Note: Descriptions are shown in the official language in which they were submitted.
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CYTOKINE ENHANCED IMMUNOTHERAPY
FOR BRAIN TUMORS
Field of the Invention
~ 5 This invention is in the field of treating cancer by administering a
combination of systemic and local immunotherapy using cytokines.
Rights of the Federal Government
The United States government has certain rights in this invention by
virtue of a grant by the National Cooperative Drug Discovery Group UO1-
CA52857 of the National Cancer Institute of the National Institutes of Health.
Bethesda, Maryland.
Background of the Invention
One-third of all individuals in the United States alone will develop
cancer. Although the five year survival rate has risen dramatically to nearly
fifty percent as a result of progress in early diagnosis and therapy, cancer
still remains second only to cardiac disease as a cause of death in the United
States. Twenty percent of Americans die from cancer, half due to lung,
breast, and colon-rectal cancer.
Designing effective treatments for patients with cancer has represented
a major challenge. The current regimen of surgical resection, external beam
radiation therapy, andlor systemic chemotherapy has been partially successful
in some kinds of malignancies, but has not produced satisfactory results in
others. In some malignancies, such as brain malignancies, this regimen
produces a median survival of less than one year. For example, 90% of
resected malignant gliomas recur within two centimeters of the original tumor
site within one year.
Though effective in some kinds of cancers, the use of systemic
chemotherapy has had minor success in the treatment of cancer of the colon-
rectum, esophagus, liver, pancreas) and kidney. A major problem with
systemic chemotherapy for the treatment of these types of cancer is that the
systemic doses required to achieve control of tumor growth frequently result
in unacceptable systemic toxicity. Efforts to improve delivery of
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chemotherapeutic agents to the tumor site have resulted in advances in organ-
directed chemotherapy, as by continuous systemic infusion, for example.
However, continuous infusions of anticancer drugs generally have not shown
a clear benefit over pulse or short-term infusions. Implantable elastomer
S access ports with self-sealing silicone diaphragms have also been tried for
continuous infusion, but extravasation remains a problem. Portable infusion
pumps are now available as delivery devices and are being evaluated for
efficacy. (See Harrison's Principles of Internal Medicine, pp. 43l-446,
Braunwald, E., et al.) ed.) McGraw-Hill Book Co. (1987)) for a general
review). Controlled release biocompatible polymers have been used
successfully for local drug delivery and have been utilized for contraception,
insulin therapy) glaucoma treatment, asthma therapy, prevention of dental
related disorders, and certain types of cancer chemotherapy. (Langer, R.,
and Wise, D., eds, Medical Applications of Controlled Release, Vol. I and
II, Boca Raton, CRC Press ( l986)).
In the brain, the design and development of effective anti-tumor
agents for treatment of patients with malignant neoplasms of the central
nervous system have been influenced by two major factors: 1) the
blood-brain barrier provides an anatomic obstruction, limiting access of drugs
to these tumors; and 2) the drugs given at high systemic levels are generally
cytotoxic. Efforts to improve drug delivery to the tumor bed in the brain
have included transient osmotic disruption of the blood brain barrier,
cerebrospinal fluid perfusion) and direct infusion into a brain tumor using
catheters. Each technique has had significant limitations. Disruption of the
blood brain barrier increased the uptake of hydrophilic substances into normal
brain, but did not significantly increase substance transfer into the tumor.
Only small fractions of agents administered into the cerebrospinal fluid
actually penetrated into the brain parenchyma. Drugs that have been used to
treat tumors by infusion have been inadequate, did not diffuse an adequate
distance from the site of infusion, or could not be maintained at a sufficient
concentration to allow a sustained diffusion gradient. The use of catheters
has been complicated by high rates of infection, obstruction, and malfunction
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due to clogging. See Tomita, T., "Interstitial chemotherapy for brain
tumors: review", J. Neuro-Oncology, 10:S7-74 (1991).
Recent advances in the understanding of the immune system and
advances in defining T cell antigens on tumor cells have shown promising
results in treating tumors with immunotherapy. Several of these new
approaches are aimed at augmenting weak host immune responses to tumor
antigens and include the use of antibodies, cellular immunotherapy, and
cytokines. The use of antibodies is difficult because it requires that the
specificity of the antibody be such that it does not significantly bind to non-
tumor cells. There are few truly tumor-specific antigens to select when an
antibody-based immunotherapy approach is designed. Cellular
immunotherapy involves the transfer of cultured immune cells that have anti-
tumor reactivity into a tumor-bearing host. Adoptive therapy with autologous
lymphokine activated killer (LAK) cells has yielded impressive results but
only in the presence of cytokines or chemotherapeutic drugs. The use of
cytokines administered directly to cells to enhance immune responses has also
been shown to be successful. See Abbas, et al. , "Immunity to Tumors" ,
page 372) Cellular and Molecular Immunolo~y, 2nd Ed., W.B. Saunders
Company ( 1994). However, the effective administration of cytokines suffers
from the same obstacles discussed above in reference to administration of
chemotherapeutic agents in that high doses of systemic administration can be
toxic and providing continuous release by Local administration can be
problematic.
It is therefore an object of the present invention to provide a
composition and method of use thereof which provides for an enhanced
immune response against tumors, especially brain tumors, improves
therapeutic efficacy and diminishes potential toxicity.
It is a further object of the present invention to provide a composition
- and method of use for the treatment of tumors, especially brain tumors,
which establishes long term memory capable of generating potent anti-tumor
responses against multiple subsequent tumor challenges.
