U.S. patent application number 11/015769 was filed with the patent office on 2005-10-20 for low dose haptenized tumor cell and tumor cell extract immunotherapy.
Invention is credited to Berd, David.
Application Number | 20050232951 11/015769 |
Document ID | / |
Family ID | 26876131 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050232951 |
Kind Code |
A1 |
Berd, David |
October 20, 2005 |
Low dose haptenized tumor cell and tumor cell extract
immunotherapy
Abstract
This invention relates to compositions comprising haptenized
tumor cells and extracts thereof, methods for preparing the
compositions, vaccines comprising such haptenized tumor cells, and
methods for treating cancer with such vaccines. In a specific
embodiment, melanoma cells are haptenized with a dinitrophenyl
group, and used for treatment of melanoma patients having
metastatic disease. Preferably, patients are given a first vaccine
dose containing haptenized cells to "prime" the immune system.
Subsequently, patients are injected with an immunomodulatory
compound such as cyclophosphamide. In a preferred embodiment, an
appropriate time period after the "priming" vaccine dose,
additional vaccine doses containing a mixture of haptenized cells
and an adjuvant are administered. The described treatment plan is
more effective for eliciting favorable anti-tumor immune
responses.
Inventors: |
Berd, David; (Wyncote,
PA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 MARKET STREET
PHILADELPHIA
PA
19103-2921
US
|
Family ID: |
26876131 |
Appl. No.: |
11/015769 |
Filed: |
December 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11015769 |
Dec 17, 2004 |
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09776250 |
Feb 1, 2001 |
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60180258 |
Feb 4, 2000 |
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60259501 |
Jan 3, 2001 |
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Current U.S.
Class: |
424/277.1 ;
514/453; 514/505; 514/649 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 2039/6012 20130101; A61P 35/00 20180101; A61K 39/0011
20130101; A61K 2039/55594 20130101; A61K 2039/5152 20130101 |
Class at
Publication: |
424/277.1 ;
514/505; 514/453; 514/649 |
International
Class: |
A61K 048/00; A61K
039/00; A61K 031/353; A61K 031/28 |
Claims
What is claimed is:
1. A composition comprising a haptenized tumor cell or tumor cell
extract comprising from about 2.times.10.sup.5 to about
2.5.times.10.sup.6 tumor cells or cell equivalents per dose,
wherein the tumor cells or cell equivalents are conjugated to a
hapten and rendered incapable of growth or multiplication in
vivo.
2. The composition of claim 1, wherein the hapten is selected from
the group consisting of dinitrophenyl, trinitrophenyl,
N-iodoacetyl-N'-(5-sulfonic 1-naphthyl) ethylene diamine,
trinitrobenzenesulfonic acid, fluorescein isothiocyanate, arsenic
acid benzene isothiocyanate, sulfanilic acid, arsanilic acid,
dinitrobenzene-S-mustard and combinations thereof.
3. The composition of claim 2, in which the hapten is
dinitrophenyl.
4. The composition of claim 1, wherein the tumor cell extract
comprises tumor cell membrane components.
5. The composition of claim 1, wherein the tumor cell extract
comprises tumor cell polypeptides.
6. The composition of claim 1, wherein the tumor cells or tumor
cell extracts originate from a tumor selected from the group
consisting of melanoma, ovarian cancer, colon cancer, breast
cancer, rectal cancer, lung cancer, kidney cancer, prostate cancer,
and leukemia.
7. The composition of claim 6, wherein the tumor is melanoma.
8. The composition of claim 6, wherein the tumor is ovarian
cancer.
9. The composition of claim 1, wherein the tumor cell or tumor cell
extract has been rendered incapable of growth by irradiation.
10. The composition of claim 1, free of any adjuvant.
11. A method for inducing an anti-tumor response in a mammalian
patient suffering from a tumor, which method comprises
administering to the patient a composition comprising a haptenized
tumor cell or tumor cell extract comprising from about
2.times.10.sup.5 to about 2.5.times.10.sup.6 tumor cells or cell
equivalents per dose, wherein the tumor cells or cell equivalents
are conjugated to a hapten, and rendered incapable of growth or
multiplication in vivo.
12. The method of claim 10, which further comprises administering a
first dose of the composition without any adjuvant.
13. The method of claim 10, wherein the composition is administered
prior to a second composition comprising an adjuvant and a tumor
cell or tumor cell extract, which second composition a) is
conjugated to a hapten, and b) contains from about 2.times.10.sup.5
to about 2.5.times.10.sup.6 tumor cells or tumor cell
equivalents.
14. The method of claim 13, wherein the adjuvant is selected from
the group consisting of Bacille Calmette-Guerin, Q-21, and
detoxified endotoxin.
15. The method of claim 11, wherein the composition is administered
prior to the administration of cyclophosphamide.
16. The method of claim 14, wherein the composition is administered
four to seven days prior to the administration of
cyclophosphamide.
17. The method of claim 10, wherein the tumor cells or tumor cell
extracts originate from a tumor selected from the group consisting
of melanoma, ovarian cancer, colon cancer, breast cancer, rectal
cancer, lung cancer, kidney cancer, prostate cancer, and
leukemia.
18. The method of claim 10, wherein the tumor cells or tumor cell
extracts are autologous.
19. The method of claim 10, wherein the tumor is melanoma.
20. The method of claim 10, wherein the patient is a human.
21. A method for inducing an anti-tumor response in a mammalian
patient suffering from a tumor, which method comprises
administering to the patient: (a) on the first day of the
treatment, a composition comprising autologous tumor cells or tumor
cell extracts, which corresponds to from about 2.times.10.sup.5 to
about 2.5.times.10.sup.6 tumor cells, free of any adjuvant; (b)
four to seven days after initiation of the treatment, an
immunomodulatory agent that potentiates protective anti-tumor
immunity or inhibits immune suppression, or both; and (c) at least
one additional composition comprising autologous tumor cells or
tumor cell extracts.
22. The method of claim 21, in which the immunomodulatory compound
is cyclophosphamide.
23. A method for inducing an anti-tumor response in a mammalian
patient suffering from a tumor, which method comprises
administering to the patient: (a) on the first day of the
treatment, a composition comprising a haptenized autologqus tumor
cell or tumor cell extract which corresponds to from about
2.times.10.sup.5 to 2.5.times.10.sup.6 tumor cells free from any
adjuvant; (b) four to seven days after initiation of the treatment,
cyclophosphamide; and (c) at least one week after initiation of the
treatment, a composition comprising an adjuvant and a haptenized
autologous tumor cell or tumor cell extract which corresponds to
from about 2.times.10.sup.5 to about 1.times.10.sup.7 tumor
cells.
24. The method in claim 22, in which the adjuvant is Bacille
Calmette-Guerin.
Description
FIELD OF THE INVENTION
[0001] The invention relates to compositions comprising haptenized
tumor cells and extracts thereof. The invention also relates to
methods for treating cancer in which a priming dose of a
hapten-modified tumor cell preparation is administered prior to any
immunomodulatory agent that potentiates protective anti-tumor
immunity or inhibits immune suppression, or both, such as, e.g.,
cyclophosphamide, and prior to compositions comprising a mixture
between an immunological adjuvant and haptenized tumor cells or
tumor cell extracts.
BACKGROUND OF THE INVENTION
Haptenized Tumor Cell Vaccines
[0002] An autologous whole-cell vaccine modified with the hapten
dinitrophenyl (DNP) has been shown to produce inflammatory
responses in metastatic sites of melanoma patients. The survival
rates of patients receiving post-surgical adjuvant therapy with
DNP-modified vaccine are markedly higher than those reported for
patients treated with surgery alone. Intact cells are preferred for
the vaccine.
[0003] U.S. Pat. No. 5,290,551, to David Berd, discloses and claims
vaccine compositions comprising haptenized melanoma cells. Melanoma
patients who were treated with these cells developed a strong
immune response. This response was detected, e.g., in a
delayed-type hypersensitivity (DTH) response to haptenized and
non-haptenized tumor cells. More importantly, the immune response
to non-haptenized cells has been associated with an increased
survival rate of melanoma patients.
[0004] Haptenized tumor cell vaccines have also been described for
other types of cancers, including lung cancer, breast cancer, colon
cancer, pancreatic cancer, ovarian cancer, and leukemia (see U.S.
patent application Ser. No. 08/203,004, filed Feb. 28, 1994; PCT
Publication Nos. WO 96/40173 and WO 98/14206, and PCT Application
No. PCT/US98/16660).
