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Cancer Patent Abstract
This invention comprises cellular vaccines and methods of using
them in cancer immunotherapy, particularly in humans. The vaccines
comprise a source of tumor-associated antigen, and a cytokine-secreting
cell line. Tumor antigen may be provided in the form of primary
tumor cells, tumor cell lines or tumor extracts prepared from the
subject. In certain embodiments of the invention, the cytokine-secreting
line is a separate tumor line that is allogeneic to the patient
and genetically altered so as to produce a cytokine at an elevated
level. Exemplary cytokines are IL-4, GM-CSF, IL-2, TNF-.alpha.,
and M-CSF in the secreted or membrane-bound form. In these embodiments,
the cytokine-producing cells provide immunostimulation in trans
to generate a specific immune response against the tumor antigen.
Vaccines may be tailored for each type of cancer or for each subject
by mixing tumor antigen with a favorable number of cytokine-producing
cells, or with a cocktail of such cells producing a plurality of
cytokines at a favorable ratio.
Cancer Patent Claims
What is claimed as the invention is:
1. A method of stimulating an anti-tumor immune response or treating
a neoplastic disease, comprising administering to a subject a composition
comprising: a cell expressing a cytokine from a recombinant polynucleotide,
wherein the cytokine comprises a heterologous transmembrane region
and is stably associated in the cell outer membrane, and wherein
the cell has been inactivated to prevent proliferation.
2. The method of claim 1, wherein the cytokine is selected from
IL-4, IL-2, TNT-.alpha., and M-CSF.
3. The method of claim 1, wherein the cell is a cancer cell.
4. The method of claim 1, wherein the cell is from a tumor of the
same tissue type as a tumor in the subject.
5. The method of claim 4, wherein the tumor is an ovarian cancer
or a brain cancer.
6. The method of claim 1, wherein the cell is allogeneic to the
subject.
7. The method of claim 1, wherein the cell is histocompatibly identical
to the subject.
8. The method of claim 1, wherein the cell has been inactivated
by irradiation.
9. The method of claim 1, wherein the cell produces a secreted
cytokine in addition to the cytokine stably associated in the outer
membrane.
10. The method of claim 1, wherein a majority of the cytokine produced
by the cell is present on the outer membrane of the cell.
11. The method of claim 1, wherein the composition comprises at
least two cells, each of which has been genetically altered to produce
a different cytokine at an elevated level, or is the progeny of
such a cell, and wherein each cytokine is stably associated in the
outer membrane of the cell.
12. The method of claim 1, wherein the cell is a human cell.
13. The method of claim 1, wherein the cell in the composition
has been transduced in vitro with a retroviral expression vector,
or is the progeny of such a cell.
14. The method of claim 1, which is a method for priming an anti-tumor
immune response.
15. The method of claim 1, which is a method for boosting or maintaining
an anti-tumor immune response.
16. The method of claim 1, which is a method for treating a neoplastic
disease.
17. The method of claim 1, further comprising providing the cytokine
expressing cell that is present in the composition.
18. The method of claim 1, further comprising preparing the composition
by transducing a cancer cell in vitro with an expression vector
encoding the membrane-associated cytokine.
19. The method of claim 1, wherein the cytokine is M-CSF.
20. A method of stimulating an anti-tumor immune response or treating
a neoplastic disease, comprising administering to a subject a composition
comprising a cell expressing an IL-4 from a recombinant polynucleotide,
wherein the IL-4 is stably associated in the cell outer membrane
and wherein the cell has been inactivated to prevent proliferation.
21. The method of claim 20, wherein the cytokine is a fusion protein
comprising a heterologous transmembrane region.
22. A method of stimulating an anti-tumor immune response or treating
a neoplastic disease, comprising administering to a subject a composition
comprising a cell expressing a GM-CSF from a recombinant polynucleotide,
wherein the GM-CSF is stably associated in the cell outer membrane,
and wherein the cell has been inactivated to prevent proliferation.
23. The method of any of claims 1, 20, or 22, wherein the composition
further comprises a tumor-associated antigen, and wherein the combination
of the cytokine and the tumor-associated antigen in the composition
is effective in treating a neoplastic disease or eliciting an anti-tumor
immunological response in the subject.
24. The method of claim 23, wherein the tumor-associated antigen
is obtained from a cell autologous to the subject.
25. The method of claim 23, wherein the tumor-associated antigen
is expressed by the same cells expressing the membrane-associated
cytokine.
26. The method of claim 23, wherein the composition comprises a
combination of: a) the cell expressing the membrane-associated cytokine;
and b) a tumor cell autologous to the subject; wherein the combination
is effective in treating a neoplastic disease or eliciting an anti-tumor
immunological response in the subject.
27. The method of claim 23, further comprising providing the tumor
associated antigen that is present in the composition.
28. The method of claim 26, wherein the tumor cell is a primary
tumor cell dispersed from a solid tumor obtained from the subject.
29. The method of claim 26, wherein the tumor cell is a glioma,
a glioblastoma, a gliosarcoma, an astrocytoma, or an ovarian cancer
cell.
30. The method of claim 26, wherein the tumor cell has been inactivated
by irradiation.
Cancer Patent Description
FIELD OF THE INVENTION
The present invention relates generally to the fields of cellular
immunology and cancer therapy. More specifically, it relates to
the generation of an anti-tumor immune response in a human by administering
a cellular vaccine, comprising cells genetically altered to secrete
a cytokine, in combination with a source of tumor antigen.
BACKGROUND
In spite of numerous advances in medical research, cancer remains
a leading cause of death throughout the developed world. Non-specific
approaches to cancer management, such as surgery, radiotherapy and
generalized chemotherapy, have been successful in the management
of a selective group of circulating and slow-growing solid cancers.
However, many solid tumors are considerably resistant to such approaches,
and the prognosis in such cases is correspondingly grave.
One example is brain cancer. Each year, approximately 15,000 cases
of high grade astrocytomas are diagnosed in the United States. The
number is growing in both pediatric and adult populations. Standard
treatments include cytoreductive surgery followed by radiation therapy
or chemotherapy. There is no cure, and virtually all patients ultimately
succumb to recurrent or progressive disease. The overall survival
for grade IV astrocytomas (glioblastoma multiforme) is poor, with
.about.50% of patients dying in the first year after diagnosis.
A second example is ovarian carcinoma. This cancer is the fourth
most frequent cause of female cancer death in the United States.
Because of its insidious onset and progression, 65 to 75 percent
of patients present with tumor disseminated throughout the peritoneal
cavity. Although many of these patients initially respond to the
standard combination of surgery and cytotoxic chemotherapy, nearly
90 percent develop recurrence and inevitably succumb to their disease.
Because these tumors are aggressive and highly resistant to standard
treatments, new therapies are needed.
An emerging area of cancer treatment is immunotherapy. The general
principle is to confer upon the subject being treated an ability
to mount what is in effect a rejection response, specifically against
the malignant cells. There are a number of immunological strategies
under development, including: 1. Adoptive immunotherapy using stimulated
autologous cells of various kinds; 2. Systemic transfer of allogeneic
lymphocytes; 3. Intra-tumor implantation of immunologically reactive
cells; and 4. Vaccination at a distant site to generate a systemic
tumor-specific immune response.
The first of the strategies listed above, adoptive immunotherapy,
is directed towards providing the patient with a level of enhanced
immunity by stimulating cells ex vivo, and then readministering
them to the patient. The cells are histocompatible with the subject,
and are generally obtained from a previous autologous donation.
One approach is to stimulate autologous lymphocytes ex vivo with
tumor-associated antigen to make them tumor-specific. Zarling et
al. (1978) Nature 274:269-71 generated cytotoxic lymphocytes in
vitro against autologous human leukemia cells. Lee et al. (1996)
abstract, Gastroenterology conducted an in vitro mixed lymphocyte
culture with inactivated leukemic blast cells and autologous lymphocytes,
and generated effector T lymphocytes cytotoxic for a tumor antigen
on autologous blast cells. An MHC D-locus incompatibility was thought
to be necessary to provide proper help in the lymphocyte culture.
Lesham et al. (1984) Cancer Immunol. Immunother. 17:117-23 developed
cytotoxic responses in vitro against murine thymoma cells by allosensitization.
Gately et al. (1982) J. Natl. Cancer Inst. 69:1245-54 found that
5 out of 9 human glioma cell lines did not elicit allogeneic cytolytic
lymphocyte responses in ex vivo cultures. However, if inactivated,
allogeneic lymphocytes were provided as stimulator cells in the
cultures, tumor-specific cytolytic T lymphocytes and non-specific
non-T effectors were generated to 4 of the nonstimulatory lines.
In U.S. Pat. No. 5,192,537, Osband suggests activating a tumor patient's
mononuclear cells by culturing them ex vivo in the presence of tumor
cell extract and a non-specific activator like phytohemagglutinin
or IL-1, and then treating the culture to deplete suppresser cell
activity.
Despite these experimental observations, systemic administration
of ex vivo-stimulated autologous tumor-specific lymphocytes has
not become part of standard cancer therapy.
Autologous lymphocytes and killer cells may also be stimulated
non-specifically. In one example, Fc receptor expressing leukocytes
that can mediate an antibody-dependent cell-mediated cytotoxicity
reaction are generated by culturing with a combination of IL-2 and
IFN-.gamma. U.S. Pat. No. 5,308,626. In another example, peripheral
blood-derived lymphocytes cultured in IL-2 form lymphokine-activated
killer (LAK) cells, which are cytolytic towards a wide range of
neoplastic cells, but not normal cells. LAK are primarily derived
from natural killer cells expressing the CD56 antigen, but not CD3.
Such cells can be purified from peripheral blood leukocytes by IL-2-induced
adherence to plastic (A-LAK cells; see U.S. Pat. No. 5,057,423).
In combination with high dose IL-2, LAK cells have had some success
in the treatment of metastatic human melanoma and renal cell carcinoma.
Rosenberg (1987) New Engl. J. Med. 316:889-897. This strategy is
labor-intensive, costly, and not suited to all patients. Schwartz
et al. (1989) Cancer Res. 49:1441-1446 showed that A-LAK cells are
superior to LAK cells at reducing lung and liver metastases of breast
cancer in experimental animal models, but this was not curative
and there were no long-term survivors.
For examples of trials conducted using LAK in the treatment of
brain tumors, see Merchant et al. (1988) Cancer 62:665-671 &
(1990) J. Neuro-Oncol. 8:173-198; Yoshida et al. (1988) Cancer Res.
48:5011-5016; Barba et al. (1989) J. Neurosurg. 70:175-182; Hayes
et al. (1988) Lymphokine Res. 7:337-345; and Naganuma et al (1989)
Acta Neurochir. (Wien) 99:157-160. Another study proposes therapy
for recurrent high-grade glioma using autologous mitogen-activated
and IL-2 stimulated (MAK) killer lymphocytes, in combination with
IL-2. Jeffes et al. (1991) Lymphokine Res. 10:89-94. While none
of these trials was associated with serious clinical complications,
efficacy was only anecdotal or transient. Induction of tumor-specific
immunity in patients receiving such treatments has not been shown.