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Summary of the Invention
A therapy for treatment of patients with cancer utilizing the
combination of a cytokine in a pharmaceutical acceptable carrier for systemic
administration and a cytokine in a pharmaceutical acceptable carrier for local
administration is described. In the most preferred embodiment, brain tumors
are treated with a cytokine such as granulocyte macrophage-colony
stimulating factor (GM-CSF) administered systemically, most preferably in
combination with tumor antigen such as replication incompetent tumor cells,
and a cytokine such as interIeukin-2 (IL-2) or IL-4 administered locally, most
preferably in a vehicle providing release over a period of time, such as
transduced cells, again most preferably replication incompetent tumor cells,
or incorporated into microparticulate vehicles such as polymeric
microspheres.
The examples demonstrate that systemic administration (vaccination)
with GM-CSF transduced tumor cells or microencapsulated GM-CSF protects
against growth of intracranial melanoma. The examples also demonstrate
that local intracranial delivery of IL-2 transduced tumor cells or
microencapsulated IL-2 generates immediate anti-tumor responses within the
central nervous system as well as long term memory capable of generating
potent anti-tumor responses against multiple subsequent tumor challenges,
including challenges outside the central nervous system. The examples
further demonstrate that combination immunotherapy using systemic
vaccination with GM-CSF transductants and local intracranial administration
of IL-2 transductants produces an anti-tumor effect that is significantly
enhanced as compared to treatment with either treatment alone.
Brief Description of the Drawings
Figure 1 is a graph showing percent survival over time in days of
experimental animal models of intracranial melanoma having received
stereotactic intracraniaI injections of wild type B 16-F10 melanoma cells in
doses of 100,000 (circles), 10,000 (squares), 1,000 (triangles), and 100
(solid
squares), and saline with no cells (solid circles).
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Figure 2 is a graph showing percent survival over time in days of
mice after systemic vaccination with a single subcutaneous injection of 106
irradiated B 16-F 10 cells engineered by gene transfer to secrete GM-CSF
(solid squares), medium (circles) or 106 irradiated B 16-F 10 wild type non-
cytokine producing cells (squares), challenged 14 days later with an injection
of 102 non-irradiated wild type B16-F10 melanoma cells in the brain,
(p<0.001).
Figure 3 is a graph showing percent survival over time in days of
mice treated with a single intracranial injection of 10S irradiated B16-F10
cells engineered by gene transfer to secrete IL-2 (solid squares), medium
(circles) or 1 OS irradiated wild type B 16-F 10 non-cytokine producing cells
(squares), challenged at the same time by stereotactic intracranial co-
injections of 10~ non-irradiated wild type B16-F10 cells, (p<0.001).
Figure 4 is a graph showing percent survival over time in days of
animals vaccinated with a subcutaneous injection of 106 irradiated GM-CSF
producing B16-F10 melanoma cells followed two weeks later with 5 x 10'
irradiated IL-2-secreting B16-F10 cells administered intracranially and
simultaneously challenged with intracranial co-injections of 10~ non-
irradiated
wild type B16-F10 cells (solid squares). Control animals received GM-CSF
vaccine alone followed by intracranial tumor challenge with wild type B16-
F10 cells (x marks), or vaccination with medium alone followed by
intracranial IL-2 therapy and tumor challenge (circles). Additional controls
received vaccination with medium followed by intracranial therapy with
medium and intracranial tumor challenge (squares).
Detailed Description of the Invention
A therapy for treatment of tumors has been developed which relies on
the combination of an initial systemic "priming" of the immune system, most
preferably through the combination of administration of a cytokine such as
GM-CSF and tumor antigen such as replication incompetent tumor cells along
with local release at the tumor site (or site of resection following tumor
removal) of a cytokine such as IL-2 which enhances the immune response
against the tumor cells. Local release can be obtained using any of several
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means, but a preferred method is using microparticles to release cytokine
over a period of at least days or transduced cells, most preferably
replication
incompetent tumor cells which are transduced with the gene encoding the
cytokine to be released. The latter is shown to release for at least five days
after implantation. Microparticies can be designed to release for between
hours and weeks or even months, as required.
The examples show the in vivo treatment of brain tumors using GM-
CSF administered systemically, and IL-2 and IL-4 administered
intracranially. However) a variety of cytokines can be used. Similar results
are expected for treatment of other tumor types.
I. Therapeutic Compositions
a. Cytokines
i. Granulocyte-macrophage colony stimulating factor.
Granulocyte-macrophage colony stimulating factor (GM-CSF) is a 22 kD
glycoprotein made by activated T cells and by activated mononuclear
phagocytes, vascular endothelial cells, and fibroblasts. GM-CSF has been
shown to prime systemic immune responses via stimulation of bone marrow
derived antigen presenting cells. See Inaba, K. , et al., J. Exp. Med. ,
175 :1157-67 ( 1992); Inaba, K. , et al. , J. Exp. Med. , 176:1693-702 (
1992);
Steinman, R., Annual Rev. Immunology, 9:271-81 (1991). In vivo studies
with GM-CSF-transduced tumor cells reveal that local release of this cytokine
results in the generation of CD4 + and CD8 + tumor-specific T lymphocytes
and systemic protection from tumor challenge: Dranoff, G. , et al. , Proc.
Natl. Acad. Sci. U.S.A., 90:3539-43 (1993).
ii. Interleukin-2. Interleukin-2 (IL-2) is produced by CD4+ T
cells, and in lesser quantities by CD8+ T cells. Secreted IL-2 is a 14 to 17
kD glycoprotein encoded by a single gene on chromosome 4 in humans. IL-
2 acts on the same cells that produce it, i. e. , it functions as an autocrine
growth factor. IL-2 also acts on other T lymphocytes, including both CD4 +
and CD8+ cells. IL-2 induces a local inflammatory response leading to
activation of both helper and cytotoxic subsets of T cells. IL-2 also
stimulates the growth of natural killer cells and enhances their cytolytic
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function. In vivo studies with IL-2-secreting tumor cells demonstrate that
powerful local and systemic antitumor immune responses are generated
leading to the destruction of the transduced tumors in the flank. Fearon, E.