[0005] Generally, the immune response to haptenized cells has been
found to be independent of the choice of hapten, but dependent on
the functional group to which the hapten is attached. In
particular, it has been reported that haptenization of
.epsilon.-amino groups of lysine and --COOH groups of aspartic acid
and glutamic acid is effective for a robust immune response (Nahas
and Leskowitz, Cellular Immunol., 1980;54:241).
[0006] It is known from animal studies that immunization of mice
with syngeneic lymphocytes modified with arsanilic acid induces
strong T cell responses against those modified cells, including DTH
(Bach et al., J. Immunol., 1978;121:1460) and cytotoxic T cells
(Sherman et al., J. Immunol., 1978;121:1432). Injection of
arsanilic acid into the rat kidney induced abrisk autoimmune
nephritis (Rennke et al., Kidney International, 1994;45:1044).
Obviously, the administration of even minute amounts of arsanilic
acid into human is unacceptable, but sulfanilic acid, a non-toxic
compound in small amounts, should induce a similar immunological
effect (Nahas and Leskowitz, supra, 1980). Both compounds can be
coupled to tyrosine and histidine after being diazotized by
treatment with sodium nitrite. Moreover, immunization of animals
with sulfanilic acid-modified protein can induce autoimmunity
(Weigle, J. Exp. Med., 1965;122:1049). A third potentially
interesting hapten in this category is phosphorylcholine (PC), in
light of the work of Kim et al. (Eur. J. Immunol., 1992;22:775).
However, it has not been established that these haptens will be
effective in humans; on the contrary, Nahas and Leskowitz, supra,
suggest otherwise.
[0007] These discoveries have led to rapid advances in the
treatment of cancer, particularly melanoma, by immunotherapy.
Nevertheless, there remains a need in the art for even more
effective therapies, since the response rates achieved with the
haptenized tumor cell vaccine technologies mentioned above, while
impressive, have not reached 100%. There is also a need in the art
for more effective treatment regimens which require substantially
fewer haptenized cells per dose, either to permit more dosages or
to provide an effective therapy with a smaller number of cells.
This is especially critical for the treatment of an early stage or
recurrent cancer, when the number of cells obtainable from a
resected tumor may be fewer than necessary for vaccine preparation
as described above.
[0008] The present invention addresses these and other needs in the
art in a surprisingly effective way.
[0009] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
SUMMARY OF THE INVENTION
[0010] The present invention advantageously provides a composition
comprising haptenized tumor cells or tumor cell extracts, which may
be used as a priming dose in a cancer treatment regimen intended to
induce an anti-tumor response in a cancer patient.
[0011] According to one aspect, the present invention relates to a
composition comprising a hapten modified mammalian, preferably
human, tumor cell or tumor cell extract.
[0012] In another aspect, the present invention is directed to a
composition comprising from about 2.times.10.sup.5 to about
2.5.times.10.sup.6 hapten modified mammalian tumor cell or cell
equivalents.
[0013] In a further aspect, the present invention is directed to a
method of treating cancer comprising administering to a mammal,
preferably a human, a composition comprising hapten modified human
tumor cell or tumor cell extract wherein said mammal suffers from a
malignant tumor of the same type as said tumor cell membrane.
[0014] In a further embodiment, the invention is directed to a
method of treating cancer comprising initiating the treatment by
administering a first dose comprising hapten modified or unmodified
tumor cells or tumor cell equivalents prior to the administration
of any immunomodulatory agent that potentiates protective
anti-tumor immunity or inhibits immune suppression, or both.
[0015] In another embodiment, the invention is directed to a method
of treating cancer comprising initiating the treatment by
administering a first dose comprising hapten modified or unmodified
tumor cells or tumor cell equivalents, free from any adjuvant,
prior to the administration of any immunomodulatory agent that
potentiates protective anti-tumor immunity or inhibits immune
suppression, or both.
[0016] In yet another embodiment, the invention is directed to a
method of treating cancer comprising initiating the treatment by
administering a first dose, comprising hapten modified or
unmodified tumor cells or tumor cell equivalents and an adjuvant,
prior to the administration of any immunomodulatory agent that
potentiates protective anti-tumor immunity or inhibits immune
suppression, or both.
[0017] In still another embodiment, the invention is directed to a
method of treating cancer comprising initiating the treatment by
administering a first dose comprising hapten modified or unmodified
tumor cells or tumor cell equivalents. After an appropriate time
period, immunomodulatory agents that potentiate protective
anti-tumor immunity or inhibit immune suppression, or both,
followed by additional vaccine preparations, which may include
immunological adjuvants, are administered according to a chosen
time schedule.
[0018] Thus, one object of the invention is to provide more
effective treatment regimens in the field of cancer vaccines by
priming the immune system with a haptenized or non-haptenized tumor
cell preparation.
[0019] This and other aspects of the invention are further
elaborated in the Detailed Description of the Invention and
Examples, infra.
DESCRIPTION OF THE DRAWING
[0020] FIG. 1. Schedule of events in a clinical treatment regimen
outlined according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The invention provides for improved treatment protocols for
cancer therapies based on the administration of tumor cell
vaccines, particularly those involving repeated injections of
haptenized tumor cell preparations. Priming the immune system by
administering a first dose containing a relatively low amount of
hapten modified tumor cells or tumor cell extracts or
non-haptenized tumor cells or tumor cell abstracts, with or without
adjuvant, before treating with cyclophosphamide and repeated doses
of haptenized tumor cell preparations with or without adjuvant, may
result in an augmented immune response to unmodified tumor cells.
This latter priming phenomenon has broad applications to improve
tumor vaccine outcomes in general, i.e., whether or not the tumor
cells or cell extracts (including purified tumor-associated
antigen) are haptenized.
[0022] The work described herein has provided strong support for
the idea that immunizing patients with hapten-modified tumor cells
can induce immunity to unmodified tumor cells. Animal and human
data indicate that a first (priming) administration of a low dose
of haptenized or non-haptenized tumor cells or tumor cell extracts
increases the efficacy of hapten-modified cells or cell extract
immunotherapy of cancer. The present invention provides a rationale
for achieving improved results in humans. More particularly, the
invention permits a more effective anti-tumor immune response,
e.g., as measured by DTH, tumor regression, prolongation of
survival, etc.
[0023] The present invention is based, in part, on data from a
newly developed animal model (described in co-pending application
Ser. No. 60/180,257, attorney docket no. 1225/0G680, filed on Feb.
04, 2000). In this model, an improvement in the therapeutic outcome
of DNP-modified, irradiated, autologous tumor cell vaccine,
preceded by low-dose cyclophosphamide, was observed when the mice
were pretreated with a single dose of DNP-modified, irradiated,
autologous tumor cells (free of the adjuvant Bacille
Calmette-Guerin) prior to the low-dose cyclophosphamide treatment,
and then subjected to vaccination with DNP-modified, irradiated,
autologous tumor cells admixed with adjuvant. These results
illustrate the potentiating effect of the pretreatment regimen for
the vaccination protocol.
[0024] The present invention is based, in part, on data derived
from human studies. An improvement in the therapeutic outcome of
DNP-modified, irradiated, autologous tumor cell vaccine, preceded
by low-dose cyclophosphamide, was observed when humans were
pretreated with a single dose of DNP-modified or non-modified,
irradiated, autologous tumor cells (free of the adjuvant Bacille
Calmette-Guerin) prior to the low-dose cyclophosphamide treatment,
and then subjected to vaccination with DNP-modified, irradiated,
autologous tumor cells admixed with adjuvant. These results
illustrate the potentiating effect of the pretreatment regimen for
the vaccination protocol
[0025] The various aspects of the invention will be set forth in
greater detail in the following sections. This organization into
various sections is intended to facilitate understanding the
invention, and is in no way intended to be limiting thereof.
Definitions
[0026] The following defined terms are used throughout the present
specification, and should be helpful in understanding the scope and
practice of the present invention.
[0027] A "hapten-modified tumor cell preparation" refers either to
haptenized tumor cells or tumor cell extract as described in
greater detail herein.
[0028] The term "corresponds" is used to describe the number of
cells in a composition or used to prepare the amount of a tumor
cell extract in a composition (i.e., cell equivalents in the
composition).
[0029] In a specific embodiment, the term "about" or
"approximately" means within 50%, preferably within 25%, and more
preferably within 10% of a given value or range. Alternatively, the
term about means within an acceptable standard error of the mean,
when considered by one of ordinary skill in the art.