Another form of adoptive therapy using autologous cells has been
proposed based on observations with tumor-infiltrating lymphocytes
(TIL). TILs are obtained by collecting lymphocyte populations infiltrating
into tumors, and culturing them ex vivo with IL-2. Finke et al.
(1990) Cancer Res. 50:2363-2370 have characterized cytolic activity
of CD4+ and CD8+ TIL in human renal cell carcinoma. TILs have activity
and tumor specificity superior to LAK cells, and have been experimentally
administered, for example, to humans with advanced melanoma. Rosenberg
et al. (1990) New Engl. J. Med. 323:570-578. The effector population
within TILs may be cytotoxic T lymphocytes (CTL) which are primed
to be tumor-specific in the host and are devoid of lytic granules,
and become transformed into cytolytic lymphoblasts when stimulated
in culture. Berke et al.(1988) J. Immunol. 129:303 ff. Unfortunately,
TILs can only be prepared in sufficient quantity to be clinically
relevant in a limited number of tumor types. These strategies remain
experimental, especially in human therapy.
The second of the strategies for cancer immunotherapy listed earlier
is adoptive transfer of allogeneic lymphocytes. The rationale of
this experimental strategy is to create a general level of immune
stimulation, and thereby overcome the anergy that prevents the host's
immune system from rejecting the tumor. Strausser et al. (1981)
J. Immunol. Vol. 127, No. 1 describe the lysis of human solid tumors
by autologous cells sensitized in vitro to alloantigens. Zarling
et al. (1978) Nature 274:269-71 demonstrated human anti-lymphoma
responses in vivo following sensitization with allogeneic leukocytes.
Kondo et al. (1984) Med Hypotheses 15:241-77 observed objective
responses of this strategy in 20-30% of patients, and attributed
the effect to depletion of suppressor T cells. The studies were
performed on patients with disseminated or circulating disease.
Even though these initial experiments were conducted over a decade
ago, the strategy has not gained general acceptance, especially
for the treatment of solid tumors.
The third of the immunotherapy strategies listed earlier is intra-tumor
implantation. This is a strategy directed at delivering effector
cells directly to the site of action. Since the transplanted cells
do not circulate, they need not be histocompatible with the host.
Intratumor implantation of allogeneic cells may promote the ability
of the transplanted cells to react with the tumor, and initiate
a potent graft versus tumor response.
Kruse et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:9577-9581
demonstrated that direct intratumoral implantation of allogeneic
cytotoxic T lymphocytes (CTL) into brain tumors growing in Fischer
rats resulted in a significant survival advantage over other populations
of lymphocytes, including syngeneic CTL, LAK cells, adherent-LAK
cells or IL-2 alone. Redd et al. (1992) Cancer Immunol. Immunother.
34:349-354 developed cytotoxic T lymphocytes specific for an allogeneic
brain tumor in rats. The lymphocytes were specific for a determinant
expressed only by the tumor, and were predicted to be useful for
therapeutic purposes in vivo. Kruse et al. (1994) J. Neurooncol.
19:161-168 prepared CTLs from four MHC incompatible rat strains,
and used them to treat Fischer rats bearing established 9L brain
tumors. CTL were administered on a biweekly schedule, a different
MHC incompatible CTL preparation being administered each time. Animals
without tumor showed minimal localized brain damage. Those with
tumors either showed: a) mononuclear cell infiltration, massive
tumor necrosis beginning 2-4 days after treatment, and total tumor
destruction by 15 days; or b) cellular infiltration, early tumor
destruction, and then tumor regrowth progressing to death of the
animal. Tumor regressor animals were resistant to intracranial rechallenge
with viable tumor cells. Kruse et al. (1994). Intratumor CTL implants
may optionally be combined with chemotherapy using cyclophosphamide.
Kruse et al. (1993) J. Neurooncol. 15:97-112.
Despite the promise of intratumor implantation techniques, several
caveats remain. First, implantation is frequently performed by surgical
techniques, which may be too invasive for routine maintenance. Second,
the strategy is directed at generating a local response, and may
not be effective against metastases. Finally, the techniques remain
unproved for use in human therapy.
The fourth of the immunotherapy strategies listed earlier is the
generation of an active systemic tumor-specific immune response
of host origin. The response is elicited from the subject's own
immune system by administering a vaccine composition at a site distant
from the tumor. The specific antibodies or immune cells elicited
in the host as a result will hopefully migrate to the tumor, and
then eradicate the cancer cells, wherever they are in the body.
Various types of vaccines have been proposed, including isolated
tumor-antigen vaccines and anti-idiotype vaccines. Mitchell et al.
(1993) Ann. N.Y. Acad. Sc. 690:153-166 have treated cancer patients
with mechanical lysates from a plurality of allogeneic melanoma
cell lines, combined with the adjuvant DETOX.TM.. These approaches
are all based on the premise that tumors of related tissue type
all share a common tumor-associated antigen. For patients with tumors
that did not acquire expression of the antigen during malignant
transformation, or that subsequently differentiated so as not to
express it, none of these vaccines will be successful.
An alternative approach to an anti-tumor vaccine is to use tumor
cells from the subject to be treated, or a derivative of such cells.
For review see, Schirrmacher et al. (1995) J. Cancer Res. Clin.
Oncol. 121:487-489. In U.S. Pat. No. 5,484,596, Hanna Jr. et al.
claim a method for treating a resectable carcinoma to prevent recurrence
or metastases, comprising surgically removing the tumor, dispersing
the cells with collagenase, irradiating the cells, and vaccinating
the patient with at least three consecutive doses of about 10.sup.7
cells. The cells may optionally be cryopreserved, and the immune
system may be monitored by skin testing. This approach does not
solve the well-established observations that many tumors are not
naturally immunogenic. Many patients from which tumors have been
resected are either tolerant or unable to respond to their own tumor
antigen, even when comprised in a vaccine preparation.
Several ways of preparing autologous or syngeneic tumor cells have
emerged that potentially enhance immunogenicity. Tumor cells may
be combined with extracts of bacillus Calmette-Guerin (BCG) or the
A60 mycobacterial antigen complex. Berd et al. (1990) J. Clin. Oncol.
8:1858-67; Maes et al. (1996) J. Cancer Res. Clin. Oncol. 122:296-300.
Tumor cells may be lysed by or mixed with vaccinia virus. Hersey
et al.; Ito et al. Tumor cells may be incubated with the Newcastle
Disease Virus (NDV). U.S. Pat. No. 5,273,745. Autologous tumor cells
may also be conjugated to haptens like dinitrophenyl. U.S. Pat.
No. 5,290,551.
In another approach to increase immunogenicity, Guo and coworkers
(WO 95/16775) suggest that tumor cells be fused with membrane components
of a second cell that has a greater immunogenic potential. Suitable
cells are an activated antigen-presenting cell such as a B cell.
The fusion partner cell may be genetically altered to express an
MHC protein, adhesion protein, or a cytokine. Rat hepatocarcinoma
cells lost tumorigenicity when fused with syngeneic B cells, and
were capable of eliciting a T-cell response. Rats injected with
the hybrid cells generated CD4+ and CD8+ T cells against subsequent
challenge, or eradicated preexisting tumors via a CD8+ T cell mediated
mechanism.
In yet another approach, autologous or syngeneic tumor cells are
genetically altered to produce a costimulatory molecule. Examples
of costimulatory molecules include cell surface receptors, such
as the B7-1 costimulatory molecule or allogeneic histocompatibility
antigens. Salvadori et al.(1995) Hum. Gene Ther. 6:1299-1306; Plaksin
et al. (1994) Int. J Cancer 59:796-801; EP 56967.
Other examples are secreted activators, including cytokines. For
review see, Pardoll et al. (1992) Curr. Opin. Immunol. 4:619-23;
Saito et al. (1994) Cancer Res. 54:3516-3520; Vieweg et al.(1994)
Cancer Res. 54:1760-1765; Gastl et al. (1992) Cancer Res. 52:6229-6236;
and WO 96/07433). Tumor cells have been genetically altered to produce
TNF-.alpha., IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IFN-.alpha.,
IFN-.gamma. and GM-CSF. Asher et al. (1991) J. Imunol. 146:3227-3234;
Blankenstein et al. (1991) J. Exp. Med. 173:1047-1052; Karp et al.
(1993) J. Imunol. 150:896-908; Douvdevani et al. (1992) Int. J.
Cancer 51:822-830; Cavallo et al. (1992) J. Immunol. Vol. 149: 3627-3635
No. 11 & (1993) Cancer Res. 53:5067-5070; Fearon et al. (1990)
Cell 60:397-403; Gansbacher et al. (1990) J. Exp. Med. 172:1217-1224;
Connor et al. (1993) J. Exp. Med. Vol. 177:1127-1134; Topalian et
al. (1988) J. Clin. Oncol. 6:838-853; McBride et al. (1992) Cancer
Res. 52:3931-3937; Golumbek et al. (1989) Science 254:713 ff &
(1991) Science 254:713-716; Tepper et al. (1989) Cell 57:503-512;
Santin et al. (1995b); Santin et al. (1995c) Int. J. Gynecol. Cancer
5:401-410; Gynecol. Oncol. 58:230-239; Santin (1996) Am. J. Obst.
Gynecol. 174:633-639; Allione et al. (1994) Cancer Res. 54:6022-6026;
EP 538952; Belldegrun et al. (1993) J. Natl. Cancer Inst. 85:207-216;
Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA Vol. 90:3539-3543.
Golumbek et al. (1989) reported that mouse renal carcinoma cells
inserted with a gene for IL-4 was strongly immunogeneic for systemic
T cell immunity, and protected mice against a subsequent lethal
challenge with unmodified, parental tumor cells. Induction of an
immune response does not depend on inherent immunogenicity; cytokines
like IL-2 induce a response that is protective against otherwise
non-immunogenic adenocarcinoma cells, including distant metastases.
Cavallo et al. 1991 & 1992. Antitumor immunity is intensified
by a cancer vaccine that produces both GM-CSF and IL-4. Wakimoto
et al. (1996) Cancer Res. 56:1828-33. The cytokine or cytokine combination
may recruit or stimulate cells of the immune system, and thereby
overcome the normal barrier to immunity. Certain cytokines also
affect the expression of major histocompatibility molecules and
intercellular adhesion molecules by cancer cells (Santin et al.
1995a, Int. J. Cancer 65:688-694), potentially improving immunogenicity.
The experiments with transduced histocompatible tumor cells have
been done chiefly in genetically restricted animal models, which
are not directly equivalent to a heterogeneous human patent population.
Colombo et al. (1995) Cancer Immunol. Immunother. 41:265-270. Not
all cancer types are responsive to the same cytokines. There are
concerns about injecting human patients with replication-competent
tumor cells, particularly after genetic alteration. In addition,
there is usually not enough time to genetically alter and grow up
sufficient cells of the the patient to be treated for use in a vaccine.