R. , et al. , Cell, 60: 397-403 ( l990) ; Gansbacher, B. , et al . , J. Exp.
Med. ,
172:1217-24 (1990).
iii. Tumor necrosis factor. Tumor necrosis factor (TNF) was
originally identified as a mediator of tumor necrosis present in the serum of
animals exposed to bacterial lipopolysaccharide (LPS) such as endotoxin.
The major endogenous source of TNF is the LPS-activated mononuclear
phagocyte, although antigen-stimulated T cells, activated natural killer
cells,
and activated mast cells can also secrete this protein. In the mononuclear
phagocyte, TNF is initially synthesized as a nonglycosylated transmembrane
protein of approximately 25 kD. TNF has potent anti-tumor effects in vitro)
although clinical trials of TNF in advanced cancer patients have been
discontinued due to toxicity. TNF-a has a diverse range of biological
properties including inducing expression of a number of cytokines such as
interleukin-6, interleukin-8, GM-CSF, and granulocye-colony stimulating
factor, as well as causing hemorrhagic necrosis in established tumors. TNF
has been reported to generate tumor suppression after tumor cell-targeted
TNF-a gene transfer. Blankenstein, T. , et al. , J. Exp. Med. , 173:1047-52
( l991 ).
iv. Interleukin-4. Interleukin-4 (IL-4) is a helper T cell-
derived cytokine of approximately 20 kD which stimulates the proliferation of
mouse B cells in the presence of anti-Ig antibody (an analog of antigen) and
causes enlargement of resting B cells as well as increased expression of class
II MHC molecules. The principal endogenous source of IL-4 is from CD4+
T lymphocytes. Activated mast cells and basophils, as well as some CD8+
T cells, are also capable of producing IL-4. IL-4 delivered intracranially
displays antitumor activity analogous to observations following administration
peripherally of IL-4 transduced tumors. Golumbek, P. T. , et al. , Science,
254:7l3-6 (I991); Yu, J.S., et al., Cancer Res., 53:3125-8 (1993).
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v. Gamma interferon. Gamma interferon (IFN-~y) is a
homodimeric glycoprotein containing approximately 21 to 24 kD subunits.
IFN-'y is produced by some CD4+ helper T cells and nearly a11 CD8+ T
cells. Transcription is directly initiated as a consequence of antigen
S activation and is enhanced by IL-2 and interleukin-12. IFN-~y is also
produced by natural killer cells. IFN-y acts as a potent activator of
mononuclear phagocytes, acts directly on T and B lymphocytes to promote
their differentiation and acts to stimulate the cytolytic activity of natural
killer
cells. IFN-Y transduced non-immunogenic sarcoma has been reported to
elicit CD8 + T cells against wild type tumor cells. Restifo) N . , et al. , J.
Exp. Med., 175:1423-28 (1992).
vi. Interleukin-3. Interleukin-3 (IL-3), also known as
multilineage colony-stimulating factor) is a 20 to 26 kD product of CD4+ T
cells that acts on the most immature marrow progenitors and promotes the
expansion of cells that differentiate into all known mature cell types. IL-3
has been reported to enhance development of tumor reactive cytotoxic T cells
by a CD4-dependent mechanism. Pulaski, B. A. , et al. , Cancer Res. ,
53:2l12-57 (1993).
vii. Interleukin-6. Interleukin-6 (IL-6) is a cytokine of
approximately 26 kD that is synthesized by mononuclear phagocytes, vascular
endothelial cells, fibroblasts, and other cells in response to IL-1 and, to a
lesser extent, TNF. It is also made by some activated T cells. IL-6
transfected into Lewis lung carcinoma tumor- cells has been reported to
suppress the malignant phenotype and to confer immunotherapeutic
competence against parental metastatic cells. Porgador, A. , Cancer Res. ,
52:3679-87 (1992).
viii. Interleukin-7. Interleukin-7 (IL-7) is a cytokine secreted
by marrow stromal cells that acts on hematopoietic progenitors committed to
the B lymphocyte lineage. IL-7 has been reported to induce CD42+ T cell
dependent tumor rejection. Hock, H. , et al. , J. Exp. Med. , 174:1291-99
( 1991}.
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ix. Granulocvte-colony stimulating factor. Granulocyte-
colony stimulating factor (G-CSF) is made by the same cells that make GM-
' CSF. The secreted polypeptide is approximately 19 kD. G-CSF gene
transfer has been reported to suppress tumorgenicity of murine
adenocarcinoma. Colombo, M. , et al. , Cancer Res. , 52:4853-57 ( 1991 ) .
x. Other cytokines. Other cytokines are known in the art to
have an anti-tumor effect and can be used in the pharmaceutical compositions
described herein. Moreover, since cytokines are known to have an effect on
other cytokines, one can administer the cytokine which elicits one of the
cytokines described above, or directly administer one of the cytokines which
is elicited. Additional cytokines are known to those skilled in the art and
are
described in Abbas, et al. , "Cytokines" , chapter 12, pp. 239-61, Cellular
and
Molecular lmmunoloey, 2nd Ed., W.B. Saunders Company (1994).
b. Combinations with other biologically active compounds
1 S The cytokines can also be administered in combination with other
cytokines, antibodies, cultured immune cells that have anti-tumor reactivity
including LAK cells, or chemotherapeutic agents, including radiation therapy.