[0030] A "formulation" refers to an aqueous medium or solution for
the preservation or administration, or both, of haptenized tumor
cells or tumor cell extracts, which is preferably directly
injectable into an organism. The aqueous medium will include salts
or sugars, or both, at about an isotonic concentration.
[0031] The phrase "pharmaceutically acceptable" refers to molecular
entities, at particular concentrations, and compositions that are
physiologically tolerable and do not typically produce an allergic
or similar untoward reaction, such as gastric upset, fever,
dizziness and the like, when administered to a human or non-human
animal. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopoeia or other
generally recognized pharmacopoeia for use in humans or non-human
animals.
[0032] As used herein, the term "isolated" means that the
referenced material is removed from the natural environment in
which it is normally found. In particular, isolated biological
material if free of cellular components. An isolated peptide may be
associated with other proteins or nucleic acids, or both, with
which it associates in the cell, or with cellular membranes if it
is membrane-associated. An isolated organelle, cell, or tissue is
removed from the anatomical site in which it is found in an
organism. An isolated material may be, but need not be,
purified.
[0033] The term "purified" as used herein refers to material that
has been isolated under conditions that reduce or eliminate
unrelated materials, i.e., contaminants. For example, a purified
protein is preferably free of other proteins or nucleic acids with
which it is associated in a cell; a purified cell is free of
unrelated cells and tissue matrix components.
[0034] A composition "free of any adjuvant" is a composition, e.g.,
a haptenized tumor cell preparation, not containing an adjuvant or
co-administered with an adjuvant, nor administered less than 24
hours before or after an adjuvant. This is also referred to as
"adjuvant free."
[0035] A "subject" is a human or a non-human animal who may receive
haptenized tumor cells formulated in a composition of the
invention. Preferably the subject is a human. However, the
invention is also contemplated for veterinary medicine,
particularly for treatment of domestic pets (dogs, cats), and
livestock (horses, cows, pigs, etc.)
[0036] An "anti-tumor response" is at least one of the following:
tumor necrosis, tumor regression, tumor inflammation, tumor
infiltration by activated T lymphocytes, delayed-type
hypersensitivity (DTH) response, and Clinical Response.
[0037] A "composition", "vaccine composition" or a "tumor cell
vaccine" are used herein interchangeably to refer to an admixture
of a hapten-modified tumor cell preparation in a formulation,
optionally with an adjuvant. In the context of an adjuvant-free
first dose (priming) embodiment of the invention, the composition
or vaccine may be modified, a mixture of modified and non-modified,
or non-modified: tumor cells, tumor cell membranes (especially the
plasma, i.e., extracellular membrane), or proteins or peptides
extracted from the tumor cell.
[0038] The terms "vaccinate", "immune therapy", and "immunotherapy"
are used herein interchangeably to refer to administration of a
composition comprising a hapten-modified tumor cell preparation to
treat a cancer, e.g., after surgical resection of the tumor.
[0039] An "anti-tumor response" includes, but is not limited to,
one or more of the following: tumor necrosis, tumor regression,
tumor inflammation, tumor infiltration by activated lymphocytes,
activation of tumor infiltrating lymphocytes, DTH response (against
tumor cells), and a Clinical Response.
[0040] The term "treat" means to attempt to elicit an anti-tumor
response against cells of the tumor, i.e., the cancer.
Haptenized Tumor Cell Preparation
[0041] The present invention is directed for use in the preparation
of haptenized tumor cell vaccines for treating cancer, including
metastatic and primary cancers. Cancers treatable with the present
invention include solid tumors and non-solid tumors, including
hematologic malignancies. Examples of solid tumors that can be
treated according to the invention include sarcomas, carcinomas,
and other tumors such as, but not limited to: fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma. Hematologic malignancies include leukemias,
lymphomas, and multiple myelomas. The following are non-limiting
preferred examples of the cancers treatable with the composition
and methods of the present invention: melanoma, including stage-4
melanoma; ovarian, including advanced ovarian; leukemia, including
but not limited to acute myelogenous leukemia; colon, including
colon metastasized to liver; rectal, colorectal, breast, lung,
kidney, and prostate cancers.
Tumor Cells
[0042] The compositions of the present invention are prepared from
tumor cells, e.g, cells obtained from tumors surgically resected in
the course of a cancer treatment regimen as described above. Tumor
cells or cell extracts to be used in the present invention are
preferably prepared as follows. Tumors are processed as described
by Berd et al., Cancer Res., 1986;46:2572, Sato, et al., Cancer
Invest., 1997;15:98, U.S. Pat. No. 5,290,551, and applications U.S.
Ser. Nos. 08/203,004, 08/479,016, 08/899,905, 08/942,794, or
corresponding PCT Publication WO96/40173, 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 non-solid tumors, blood or bone marrow samples may be
collected and tumor cells isolated by density gradient
centrifugation.
[0043] The tumor cells of the present invention may be intact,
attenuated, or killed cells. Tumor cells incapable of growth and
division after administration into the subject, such that they are
substantially in a state of no growth, are preferred for use in the
present invention. It is to be understood that "cells in a state of
no growth" means intact cells that will not divide. Conventional
methods of rendering cells incapable of division are known to
skilled artisans and may be useful in the present invention. For
example, cells may be irradiated prior to use. Tumor cells may be
irradiated to receive a dose of about 2500 cGy to prevent the cells
from multiplying after administration. Alternatively,
haptenization, and particularly dual haptenization, can render the
cells incapable of growth.
[0044] The tumor cells should preferably originate from the same
type of cancer as that to be treated, and are even more preferably
syngeneic (e.g., autologous or tissue-type matched). For purposes
of the present invention, syngeneic refers to tumor cells that are
closely enough related genetically that the immune system of the
intended recipient will recognize the cells as "self", e.g., the
cells express the same or almost the same complement of MHC
molecules. Another term for this is "tissue-type matched." For
example, genetic identity may be determined with respect to
antigens or immunological reactions, and any other methods known in
the art. A syngeneic tumor cell can be created by genetically
engineering a tumor cell to express the required MHC molecules.
[0045] Preferably the cells originate from the type of cancer which
is to be treated, and, more preferably, from the same patient who
is to be treated. The tumor cells may be, but are not limited to,
autologous cells dissociated from biopsy or surgical resection
specimens, or from tissue culture of such cells. Nonetheless,
allogeneic cells and stem cells are also within the scope of the
present invention.
Tumor Cell Membranes
[0046] 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, e.g., from a feline, canine,
equine, bovine, or porcine family. Isolation and preparation of
haptenized tumor cell membranes is described in U.S. patent
application Ser. No. 08/479,016, filed Jun. 7, 1995 and U.S.
application Ser. No. 90/025,012, filed Feb. 17, 1998.
[0047] The tumor cells from which membranes are isolated may be
intact, attenuated, or killed cells. Tumor cells rendered incapable
of growth and division prior to administration into the patient,
such that the cells are substantially in a state of no growth, can
be used in the present invention. Alternatively, tumor cell
membranes may also be isolated from tumor cells capable of in vivo
growth and division, since the membranes by themselves cannot
multiply. Preferably, in such a case, the tumor cell membrane
preparation is not contaminated with tumor cells capable of
multiplying in vivo.
[0048] As with tumor cells, tumor cell membranes are preferably
isolated from the tumor cells of the same type of cancer as that 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. autologous), but may
also be allogeneic to that subject. There may be genetic identity
between a particular antigen on the tumor cell used as a membrane
source and an antigen present on the patient's tumor cells. The
tumor cells may be, but 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.
[0049] Tumor 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, more than about 50% of the membranes are tumor cell
outer membranes. Preferably, more than about 60% of the membranes
consist of tumor cell outer membranes, with more 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.
[0050] Preferably, the isolated membranes are substantially free of
nuclei and intact cells. For example, a membrane preparation is
substantially free of nuclei or intact cells if it contains less
than about 100 cells and/or nuclei in about 2.times.10.sup.8 cell
equivalents (c.e.) of membrane material. A cell equivalent is that
amount of membrane isolated from the indicated number of cells. An
isolated tumor cell membrane which is substantially free of cells
and/or nuclei may contain lymphocytes and/or lymphocyte
membranes.
[0051] 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,
preferably including a fraction of the outer membrane, contains an
MHC molecule fraction and/or a heat shock protein fraction. The
size of the membrane fragments is not critical.
[0052] 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.
[0053] A tumor cell membrane (modified or un-modified) as referred
to in this specification includes any form in which such a membrane
preparation may be stored or administered, such as, for example, a
membrane resuspended in a diluent, a membrane pellet, or a frozen
or a lyophilized membrane.