Blumbach (WO 96/05866) has suggested vaccines of live tumor cells
transduced with: a) a gene coding for an immunostimulatory protein;
b) a cytokine; and c) a thymidine kinase gene. The composition is
provided as live cells which can grow in vivo and stimulate a response,
and then be selectively killed via the thymidine kinase. The possibility
of escape mutants is likely to be a subject of regulatory concern
for this approach in human therapy. Golumbek et al. (1992) J. Immunother.
12:224-230 have shown that proliferating tumor cells with suicide
genes can also survive toxin treatment when they exit the cell cycle
temporarily or are sequestered pharmacologically.
As an alternative, Cohen (WO 95/31107) suggested that neoplastic
disease can be treated with a cellular immunogen comprising allogeneic
cells genetically altered to express one or more cytokines, and
also to express tumor-associated antigens encoded by autologous
genomic tumor DNA. In this approach, an allogeneic cell (exemplified
as a mouse LM cell) is genetically altered to express: a) a cytokine;
and b) a tumor-associated antigen autologous to the subject to be
treated. Accordingly, the vaccine need not comprise live tumor cells.
However, application of the Cohen invention to human subjects would
require prior knowledge for each patient of a particular tumor-associated
antigens expressed by the particular tumor. Many human cancers of
widespread clinical interest do not have reliable commonly-shared
markers. Once a relevant marker is identified for a particular patient,
a cell line must be engineered accordingly, and cultured to the
required density prior to treatment. Thus, each patient would become
their own research and development project. Since the immune response
would be focused only at the particular tumor-associated antigen
used, it may be less effective than one directed against the spectrum
of antigen expressed by a complete tumor cell. Furthermore, the
vaccine comprises a live genetically altered cell line, raising
the concerns outlined earlier. Cohen demonstrated only a modest
improvement in survival in the animal studies, and failed to provide
any evidence that his formulation would be effective in human cancer
patients.
A suitable strategy for a human anti-tumor cellular vaccine has
to contend with the following problems: a) heterogeneity amongst
tumors (even tumors of the same type) in the display of tumor-associated
antigens; b) heterogeneity in the immune response between individuals
with regards to both antigens and cytokines; c) ethical and regulatory
concerns about compositions that may be used in humans; and d) lack
of development time in most clinical settings, limiting the ability
to engineer new cell lines or otherwise tailor the vaccine to each
patient.
SUMMARY OF THE INVENTION
This invention provides compositions and methods for eliciting
an anti-tumor immune response in a human patient in need thereof.
The compositions of the invention are cells or cell mixtures in
a compatible excipient, and are referred to herein as a vaccine
or an immunogenic composition. They may be administered to patients
either to treat or palliate a clinically detectable tumor, or for
prophylaxis, particularly after surgical debulking, chemotherapy
or radiation therapy of a previously detectable tumor. The compositions
are typically administered at a location distant from the original
tumor, with the objective of stimulating a systemic reactivity against
the tumor. The reactivity may in turn eradicate or slow the development
of tumor cells, either at the primary site, within metastases (if
there are any), or both.
Minimally, the vaccines of this invention comprise two components.
The first is a source of tumor antigen, preferably a plurality of
antigens, which is associated with the cancer for which the patient
is at risk. A convenient source of tumor-associated antigen is tumor
cells previously obtained from the patient, such as during surgical
resection. The second component is a cytokine producing cell capable
of stimulating the patient's immune system to produce an anti-tumor
response.
In one series of preferred embodiments, the cytokine producing
cell is a cell from an allogeneic donor, typically a tumor cell
and preferably a tumor cell of the same type as the subject being
treated, that has been genetically altered to express the cytokine
at an elevated level. A preferred source of tumor antigen is the
patient, and the vaccine is typically assembled by mixing tumor
cells from the patient (or antigen therefrom) with the allogeneic
cytokine-producing cells.
In another series of preferred embodiments, the cytokine producing
cell is a cell that is autologous or syngeneic to the patient that
has been genetically altered to produce a cytokine. Typically, the
cell will be a cell obtained from the patients tumor (or its progeny)
that was subsequently altered so as to produce an effective amount
of the cytokine. In this form, the same cell provides both components
necessary to evoke the desired response: i.e., both the tumor antigen
and the stimulatory cytokine.
Cytokines useful for expression by the cytokine-producing cells
according to either of these series of embodiments include those
that promote immunostimulation against the tumor antigen by any
mechanism. Preferred and non-limiting examples are IL-4, GM-CSF,
IL-2, TNF-.alpha., and M-CSF. In certain embodiments, the cytokine
is primarily secreted by the cell. In other embodiments, the cytokine
is produced by the cell as a transmembrane protein, and provides
immunostimulation by a mechanism that may involve intercellular
contact. Transmembrane cytokines include those such as mM-CSF, that
naturally occur in a transmembrane form, and cytokines that naturally
occur in a secreted form that are engineered to incorporate a region
that allows them to be retained in the cell membrane.
Also embodied in this invention are compositions and methods for
treating a neoplastic disease such as cancer, comprising administering
any one of the compositions or vaccines of this invention, or eliciting
an anti-tumor immunological response according to any one of the
methods of this invention. The treatment is effective in palliating
the disease condition according to any clinically acceptable criteria
for improvement, such as inhibition of tumor growth, increase in
life expectancy of the patient, or improvement in quality of life
or performance activity score.
Further embodiments of the invention include kits and methods for
assembling the immunological and pharmaceutical compositions of
this invention for use according to the descriptions provided in
this disclosure.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a two-panel graph depicting the growth properties (Panel
A) and cytokine secretion (Panel B) by an ovarian cancer cell line
genetically altered to express IL-4, designated UCI 107E IL-4 GS.
FIGS. 2A-C are graphs showing the effects of irradiation on the
IL-4 secreting tumor cell line UC1 107E IL-4 GS. FIG. 2A shows the
growth paffem of cells given 5,000 (.quadrature.) or 10,000 (.box-solid.)
rads. FIGS. 2B & 2C show IL-4 detected by ELISA in the culture
medium expressed as total concentration (FIG. 2B) or per cell (FIG.
2C) Various times after irradiation.
FIG. 3A-C are a series of FACS analysis profiles (incidence versus
fluorescence intensity) revealing expression of various surface
antigen by UCI 107E IL-4 GS, before or after irradiation with 5,000
or 10,000 rads.
FIG. 4 is a four-panel graph depicting cytokine secretion by human
glioblastoma clones genetically altered to express TNF-.alpha.,
IL-4, IL-2, or GM-CSF.
FIG. 5 is a three-panel graph showing the effects of irradiation
on viability and TNF-.alpha. secretion by the glioblastoma cell
line ACBT/TNF-G at 10,000 (.quadrature.) or 20,000 (.box-solid.)
rads.
FIG. 6 is a graph showing that rats surviving intracranial implantation
with live syngeneic glioblastoma cells expressing membrane M-CSF
(.box-solid.) but not naive controls (.circle-solid.) survive a
subsequent intracranial challenge with parental glioblastoma cells
(Panel A), or glioma cells (Panel B), but not adenocarcinoma cells
(Panel C).
FIG. 7 is two-panel graph showing that syngeneic glioblastoma cells
expressing membrane M-CSF induce a systemic cell-mediated immunity
in animals. The Upper Panel shows that splenocytes transfer tumor
resistance between animals (.circle-solid.) unless depleted using
anti-T-lymphocyte antibody (.tangle-solidup.). The Lower Panel shows
that administration of the M-CSF expressing cell line (.box-solid.)
but not vector control cells (.circle-solid.) at a site outside
the brain causes regression of the tumor cells injected subcutaneously.
DETAILED DESCRIPTION
A central feature of the cellular vaccines of this invention is
the use of multiple components that act in concert once inside the
host to produce the desired effect. In other words, the strategy
is more than just an injection of cancer cells.
The strategy is a significant departure from previous approaches
to cancer immunotherapy in humans. Several important embodiments
of this invention differ from other compositions comprising cancer
cells, in that it contains separately: a) tumor associated antigen;
and b) a cytokine expressing cell line that acts in trans to induce
a beneficial response against the antigen.
Tumor antigen is preferably provided from a cancer cell or the
progeny thereof, preferably a cell autologous to the subject to
be treated, typically obtained from the subject either by surgical
resection, biopsy, blood sampling, or other suitable technique.
The cytokine-producing cell is from a different donor which is genetically
altered and characterized ahead of time for properties relating
to its ability to stimulate an enhanced immune response when used
in a vaccine of this invention. The genetically altered cell is
allogeneic to the subject being treated, and is typically the same
type of cancer as is borne by the subject.
A separately filed patent application shows that mixed lymphocytes
implanted directly into a tumor bed limits or even reverses tumor
growth. These experiments and results are described in a PCT patent
application published as WO 96/29394 (corresponding to PCT/US96/03621),
which is hereby incorporated herein by reference in its entirety.
The effect on tumor mass appeared in part to be due to an active
immunological reaction of host origin, which appears to be a long-lasting
one. It was hypothesized that increased expression transplantation
antigens stimulated by allogeneic lymphocytes in the implant resulted
in the massive recruitment of lymphoid cells near the tumor site,
and that certain of the recruited cells played a role in reacting
specifically against the tumor. A significant element of this hypothesis
is that the cells stimulating the host immune response (the mixed
lymphocytes) are different from the source of tumor antigen, but
lead to reactivity against tumor antigen.
Observations of this kind contributed partly to the inspiration
for additional vaccine compositions. A second-generation vaccine
would encompass a number of improvements over previously disclosed
compositions. Desirable improvements include: a composition that
could stimulate an active response against a plurality of tumor-associated
antigens in any subject treated; the ability to prepare a vaccine
without preculturing of the subject's cells, preferably at the instant
that tumor cells are available from the subject; the use of cells
of minimal proliferative capacity; a well-defined and reproducible
immunostimulatory capacity; the ability to tailor the immunostimulatory
capacity to the patient, as required; and a capability to administer
the vaccine at a site distant from the primary tumor, preferably
with minimal invasiveness.
In order to meet these requirements, it was decided that the immunostimulatory
cells and the source of tumor antigen should be different. Cells
genetically altered to produce cytokines are strongly immunostimulatory.
When cells are obtained from a donor other than the subject, they
can be genetically altered in advance, cloned to stabilize the characteristics,
selected for high levels of expression, and further selected for
an ability to express cytokines even after inactivation. This eliminates
the need to culture each autologous cell line, and has the benefit
of careful standardization and quality control. The fact that the
immunostimulatory cells are typically HLA-incompatible is probably
irrelevant, since their main role is to initiate an immunological
reaction in the host, which can then mature after the immunostimulatory
cells are depleted. HLA-incompatibility can even be an advantage
in the immunostimulatory potential of the cells.