The active compounds can be incorporated into the same vehicle as the
cytokines for administration, or administered in a separate vehicle.
i. Chemotherapeutic agents.
The cytokines can be used alone, or in combination with other
chemotherapeutic agents. Examples of chemotherapeutic agents include
cytotoxic agents such as paclitaxel) camptothecin, ternozolamide, 1,3-bis(2-
chloroethyl)-1-nitrosourea (BCNU), adriamycin, platinum drugs such as
cisplatin, differentiating agents such as butyrate derivatives, transforming
growth factor such as factor-alpha-Pseudomonas exotoxin fusion protein, and
antibodies to tumor antigens, especially glioma antigens, such as monoclonal
antibody 81 C6. These agents can be incorporated into polymeric matrices for
delivery along with the cytokines. See, for example, Domb, et al., Polym.
Prepr., 32(2):219-220 (1991), reported incorporating the water soluble
chemotherapeutic agents carboplatin, an analog of cisplatin, and 4-
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hydroperoxycyclophosphamide into a biodegradable polymer matrix for
treating tumors, with promising results in.
ii. Other pharmaceutically active compounds.
In variations of these embodiments) it may be desirable to include
other pharmaceutically active compounds, such as steroidal
antiinflammatories which are used to reduce swelling, antibiotics, antivirals,
or anti-angiogenic compounds. For example, dexamethasone, a synthetic
corticosteroid used systemically to control cerebral edema, has been
incorporated into a non-biodegradable polymer matrix and tested in rat brain
in vitro and in vivo for efficacy in reversing cerebral edema. Other
compounds which can be included are preservatives, antioxidants, and fillers,
coatings or bulking agents which may also be utilized to alter polymeric
release rates.
c. Cytolcine Formulations
The cytokines can be administered in a pharmaceutically acceptable
carrier such as saline, phosphate buffered saline, cells transduced with a
gene
encoding the cytokine, microparticles, or other conventional vehicles.
i. Polymeric formulations
The cytokines can be encapsulated into a biocompatible polymeric
matrix, most preferably biodegradable, for use in the treatment of solid
tumors. The cytokine is preferably released by diffusion andlor degradation
over a therapeutically effective time) usually eight hours to five years,
preferably one week to one year. As used herein, microencapsulated
includes incorporated onto or into or on microspheres, microparticles, or
microcapsules. Microcapsules is used interchangeably with microspheres and
nvcroparticles) although it is understood that those skilled in the art of
encapsulation will recognize the differences in formulation methods, release
characteristics, and composition between these various modalities. The
microspheres can be directly implanted or delivered in a physiologically
compatible solution such as saline.
Biocompatible polymers can be categorized as biodegradable and non-
biodegradable. Biodegradable polymers degrade in vivo as a function of
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chemical composition, method of manufacture, and implant structure.
Synthetic and natural polymers can be used although synthetic polymers may
be preferred due to more uniform and reproducible degradation and other
physical properties. Examples of synthetic polymers include polyanhydrides,
polyhydroxyacids such as polyiactic acid, polyglycolic acid and copolymers
thereof, polyesters, polyamides, polyorthoesters, and some polyphosphazenes.
Examples of naturally occurring polymers include proteins and
polysaccharides such as collagen, hyaluronic acid, albumin and gelatin. The
ideal polymer must also be strong, yet flexible enough so that it does not
crumble or fragment during use.
Cytokines and optionally) other drugs or additives, can be
encapsulated within, throughout, andlor on the surface of the implant. The
cytokine is released by diffusion, degradation of the polymer, or a
combination thereof. There are two general classes of biodegradable
polymers: those degrading by bulk erosion and those degrading by surface
erosion. The latter polymers are preferred where more linear release is
required. The time of release can be manipulated by altering chemical
composition; for example, by increasing the amount of an aromatic monomer
such as p-carboxyphenoxy propane (CPP) which is copolymerized with a
monomer such as sebacic acid (SA). A particularly preferred polymer is
CPP-SA (20:80). Use of polyanhydrides in controlled delivery devices has
been reported by Leong, et al. , J. Med. Biomed. Mater. Res. , 19:941 ( 1985);
J. Med. Biomed. Mater. Res. , 20: S 1 ( 1986); and Rosen, et al. ,
Biomaterials,
4:131 (1983). U.S. Patents that describe the use of polyanhydrides for
controlled delivery of substances include U.S. Patent 4,857,311 to Domb and
Langer, U . S . Patent 4, 888,176 to Langer, et al. , and U . S . Patent 4 ,
789, 724
, to Domb and Langer. Other polymers such as polylactic acid, polyglycolic
acid, and copolymers thereof have been commercially available as suture
materials for a number of years and can be readily formed into devices for
drug delivery.
Non-biodegradable polymers remain intact in vivo for extended
periods of time (years). Agents loaded into the non-biodegradable polymer
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matrix are released by diffusion through the polymer's micropore lattice in a
sustained and predictable fashion, which can be tailored to provide a rapid or
a slower release rate by altering the percent cytokine loading, porosity of
the
matrix, and implant structure. Ethylene-vinyl acetate copolymer (EVAc) is
an example of a nonbiodegradable polymer that has been used as a local
delivery system for proteins and other macromolecules, as reported by
Larger, R. , and Follanan, J . , Nature (London) , 263 : 797-799 ( 1976) .
Others
include polyurethanes, poiyacrylonitriles, and some polyphosphazenes.
In the preferred embodiment, only polymer and cytokines to be
released are incorporated into the delivery device, although other
biocompatible, preferably biodegradable or metabolizable, materials can be
included for processing purposes as well as additional therapeutic agents.
Buffers, acids and bases are used to adjust the pH of the composition.