[0054] The tumor cell membranes can be obtained from haptenized
cells, or may be haptenized after extraction from the cells using
the techniques described infra.
[0055] Tumor cell membranes are prepared from tumor cells, e.g.,
obtained as described above, 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 coupling 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.
[0056] 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 or nuclei remain in about 2.times.10.sup.8 cell equivalents
(c.e.) of membrane material. The retrieved supernatant contains
membranes which, for example, may be pelleted by
ultracentrifugation at about 100,000 g for about 90 minutes. The
pellet contains mainly membranes. Membranes may be resuspended, for
example, in about 8% sucrose, 5 mM Tris, pH 7.6 and frozen at about
-80.degree. C. until use. Any diluent may be used, preferably one
that acts as a stabilizer. To determine the quality of membrane
preparation, a fraction (about 6.times.10.sup.7 c.e. membranes) may
be cultured regularly. Cell colonies should not develop and cells
or nuclei should not be detected by light microscopy.
[0057] 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) which
involves a 30 minute incubation of tumor cells or membranes with a
hapten under sterile conditions, followed by washing with sterile
saline. Hapten-modification may be confirmed by flow cytometry
using a monoclonal anti-hapten antibody.
[0058] 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, the cells or membranes may be thawed on the day that a
patient is to be skin tested or treated.
[0059] Allogeneic tumor cell membranes may be prepared as described
above. However, prior to administration to a subject the
preparation may be 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 a couple of hours to about several
days. The membrane-pulsed antigen presenting cells are then washed
and injected into the patient.
[0060] Antigen-presenting cells may be prepared in a number of ways
including for example the methods of Grabbe et al. (Immunol. Today,
1995;16:117-121) and Siena et al. (Exp. Hematol.,
1995;23:1463-1471). Briefly, blood is obtained, for example by
venipuncture, from the patient to be immunized. Alternatively, a
sample of bone marrow may be collected. 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. The
purified leukocytes are 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 but 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).
[0061] The antigen-presenting cells isolated and expanded in
culture, for example, may be characterized as dendritic cells,
monocytes, macrophages, and Langerhans cells.
Tumor Cell Peptides
[0062] The isolation of peptides to be used in hapten-modified
anti-cancer vaccines is described in U.S. patent application Ser.
No. 08/479,016, filed Jun. 7, 1995 and patent application Ser. No.
09/447,897, filed Nov. 24, 1998. Both applications disclose
extraction and isolation of hapten-modified peptides, which can be
adapted for the present invention. Peptides can also be synthesized
based on known sequences, or isolated prior to haptenization. The
isolated peptides can then be modified by dual-haptenization.
[0063] For purposes of the present invention, peptides are
compounds of two or more amino acids and include proteins. Peptides
will preferably be of low molecular weight, of about 1,000 kD to
about 10,000 kD, more preferably about 1,000 kD to about 5,000 kD,
which are isolated from a haptenized tumor cell and which stimulate
T cell lymphocytes to produce gamma interferon. The peptide of the
invention may be from about 8 to about 20 amino acids, preferably
from about 8 to about 12 amino acids. In addition, the peptide is
preferably 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 peptides of the present invention include but are not
limited to peptides which bind to MHC molecules, a cell
surface-associated protein, a peptide associated with a heat shock
protein/chaperonin, a protein encoded by cancer oncogenes, or
mutated anti-oncogenes. In one preferred embodiment of the
invention, peptides are bound to the MHC molecules. For purposes of
the present invention "a peptide equivalent" is the peptide having
the same amino acid sequence as the peptide isolated from an MHC
molecule, although prepared either by degradation of a protein
comprising the peptide, synthesized in vitro or recombinant DNA
technology.
[0064] Preferably, the peptides are derived from tumor specific
antigens. There is substantial evidence that the sarne
T-cell-defined tumor antigens are expressed by different human
melanoma tumors, suggesting that transformation-associated events
may give rise to recurrent expression of the same tumor antigen in
tumors of related tissue and/or cellular origin (Sahasrabudhe et
al., J. Immunol., 1993;151:6302-6310; Shamamian et al., Cancer
Immunol. Immunother., 1994;39:73-83; Cox et al., Science,
1994;264:716; Peoples et al., J. Immunol., 1993;151:5481-5491;
Jerome et al., Cancer Res., 1991;51:2908-2916; Morioke et al., J.
Immunol., 1994;153:5650-5658). Examples of such antigens include,
but are not limited to, MART 1/Melan A, gp-100, and tyrosinase
(melanoma); MAGE-1 and MAGE-3 (bladder, head and neck, non-small
cell carcinoma); HPV E6 and E7 proteins (cervical cancer);
HER2/neu/c-erbB-2 (breast cancer); HER3, HER4, Mucin (MUC-1)
(breast, pancreas, colon, prostate); prostate specific antigen
(PSA) (prostate); and CEA (colon, breast, GI).
[0065] The cell extracts of the invention, including peptides
originally isolated from MHC molecules located on tumor cell plasma
membranes, have the property of stimulating T cells. For purposes
of the present invention, stimulation refers to proliferation of T
cells as well as production of cytokines by T cells in response to
the cell extract. Proliferation of T cells may be observed by
uptake by T cells of modified nucleic acids, such as but not
limited to .sup.3H thymidine, .sup.125IUDR (iododeoxyuridine); and
dyes such as 3-(4,5-dimethylthiazol-2-yl)-2,5-dip- henyltetrazolium
bromide (MTT) which stains intact cells. In addition, production of
cytokines such as but not limited to .gamma.-interferon
(INF.gamma.), tumor necrosis factor (TNF), and interleukin-2 (IL-2)
may be tested. Production of cytokines is preferably in an amount
greater than 15 picograms/ml, more preferably about 20 to about 30
picograms/ml, even more preferably about 50 picograms/ml.
Alternatively, cytotoxicity assays can be used to evaluate T cell
stimulation.
[0066] From the hapten-modified cells, peptides may be extracted,
some of which are hapten-modified as a result of modifying the
cells. Alternatively, extracted or synthetic peptides can be
reacted with a hapten after isolation or synthesis. Protein
extraction techniques known to those of skill in the art may be
followed by antigen assays to isolate proteins or peptides
effective for patient treatment. The methods of isolating cell
extracts are readily known to those skilled in the art. Briefly,
cancer cells are isolated from a tumor and cultured in vitro. A
hapten preparation is added to the cultured cells in accordance
with the method set forth above. Peptides are isolated from cells
according to an established technique, e.g., the 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 but not limited to trifluoroacetic
acid (TFA). The cells are thereafter 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 reversed-phase HPLC column (Superpac Pep S,
Pharmacia LKB) in 0.1% TFA using a gradient of increasing
acetonitrile concentration; flow rate=1 ml/min, fraction size=1 ml.
Fractions containing small peptides are collected 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, N.Y. (1989), concentrated, and frozen.
[0067] The HPLC fractions containing small peptides are screened
for immunological activity, e.g, by allowing them to bind to
autologous B lymphoblastoid cells which are then tested for their
ability to stimulate tumor-specific T lymphocytes. T cells used for
this testing are isolated from a human patient and propagated in
vitro as described in PCT Publication No. WO98/14206. The peptides
that stimulate T cells are then analyzed for their structure. For
example, the peptides are sequenced using methods known in the art
to determine their amino acid sequence. In one embodiment of the
invention, the peptides are sequenced as a pool as described by
Burrows et al. (J. NeuroSci. Res., 1997;49:107-116) and Gavin et
al. (Eur. J. Immunol., 1994;24:2124-33) to determine prevailing
motifs. In another embodiment of the invention, the peptides are
further separated using methods known in the art, such as HPLC, as
described in U.S. Pat. Nos. 5,747,269; 5,487,982; 5,827,516 and
5,820,862 and sequenced. Sequencing is performed by using Edman
degradation as described in Edman and Berg, Eur. J. Biochem.,
1967;80:116-132, or any modification thereof known in the art. One
powerful technique for characterizing isolated peptides is mass
spectrometry.
[0068] Once the sequence of the peptides isolated from the MHC
molecules is known, synthetic peptides having the same sequence are
synthesized and used as a vaccine alone, presented on an antigen
presenting cell and/or in combination with other extracts or whole
cells using the methods described above. The equivalent peptides
may also be produced recombinantly or by chemical degradation of
proteins containing the isolated peptides.
[0069] In another embodiment, the structure of known peptides is
altered by changing at least one amino acid and the so altered
peptides are tested for their ability to stimulate T cells.