Stimulation is provided to generate an immune reaction against
tumor antigen as a bystander. When used in an implant setting, a
nexus of tumor antigen is supplied by residual primary tumor cells.
For a systemic or distally administered composition, it is necessary
to provide the nexus of tumor antigen by mixing it into the preparation.
Preferably, a plurality of tumor antigen associated with the subject's
own tumor is used. This is conveniently provided by using cells
obtained from the subject's tumor, progeny thereof, or an extract
of either the primary tumor cells or their progeny. These cells
may also be inactivated, since they generally do not need to proliferate
to provide tumor antigen. Using autologous tumor cells confers the
additional advantage of being HLA-compatible, meaning the cells
may persist near the injection site or at another site of ongoing
immunological activity, to assist in the maturation of the response.
A hallmark of the cellular vaccines of the present invention is
that the effect is substantially greater than is obtained using
tumor cells alone, or tumor cells mixed with previously used adjuvants
or cofactors. Interaction between the tumor cells and the stimulated
lymphocytes of the vaccines is probably complex. While not wishing
to be bound by theory, it is envisaged that the cytokine expressed
by the genetically altered cell is effective in recruitment, activation,
or stimulating the interaction of host immune cells. The recruited
and stimulated host cells may then respond to atypical (but otherwise
less immunogenic) components in the vicinity, including any antigens
present upon or within or secreted by the autologous tumor cells.
The cytokine-producing cells may also play a role in promoting antigen
processing and presentation, or provide co-stimulation for antigen
being presented. In addition, the cytokine-producing cells may also
provide specific immunostimulation in cis for the tumor antigens
expressed by the cytokine-producing cells. Accordingly, in certain
preferred embodiments of this application, the cells used to generate
the cytokine-producing cells are derived from a tumor type that
is closely related to that of the subject being treated.
An immunological response resulting from administration of the
vaccine may comprise both humoral and cellular components, but a
cellular response is especially preferred. Cellular immunity (either
cytotoxic lymphocytes, or helper-inducer cells recruiting other
effector mechanisms) are believed to be important in providing a
specific effect against the cells of the target neoplasia. The presence
of an immunological response may be monitored by standard immunological
techniques. However, in human therapy, a primary objective is an
improvement in the clinical condition of the patient. Clinical outcome
is therefore a superior assay for the effectiveness of the compositions
and methods of this invention when directed towards cancer treatment.
The present invention is superior to strategies used or suggested
previously. Advantages of the vaccine compositions of this invention
include the following: The vaccines improve the clinical condition
or prognosis of human cancer patients, even though tumor cells residing
in cancer patients are apparently poorly immunogenic on their own.
Although the response is presumably mediated by a tumor-associated
antigen, there is no need to confirm the presence of any particular
antigen on the tumor of a treated subject. Use of patent's own tumor
cells or an extract of such cells ensures a spectrum of relevant
antigens. There is no need to genetically alter patients' cells,
or use patients' DNA to genetically alter cells of the vaccine.
The strategy is aimed at generating a long-lived systemic immune
response, and may therefore be effective not only against the primary
tumor, but also against metastatic cells sharing tumor antigen with
the primary tumor. With the exception of the initial sampling of
the tumor cells, the protocol may be performed with minimally invasive
procedures. The vaccine compositions are preferably administered
at a site distant from the tumor. Subcutaneous routes of administration
are preferred.
A particularly beneficial feature of certain vaccines of the invention
is the fact that vaccine compositions can be tailored to particular
cancer types, clinical features, and even to an individual subject,
as necessary.
This is important where different tumor types respond to different
cytokines and cytokine mixtures. For example, one tumor type can
respond more frequently to IL-4 in combination with GM-CSF, whereas
another tumor can respond to IL-4 in combination with TNF-.alpha..
Accordingly, a vaccine for the first tumor type is prepared by mixing
cells genetically altered to express IL-4, and cells genetically
altered to express GM-CSF with tumor-associated antigen from the
subject to be treated. A vaccine for the second type is prepared
by mixing cells expressing IL-4 with cells expressing TNF-.alpha.
and tumor-associated antigen. There is no need to genetically alter
a cell line to express multiple cytokines (although this is included
in the invention) since lines expressing different cytokines may
be combined. In another example, one tumor type can respond slightly
better to IL-4 and GM-CSF at a 2:1 molar ratio, while another can
respond slightly better to IL-4 and GM-CSF at a 1:2 molar ratio.
The cells can be mixed together in a suitable proportion to provide
a molar ratio suited for the tumor being treated. In a third example,
the ratio of cytokine-secreting cells to tumor antigen or autologous
tumor cells is also adjusted according to the tumor being treated.
Fine-tuning of the components of the vaccine can be done according
to previous observations on the effectiveness of the vaccine in
various clinical settings, in the context of features of the tumor
in the subject being treated. A principal feature is the type of
cancer being treated, with optional secondary features including,
but not limited to, the location of the tumor in the body, staging,
invasiveness, morphological features, results of biochemical tests
for antigen expression or genetic alteration conducted on patient's
serum or a tumor sample, clinical features, and response to previous
therapy.
Fine-tuning the vaccine is an added benefit of the nature of the
composition, but is not required. Many combinations of cytokine-producing
cells and autologous tumor cells are effective, and are encompassed
by the claimed invention. Effective combinations are readily determined
by a practitioner of ordinary skill in the art by following the
guidelines provided herein. The availability of a plurality of allogeneic
cytokine-producing cells for admixing into a vaccine considerably
facilitates not only the adjustment of the composition in accordance
with previous experience, but also the initial testing of various
potential combinations.
Other embodiments of this invention involve genetically altering
a patient's own tumor cells so as to produce a stimulatory cytokine,
especially a membrane-bound cytokine. The same cell thus provides
both the cytokine and the tumor antigen. These cells can be administered
alone, or are optionally mixed with allogeneic cells producing additional
cytokines in order to increase stimulation, again providing an opportunity
to fine-tune the relative amount and mixture of cytokines provided.
A further description of preferred ways to prepare and use the
vaccine compositions of this invention are provided in the sections
that follow.
Definitions
The terms "vaccine", "immunogen", or "immunogenic
composition" are used herein to refer to a compound or composition,
as appropriate, that is capable of conferring a degree of specific
immunity when administered to a human or animal subject. As used
in this disclosure, a "cellular vaccine" or "cellular
immunogen" refers to a composition comprising at least one
cell population, which is optionally inactivated, as an active ingredient.
The vaccines, immunogens, and immunogenic compositions of this invention
are active vaccines, which means that they are capable of stimulating
a specific immunological response (such as an anti-tumor antigen
or anti-cancer cell response) mediated at least in part by the immune
system of the host individual. The immunological response may comprise
antibody, immunoreactive cells (such as helper/inducer or cytotoxic
cells), or any combination thereof, and is preferably directed towards
an antigen that is present on a tumor towards which the treatment
is directed. The response may be elicited or restimulated in a subject
by administration of either single or multiple doses. Nothing further
is required of a composition in order for it to qualify as a vaccine,
unless otherwise specified.
A compound or composition is "immunogenic" if it is capable
of either: a) generating an immune response against an antigen (such
as a tumor antigen) in a naive individual; or b) reconstituting,
boosting, or maintaining an immune response in an individual beyond
what would occur if the compound or composition was not administered.
A composition is immunogenic if it is capable of attaining either
of these criteria when administered in single or multiple doses.
"Stimulating" an immune or immunological response refers
to administration of a compound or composition that initiates, boosts,
or maintains the capacity for the host's immune system to react
to a target substance, such as a foreign molecule, an allogeneic
cell, or a tumor cell, at a level higher than would otherwise occur.
Stimulating a "primary" immune response refers herein
to eliciting specific immune reactivity in a subject in which previous
reactivity was not detected; for example, due to lack of exposure
to the target antigen, refractoriness to the target, or immune suppression.
Stimulating a "secondary" response refers to the reinitiation,
boosting, or maintenance of reactivity in a subject in whom previous
reactivity was detected; for example, due to natural immunity, spontaneous
immunization, or treatment using one or several compositions or
procedures.
A "cell line" or "cell culture" denotes higher
eukarvotic cells grown or maintained in vitro. "Progeny"
of a cell include any cells formed by cell division of a progenitor,
either in vivo or in vitro. It is understood that the descendants
of a cell may not be completely identical (either morphologically,
genotypically, or phenotypically) to the parent cell.
"Inactivation" of a cell is used herein to indicate that
the cell has been rendered incapable of cell division to form progeny.
The cell may nonetheless be capable of response to stimulus, or
biosynthesis and/or secretion of cell products such as cytokines.
Methods of inactivation are known in the art. Preferred methods
of inactivation are treatment with toxins such as mitomycin C, or
irradiation. Cells that have been fixed or permeabilized and are
incapable of division are also examples of inactivated cells.
"Genetic alteration" refers to a process wherein a genetic
element is introduced into a cell other than by mitosis or meiosis.
The element may be heterologous to the cell, or it may be an additional
copy or improved version of an element already present in the cell.
Genetic alteration may be effected, for example, by transducing
a cell with a recombinant plasmid or other polynucleotide through
any process known in the art, such as electroporation, calcium phosphate
precipitation, or contacting with a polynucleotide-liposome complex.
Genetic alteration may also be effected, for example, by transduction
or infection with a DNA or RNA virus or viral vector. It is preferable
that the genetic alteration is inheritable by progeny of the cell,
but this is not necessarily required for the practice of this invention,
particularly when altered cells are used in a pharmaceutical composition
without further proliferation. A cell is described as "genetically
altered" if it has itself been subjected to genetic alteration,
or if it is the progeny of a cell that was subjected to genetic
alteration, providing it retains the alteration of the progenitor.
A cell is said to be "inheritably altered" if a genetic
alteration is present which is inheritable by progeny of the altered
cell.
The terms "tumor cell" or "cancer cell", used
either in the singular or plural form, refer to cells that have
undergone a malignant transformation that makes them pathological
to the host organism. Primary cancer cells (that is, cells obtained
from near the site of malignant transformation) can be readily distinguished
from non-cancerous cells by well-established techniques, particularly
histological examination. The definition of a cancer cell, as used
herein, includes not only a primary cancer cell, but any cell derived
from a cancer cell ancestor. This includes metastasized cancer cells,
and in vitro cultures and cell lines derived from cancer cells.
The term "tumor-associated antigen" or "TAA"
is used herein to refer to a molecule or complex which is expressed
at a higher frequency or density by tumor cells than by non-tumor
cells of the same tissue type. Tumor-associated antigens may be
antigens not normally expressed by the host; they may be mutated,
truncated, misfolded, or otherwise abnormal manifestations of molecules
normally expressed by the host; they may be identical to molecules
normally expressed but expressed at abnormally high levels; or they
may be expressed in a context or milieu that is abnormal. Tumor-associated
antigens may be, for example, proteins or protein fragments, complex
carbohydrates, gangliosides, haptens, nucleic acids, or any combination
of these or other biological molecules. Knowledge of the existence
or characteristics of a particular tumor-associated antigen is not
necessary for the practice of the invention.