Agents to increase the diffusion distance of agents released from the
implanted polymer can also be included.
Fillers are water soluble or insoluble materials incorporated into the
formulation to add bulk. Types of fillers include sugars, starches and
celluloses. The amount of filler in the formulation will typically be in the
range of between about 1 and about 90% by weight.
Spheronization enhancers facilitate the production of spherical
implants. Substances such as zein, microcrystailine cellulose or
microcrystalline cellulose co-processed with sodium carboxymethyl cellulose
confer plasticity to the formulation as well as .implant strength and
integrity.
During spheronization, extrudates that are rigid, but not plastic, result in
the
formation of dumbbell shaped implants andlor a high proportion of fines.
Extrudates that are plastic, but not rigid, tend to agglomerate and form
excessively large implants. A balance between rigidity and plasticity must be
maintained. The percent of spheronization enhancer in a formulation depends
on the other excipient characteristics and is typically in the range of IO to
90% (wlw).
Disintegrants are substances which, in the presence of liquid, promote
the disruption of the implants. The function of the disintegrant is to
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counteract or neutralize the effect of any binding materials used in the
formulation. The mechanism of disintegration involves, in large part,
moisture absorption and swelling by an insoluble material. Examples of
disintegrants include croscarmellose sodium and crospovidone which are
typically incorporated into implants in the range of 1 to 20 % of total
implant
weight. In many cases, soluble fillers such as sugars (mannitol and lactose)
can also be added to facilitate disintegration of the implants.
Surfactants may be necessary in implant formulations to enhance
wettability of poorly soluble or hydrophobic materials. Surfactants such as
polysorbates or sodium lauryl sulfate are, if necessary, used in low
concentrations, generally less than 5 % .
Binders are adhesive materials that are incorporated in implant
formulations to bind powders and maintain implant integrity. Binders may be
added as dry powder or as solution. Sugars and natural and synthetic
polymers may act as binders. Materials added specifically as binders are
generally included in the range of about 0.5 to 15 % w/w of the implant
formulation. Certain materials) such as microcrystalline cellulose, also used
as a spheronization enhancer, also have additions! binding properties.
Various coatings can be applied to modify the properties of the
implants. Three types of coatings are seal, gloss and enteric. The seal coat
prevents excess moisture uptake by the implants during the application of
aqueous based enteric coatings. The gloss coat improves the handling of the
finished product. Water-soluble materials such as hydroxypropyl cellulose
can be used to seal coat and gloss coat implants. The seal coat and gloss
coat are generally sprayed onto the implants until an increase in weight
between about 0.5 % and about 5 % , preferably about 1 % for seal coat and
about 3 % for a gloss coat, has been obtained.
Enteric coatings consist of polymers which are insoluble in the low
pH (less than 3.0) of the stomach, but are soluble in the elevated pH (greater
than 4.0) of the small intestine. Polymers such as Eudragit , RohmTech,
Inc., Malden, MA) and Aquateric , FMC Corp., Philadelphia, PA, can be
used and are layered as thin membranes onto the implants from aqueous
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solution or suspension. The enteric coat is generally sprayed to a weight
increase of about one to about 30 % , preferably about 10 to about 15 %o , and
can contain coating adjuvants such as plasticizers, surfactants, separating
agents that reduce the tackiness of the implants during coating, and coating
permeability adjusters. Other types of coatings having various dissolution or
erosion properties can be used to further modify implant behavior. Such
coatings are readily known to one of ordinary skill in the art.
Controlled release devices are typically prepared in one of several
ways. For example, the polymer can be melted, mixed with the substance to
be delivered, and then solidified by cooling. Such melt fabrication processes
require polymers having a melting point that is below the temperature at
which the substance to be delivered and polymer degrade or become reactive.
Alternatively, the device can be prepared by solvent casting, where the
polymer is dissolved in a solvent, and the substance to be delivered dissolved
or dispersed in the polymer solution. The solvent is then evaporated, leaving
the substance in the polymeric matrix. Solvent casting requires that the
polymer be soluble in organic solvents and that the agents to be encapsulated
be soluble or dispersible in the solvent. Similar devices can be made by
phase separation or emulsification or even spray drying techniques. In still
other methods, a powder of the polymer is mixed with the cytokine and then
compressed to form an implant.
Methods of producing implants also include granulation, extrusion,
and spheronization. A dry powder blend is produced including the desired
excipients and microspheres. The dry powder is granulated with water or
other non-solvents for microspheres such as oils and passed through an
extruder forming "strings" or "fibers" of wet massed material as it passes
through the extruder screen. The extrudate strings are placed in a
spheronizer which forms spherical particles by breakage of the strings and
repeated contact between the particles, the spheronizer walls and the rotating
spheronizer base plate. The implants are dried and screened to remove
aggregates and fines. These methods can be used to make micro-implants
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(microparticles, microspheres, and microcapsules encapsulating cytokines to
be released), slabs or sheets, films, tubes, and other structures.
ii. Cytokine secreting Genetically engineered cells
Cytokine secreting cells can be genetically engineered by methods
known in the art to deliver cytokines systemically or locally. Cells can
either
be transformed or transduced, with bacterial or viral DNA vectors,
respectively. In a preferred embodiment the cells are tumor cells. To
prevent replication and preserve cytokine production, the transduced cells can
be irradiated prior to in vivo injection. The advantage of the tumor cells is
that the patient is exposed to antigen in combination with cytokine
stimulating
the immune response to the cytokine. Examples of the preparation of tumor
cells engineered to secrete IL-4 are described in Golumbek, P. T. , et al. ,
Science, 2S4:713-6 ( 1991 ) and Yu, J. S. , et al. , Cancer Res. 53:3125-8
(1993). IL-2 gene transfer into tumor cells is described in Gansbacher, B.,
et al., J. Exp. Med. , 172:1217-24 ( 1990). Tumor cells engineered to secrete
GM-CSF are described in Dranoff, G. , et al. , Proc. Natl. Acad. Sci. U. S.A.