Haptenization
[0070] The tumor cells, membranes, or peptides can be haptenized.
For purposes of the present invention, virtually any small
molecule, including peptides, that can induce an immune response
when conjugated to a carrier, may function as a hapten. A variety
of haptens of different chemical structure have been shown to
induce similar types of immune responses: e.g., dinitrophenyl
(DNP); trinitrophenyl (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.,
1995;105:92); and arsenate (Nalefski and Rao, J. Immunol.,
1993;150:3806). Conjugation of a hapten to a cell may preferably be
accomplished by conjugation via .epsilon.-amino groups of lysine or
--COOH groups. This group of haptens include a number of chemically
diverse compounds: halonitrobenzenes (including
dinitrofluorobenzene, difluorodinitrobenzene,
trinitrofluorobenzene), N-iodoacetyl-N'-(5-sulfon- ic-1-naphthyl)
ethylene diamine, nitrobenzene sulfonic acids (including
trinitrobenzenesulfonic acid and dinitrobenzene sulfonic acid),
fluorescein isothiocyanate, arsenic acid benzene isothiocyanate,
and dinitrobenzene-S-mustard (Nahas and Leskowitz, Cellular
Immunol., 1980;54:241). Once familiar with the present disclosure,
skilled artisans would be able to choose haptens for use in the
present invention.
[0071] Haptens generally include a reactive group for conjugation
to a substituent on an amino acid side chain of a protein or
polypeptide (e.g., a free carboxylic acid group as in the case of
aspartic acid or glutamic acid; the .epsilon.-amino group of
lysine; the thiol moiety of cysteine; the hydroxyl group of serine
or tyrosine; the imidazole moiety of histidine; or the aryl groups
of tryptophan, tyrosine, or phenylalanine). As used herein, the
term "reactive group" refers to a finctional group on the hapten
that reacts with a functional group on a peptide or protein. The
term "functional group" retains its standard meaning in organic
chemistry. These reactive groups on a hapten are termed herein the
"hapten reactive group". Numerous hapten reactive groups are known,
which interact with the substituents present on the side chains of
amino acids that comprise peptides and proteins. Preferred examples
of such reactive groups for conjugation to specific polypeptide
substituents are carboxylic acid or sulfonic acid derivatives
(including acid chlorides, anhydrides, and reactive carboxylic
esters such as N-hydroxysuccinimide esters), imidoesters, diazonium
salts, isocyanates, isothiocyanates, halonitrobenzenes,
.alpha.-halocarbonyl compounds, maleimides, sulfur mustards,
nitrogen mustards, and aziridines.
[0072] Functional groups reactive with primary amines. Hapten
reactive groups that would form a covalent bond with primary amines
present on amino acid side chains would include, but not be limited
to, acid chlorides, anhydrides, reactive esters,
.alpha.,.beta.-unsaturated ketones, imidoesters, and
halonitrobenzenes. Various reactive esters with the capability of
reacting with nucleophilic groups such as primary amines are
available commercially, e.g., from Pierce (Rockford, Ill.).
[0073] Functional groups reactive with carboxylic acids. Carboxylic
acids in the presence of carbodiimides, such as EDC, can be
activated, allowing for interaction with various nucleophiles,
including primary and secondary amines. Alkylation of carboxylic
acids to form stable esters can be achieved by interaction with
sulfur or nitrogen mustards, or haptens containing either an alkyl
or aryl aziridine moiety.
[0074] Functional groups reactive with aromatic groups. Interaction
of the aromatic moieties associated with certain amino acids can be
accomplished by photoactivation of aryl diazonium compound in the
presence of the protein or peptide. Thus, modification of the aryl
side chains of histidine, tryptophan, tyrosine, and phenylalanine,
particularly histidine and tryptophan, can be achieved by the use
of such a reactive functionality.
[0075] Functional groups reactive with sulfhydryl groups. There are
several reactive groups that can be coupled to sulfhydryl groups
present on the side chains of amino acids. Haptens containing an
.alpha.,.beta.-unsaturated ketone or ester moiety, such as
maleimide, provide a reactive functionality that can interact with
sulfhydryl as well as amino groups. In addition, a reactive
disulfide group, such as 2-pyridyldithio group or a
5,5'-dithio-bis-(2-nitrobenzoic acid) group is also applicable.
Some examples of reagents containing reactive disulfide bonds
include N-succinimidyl 3-(2-pyridyl-dithio) propionate (Carlsson,
et al., Biochem J., 1978;173:723-737), sodium
S-4-succinimidyloxycarbonyl- -alpha-methylbenzylthiosulfate, and
4-succinimidyloxycarbonyl-alpha-methyl- -(2-pyridyldithio)toluene.
Some examples of reagents comprising reactive groups having a
double bond that reacts with a thiol group include succinimidyl
4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidyl
m-maleimidobenzoate.
[0076] Other functional molecules include succinimidyl
3-(maleimido)propionate, sulfosuccinimidyl
4-(p-maleimido-phenyl)butyrate- , sulfosuccinimidyl
4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,
maleimidobenzoyl-N-hydroxy-succinimide ester. Many of the
above-mentioned reagents and their sulfonate salts are available
from Pierce.
[0077] Haptens also include a hapten recognition group that
interacts with antibody. The recognition group is irreversibly
associated with the hapten reactive group. Thus, when the hapten
reactive group is conjugated to a functional group on the target
molecule, the hapten recognition group is available for binding
with antibody. By selecting an appropriate hapten reactive group,
antibody recognition of, and binding to, a hapten recognition group
can be made independent of the functional group to which the hapten
is conjugated. When this is the case, the haptens are functionally
equivalent, and are said to share antibody binding features.
Naturally, in cases where the recognition groups of two haptens
differ chemically, the reactive groups may be the same or
different, i.e., reactive with the same or different functional
groups on the target molecule.
[0078] Examples of different hapten recognition groups include
without limitation to dinitiophenyl, trinitrophenyl, fluorescein,
other aromatics, phosphorylcholine, peptides, advanced
glycosylation endproducts (AGE), carbohydrates, etc.
[0079] In a specific embodiment, the same hapten recognition group
can be coupled to different amino acids through different hapten
reactive groups. For example, the reagents dinitrobenzene sulfonic
acid, dinitro-phenyldiazonium, and dinitrobenzene-S-mustard, all
form the dinitrophenyl hapten coupled to amino groups, aromatic
groups, and carboxylic acid groups, respectively. Similarly, an
arsonic acid hapten can be coupled by reacting arsonic acid benzene
isothiocyanate to amino groups or azobenzenearsonate to aromatic
groups. In another specific embodiment, tumor cells or cell
extracts are conjugated with two haptens by derivitization of two
different functional groups. For example, a tumor cell preparation
may be dual-haptenized with a DNP group coupled to .epsilon.-amino
groups, and with a sulfanilic acid group coupled to aromatic side
chains of histidine and tyrosine.
Isolation and Haptenization of Tumor Cells
[0080] The dissociated cells, cell membranes, or peptides may be
stored frozen in a freezing medium (e.g., prepared from a
sterile-filtered solution of 50 ml Human Serum Albumin (HSA)
(American Red Cross) added to 450 ml of RPMI 1640 (Mediatech)
supplemented with L-glutamine and adjusted to an appropriate pH
with NaOH), such as in a controlled rate freezer or in liquid
nitrogen until needed. The cells are ready for use upon thawing.
Preferably, the cells are thawed shortly before haptenization.
Optionally, the cells may be washed, and optionally irradiated to
receive a dose of about 2500 cGy. They may then be washed again and
suspended in Hanks Balanced Salt Solution (HBSS) without phenol red
and without HSA.
[0081] Modification of the prepared cells with DNP or another
hapten may be performed by known methods, e.g. by the method of
Miller and Clanian (J. Immunol., 1976;117:151), incorporated herein
by reference in its entirety, which involves a 30 minute incubation
of tumor cells with DNFB under sterile conditions, followed by
washing with sterile saline or HBSS/HSA.
Vaccine Preparations
[0082] The compositions of the invention may be administered in a
mixture or in combination with a pharmaceutically-acceptable
carrier, selected with regard to the intended route of
administration and standard pharmaceutical practice. Dosages may be
set with regards to weight, and the clinical condition of the
patient. The proportional ratio of active ingredient to carrier
naturally depend on the chemical nature, solubility, and stability
of the compositions, as well as the dosage contemplated. The amount
of the tumor cells of the invention to be used depend on such
factors as the affinity of the compound for cancer cells, the
amount of cancer cells present, and the solubility of the
composition. The compounds of the present invention may be
administered by any suitable route, including inoculation and
injection via, for example, intradermal, intravenous,
intraperitoneal, intramuscular, and subcutaneous routes.
