition contains
melanoma cell
peptides isolated from about 105 to about 2.5 x 10' cells, c.e. membranes, or
a combination
thereof per dose, more preferably from about S x 1 O6 cells/c. e. The amount
of the tumor cells,
membranes or peptides of the invention to be used generally depends on such
factors as the
affinity of the compounds for cancerous cells, the amount of cancerous cells
present and the
solubility of the 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
administration. Alternatively, the dosage form may contain the isolated
preparations of the
invention thereof to be reconstituted at the time of the administration with,
for example, a
suitable diluent.
Hapten
The melanoma cells, melanoma cell membranes and melanoma peptides of the
present invention may be used as modified, unmodified, or a combination of
modified and
unmodified. 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 (Jung et al., Eur. J.
Immunol. 1991
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21:1303); nickel (Pistoor et al., J. Invest. Dermatol.1995105:92); arsenate
(Nalefski and Rao,
J. Immunol. 1993 150: 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 -COOH groups.
Additionally, hapten that
can bind to hydrophobic amino acids such as tyrosine and histidine via diazo
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, Cellularlmmunol.1980 54:241 ) and combinations
thereof.
In view of the present disclosure, a 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.
Adjuvant
In one embodiment, the melanoma cell, melanoma cell membrane, peptide or
T cell is administered with an immunological adjuvant. The adjuvant has the
property of
augmenting an immune response to the preparations of the present invention.
Representative
examples of adjuvants are 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 (1FN-~), 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 the Preparations of the Invention
The melanoma 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),
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U.S. Patent No. 5,290,551, and applications U.S. Serial Nos. 08/203,004,
08/479,016,
08/899,905, 08/942,794, or corresponding PCT application PCT/LJS96/09511, each
ofwhich
is incorporated herein by reference in its entirety. Briefly, the cells are
extracted by
dissociation, such as for example by enzymatic dissociation with collagenase
and DNase, by
5 mechanical dissociation in a blender, by teasing with tweezers, using mortar
and pestle,
cutting into small pieces using a scalpel blade.
Melanoma cell membranes are prepared from melanoma 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
10 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.
15 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 rnay be centrifuged at
low speed, such
as for example, at about 500-2,000 g for about five minutes. The separation
procedure may
be such that less than about 100 cells and/or nuclei remain in about 2 x 108
cell equivalents
(c.e.) of membrane material. The postnuclear supernatant containing membranes
may be
pelleted by ultracentrifugation, for example 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 cultured under
standard cell
culture conditions. 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.
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
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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 melanoma cell membranes may be prepared as described above.
However, prior to administration 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 melanoma cell membranes
originating from
a source other than the patient's own tumor. Allogeneic melanoma 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
washed 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 Siena et al.,1995. Briefly,
blood is obtained,
for example by venipuncture or by leukapheresis, from the patient to be
immunized.
Alternatively, bone marrow may be obtained. 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. The
purified leukocytes
may be cultured and expanded in tissue culture medium (for example, RPMI-1640
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), TIVF (tumor necrosis factor), interleukin 3 (IL3), FLT3 Iigand and
granulocyte colony
stimulating factor (G-CSF).
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The antigen-presenting cells isolated and expanded in culture may be dendritic
cells, monocytes, macrophages, and Langerhans cells, for example.
The peptides of the invention may be isolated from cells according to an
established technique of Rotzschke et al., Nature, 1990, 348, 252, the
disclosure of which is
hereby incorporated by reference in its entirety. The cells are treated with a
weak acid, such
as and not limited to trifluoroacetic acid. The cells are then centrifuged and
the supernatant
is saved. Compounds having a molecular weight greater than 5,000 are removed
from the
supernatant by gel filtration (G25 Sepharose, Pharmacia). The remainder of the
supernatant
is separated on a reverse-phase HPLC column (Superpac Pep S, Pharmacia LKB) in
0.1 %.
trifluoroacetic acid (TFA) using a gradient of increasing acetonitrile
concentration; flow rate
1 ml/min, fraction size 1 ml. Fractions containing small peptides are obtained
by HPLC
according to the method of Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ( 1989),
concentrated, and
frozen. The fractions are screened for immunological~ activity by allowing
them to bind to
autologous B lymphoblastoid cells which are then tested for ability to
stimulate melanoma-
specific T lymphocytes.
T cells are isolated from biopsies by known techniques, such as preparation of
single cell suspension, filtration, depletion of monocytes and isolation of a
subset expressing
a particular TCR type by causing that subset to expand in the presence of a
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 vitro using methods known in the art.
Specifically, T lymphocytes may be prepared from tumors as follows. Single
cell
suspensions may be prepared from tumors by digestion with a mixture of 0.14%
collagenase,
0.03% DNase and optionally 2.5 U/ml hyaluronidase (Sigma Chemical CO., St.