,
90:3539-43 (l993). TNF gene transfer is described in Hlankenstein, T., et
al. , J. Exp. Med. ( 73 :1047-52 ( 1991 ) . IL-6 transfection into lung
carcinoma
tumor cells is described in Progador, A, et al. , Cancer Res. , S2:3679-87
( 1992) . yIFN cDNA transduced into non-immunogenic sarcoma is described
in Restifo, N., J. Exp. Med., 175:1423-28 (1992). G-CSF gene transfer is
described in Colombo, M. , et al . , Cancer Res. , 52:4853-57 ( Z 991 ) .
Other
cytokines including IL-3 and IL-7 can be secreted from cells by applying the
methods used for the cytokines described above or by the general procedures
for genetically modifying nonimmunogenic murine fibrosarcoma described in
Karp, S . E. , et al. , J. Immunol. , 150: 896-908 ( 1993) and Jaffee, E. et
al. , J.
Immunotherapy) in press (l995). The publications cited above describe
methods of genetically engineering cells to secrete cytokines. Additionally,
cancer vaccines expressing two or more cytokines from engineered vectors
containing genes encoding two different cytokines or by sequential
recombinant retrovirus-mediated genetic transductions can be prepared.
Suitable pharmaceutical vehicles are known to those skilled in the art.
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Inducers can also be used to produce cells which secrete cytokines.
Inducers may be used alone in some cases or in combination with
transforming agents, and include tumor necrosis factor (TNF), endotoxin) and
other agents known to those skilled in the art. In general, the cultured cells
are exposed to an amount effective to activate the cells, as determined by
cytokine expression, immunoglobulin secretion, and/or other indicators such
as proliferation or alteration of cell surface properties or markers. In some
cases, cells are initially exposed to a small amount to "prime" the cells,
then
to a subsequent dose to elicit greater activation of the cells.
Cells can be administered in medium or washed and administered in
saline. Alternatively, cells can be encapsulated in a polymeric matrix and
administered.
II. Administration to Patients
The cytokines described herein or their functionally equivalent
derivatives can be administered alone or in combination with, either before,
simultaneously ) or subsequent to, treatment using other chemotherapeutic or
radiation therapy or surgery. A preferred embodiment is systemic
administration of a first cytokine such as GM-CSF, most preferably in
combination with tumor antigen in the form of replication incompetent tumor
cells, followed by local administration during surgery or by injection of
either a biocompatible polymeric matrix loaded with the selected cytokine or
cytokine secreting cells, using dosages determined as described herein. Local
administration can be at a site adjacent to the tumor to be removed, or in the
tumor, or in the place where the tumor was removed. The dosages for
functionally equivalent derivatives can be extrapolated from the in vivo data.
An effective amount is an amount sufficient to induce an anti-tumor effect as
measured either by a longer survival time or decrease in tumor size.
Local administration can also be accomplished using an infusion
pump, for example, of the type used for delivering insulin or
chemotherapeutic agents to specific organs or tumors.
In the preferred method of administration, the polymeric implants or
cytokine secreting cells are implanted at the site of a tumor, two weeks after
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systemic administration of the polymeric implants or cytokine secreting cells,
for example, by subcutaneous injection at a distant site. If biodegradable
polymers are used, preferably they are less than about 100 to 200 microns in
diameter and injected by means of a catheter or syringe; it is not necessary
to
remove the implant following release of the immunotherapeutic agent.
The cytokines can also be combined with other therapeutic modalities,
including radiotherapy, chemotherapeutic agents administered systemically or
locally, and other immunotherapy.
The cytokines are preferably administered to brain cancer patients.
However, the cytokine combination therapy can also be applied to all cancer
patients including those having breast cancer, lung cancer, and colon cancer.
The treatment will provide an immediate response at the site of the local
administration as well as provide long term memory protection against cancer
at the site of local administration and at a site distant from the site of
local
administration. The immunotherapy described herein is therefore useful in
preventing or treating metastases.
III. Examples.
The present invention will be further understood by reference to the
following non-limiting examples.
Example 1: Systemic vaccination with GM-CSF-transduced tumor cells
Tumor cell lines and animals. B16-F10 melanoma cells were
maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing
10% fetal calf serum and penicillinlstreptomycin. Animals used for a11
experiments were 6 to 12 week old C57BL/6 female mice obtained from
Harlan (Indianapolis, IN). B16-F10 melanoma cells were transduced with
murine GM-CSF gene by using the replication-defective MFG retroviral
vector as previously described in Dranoff, G. , et al. , Proc. lVatl. Acad.
Sci.
U.S.A., 90:3539-43 (1993). The amount of cytokine produced by the
transformed tumor cells was quantified routinely by a standard ELISA
technique (Endogen, Cambridge, MA). Cultured tumor monolayers were
harvested with trypsin and resuspended in DMEM before injection. Tumor
cells were exposed to 5000 rads from a '3'cesium source (Gammacell Model
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#62 irradiator, Nordin International, Inc. , I~anata, Ontario, Canada)
discharging 1378 rads per minute, immediately before injection in order to
render them replication incompetent.