[0083] In a preferred embodiment of the invention, the composition
to be used for the first "priming" dose comprises a vaccine
comprising about 2.times.10.sup.5 to 2.5.times.10.sup.6, more
preferably less than about 2.times.10.sup.6, even more preferably
less than about 1.times.10.sup.6, growth-incapacitated tumor cells
or tumor cell equivalents suspended in a pharmaceutically
acceptable carrier or diluent, such as but not limited to Hanks
solution, saline, phosphate-buffered saline (PBS), and water, and
optionally with adjuvant. The composition may be administered by
intradermal injection into from one to about three contiguous sites
per administration on the upper arms or legs, excluding limbs
ipsilateral to a lymph node dissection. Vaccine preparations for
subsequent administrations can comprise from about 2.times.10.sup.5
to about 1.times.10.sup.7 tumor cells or tumor cell equivalents,
preferably from about 1.times.10.sup.6 to about 2.5.times.10.sup.6
tumor cells or tumor cell equivalents.
Formulations
[0084] The formulations according to the invention may be prepared
in various ways. The different components may be mixed together,
and then added to haptenized tumor cells or tumor cell equivalents.
It is also possible to mix one or several of the components with
the haptenized tumor cell preparation and then add the remaining
component(s). The preparation of the formulation and its addition
of the haptenized tumor cells are preferably performed under
sterile conditions.
[0085] The respective proportions of the components of the media
according to the invention may be adapted by persons skilled in the
art.
[0086] Generally, for human tumor cells, HSA will be added to an
appropriate buffered cell culture medium. "Human serum albumin" or
"HSA" refers to a non-glycosylated monomeric protein consisting of
585 amino acid residues, having a molecular weight of about 66 kD.
Its globular structure is maintained by 17 disulfide bridges, which
create a sequential series of 9 double loops (Brown, "Albumin
structure, function and uses", Rosenoer, V. M. et al. (eds.),
Pergamon Press:Oxford, pp. 27-51, 1977). The genes encoding for HSA
are known to be highly polymorphic, and more than 30 apparently
different genetic variants have been identified by electrophoretic
analysis (Weitkamp, L. R. et al., Ann. Hum. Genet.,
1973;37:219-226). The HSA gene comprises 15 exons and 14 introns
corresponding to 16,961 nucleotides from the putative mRNA
"capping" site up to the first site of addition of poly(A).
Autologous serum albumins can be used in the preparation of tumor
cells from other animal species.
[0087] In its essence, a buffered cell culture medium is an
isotonic buffered aqueous solution, such as phosphate buffered
saline, Tris-buffered saline, or HEPES buffered saline. In a
preferred embodiment, the medium is plain Hank's medium (no phenol
red), e.g., as sold commercially by Sigma Chemical Co. (St. Louis,
Mo., USA). Other tissue culture media can also be used, including
basal medium Eagle (with either Earle's or Hank's salts),
Dulbecco's modified, Eagle's medium (DMEM), Iscove's modified
Dulbecco's medium (IMDM), Medium 199, Minimal Essential Medium
(MEM) Eagle (with Earle's or Hank's salts), RPMI, Dulbecco's
phosphate buffered salts, Earle's balanced salts (EBSS), and Hank's
Balanced Salts (HBSS). These media can be supplemented, e.g., with
glucose, Ham's nutrients, or HEPES. Other components, such as
sodium bicarbonate and L-glutamine, can be specifically included or
omitted. Media, salts, and other reagents can be purchased from
numerous sources, including Sigma, Gibco, BRL, Mediatech, and other
companies. For use in humans, an appropriate medium is
pharmaceutically acceptable.
[0088] Preferably, a formulation of whole, intact cells comprises
an optimized HSA concentration in a buffered cultured medium,
preferably HBSS. In a specific embodiment, the final concentration
of HSA is about 1.0% in a HBSS. However, an unexpected improvement
in cell viability can be achieved using at least about 0.25% HSA, a
greater improvement in cell viability with 0.3% HSA (as compared to
0.1% HSA), and an even greater improvement is possible using at
least about 0.5% HSA. Upper limits to the concentration are
determined by the need to avoid contaminants that may be present in
naturally-derived HSA, or alternatively to avoid allergic reactions
to recombinant HSA. Preferably, the concentration of HSA in a
formulation of the invention is no more than about 10%. More
preferably, the concentration is less than or equal to about 5%
and, more preferably still, less than or equal to about 2%.
[0089] Also, a composition or formulation of the invention may
contain other components in addition to HSA to further stabilize
the haptenized tumor cells. Examples of such components include,
but are not limited to, carbohydrates and sugars, such as dextrose,
sucrose, glucose, and the like, e.g., at a 5% concentration; medium
to long chain polyols, such as glycerol, polyethylene glycol, and
the like, e.g., at 10% concentration; other proteins; amino acids;
nucleic acids; chelators; proteolysis inhibitors; preservatives;
and other components. Preferably, any such constituent of a
composition of the invention is pharmaceutically acceptable.
Adjuvant
[0090] In preferred embodiment, the tumor cell vaccines
administered, including the "priming" dose, may be administered
with an immunological adjuvant. The term "adjuvant" refers to a
compound or mixture that enhances the immune response to an antigen
(Hood et al., Immunology, Second Edition, 1984, Benjamin-Cummings:
Menlo Park, Calif., p. 384). While commercially available
pharmaceutically acceptable adjuvants are limited, representative
examples of adjuvants include Bacille Calmette-Guerin (BCG) the
synthetic adjuvant QS-21 comprising a homogeneous saponin purified
from the bark of Quillaja saponaria and Corynebacterium parvum
(McCune et al., Cancer, 1979;43:1619). Other adjuvants include
Complete and Incomplete Freund's Adjuvant, detoxified endotoxins,
mineral oils, surface active substances such as lipolecithin,
pluronic polyols, polyanions, peptides, and oil or hydrocarbon
emulsions. In some cases, immunostimulatory compounds, as
exemplified below, may function as adjuvants.
[0091] It will be understood that the adjuvant is subject to
optimization. In other words, the skilled artisan can engage in
experimentation that is no more than routine to determine the best
adjuvant to use.
Immunomodulators and Combination Therapies
[0092] Treatment regimens for haptenized tumor cells or tumor cell
extracts may include immunodulators, i.e. drugs that alter,
suppress or strengthen the body's immune system. The
immunomodulators can be generally subdivided into groups according
to function, as listed below. However, it is to be understood that
exemplified immunomodulatory compounds may serve different
functions depending on cell-type, dosage, formulation,
administration route, treatment regimen, and the patients
condition. Immunopotentiators herein refers to compounds that
potentiate the immune system for treatment with a tumor vaccine.
Preferably, immunopotentiators at least temporarily diminish any
down-regulation of anti-tumor responses evoked by haptenized tumor
cells or cell extracts according to the present invention, the
down-regulation involving, e.g., T suppressor cells; while to a
lesser extent affecting other parts of the immune system. A
preferred example of such a compound included is cyclophosphamide.
Cyclophosphamide is preferably administered at doses lower than
about 1000 mg/m.sup.2, or, more preferably, at doses of about 300
mg/m.sup.2. Immunosuppressants include, but is not limited to,
chemotherapeutic agents known in the art, preferably administered
at doses inducing a general suppression of the immune system.
Immunostimulants is a general term encompassing endotoxin and
endogenous agents, e.g. cytokines and lymphokines, including but
not limited to IL-2, IL-4, INF.gamma., IL-12, and GM-CSF. According
to the present invention, immunomodulators may be administered
alone, mixed, or co-administered with the haptenized tumor cell
vaccine, as appropriate. The tumor cells and extracts of the
invention may also be used in conjunction with other cancer
treatments including, but not limited to, chemotherapy, radiation
therapy, immunotherapy, and gene therapy.
Clinical Response Criteria
[0093] Standard criteria for evaluating treatment response include:
Complete response, which indicates complete disappearance of all
metastases for at least about one month, more preferably for at
least about three months, without development of new metastases;
Partial response, which indicates at least about 50% reduction in
the mean diameter of a measurable metastasis for at least about one
month, more preferably for at least about three months, without
development of new metastases; and Mixed response, which indicates
at least about 50% reduction in the mean diameter of a measurable
metastasis with concomitant growth of another metastasis. Stable
disease indicates more than about 25% increase in the mean diameter
of any measurable metastasis. Prolongation of time to relapse or of
survival are both also examples of possible clinical response
criteria.