A protein such as a cytokine is referred to as a "transmembrane"
protein if it normally remains stably associated in the membrane
of the cell in which it is produced. The term does not require any
particular configuration of the protein in the lipid bilayer of
the membrane.
As used herein, "treatment" refers to clinical intervention
in an attempt to alter the natural course of the individual or cell
being treated, and may be performed either for prophylaxis or during
the course of clinical pathology. Desirable effects include preventing
occurrence or recurrence of disease, alleviation of symptoms, diminishment
of any direct or indirect pathological consequences of the disease,
preventing metastasis, lowering the rate of disease progression,
amelioration or palliation of the disease state, and remission or
improved prognosis.
The "pathology" associated with a disease condition is
anything that compromises the well-being, normal physiology, or
quality of life of the affected individual. This may involve (but
is not limited to) destructive invasion of affected tissues into
previously unaffected areas, growth at the expense of normal tissue
function, irregular or suppressed biological activity, aggravation
or suppression of an inflammatory or immunological response, increased
susceptibility to other pathogenic organisms or agents, and undesirable
clinical symptoms such as pain, fever, nausea, fatigue, mood alterations,
and such other features as may be determined by an attending physician.
An "effective amount" is an amount sufficient to effect
a beneficial or desired clinical result, particularly the generation
of an immune response, or noticeable improvement in clinical condition.
An immunogenic amount is an amount sufficient in the subject group
being treated (either diseased or not) to elicit an immunological
response, which may comprise either a humoral response, a cellular
response, or both. In terms of clinical response for subjects bearing
a neoplastic disease, an effective amount is amount sufficient to
palliate, ameliorate, stabilize, reverse or slow progression of
the disease, or otherwise reduce pathological consequences of the
disease. An effective amount may be given in single or divided doses.
Preferred quantities and cell ratios for use in an effective amount
are given elsewhere in this disclosure.
An "individual" or "subject" is a vertebrate,
preferably a mammal, more preferably a human. Non-human mammals
include, but are not limited to, farm animals, sport animals, and
pets.
General Techniques
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of molecular biology, microbiology,
cell biology, biochemistry and immunology, which are within the
skill of the art. Such techniques are explained fully in the literature,
such as, "Molecular Cloning: A Laboratory Manual", second
edition (Sambrook et al., 1989); "Oligonucleotide Synthesis"
(M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I.
Freshney, ed., 1987); "Methods in Enzymology" (Academic
Press, Inc.); "Handbook of Experimental Immunology" (D.
M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors
for Mammalian Cells" (J. M. Miller & M. P. Calos, eds.,
1987); "Current Protocols in Molecular Biology" (F. M.
Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction",
(Mullis et al., eds., 1994); "Current Protocols in Immunology"
(J. E. Coligan et al., eds., 1991). See also Gately et al., Lee
et al., and Zarling et al. (infra) for examples of techniques in
mixed lymphocyte cultures.
General procedures for the preparation and administration of pharmaceutical
compositions are outlined in Remington's Pharmaceutical Sciences
18th Edition (1990), E. W. Martin ed., Mack Publishing Co., PA.
All patents, patent applications, articles and publications mentioned
herein, both supra and infra, are hereby incorporated herein by
reference.
Preparation of Cellular Vaccines
The cellular vaccines of this invention are typically assembled
by preparing each cell population or equivalent thereof in an appropriate
fashion, combining the components, and optionally coculturing or
storing cell mixtures before administration to a subject.
Tumor-associated antigen: The source of tumor-associated antigen
is most usually a tumor cell or cell line that is close in phenotype
to that for which the patient is being treated. Tumors from the
same tissue type and with similar histological characteristics tend
to share tumor-associated antigens. While the complete spectrum
of antigens may vary between individual tumors, there is a substantial
probability that at least one will be shared. Preferably, the tumor
cells are histocompatible with the subject to be treated.
Generally, when it is possible to obtain tumor cells in advance
from the subject to be treated, these cells are preferred as more
likely to bear a full complement of relevant tumor-associated antigens.
Circulating tumors such as leukemias and lymphomas may be readily
sampled from peripheral blood. Otherwise, tumor cells are generally
sampled by a surgical procedure, including but not limited to biopsy,
or surgical resection or debulking. Tumor cells may also be collected
from metastatic sites. Solid tumors may be dissociated into separate
cells by physical manipulation optionally combined with enzymatic
treatment with such proteases as collagenase and the like. The cells
are then transferred into fresh medium. Cells may be stored until
further use, for example, by freezing in liquid N.sub.2. Optionally,
and especially when the original tumor mass is small, it is preferable
to expand the tumor cell population to ensure an adequate supply.
Cells are cultured in a growth medium suitable for propagation,
optionally supplemented with growth factors.
Preferably, a stable cell population comprising features of the
tumor cells is obtained without further transformation, although
transformation is permissible where required. The cell population
can be optionally cloned to enhance its stability or refine its
characteristics, although this is generally not necessary. Conditions
for reliably establishing short-term cultures and obtaining at least
10.sup.8 cells from a variety of tumor types is described in Dillman
et al. (1993) J. Immunother. 14:65-69. If possible, the original
tumor cell preparation is used without proliferation, since it is
possible that a critical tumor antigen will be lost through the
proliferative process.
Cancer cells or cell lines obtained as described may be combined
directly with the other components of the vaccine. However, it is
preferable to inactivate the cancer cells to prevent further proliferation
once administered to the subject. Any physical, chemical, or biological
means of inactivation may be used, including but not limited to
irradiation (preferably with at least about 5,000 cGy, more preferably
at least about 10,000 cGy, even more preferably at least about 20,000
cGy); or treatment with mitomycin-C (preferably at least 10 .mu.g/mL;
more preferably at least about 50 .mu.g/mL).
Cancer cells for use as a tumor antigen source may alternatively
be fixed with such agents as glutaraldehyde, paraformaldehyde, or
formalin. They may also be in an ionic or non-ionic detergent, such
as deoxycholate or octyl glucoside, or treated, for example, using
Vaccinia Virus or Newcastle Disease Virus. If desired, solubilized
cell suspensions may be clarified or subject to any of a number
of standard biochemical separation procedures to enrich or isolate
particular tumor-associated antigens or plurality of antigens. Preferably,
tumor antigen associated with the outer membrane of tumor cells,
or a plurality of tumor associated antigens is enriched. The degree
of enrichment may be 10-fold or more preferably 100-fold over that
of a whole-cell lysate. Isolated antigens, recombinant antigens,
or mixtures thereof may also be used. Before combination with other
components of the vaccine, the tumor antigen preparation is depleted
of the agent used to treat it; for example, by centrifuging and
washing the fixed cells, or dialysis of the solubilized suspension.
Preparation of tumor antigen, particularly beyond inactivation of
the source tumor cell, may be viewed as optional and unnecessary
for the practice of the embodiments of the invention, unless specifically
required.
Cytokine-producing cells: The cellular vaccines of this invention
also comprise a second cell population, of which at least a portion
are cells producing a soluble or membrane-bound factor capable of
potentiating an immunological response against the tumor-associated
antigen or autologous cell of the vaccine.
Any cytokine or chemokine may be used for this purpose, especially
those that have amongst their biological activities one or more
of the following: a) the ability to recruit, enhance proliferation,
enhance cytokine secretion by, or otherwise activate cells of the
lymphocyte lineage; b) the ability to enhance uptake into antigen-presenting
cells, the subsequent processing and display of antigen, or the
concurrent production of cytokines; c) the ability to enhance display
of histocompatibility antigens; d) the ability to enhance display
of tumor-associated antigen by tumor cells; e) the ability to recruit
other cells or soluble components that may participate in inflammation;
or f) any other effect that results in a localized immune stimulation.
The effect or effects can be measured in vitro according to standard
immunological techniques, but should come into play in sufficient
proximity to the tumor-associated antigen to provide an immunostimulatory
effect that is at least partly specific for the antigen. A cytokine
capable of mediating a plurality of the above-listed effects are
particularly preferred.
Preferred cytokines include, but are not limited to, tumor necrosis
factors, exemplified in TNF-.alpha.; interleukins, exemplified in
IL-2, IL-4, IL-6, IL-7, and IL-10; interferons, exemplified in IEN-.alpha.
and IFN-.gamma.; hematopoetic factors; and colony stimulating factors,
exemplified in GM-CSF and M-CSF. Different cytokines are more effective
in certan cancers than others, and may vary between different cancers
and patient groups. TNF and IL-2 are effective in cancers like adenocarcinoma
in syngeneic animal vaccines, but less effective in ovarian or brain
cancer, while M-CSF is especially potent in syngeneic animal vaccines
for brain cancer.
Amongst the possible cytokines that can be used with this invention,
GM-CSF and M-CSF are especially preferred because of their important
role in the maturation and function of specialized antigen-presenting
cells. This is believed to be important because many tumor cells,
such as those of epithelial origin, do not express detectable MHC
class II molecules. IL-4 is also preferred, as a pluripotent cytokine
endowed of a broad range of stimulating activities on both B and
T lymphocytes, as well as on hematopoietic cells. Its roles include
the recruitment and activation of CD4+ antigen-presenting cells,
as well as induction of cytotoxic T lymphocytes. TNF-a is a fourth
cytokine which is preferred, in part because of its broad range
of effects in the immune and inflammatory responses. Amongst cytokine
combinations, the combination of GM-CSF and IL-4 is especially preferred.
Embodiments of the invention with both of these cytokines are vaccines
comprising autologous cancer cells and allogeneic cells genetically
altered to express both GM-CSF and IL-4, or even more preferably
vaccines comprising autologous cancer cells, allogeneic cells genetically
altered to express GM-CSF, and different allogeneic cells genetically
altered to express IL-4.
The majority of cytokine produced by the cells used in this invention
may be secreted from the cells, or present on the outer membrane
of the cells. Where the cytokine has a local immunostimulatory effect,
it can be preferable that it be primarily attached to the cell membrane
to keep it in the vicinity of bystander tumor antigen comprised
in the vaccine. Where the cytokine has a recruitment effect, it
can be preferable that it be primarily secreted. As a third option,
the cytokine can be synthesized by the cell in both membrane-associated
and secreted form. As illustrated in Example 5, the preferable form
of a particular cytokine can be determined by simple side-by-side
comparison. M-CSF may be used in either form, and in certain vaccine
compositions may be more effective in the membrane-associated form.
While not wishing to be bound by theory, it is possible that the
membrane-associated form creates a bridge between the allogeneic
tumor cell and antigen-presenting cells or responder lymphocytes;
in effect a forced antigen presentation. M-CSF may have an advantage
over many other cell-surface receptor ligands in this regard, because
of an ability to simultaneously bridge cells, and provide a stimulatory
signal through its cytokine effect.