Technique for intracranial cell injections. Mice were anesthetized
with an intraperitoneaI injection of 0.1 ml of a stock solution containing
ketamine hydrochloride 25 mg/ml, xylazine 2.5 mglml, and 14.2S % ethyl
alcohol diluted 1:3 in 0.9 % NaCI solution. For stereotactic intracranial
injections of tumor cells, the surgical site was shaved and prepared with 70 %
ethyl alcohol and prepodyne solution. After a midline incision, a 1 mm burr
hole center 2 mrn posterior to the coronal suture and 2 mm lateral to the
sagittal suture was made. Animals were then placed in a stereotactic frame
and cells were delivered by a 26 gauge needle to a depth of 3 mm over a
period of 3 minutes. The total volume of injected cells was 10 ~d. The
needle was removed, the site irrigated with sterile 0.9 % NaCI solution, and
the skin was sutured closed with 4.0 vicryl.
Development of an intracranial B16-F10 melanoma model in C57BLl6
mice. To assess the intracrarual growth characteristics of B16-F10
melanoma, animals were divided into five groups of at least eight animals
and received stereotactic intracranial injections of either IOz, 103, 104, or
10S
wild type B16-FIO melanoma cells into the left parietal region by the method
described above. Control animals received similar intracranial injections of
0.9 % NaCI solution. Animals were assessed daily for survival. For
histologic analysis) brains were removed at the time of death and fixed in
10% formalin for at least 5 days, sectioned, embedded in paraffin, and
stained with hematoxylin and eosin.
All animals receiving the highest dose of B16-F10 (105 cells) died
within 15 days of treatment, with a median survival of 12 days. The results
are shown in Figure 1. At doses of l03 and 10~ cells the median survival
was 20 and 16 days, respectively. Intracranial does of 102 cells were
uniformly fatal (median survival 20 days, range 16 to 21 days). These
kinetics emphasize the highly tumorigenic, poorly immunogenic character of
r.._ .
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B16-F10. A11 control animals (treated with stereotactic injections of 0.9%
NaCI) were alive at 120 days.
Histologic analysis of the brains revealed that a11 animals treated with
intracranial wild type B16-F10 developed large, solid tumor masses at the
injection in the parietal region. Metastatic deposits of tumor were
occasionally observed in the brain parenchyma distant from the injection site,
especially in those animals receiving large tumor inoculations. Tumor
deposits were also observed in the subarachnoid space surrounding the spinal
cord, suggesting that cell migration occurred along cerebrospinal fluid
pathways. No tumor was apparent outside the craniospinal axis. This new
model has proven to be highly reliable and reproducible. With intracranial
doses of 10' tumor cells, animals died consistently over a 3 to 4 day period
within 20 days of injection.
Vaccination studies. Cytokine-secreting B16-F10 melanoma cells
were evaluated for their vaccination properties and ability to protect against
intracranial challenge with wild type B16-F10 melanoma. For these studies,
animals were treated with a single subcutaneous flank injection of 10b
irradiated GM-CSF secreting B16-F10 cells using a tuberculin syringe with a
27 gauge needle. The total amount of GM-CSF secreted by the transduced
cells in vitro prior to injection was between 50 and 60 ng per 106 cells per
day. To prevent replication and preserve cytokine production, the transduced
cells were irradiated prior to in vivo injection. Previous studies had shown
that B 16-F 10 melanoma cells exposed to 5000 rads are rendered replication
incompetent, but maintain their metabolic viability and continue to secrete
cytokine in vivo for up to 5 days, Jaffee, E. et al. , "Use of murine models
of
cytokine-secreting tumor vaccines to study feasibility and toxicity issues
critical to designing clinical trials", J. Immunotherapy) in press (1995). The
experiments were conducted with two control groups: one group received
flank injections of 106 irradiated wild type (non-cytokine-producing) B16
cells
and the other was treated with a subcutaneous injection of either cell growth
medium or 0.9 % NaCI. There were at least 10 animals per group for all
experiments. After 2 weeks a11 animals were challenged with stereotactic
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intracranial injections of 10Z non-irradiated wild type B16-F10 melanoma
cells by the method outlined above. Animals were assessed for survival.
Statistical analysis. For all efficacy studies, survival was plotted
using a Kaplan-Meier survival analysis and statistical significance was
determined by the Kruskal-Wallis nonparametric analysis of variance as
described in Zar, J. , eds. , Biostatistical Analysis, Englewood Cliffs, New
Jersey, Prentice-Hall, Inc. , pp. 140-50 ( 1984). Percentages of long term
survivors were analyzed by comparison of two proportions.
Results
Figure 2 shows the results of the systemic vaccination studies
performed on animals receiving single subcutaneous injections of 106
irradiated B16-F10 cells engineered by gene transfer to secrete GM-CSF.
GM-CSF secreting cells administered in this manner were found to protect
against intracranial challenge with the wild type tumor. Median survival for
IS animals treated with the GM-CSF vaccine and then challenged 2 weeks later
with wild type B16 in the brain was 27 days, compared to 19 and 17 days for
control animals treated with a vaccine of irradiated) wild type cells (which
did not secrete GM-CSF) or medium, respectively (p < 0.0001). These
results have been replicated 4 times) each with similar and statistically
significant results.
Example 2: Systemic vaccination with microencapsulated GM-CSF
Thirty-two C57BL/6 mice were divided into four groups of eight and
given flank vaccinations with either: 1) saline, 2) 2S00 ng bolus GM-CSF
and 1 x 106 irradiated B16 melanoma cells, 3) 2S00 ng GM-CSF
microspheres and 1 x I06 irradiated melanoma cells, or 4) 1 x 106 irradiated
melanoma cells transduced to produce GM-CSF. Fourteen days later all
animals received a challenge of B16 melanoma in the left frontal lobe.