EXAMPLES
[0094] The following examples are illustrative of the invention,
but not limiting thereof.
Example 1
Treatment Protocol for Melanoma Patients
[0095] Patient group. The treatment efficacy of DNP-modified
autologous melanoma cells (vaccine) is studied in post-surgical
stage III melanoma patients having metastatic lymph node
involvement, or stage IV melanoma patients with lung metastases.
Prior to the initiation of the vaccine treatment, preferably within
two months from the starting point, one or more tumor masses are
surgically resected from each patient. For axillary nodes, a formal
axillary node dissection is performed. For inguinal nodes, a
superficial inguinal dissection is performed, with a deep
dissection at the discretion of the surgeon. For other lymph
node-bearing areas, an appropriate node dissection is performed. An
adequate tumor sample, approximately 2 cm or 5 g, yielding at least
about 50.times.10.sup.6 cells, is kept under sterile conditions to
be used for vaccine preparation. Patients are preferably confirmed
melanoma-free by computed tomography (CT) or magnetic resonance
imaging (MRI).
[0096] Vaccine preparation. The vaccine is prepared from a
cryopreserved cell suspension, previously prepared from a portion
of the patient's excised lymph node tumor mass, for delivery on the
same day as the treatment. Cells are prepared and haptenized as
described previously (see "Detailed Description" in the instant
application; Berd et al., Cancer Res 1986;46:2572-7; U.S. Pat. No.
5,290,551; U.S. patent applications Ser. No. 08/203,004; Ser. No.
08/475,016; and Ser. No. 08/899,905). After haptenization and
washing, the cells are suspended in HBSS supplemented with 1% HSA
and stored at 4.degree. C.
[0097] Vaccine dose 1 consists of (0.75.+-.0.25).times.10.sup.6
DNP-modified, intact tumor cells suspended in 0.2-0.3 ml Hank's
solution.
[0098] Vaccine doses 2-8 consists of (2.5.+-.0.75).times.10.sup.6
DNP-modified, intact tumor cells suspended in 0.2-0.3 ml Hanks
solution. Vaccines for doses 2-8 are gently mixed with Tice
Bacillus Calmette-Gurin (Tice BCG; substrain of the Pasteur
Institute strain; Organon Teknika Corp.). BCG in the form of a
lyophilized powder, is reconstituted with 1.0 ml of saline for
injection without preservative to make the "stock Tice BCG"
solution. The stock solution is used the same day as prepared. The
second and third doses of vaccine will be mixed with 0.1 ml of a
1:10 dilution (in saline for injection without preservative) of the
"stock Tice BCG" solution (Tice-A). The fourth and fifth vaccines
will be mixed with 0.1 ml of a 1:100 dilution of the "stock Tice
BCG" solution (Tice-B). The sixth and seventh vaccines, as well as
the vaccine booster at 6 months, will be mixed with 0.1 ml of a
1:1000 dilution of the "stock Tice BCG" solution (Tice-C). The
expected BCG reaction is an inflammatory papule with no more than
small (<5 mm) central ulceration. If reactions are larger than
this (greater than Grade 2; see Section 11.1), the dose of BCG for
the next doses will be further reduced by one 10-fold increment
(Tice-D). No more than two consecutive doses of Tice-A or Tice-B
should be given.
[0099] Treatment outline. On Day 1, the first vaccine dose,
containing haptenized cells free from adjuvant, is administered as
a single intradermal injection into the same limb as for all
subsequent vaccine doses: if into an arm, into the ventral region
of the forearm; if into a leg, into the anterior region just above
the knee. A single dose of cyclophosphamide (300 mg/m.sup.2) is
given as a rapid (5-10 min) intravenous infusion on Day 7.
Beginning on Day 10, subsequent vaccine doses 2-8 are administered
weekly, followed by one booster at month 6. The vaccine is equally
distributed into three intradermal sites, 1-2 cm apart. All vaccine
injections will be given into the same region, separated from
previous injections by 1-2 cm. Ordinarily, the injection site will
be the upper dorsal arm, but not on the side of a lymph node
dissection. Patients who have undergone bilateral axillary node
dissection will be injected in the upper lateral thigh. Routine
laboratory assays (e.g., hematology (CBC with differential platelet
count), chemistry (BUN or creatinine, LDH, SGOT, alkaline
phosphatase, electrolytes), and hepatitis), physical examinations,
and clinical evaluations (e.g., CT/MRI of
head-chest-abdomen-pelvis) are conducted regularly over a period of
5 years. The vaccine study is shown in FIG. 1.
[0100] Evaluation. An analysis evaluating relapse-free survival
will be the primary efficacy endpoint. In addition, overall
survival and tolerance will be evaluated. The incidence of relapse
is calculated with the Kaplan-Meier's product-limit method.
Example 2
Pretreatment with BCG-free Haptenized Tumor Cells Improves
Therapeutic Outcome
[0101] Anti-metastatic effects are induced by DNP-modified,
irradiated, autologous tumor cell vaccine (preceded by low-dose
cyclophosphamide) in a newly developed animal model, (described in
co-pending application Ser. No. 60/180,257, attorney docket No.
1225/0G680, filed on Feb. 4, 2000). This animal model,
particularily useful for providing information on the effectiveness
of postsurgical immunotherapies for recurrence of metastatic
disease, was used to evaluate whether a modified protocol could
offer even greater therapeutic benefits. Specifically, treatment
regimen (A), consisting of low-dose cyclophosphamide followed by
unmodified or DNP-modified autologous (syngeneic) tumor cell
vaccine, was compared to a new pretreatment regimen (B), in which a
single dose of autologous DNP-modified tumor cells (free of BCG)
was administered prior to initiation of the low-dose
cyclophosphamide treatment, followed by unmodified or DNP-modified
autologous tumor cell vaccine.
Materials and Methods
[0102] Tumor cells. The highly metastatic 410.4 tumor (See, e.g.,
Miller et al., Invasion Metastasis 1981;1:220, Pulaski et al.,
Cancer Res 1998;58:1486, and Miller, Cancer Res 1978;38:3758),
originating from a spontaneously arising murine mammary carcinoma
(See, e.g., Heppner et al., Cancer Res 1978;38:3758, and Miller et
al., Cancer Res 1981;41:3863), was used. Tumor cells were
maintained in vitro at 37.degree. C. in 5% CO.sub.2 in Falcon 75
cm.sup.2 polystyrene tissue culture flasks (Becton Dickinson
Labware, Franklin lakes, N.J.) in Dulbecco's modified Eagle medium
(DMEM) supplemented with 10% fetal bovine serum and 100 units/ml
penicillin and 100 .mu.g/ml streptomycin. Every 2-3 days the tumor
cells were detached with trypsin-EDTA solution (Life Technologies
Inc., Grand Island, N.Y.) and 2.0.times.10.sup.6 cells in 20 ml of
medium were seeded per new flask.
[0103] In vivo tumor model In vitro cultured 410.4 tumor cells were
detached with trypsin-EDTA, and 3.times.10.sup.5 410.4 tumor cells
in 0.2 ml RPMI-1640 medium (Life Technologies Inc.) were injected
into the mammary fatpads of female BALB/cAnNCrlBR mice, 7-10 weeks
old (Charles Rivers Breeding Laboratories, Wilmington, Mass.). When
tumors reached 6-8 mm in diameter, the tumors were surgically
excised. Unless otherwise stated, 5-8 days after tumor excision,
the mice were divided into groups and subjected to our experimental
design.
[0104] Vaccine preparation. On the day of vaccination, in vitro
cultured 410.4 tumor cells were detached with 0.02% EDTA solution
(without trypsin) (Sigma Chemical Co., St Louis, Mo.) followed by
forceful pipetting, and the tumor cells were then subjected to
.gamma.-irradiation (2500 cGY from a Cesium-137 source (J. L.
Sepherd and Associates, Model 143-68 irradiator). Subsequently, an
aliquot of the .gamma.-irradiated 410.4 cells was DNP modified by
exposure to dinitrofluorobenzene (DNFB, Sigma Chemicals Co., St.
Louis, Mo.), according to the protocol of Berd et al. (J Clin Oncol
1997;15:2359). Each vaccine was administered in a total volume of
0.2 ml and consisted of 3-5.times.10.sup.6 unmodified or
DNP-modified, .gamma.-irradiated, tumor cells admixed with 0.5 to
4.times.10.sup.6 colony-forming units (CFU) of BCG, Tice
strain.