Louis, MO)
for 3 hours at room temperature. The cells may be filtered through a layer of
no. 100 nylon
mesh then washed and resuspended in buffer, e.g., Hanks Buffered Saline. The
mixture of
cells may be depleted of monocytes by panning of the mixture over plastic
dishes in a final
volume of 2ml RPMI-1640 supplemented with 10% pooled human serum and cultured
for one
week. T cells can be expanded by exposure to antibodies and/or an to
immunostimulatory
cytokine (such as, IL-2) andlor superantigens as disclosed e.g., in
PCT/US93/05213. Activity
of the T cells may be measured after four to five weeks of in vitro
stimulation. T cells can
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also be purified using Dynabeads (DYNAL, Lake Success, New York) coated with
various
antibodies, e.g., anti-BV 14 or anti-CD8+, to enhance the degree and speed of
purification of
the T cells prior to or after expansion of the T cells. Alternative methods of
T cells expansion
in vitro involve use of superantigens or monoclonal antibodies to a T cell
receptor expressed
by the tumor infiltrating cells or use of an immunostimulatory cytokines such
as TNF, gamma
interferon, and an interleukin (IL-1, IL-2, IL-12 etc.).
Methods of Inducing an Anti-tumor Response
The present invention is directed to a method of inducing an anti-tumor
response
against a melanoma by administering an effective amount of a composition
comprising at
least one of the following: (i) a hapten-modified syngeneic mammalian melanoma
cell
substantially in a no growth phase, (ii) a hapten-modified melanoma cell
membrane, (iii) a
peptide isolated from said hapten-modified melanoma cell or membrane and (iv)
a T cell
capable of mediating an anti-tumor response such as for example regression of
a melanoma.
The melanoma treated according to the present invention includes metastatic
melanoma which
may be lung, lymph node or subcutaneous metastasis, and is preferably a lung
metastasis.
The lung metastasis to be treated according to the invention is preferably a
small lung
metastasis. In one preferred embodiment of the invention, a melanoma
metastasis in a
mammal is limited to the lung of said mammal. Primary cancers may also be
treated
according to the invention. Stage I, II, III, or IV cancer may be also be
treated according to
the invention, and preferably stages III and IV. In one embodiment of the
invention, domestic
animals may be treated.
In one preferred embodiment, a mammal, preferably a human, having a single or
multiple lung metastases is treated. Small metastases are particularly suited
for treatment
according to the present invention. The size of such metastases may be about 2
cm in
diameter, preferably less than about 1.5 cm, and most preferably less than
about 1 cm. An
anti-tumor response resulting from such a treatment may be a partial or a
complete regression
of the metastatic tumor or a stable disease. A "complete" regression indicates
about 100%
regression for a period of at least one month, more preferably for a period of
at least three
months. A "partial" regression indicates more than about SO % regression for a
period of at
least one month, more preferably for a period of at least three months. A
"stable" disease
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indicates a condition in which there is no significant growth of the tumor
after the vaccine
treatment. Another anti-tumor response that may be observed upon following the
treatment
of the invention is prolongation of survival.
Prior to administration of the vaccine composition of the invention, the
subject
may be immunized to the hapten used to modify tumor cells and membranes by
applying it
to the skin. For example, dinitrofluorobenzene (DNFB) may be used to immunize
the patient
against DNP. Subsequently (about two weeks later, for example), the subject
may be injected
with a preparation of the invention. In one embodiment of the invention, the
patient is not
immunized prior to administration of the vaccine.
A pharmaceutically acceptable amount of a low-dose cyclophosphamide or
another low-dose chemotherapy may be administered preceding the administration
of the
composition. A haptenized vaccine composition may optionally be followed by
administration of a pharmaceutically acceptable amount of a non-haptenized
composition.
A non-haptenized composition may also be administered in accordance with the
methods of
the present invention.
The composition may be administered (such as by reinjection) for a total of at
least three and preferably at least six treatments. In one 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
week, every 2 weeks, or every 4 weeks. 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 a$er the initial
administration.
The present invention may be used following conventional treatment for cancer,
such as surgery. Excised tumors or collected tumor cells may be used to
prepare tumor cells
and membranes as described above.
The preparations of the invention may be administered by any suitable route,
including inoculation and injection, for example, intradermal, intravenous,
intraperitoneal,
intramuscular, and subcutaneous. There may be multiple sites of administration
per each
vaccine treatment. For example, the vaccine composition may be administered by
intradennal
injection into at least two, and preferably three, contiguous sites per
administration. In one
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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
5 immune response. Biological response modifiers of the present invention
include and are not
limited to interleukin-12, interleukin-15 and gamma interferon. In one
embodiment, IL12 is
given following each vaccine inj ection. 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
10 destruction and regression of the tumors.
The modified tumor cells, membranes and peptides each have the property of
stimulating T cells. "Stimulation" for purposes of the present invention
refers to inducing
proliferation of T cells as well as production of cytokines by T cells in
vitro. Proliferation of
T cells may be detected and measured by the uptake of modified nucleotides,
such as and not
15 limited to 3H thymidine, 'ZSILIDR (iododeoxyuridine); and dyes such as 3-
(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) which stain live
cells. In
addition, production of cytokines such as and not limited to IFNy, tumor
necrosis factor
(TNF), and IL-2 may 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
20 above the background level, which is generally above 25 picograms/ml, and
is preferably
above 100 picograms/ml.