Other cytokines that have both of these properties may be particularly
effective in tumor vaccine compositions, and cells of the vaccine
are preferably altered to express them in the membrane-associated
form. Where particular cytokines have potent immunostimulatory activity
but do not occur naturally in a membrane-bound form, it is possible
to create a membrane-bound form as a fusion protein. Allogeneic
cells are genetically altered with a vector comprising a cytokine
encoding region and a transmembrane region in the same open reading
frame, the transmembrane region being either upstream or downstream
from the cytokine encoding region and optionally separated by an
in-frame spacer region. The transmembrane region may be modeled
on other known transmembrane proteins, or be an artificially designed
polypeptide segment with a high degree of lipophillicity.
The protein and DNA encoding sequences of human IL-4 and TNF-.alpha.
are known, and vectors comprising encoding sequences are available.
For the IL-4 sequences and vectors, see U.S. Pat. No. 5,017,691
and EP 230107. Genetically altered CHO cells are described in U.S.
Pat. No. 5,034,133. The use of IL-4 (either as the isolated recombinant
or in a genetically altered cell) in treating solid tumors are described
in U.S. Pat. No. 5,382,427. TNF polypeptides, encoding sequences,
vectors, and genetically altered host cells are described in U.S.
Pat. No. 5,288,852, EP 155549, and U.S. Pat. No. 4,879,226. Variants
of TNF, which may also be used in this invention, are described
in U.S. Pat. No. 4,677,063. Compositions comprising TNF-.alpha.
and interferon are taught in EP 131789. Synergism of TNF and IL-4
in the inhibition of cancer cell growth is described in WO 92/05805.
Other cytokines and cytokine-encoding polynucleotides are described
further in the example section below, or may be readily obtained
through publicly available biological deposits, or may be prepared
according to publicly available disclosures.
The cell used to produce the cytokine for the vaccines of this
invention is obtained from a different donor than the subject being
treated. The donor is of the same species as the subject. Consequently,
except in unusual circumstances, the cell is allogeneic to the subject.
For the general practice of this invention, this definition is satisfied
by at least one allelic difference at the amino acid level in a
major histocompatibility complex (MHC) Class I or Class II antigen
between the cytokine-secreting cell and the subject to be treated.
Typically, a plurality of differences will be present in both Class
I and Class II antigens will be present, and these differences will
be recognizable either in an antibody-mediated tissue typing cytotoxicity
test, or a mixed lymphocyte reaction between the cytokine-secreting
cells and lymphocytes from the subject to be treated. Differences
in Class I antigens are generally more relevant, since most cell
types do not express Class II. In certain embodiments of this invention,
the number of MHC differences is irrelevant, as long as the cells
are allogeneic. In other embodiments of this invention, MHC differences,
particularly Class I differences are preferred as a potentiating
factor in immune stimulation. In this context, at least 2 differences
are preferred; at least about 3 differences are even more preferred.
When using human cells, differences are especially preferred in
the HLA-A -B and -C loci.
The cell will also generally be a cell that can be maintained in
culture for a large number of replications and genetically desired,
if necessary. Typically, the cell will be a neoplastic cell, a malignantly
transformed cell, or the progeny of such cells. Cells may be deliberately
transformed into long-lived cell lines by any method, including,
but not limited to, fusion with other cell lines, treatment with
a chemical carcinogen, or infection with a suitable virus such as
Epstein-Barr virus or oncogenic virus. More usually, the cell will
be the progeny of a primary tumor occurring in the appropriate species,
that has been established in ex vivo culture.
In certain embodiments of this invention, tumor cells are used
as the allogeneic cytokine-expressing cell, wherein the tumor is
a different type from that of the subject being treated. This may
provide additional bystander effect by providing a plurality of
novel immunogenic antigens. In this context, the tumor cell is preferably
selected so as to comprise a large proportion or particularly high
level of an immunogenic epitope. In other embodiments of this invention,
tumor cells are used from a tumor type similar or identical to that
of the subject in terms of its tissue source, morphological characteristics,
surface antigen expression, clinical manifestations, and any other
relevant criteria. This is preferred when it is desirable to increase
the probability that a tumor-associated antigen, or a plurality
of such antigens may be overexpressed both on the cytokine-expressing
tumor cell of the vaccine, and tumor cells in the subject being
treated. Shared tumor-associated antigens may permit cis stimulation
of therapeutically relevant immune reactivity.
Cells may be stimulated to secrete cytokines at suitable levels
according to any method known in the art, including, but not limited
to, coculturing with other cells or treatment of the cells with
the same or different cytokines.
Most typically, in order to provide a high and reliable level of
cytokine expression, the cells are genetically altered so as to
synthesize the cytokine at an elevated level. It is recognized that
certain cells such as lymphocytes and macrophages may already produce
detectable levels of certain cytokines. "Elevated levels"
of expression that occur as a result of genetic alteration exceed
levels observed in cells not genetically altered or otherwise manipulated
in the same way, but that are otherwise similar.
Genetic alteration may be effected by any method known in the art.
Typically, an encoding sequence for the desired cytokine is operatively
linked to a heterologous promoter that will be constitutively or
inducibly active in the target cell, along with other controlling
elements and a poly-A sequence necessary for transcription and translation
of the protein. The expression cassette thus composed is introduced
into the cell by any method known in the art, such as calcium-phosphate
precipitation, insertion using cationic liposomes, or using a viral
vector tropic for the cells. Methods of genetic alteration are described
in the patent publications cited in relation to some of the cytokines
listed earlier.
One preferred method is the use of adenovirus vectors. For example
see, Graf et al. (1995) Soc. Neuroscience 21:838.5. Briefly, adenoviral
recombinant expression vectors prepared by genetic engineering of
commercially available plasmids such as those supplied by Microbix,
Canada. Suitable infection conditions and multiplicities of infection
(MOI) may be determined in preliminary experiments using a reporter
gene such as .beta.-galactosidase, and then used for cytokine transfer
(Kammersheidt et al.). An advantage of using a viral vector is that
the vector may first be replicated, and then an entire population
of cells may be infected and altered. Accordingly, genetically altered
cytokine secreting cells may be established as a cell line, or a
freshly obtained cell isolate or cell culture is altered de novo
just prior to use in a vaccine of this invention. In the latter
instance, preparation of the vaccine would additionally comprise
the step of transducing a population of cells allogeneic to the
intended recipient with a vector comprising an encoding region for
a particular cytokine of interest. Transduction using adenoviral
vectors and the like is especially preferred when it is desirable
to achieve very high levels of cytokine expression by the genetically
altered cells.
An even more preferred preferred method of genetic alteration is
the use of a retroviral vector comprising a suitable expression
cassette. Non-limiting illustrations are provided in the example
section below, and may also be found in Santin 1995b, 1995c &
1996. Although this approach may not achieve quite the same level
of expression available in some other systems, a particular benefit
is that the genetically altered cell is highly stable in the amount
of cytokine produced. This means that the level of expression can
be characterized exactly, and relied upon as a reagent composition
through multiple passages and different storage conditions. In addition,
genetically altered cells may be prepared which are capable of producing
cytokine even after inactivation by irradiation. The levels of cytokine
can be adjusted upwards, where necessary, simply by increasing the
number of genetically altered cells in the dosage.
As shown in Examples 1-4, tumor lines can be created using the
LXSN retroviral vector that produce cytokine at a stable and reliable
level through multiple cell divisions. Levels of cytokine secretion
may be determined by immunoassay or bioassay. Cells with these properties
are generally preferred, since they can be biochemically characterized
and clinically tested in advance. Accordingly, it is generally preferable
to clone genetically-altered cells and select high-producer clones.
Supernatant of 1.times.10.sup.6 cells/ml cultured in 10 ml medium
for 48 hours at 37.degree. C. may contain the following preferred
levels of cytokines: IL-4, IL-2, TNF-.alpha., the secreted form
of M-CSF, or most other cytokines of about the same molecular mass
are preferably produced at least 500, more preferably at least about
1000, even more preferably at least about 2000 pg/mL. GM-CSF is
preferably produced at least 100, more preferably at least about
200, even more preferably at least about 400 pg/mL. Membrane-associated
cytokines, where the majority produced by the cell, should be biosynthesized
at a rate preferably 25%, more preferably 50%, and even more preferably
100% of the range obtained for high-level producers of the secreted
form.
It is also highly desirable that a substantial proportion of cytokine-producing
cells remain viable and be able to secrete the cytokine of interest
after inactivation to prevent proliferation. Preferred treatments
halt development of at least about 95%, or more preferably at least
about 99% of the cells. Typically, when using irradiation, the levels
required are 2,500 rads, more preferably 5,000 rads, even more preferably
10,000 rads, and still more preferably 20,000 rads. The cells preferably
produce cytokine 2 days after irradiation at a rate that is at least
about 10%, more preferably at least about 20%, more preferably at
least about 50%, still more preferably at least about 100% of the
pre-irradiated level, when standardized for viable cell number.
The cytokine producing cells can also be modified in other ways,
if desired. In particular, they can be genetically altered to express
additional proteins, including but not limited to additional cytokines,
additional tumor-associated antigens, or additional cell-surface
markers, such as adhesion molecules like ICAM-1, histocompatibility
antigens, or costimulation markers like the B-cell marker B7-1 or
B7-2. Alternatively or in addition, they may be modified so as to
produce multiple copies of the same or similar proteins, including
multiple copies of the same cytokine in membrane-associated or secreted
form, or both. Transduction for expression of multiple proteins
or multiple protein copies may be conducted concurrently or sequentially.
More than one genetic alteration may be viewed as optional, and
is not required for the practice of this invention.
Assembly of the vaccine: The vaccines of this invention comprise
autologous tumor cells (or an alternative source of tumor-associated
antigen) and at least one cell allogeneic to the host that produces
a cytokine of therapeutic importance. As described earlier, certain
embodiments of this invention comprise a plurality of different
allogeneic cells, each of which produces a different cytokine. Preferably,
the cytokines produced by each different cell are amongst those
listed herein.
In one method, cell components of the vaccine are prepared and
combined in bulk at the desired ratio(s) to provide sufficient cells
for the entire course of treatment envisioned. The mixture is stored
frozen, and aliquots are thawed seriatim for each administration.
This ensures a consistency amongst the cell ratio.
To allow adjustments to components of the vaccine or the ratios
used, it is generally preferable to assemble the vaccine close to
the time of administration. Various cell populations may be collected
in advance, and cultured or cryopreserved as necessary to ensure
sufficient numbers of cells for administration and testing throughout
the planned protocol.