[0105] Treatment protocols. (A) Non-pretreatment protocol. Unless
otherwise stated, on day five to eight after tumor excision mice
received an intraperitoneal (i.p.) injection of 15 mg/kg
cyclophosphamide. Three days after the low-dose cyclophosphamide
treatment, the mice received a subcutaneous (s.c.) injection of
unmodified, or DNP-modified, irradiated, autologous tumor cell
vaccine close to the site of tumor excision. This protocol was
repeated every 10 days for the duration of the experiment. The mice
were monitored twice a week for the appearance of visible
metastases and the results are presented as percentage of
relapse-free survival among all mice subjected to the same
treatment protocol.
[0106] (B) Pretreatment protocol. The pretreatment protocol
consisted of a single s.c. injection of 1.times.10.sup.6
DNP-modified, irradiated, autologous tumor cell (free of BCG)
administered on the back of the mice on day 3-7 after the excision
of the primary tumor. Three to seven days later, the mice received
an i.p. injection of 15 mg/kg cyclophosphamide (Mead Johnson-A
Bristol-Myers Squibb Co., Princeton, N.J.). Three days after the
low-dose cyclophosphamide treatment, the mice received a s.c.
injection of unmodified, or DNP-modified, irradiated, autologous
tumor cell vaccine close to the site of tumor excision. This
protocol was repeated every 10 days for the duration of the
experiment. The mice were monitored twice a week for the appearance
of visible metastases and the results are presented as percentage
of relapse-free survival among all mice subjected to the same
treatment protocol.
[0107] Statistical analysis. The percentages of relapse-free
survival of mice subjected to different treatment protocols were
compared at various time points after tumor excision by the use of
a paired Student's T-test. A p value of 0.05 or lower was
considered significant.
Results
[0108] Protocol A only. Treatment of mice (from which the primary
tumor was surgically excised) with low-dose cyclophosphamide
followed by DNP-modified, irradiated, autologous tumor cell vaccine
led to a significantly better relapse-free survival than treatment
of such mice with low-dose cyclophosphamide followed by unmodified,
irradiated, autologous tumor cell vaccine (p=0.005).
[0109] Protocol A vs Protocol B. Pretreatment of mice with
DNP-modified, irradiated, autologous tumor cells (without BCG) 3-7
days prior to the initiation of the low-dose cyclophosphamide
treatment followed by DNP-modified, -irradiated, autologous tumor
cell vaccine resulted in a significant improvement in the
relapse-free survival relative to that observed in mice that
received low-dose cyclophosphamide followed by DNP-modified,
irradiated, autologous tumor cell vaccine without the pretreatment
regimen (p=0.002).
[0110] Conclusion. The results illustrated that Protocol B offered
additional therapeutic benefits in the described tumor model of
metastatic disease as compared to Protocol A, suggesting that a
pretreatment protocol, where haptenized tumor cells are
administered prior to cyclophosphamide and additional vaccine
doses, may offer further therapeutic benefits also in patients with
metastatic melanoma.
Example 3
The Timing of an Haptenized or Non-Haptenized, Irradiated,
Autologous, Melanoma, Tumor Cell Treatment Prior to Administration
of an Immunomodulator and an Haptenized Vaccine Improves
Therapeutic Outcome
[0111] The anti-metastatic effects of hapten-modified vaccines are
increased by the administration of a "priming" induction dose
administered several days before administration of a low dose of
the immunomodulator cyclophosphamide (CY) and DNP vaccine mixed
with BCG. The induction (or "priming") dose comprised of one of the
three following compositions: (1) 10.sup.6 irradiated, autologous,
tumor cells (AU TC); (2) a mixture of irradiated, autologous, tumor
cells and irradiated, autologous, DNP-modified tumor cells 10.sup.6
of each; or (3) 10.sup.6 irradiated, autologous, DNP-modified tumor
cells.
Material and Methods
[0112] Patient group. The treatment efficacy of administering a
"priming" induction dose of haptenized or non-haptenized or a
mixture of haptenized and non-haptenized, irradiated, autologous,
tumor-cells before administration of a haptenized vaccine was
studied in 214 post-surgical melanoma patients having bulky,
regional, lymph-node metastases. Prior to the initiation of the
vaccine treatment, preferably within two months from the starting
point, one or more of the tumor masses was surgically resected from
each patient. An adequate tumor sample, approximately 2 cm or 5 g,
yielding at least about 50.times.10.sup.6 cells, was kept under
sterile conditions to be used for vaccine preparation. Vaccines
were prepared according to methods outlined in Example 1. Patients
were preferably confirmed melanoma-free by computed tomography (CT)
or magnetic resonance imaging (MRI).
[0113] Treatment protocols. Three protocols were used to evaluate
whether a pre-treatment induction dose administered several days
before the administration of low-dose cyclophosphamide and
DNP-vaccine mixed with BCG offers greater therapeutic effects than
protocols without a pre-treatment induction dose. The induction (or
"priming") dose comprised one of the three following compositions:
(1) 10.sup.6 irradiated, autologous, tumor cells (AU TC); (2) a
mixture of irradiated, autologous, tumor cells and irradiated,
autologous, DNP-modified tumor cells (10.sup.6 of each); or (3)
10.sup.6 irradiated, autologous, DNP-modified tumor cells;
[0114] Protocol A (124 patients): All patients were administered a
low-dose of cyclophosphamide (300 mg/m.sup.2) intravenously,
followed by multiple, intradermal injections of DNP-vaccine (dose
range: 2.5-25.0.times.10.sup.6 irradiated AUTC) mixed with BCG
three days after the low-dose of cyclophosphamide. The "priming"
dose was administered as an intradermal injection into the ventral
forearm of the patient 5-7 days prior to CY. Protocol B (27
patients): The primary dose was administered on the same day as CY.
Protocol C (43 patients): The primary dose was administered at the
time points (A+B).
Results
[0115] Protocol A, which included administration of a "priming"
induction dose comprising one of the three following compositions:
(1) 10.sup.6 irradiated, autologous, tumor cells (AU TC); (2) a
mixture of 10.sup.6 each of irradiated autologous, tumor cells and
irradiated, autologous, DNP-modified tumor cells; or (3) 10.sup.6
irradiated, autologous, DNP-modified tumor cells (the preferred
reduction composition) several days before administration of a low
dose of cyclophosphamide (CY) and DNP-modified, irradiated,
autologous tumor cells mixed with BCG led to a significantly better
relapse-free survival rate than with protocols in which a primary
composition was administered on the same day as cyclophosphamide
(or omitted altogether). For Protocol A, the 5 year relapse-free
survival rate was 41% versus 18% in Groups B and C, which received
a primary dose on the same day as CY (p=0.1, log rank test).
Multi-variate analysis (Cox regression) showed that the difference
in relapse-free survival was not attributable an imbalance of known
prognostic variables (number of (+) nodes) between the patient
groups treated under each protocol.
[0116] Furthermore, the proportion of patients who developed (+)
delayed-type sensitivity (.gtoreq.5 mm diameter induration) was
81/115=70.4% (p<0.001, Fisher's exact test), which is
significantly higher than the (+) DTH response rates of protocols
with the "priming" dose the same day as CY. The proportion of
patients who developed (+) delayed-type sensitivity (.gtoreq.5 mm
diameter induration) under Protocol C was 10/51=19.6% (p<0.001,
Fisher's exact test). Since it has been shown that development of
(+) DTH response to DNP modified or unmodified AU TC following
vaccine treatment was significantly associated with longer
survival, the "priming" induction dose comprising one of the three
following compositions: (1) irradiated, autologous, tumor cells
(AUTC); (2) a mixture of irradiated, autologous, tumor cells and
irradiated, autologous, DNP-modified tumor cells; and (3)
irradiated, autologous, DNP-modified tumor cells irradiated,
autologous, tumor cells as in Protocol A, increases the chances of
patient survival by increasing (+) DTH response rates.
[0117] The timing of the "priming" induction dose several days
prior to administration of a low-dose of cyclophosphamide and not
near or on the same day as CY apparently determines whether the
subsequent course of DNP-vaccine results in tumor immunity or
unresponsiveness.
[0118] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0119] It is further to be understood that all numerical values are
approximate and are provided for description only.
[0120] Patents, patent applications, and publications cited
throughout this application are incorporated herein by reference in
their entireties.
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