The following non-limiting examples further illustrate the invention.
EXAMPLES
Example 1:
Sixteen patients suffering from melanoma and having lung metastases were
treated according to the present invention. All treated patients had
measurable lung
metastases. For purposes of the present invention, the term "measurable" lung
metastasis
refers to a metastasis that is visible on an X ray. Melanoma cells were
isolated from lymph
node and lung metastases and prepared in accordance with the methods set forth
above. The
cells were obtained by dissociation of metastatic masses enzymatically (with
collagenase and
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DNAse). The cells were conjugated to DNP as described herein. Some of the
patients were
sensitized to DNP by topical application of 5 % dinitrochlorobenzene to the
upper arm. The
first vaccine was preceded by a low dose cyclophosphamide (CY). On day 0, the
patients
were given cyclophosphamide 300 Mg/MZ i.v. Three days later, they were
injected
intradermally with a vaccine containing 2.5 X 106 to 25 X 10~ autologous,
cryopreserved,
irradiated (2500 R) tumor cells mixed with BCG. Mo$t patients were treated
every week for
6 treatments. The patients were compared to their condition prior to treatment
with the
vaccine. The patients treated with other cancer therapies prior to the vaccine
study were
removed from such treatments at least two months prior to starting the vaccine
study.
Accordingly, the patients were untreated beginning the vaccine study.
Partial responses (PR) (documented by computerized tomography) were observed
in 3/16 patients, and a fourth patient has exhibited ongoing tumor regression
with <50%
regression to date (stable disease). The details of the PR's were as follows:
Patient I : 90%
regression of multiple (>50) small (5-10 mm diameter) lung nodules; Patient 2:
75%
regression of a solitary 1 cm diameter nodule; and Patient 3: complete
regression of a 1 cm
nodule with partial regression of two accompanying nodules. All responses were
in patients
whose metastases were limited to the lung. Tumor regression was not evident in
any
responder until 4-6 months after beginning vaccine treatment. The survival of
the 4 patients
with PR or stable disease is 34.5, 8.5+, 9.2+, and 17+ months, respectively.
Accordingly, a
DNP-melanoma cell vaccine can induce radiographically documented regression of
melanoma
metastases. Small lung metastases may be particularly susceptible to
immunological
destruction.
Example 2:
Tumor masses for the preparation of a vaccine composition of the invention may
be obtained from lymph node, lung or subcutaneous metastatic masses and
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.
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Modification with DNP may be performed by the method of Miller and Claman (
1976). This
involves a 30 minute incubation of melanoma cells with dinitrofluorobenzene
(DNFB) under
sterile conditions, followed by washing with sterile saline.
Melanoma patients having small multiple lung metastases and melanoma patients
with metastases localized to the lung may be treated as follows. The vaccine
composition
may contain a minimum of 2.5x 1 O6 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 injections 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 ideal vaccine reaction
is an
inflammatory papule with no more than small (< 5mm) 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) 1x106 autologous
melanoma cell
membranes umnodified and modified with DNP; both enzymatically-dissociated
(TCE) and
mechanically-dissociated (TCM) melanoma 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 pg DNFB to the skin of the ventral surface of the upper arm and examining
the area for
a circle of induration 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: I) sensitizing patients to DNP-modified lymphocytes and 2) tolerizing
patients by
injection of unmodified melanoma cells.
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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 measured by proliferation,
cytokine release, and
cytotoxicity.
Patients may be evaluated for metastatic disease before vaccine therapy
begins.
After the end of the first six weeks of vaccine therapy, evaluations may be
performed every
three months. Evaluations may continue through year two, every four months in
year three,
and every six months thereafter. Patients who are responders at the 1 year
evaluation may
receive a final booster injection of vaccine. Then their condition may be
followed without
further treatment.
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.
Example 3:
Four patients that responded to the vaccine of the invention as described in
Example 1, were continued to be monitored to assess the effectiveness of the
vaccine. Three
patients (Patients 2, 3 and 4 in Example 1 ) received a total of 6 vaccines, a
vaccine per week,
with a booster vaccine at six and 12 months from the first vaccine. One
patient (Patient 1)
received weekly vaccines for 12 weeks without booster vaccine. Two patients
had complete
remission and two patients had partial remission. Patient 1 (of Example 1 )
had a partial
remission for a duration of 12 months. Patient 2 (of Example 1 ) had partial
remission for a
period of 8 months. Patient 3 (of Example 1) had a complete remission for a
period of 29
months, and the fourth patient (of Example 1 ) had a complete remission in
excess of 27
months. The term "complete remission" indicates that all detectable tumors
were completely
gone. The term "partial remission" generally indicates that there was at least
a 50% decrease
in the diameter of the tumors. However, in the present case, with respect to
Patient 1 and
Patient 2, about 90% decrease in the diameter of the tumors was observed. The
results are
represented in Figures A-I.
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26
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SUBSTITUTE SHEET (RULE 26~