It is important to remove any additional components used in preparing
the cells which may have an unwanted effect in the subject. In particular,
fetal calf serum, bovine serum components, or other biological supplements
in the culture medium are typically removed so as to avoid an immunological
side reaction against them. Typically, the cell components of the
vaccine are washed, such as by repeated gentle centrifugation, into
a suitable pharmacologically compatible excipient. Compatible excipients
include isotonic saline, with or without a physiologically compatible
buffer like phosphate or Hepes and nutrients such as dextrose, physiologically
compatible ions, or amino acids, and various culture media suitable
for use with lymphocyte populations, particularly those devoid of
other immunogenic components. Carrying reagents, such as albumin
and blood plasma fractions and nonactive thickening agents, may
also be used. Non-active biological components, to the extent that
they are present in the pharmacological preparation, are preferably
derived from the same species, and are even more preferably obtained
previously from the subject to be treated.
The vaccine compositions of this invention may optionally include
additional active components working independently or in concert
with the tumor associated antigen and activated allogeneic cells.
Such optional components include but are not limited to isolated
or recombinant cytokines, particularly those explicitly referred
to in this disclosure, adjuvants, and other cell types. Preferred
additional components are bacillus of the M. bovis strain Calmette-Guerin
(BCG) or extracts thereof, or alternatively, the A60 mycobacterial
antigen complex (Maes et al.).
A vaccine composition of this invention is deemed "suitable"
for administration to a human if reasonable and acceptable standards
have been taken to ensure that the vaccine itself will not confer
additional major pathology on the recipient. Side effects such as
local inflammation, induration, or pain, or a febrile response may
be unavoidable and are generally acceptable if the treatment is
otherwise successful in a substantial proportion of patients. However,
the composition should be reasonably free of: a) unrelated and pathological
infectious or chemical agents, particularly from the donor of the
allogeneic lymphocytes; b) undesirable growths as may be generated
or propagated in tissue culture, such as bacteria or bacterial toxins,
mycobacteria, and viruses; c) unacceptable levels of oncogenic agents
or aggressively growing cancer cells not originating from the subject
being treated; and d) components liable to initiate or effect an
undesirable immune reaction, particularly anaphylactic shock. Particular
tests that can be used are listed in the example section of this
disclosure.
The compositions of the present invention, and subcomponents thereof
may be supplied in unit dosage or kit form. Kits of this invention
can comprise various components of a cellular vaccine or pharmaceutical
composition therefor provided in separate containers. The containers
may separately contain cells or antigens such that when mixed together
constitute a vaccine of this invention in unit dosage or multiple
dosage form. Preferred kits comprise in separate containers: cytokine-secreting
allogeneic cells; and tumor-associated antigen from the human, particularly
primary tumor cells from the human, or progeny thereof. Alternatively,
the kits may comprise a cell or cell mixture in one container and
a pharmaceutical excipient in another container. Preferred kits
of this nature comprise cytokine-secreting allogeneic cells in one
container, and an excipient in another. The user can employ the
excipient to prepare their own tumor antigen or autologous tumor
cells, said preparation then being combined with the cytokine-secreting
cells for administration to a subject. Packaged compositions and
kits of this invention typically include instructions for storage,
preparation and administration of the composition.
Use of Cellular Vaccines in Cancer Treatment
The compositions of this invention may be administered to subjects,
especially but not limited to human subjects. They are particularly
useful for eliciting an immune response against a tumor-associated
antigen, or for treating cancer.
Objectives of treatment: One purpose of administering the vaccine
is to elicit an immune response. The immune response may include
either humoral or cellular components, or both. Humoral immunity
may be determined by a standard immunoassay for antibody levels
in a serum sample from the treated individual.
Since cellular immunity is thought to play an important role in
immune surveillance of cancer, generating a cellular immune response
is frequently a particular objective of treatment. As used herein,
a "cellular immune response" is a response that involves
T cells, and can be observed in vitro or in vivo.
A general cellular immune response may be measured as the T cell
proliferative activity in cells (particularly PBL) sampled from
the subject after vaccine administration. Inactivated tumor cells,
preferably derived from the subject, are used as stimulators. A
non-specific mitogen such as PHA serves as a positive control; incubation
with an unrelated stimulator cell serves as a negative control.
After incubation of the PBMCs with the stimulators for an appropriate
period (typically 5 days), [.sup.3H]thymidine incorporation is measured.
If desired, determination of the subset of T cells that is proliferating
can be performed using flow cytometry. T cell cytotoxicity (CTL)
can also be measured. In this test, an enriched T cell population
from the subject are used as effectors in a standard .sup.51Cr release
assay. Tumor cells are radiolabeled as targets with about 200 .mu.Ci
of Na.sub.2.sup.51CrO.sub.4 for 60 minutes at 37.degree. C., followed
by washing. T cells and target cells (.about.1.times.10.sup.4/well)
are then combined at various effector-to-target ratios in 96-well,
U-bottom plates. The plates are centrifuged at 100.times.g for 5
minutes to initiate cell contact, and are incubated for 4-16 hours
at 37.degree. C. with 5% CO.sub.2. Release of .sup.51Cr is determined
in the supernatant, and compared with targets incubated in the absence
of T cells (negative control) or with 0.1% TRITON.TM. X-100 (positive
control).
Another purpose of administering the vaccine is for treatment of
a neoplastic disease, particularly cancer. Beneficial effect of
the vaccine will generally be at least in part immune mediated,
although an immune response need not be positively demonstrated
in order for the compositions and treatment methods to fall within
the scope of this invention, unless otherwise required.
Suitable subjects: The compositions of this invention may be used
for administration to both human and non-human vertebrates. They
provide advantages over previously available compositions particularly
in outbred populations, and particularly in spontaneous tumors.
Veterinary applications are contemplated within the scope of the
invention.
Cellular vaccines are designed for use in human subjects, and are
especially suitable for human treatment. The vaccines may be given
to any human subject with the discretion of the managing physician.
Typically, the subject will either have cancer, or be at substantial
risk of developing cancer.
Typical human subjects for therapy comprise two groups, which may
be distinguished by clinical criteria. Patients with "advanced
disease" or "high tumor burden" are those who bear
a clinically measurable tumor. A clinically measurable tumor is
one that can be detected on the basis of tumor mass (e.g., by palpation,
MRI, CAT scan, X-ray, or radioscintigraphy; positive biochemical
or histopathological markers on their own are insufficient to identify
this population).
A vaccine composition embodied in this invention is administered
to patients with advanced disease with the objective of palliating
their condition. Ideally, reduction in tumor mass occurs as a result,
but any clinical improvement constitutes a benefit. Clinical improvement
includes decreased risk or rate of progression or reduction in pathological
consequences of the tumor.
A second group of suitable subjects is known in the art as the
"adjuvant group". These are individuals who have had a
history of cancer, but have been responsive to another mode of therapy.
The prior therapy may have included (but is not restricted to) surgical
resection, radiotherapy, traditional chemotherapy, and other modes
of immunotherapy. As a result, these individuals have no clinically
measurable tumor by the definition given above. However, they are
suspected of being at risk for recurrence or progression of the
disease, either near the original tumor site, or by metastases.
The adjuvant group may be further subdivided into high-risk and
low-risk individuals. The subdivision is made on the basis of features
observed before or after the initial treatment. These features are
known in the clinical arts, and are suitably defined for each different
cancer. Features typical of high risk subgroups are those in which
the tumor has invaded neighboring tissues, or which show involvement
of lymph nodes.
A vaccine composition embodied in this invention is administered
to patients in the adjuvant group in order to elicit an anti-cancer
response primarily as a prophylactic measure against recurrence.
Ideally, the composition delays recurrence of the cancer, or more
preferably, reduces the risk of recurrence (i.e., improves the cure
rate). Such parameters may be determined in comparison with other
patient populations and other modes of therapy.
Of course, crossovers between these two patient groups occur, and
the vaccine compositions of this invention may be administered at
any time that is appropriate. For example, therapy may be conducted
before or during traditional therapy of a patient with high tumor
burden, and continued after the tumor becomes clinically undetectable.
Therapy may be continued in a patient who initially fell in the
adjuvant group, but is showing signs of recurrence.
Examples of tumors that can be treated by the compositions and
methods of this invention include the following: pancreatic tumors,
such as pancreatic ductal adenocarinomas; lung tumors, such as small
and large cell adenocarcinomas, squamous cell carcinoma, and brionchoalveolar
carcinoma; colon tumors, such as epithelial adenocarcinoma and their
metastases; and liver tumors, such as hepatoma and cholangiocarcinoma.
Also included are breast tumors, such as ductal and lobular adenocarcinoma;
gynecologic tumors, such as squamous and adenocarcinoma of the uterine
cervix, and uterine and ovarian epithelial adenocarcinoma; prostate
tumors, such as prostatic adenocarcinoma; bladder tumors, such as
transitional squamous cell carcinoma; tumors of the RES system,
such as nodular or diffuse B or T cell lymphoma, plasmacytoma, and
acute or chronic leukemia; skin tumors, such as malignant melanoma;
and soft tissue tumors, such as soft tissue sarcoma and leiomyosarcoma.
Of especial interest are brain tumor, such as astrocytoma, oligodendroglioma,
ependymoma, medulloblastomas, and primitive neural ectodermal tumor.
Included in this category are gliomas, glioblastomas, and gliosarcomas.
Also of especial interest is ovarian carcinoma.
The immune status of the individual may be any of the following:
The individual may be immunologically naive with respect to certain
tumor-associated antigens present in the composition, in which case
the compositions may be given to initiate or promote the maturation
of an anti-tumor response. The individual may not be currently expressing
anti-tumor immunity, but can have immunological memory, particularly
T cell memory relating to a tumor-associated antigen comprised in
the vaccine, in which case the compositions can be given to stimulate
a memory response. The individual can also have active immunity
(either humoral or cellular immunity, or both) to a tumor-associated
antigen comprised in the vaccine, in which case the compositions
may be given to maintain, boost, or maturate the response, or recruit
other arms of the immune system. The subject should be at least
partly immunocompetent, so as to minimize a graft versus host reaction
of pathological scope. However, it is recognized that cancer patients
often show a degree of immunosuppression, and this does not necessarily
prevent the use of the compositions of the invention, as long as
the compositions may be given safely and effectively. Immunocompetence
in the subject may be of host origin, or may be provided by way
of a concurrent adoptive transfer treatment.
Modes of administration and dose: The compositions of this invention
may be administered to the subject at any site, particularly a site
that is "distal" to or "distant" from the primary
tumor.
The route of administration of a pharmaceutical composition may
be parenteral, intramuscular, subcutaneous, intradermal, intraperitoneal,
intranasal, intravenous (including via an indwelling catheter),
via an afferent lymph vessel, or by another route that is suitable
in view of the tumor being treated and the subject's condition.
Because of low-level inflammation or induration that may occur for
the few days after administration, relatively non-invasive methods
are preferred, particularly subcutaneous routes.
The dose given is an amount "effective" in bringing about
a desired therapeutic response, be it the stimulation of an immune
response, or the treatment of cancer as defined elsewhere in this
disclosure. For the pharmaceutical compositions of this invention,
effective doses typically fall within the range of about 10.sup.5
to 10.sup.10 cells, including allogeneic cytokine-producing cells,
and autologous tumor cells or an equivalent thereof. Where a tumor
antigen preparation or tumor cell extract is used in place of autologous
tumor cells, the amount of tumor antigen present should be equivalent
to what would be provided in the level of cells indicated. The number
of autologous tumor cells may be adjusted to accommodate unusually
high or low levels of tumor antigen expression. Where a plurality
of allogeneic cells genetically altered to produce different cytokines
is used, the range referred to includes the total number of such
cells. The number of allogeneic cytokine-producing cells is adjusted
according to the level of cytokines produced by the cell population.
Preferably, between about 10.sup.6 to 10.sup.9 of allogeneic cytokine-producing
cells and about 10.sup.6 to 10.sup.9 autologous tumor cells are
used; more preferably between about 2.times.10.sup.6 and 5.times.10.sup.8
cells in each cell population is used; more preferably between about
5.times.10.sup.6 and 2.times.10.sup.8 cells in each population are
used; even more preferably between about 1.times.10.sup.7 and 1.times.10.sup.8
cells in each population are used. Multiple doses, when used in
combination to achieve a desired effect, each fall within the definition
of an effective amount.
The various components of the cellular vaccine are present in an
"effective combination", which means that there are sufficient
amounts of each of the components for the vaccine to be effective.
This will depend not only on the absolute number of cells, but also
on the ratio of the various components of the vaccine one to another.
Preferred ratios of total allogeneic cytokine-secreting cells to
autologous tumor cells or equivalent are 100:1 to 1: 100, more typically
they are between about 25:1 and 1:25, even more preferably they
are between about 10:1 and 1: 10, still more preferably they are
between about 3:1 and 1:3. Often more important than the actual
number of cytokine-producing cells used is the biosynthetic capability
of the cells; fewer cells being required where the biosynthetic
capability is higher. Preferably, the allogeneic cells in a dose
of the vaccine are capable of synthesizing at least about 0.1 ng,
more preferably at least about 0.5 ng, more preferably at least
about 2 ng, even more preferably at least about 10 ng of the cytokine
of interest during a 1 hour incubation under physiological conditions.
Where a plurality of different cytokine-producing cells are used,
ratios are chosen to give appropriate levels of biological activity;
typically between 25:1 and 1:25, more usually between 5:1 and 1:5
on a molar basis. Determination of optimal cell dosage and ratios
is a matter of routine determination, as described in the example
section below, and within the skill of a practitioner of ordinary
skill, in light of the instructions provided herein.
For embodiments of the invention where the vaccine consists essentially
of cells autologous to the patient expressing a membrane cytokine,
the number of cells is between 10.sup.5 and 10.sup.10 per dose;
more preferably between about 4.times.10.sup.6 and 1.times.10.sup.9
cells per dose; more preferably between about 1.times.10.sup.7 and
4.times.10.sup.8 cells per dose even more preferably between about
2.times.10.sup.7 and 2.times.10.sup.8 cells per dose. Multiple doses,
when used in combination to achieve a desired effect, each fall
within the definition of an effective amount.
The pharmaceutical compositions of this invention may be given
following, preceding, in lieu of, or in combination with, other
therapies relating to generating an immune response or treating
cancer in the subject. For example, the subject may previously or
concurrently be treated by chemotherapy, radiation therapy, and
other forms of immunotherapy and adoptive transfer. Example 7 describes
the use of a vaccine of this invention in combination with such
chemotherapeutic agents as Cisplatin, combination Cisplatin/Cyclophaphamide,
Cisplatin/Cyclophosphamidel Doxorobicin or Taxol. Where such modalities
are used, they are preferably employed in a way or at a time that
does not interfere with the immunogenicity of the compositions of
this invention. The subject may also have been administered another
vaccine or other composition in order to stimulate an immune response.
Such alternative compositions may include tumor antigen vaccines,
nucleic acid vaccines encoding tumor antigens, anti-idiotype vaccines,
and other types of cellular vaccines, including cytokine-expressing
tumor cell lines.
In a particular embodiment, the subject will have previously been
treated with an intra-tumor implant of stimulated allogeneic lymphocytes,
such as is described in International Patent Application WO 96/29394.
Combination protocols wherein another mode of vaccination or other
therapy preceding or following administration of an autologous tumor
cell/allogeneic cytokine-secreting cell vaccine, are embodied in
the present invention.
Timing of administration is within the judgment of the managing
physician, and depends on the clinical condition of the patient,
the objectives of treatment, and concurrent therapies also being
administered. At an appropriate time in patient management, an initiating
dose is given, and the patient is monitored for either an immunological
or clinical response, often both. Suitable means of immunological
monitoring include a one-way MLR using patient's PBL as responders
and primary tumor cells as stimulators. An immunological reaction
may also be manifest by a delayed inflammatory response at the injection
site. Suitable means of monitoring the tumor are selected depending
on the tumor type and characteristics, and may include magnetic
resonance imaging (MRJ), radioscintigraphy with a suitable imaging
agent, monitoring cf circulating tumor marker antigens, and the
subject's clinical response. An example of an appropriate clinical
marker is serum CA-125 for the monitoring of advanced ovarian cancer.
Hempling et al. (1993) J. Surg. Oncol. 54:38-44. Additional doses
may be given as appropriate, typically on a monthly, semimonthly,
or preferably a weekly basis, until the desired effect is achieved.
Thereafter, and particularly when the immunological or clinical
benefit appears to subside, additional booster or maintenance doses
may be given as required.
When multiple doses of a cellular vaccine are given to the same
patient, some attention should be paid to the possibility that the
allogeneic lymphocytes in the vaccine may generate an anti-allotype
response. The use of a mixture of allogeneic cells from a plurality
of donors, and the use of different allogeneic cell populations
in each dose, are both strategies that can help minimize the occurrence
of an anti-allotype response.
During the course of therapy, the subject is evaluated on a regular
basis for side effects at the injection site, or general side effects
such as a febrile response. Side effects are managed with appropriate
supportive clinical care.
Cell Lines
This invention includes the cell lines listed in the Table below.
These cell lines are provided and described for the convenience
of the practitioner, and may be used, inter alia, for the preparation
of certain vaccines of this invention, or for methods of treatment
of this invention. None of the cell lines listed is required for
the general practice of the invention, except in particular embodiments
where a cell line is explicitly required.
TABLE-US-00001 TABLE 1 ATCC Accession Designation Origin Description
No. UCI-107E IL-4 GS Ovarian genetically altered carcinoma to express
IL-4 UCI-107M GM-CSF-MPS cell line genetically altered UCI-107 to
express GM-CSF UCI-107A IL-2 AS genetically altered to express IL-2
ACBT Glioblastoma parental cell line cell line ACBT/TNF-G genetically
altered to express TNF-.alpha. ACBT/IL-4-T genetically altered to
express IL-4 ACBT/IL-2-C2 genetically altered to express IL-2 ACBT/GM-CSF-M4
genetically altered to express GM-CSF
Upon allowance and issuance of this application as a United States
Patent, all restriction on the availability of the deposits will
be irrevocably removed, and access to the designated deposits will
be available during pendency of the above-named application to one
determined by the Commissioner to be entitled thereto, under 37
CFR .sctn.1.14 and 35 USC .sctn.1.22. Moreover, the designated deposits
will be maintained for a period of thirty (30) years from the date
of deposit, or from five (5) years after the last request for the
deposit; or for the enforceable life of the U.S. patent, whichever
is longer.
The examples presented below are provided as a further guide to
a practitioner of ordinary skill in the art, and are not meant to
be limiting in any way.
EXAMPLES
Example 1
An Ovarian Cancer Cell Line Transduced to Express IL-4
A human ovarian cancer cell line was genetically altered to secrete
IL-4, using a retroviral vector comprising an IL-4 encoding construct.
The cell line was stable, and capable of IL-4 biosynthesis even
after an inactivating dose of radiation. The cell line expressed
MHC Class I and IIcr-2/neu antigens, but no MHC Class II antigens,
ICAM-1, CA-125, or IL-4 receptors.
The human ovarian cell line UCI-107 was established from a previously
untreated patient with a primary Stage III serous papillary adenocarcinoma
of the ovary. The UCI-101 and UCI-107 cell lines have been previously
characterized (Gamboa-Vujicic et al. Submitted, Gynecol. Oncol.)
and were kindly provided by Dr. Alberto Manetta (University of California,
Irvine Medical Center). Cells were maintained at 37.degree. C.,
5% CO.sub.2 in complete media (CM) containing RPMI 1640 (Gibco Life
Technologies), 10 percent fetal bovine serum (FBS, Gemini Bioproducts,
Calabassas, Calif.), and 1 percent penicillin/streptomycin sulfate
(Irvine Scientific, Santa Ana, Calif.).
Retroviral vectors were constructed as follows: The pLXSN plasmid
was kindly provided by Dr. A. Dusty Miller (Fred Hutchinson Cancer
Center, Seattle, Wash.). This plasmid, derived from a Maloney murine
leukemia virus (MMLV) contains the neophosphotransferase gene whose
constitutive expression is driven by the SV40 enhancer/promoter,
the 5' retroviral LTR of the integrated vector drives the expression
of an inserted gene. The human IL-4 cDNA was obtained from ATCC
in the Okayama and Berg pCD cloning vector, and was excised using
BamHI restriction enzyme. Okayama et al. (1983) Mol. Cell. Biol.
3:228-289. The cDNA was then cloned into the BamHI restriction site
in the multiple cloning region of pLSXN. Proper orientation of the
cDNA was determined by diagnostic restriction endonuclease digests.
Once constructed, retroviral plasmid DNA was then purified by CsCl
gradient density centrifugation.
Purified retroviral plasmid DNA (LXSN/IL-4) was used to transduce
the murine esotropic packaging cell line GP-E86 by the calcium phosphate
method. Forty-eight-hour supernatant from these cells was then used
to infect the murine amphotropic-packaging cell line, PA317. The
PA-317-packaging cell line was obtained from the ATCC and maintained
in CM. Transduced PA317 cells were selected by resistance to G418.
Isolated clones were expanded, aliquoted, and frozen under liquid
nitrogen in a master cell bank. The supernatant from a transduced
PA317 clone, containing infectious, replication-incompetent retrovirus,
was used to infect the human carcinoma cell lines. Briefly, human
ovarian carcinoma cell lines were seeded in 100-mm tissue culture
dishes at densities of 1.times.10.sup.6 cells in 10 ml CM and incubated
for 4 hr at 37.degree. C., 5% CO.sub.2 to allow adherence. After
incubation, the medium was aspirated and replaced with 5 ml of 2%
polybrene in phosphate-buffered saline (PBS), (Aldrich Chemical
Co. Inc., Milwaukee, Wis.). After 30 min at 37.degree. C., 5% CO.sub.2,
10 ml of retroviral supernatant was added, and retroviral-mediated
gene transfer was accomplished by overnight incubation. Supernatants
were then aspirated and replaced with CM. After an additional 48- |