Results
The median survival of the GM-CSF microsphere group and the
transduced GM-CSF secreting group was 27.5 days each. These survival
periods are significantly prolonged compared to the control animals receiving
saline which survived 16.5 days, (p < 0.001). The GM-CSF bolus group
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survived 15.5 days, therefore having no effect on survival. No GM-CSF
treated animal showed any sign of toxicity from the vaccination.
This study demonstrates that a significant anti-tumor immune response
is generated using a systemic vaccination with GM-CSF microspheres. This
response is comparable to that generated by GM-CSF transduced tumor cells
and represents a practical method for delivering GM-CSF in treatments
employing GM-CSF immunotherapy.
Example 3: Local intracranial delivery of IL-2-transduced tumor cells
Animals received intracranial stereotactic co-injections of either 103,
10~, l05, or 106 irradiated cytokine-producing B16-F10 melanoma cells,
which produce 80 ng of IL-2 per 106 cells every twenty-four hours as
determined by ELISA. Control animals received co-injections of irradiated
non-IL-2 producing wild type B16-F10 at the equivalent doses of 103) 104,
105, or 10~ cells. Simultaneously, all animals were challenged with
stereotactic intracranial injections of 10~ live non-irradiated B16-F10
melanoma cells. For these experiments, there were at least 10 animals per
group.
Results
IL-2 transduced cells were found to be highly effective in treating
intracranial tumor when they were delivered directly to the brain. Initial
studies showed that the IL-2 effect was dose-dependent. The results are
shown in Figure 3. Doses of 0.08 ng per day of IL-2 ( 103 IL-2 producing
cells) showed no enhanced survival compared. to controls) whereas a dose of
0. 8 ng per day ( 10~ IL-2 producing cells) showed a trend toward prolonged
survival that did not reach statistical significance. When tested at a dose of
8.0 ng per day ( 105 IL-2 producing cells) ) a significant prolongation in
survival was seen. While untreated control animals had died with intracranial
tumor by day 22 (median survival, 20 days), median survival for the IL-2
group was 31 days with 33 % of the mice living more than 80 days,
(p < 0.001 ) . Interestingly, several animals that died in the IL-2 treatment
group were found to have no tumor in the brain; rather, tumor was observed
surrounding the spinal cord suggesting that the protective effect of IL-2 was
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localized to the site of cytokine delivery. At higher doses of IL-2 (80.0 ng
per day), signs of IL-2 toxicity were observed including ataxia and death.
These animals died before controls {median survival, 12 days). These studies
have been replicated 7 times with similar doses of IL-2 transductants. In
each experiment, there was a significant prolongation of survival in animals
treated with intracranial IL-2 transductants as compared to controls.
On histologic examination at the time of death, inflammation was
notably absent in the brain parenchyma of the treated animals. Lymphocytes
were observed, however, in the cerebrospinal fluid spaces of the IL-2 treated
animals.
Example 4: Intracranial delivery of microencapsulated interleukin-2
produces long term memory and protects against re-
challenges
IL-2 was incorporated into biodegradable sustained release
microspheres having an average diameter of 4 ~,m. These microspheres were
stereotactically injected into the brains of mice to deliver 100 ng of IL-2
over
a 7 day period. Safety testing showed no evidence of clinical or histologic
toxicity from this dose over a 120 day period. To evaluate efficacy as a
primary treatment, a lethal challenge of B 16 melanoma was stereotactically
co-injected into the left parietal lobe of 25 mice with either saline, empty
microspheres, or the IL-2 containing microspheres. In a similar experiment,
9L glioma cells were co-injected into the left parietal lobe of rats.
To evaluate the potential brain vaccine effect of this therapy, a group
of non tumor-bearing mice were co-injected intracranially with IL-2
microspheres and melanoma cells which had been irradiated to prevent
replication. Thirty days later these animals and a group of control mice
having no previous treatment were challenged intracranially with the lethal
dose of live melanoma with no additional IL-2 therapy.
Results
Seventy-five percent of the IL-2 treated mice (6 of 8) were alive with
no sign of tumor 90 days after initial treatment while median survival of
untreated controls was 18 days. No animal survived more than 21 days.
The empty microspheres had no effect on survival. In a similar mouse
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melanoma experiment, no impact on survival from either a bolus injection of
IL-2 to the tumor site or systemic IL-2 administration was observed. In the
glioma experiments, microsphere delivery of IL-2 prolonged median survival
in rats to 55 days compared to 25 days for the controls.
Previous IL-2 vaccination extended median survival by 45 percent,
with 2 to 5 animals alive at 160 days after tumor challenge with all controls
dead by 23 days.
These results indicate that brain interstitial immunotherapy with IL-2
delivered by microspheres is an effective and safe treatment against brain
metastases and inhibits tumor recurrence. Additional efficacy results and
release kinetics of IL-2 from microspheres are reported in Sills, A. K. , Jr.
,
et al., Proceed. Intern. Symp. Control. Rel. Biomater. , 23 (July, 1996), and
Kalyanasundaram, S. , et al. , Proceed. Intern. Symp. Control. Rel. Biomater.
,
23 (July, 1996), respectively.
Example 5: Intracranial delivery of II,-2 transduced cells produces long
term memory and protects against re-challenges
A group of 11 C57BLI6 mice were initially treated with intracranial
IL-2 and co-injected with a dose of B 16 melanoma that was uniformly fatal in
control animals. Unlike controls, a11 11 animals who received intracranial
IL-2 survived the initial tumor challenge in the brain and were rechallenged
at the same site on day 70. Six of the 11 were rechallenged with wild-type
melanoma cells. The other five received injections of media alone. Twelve
additional mice, previously untreated, were similarly challenged with B 16
melanoma in the brain.
Results
The melanoma challenge was fatal to a11 previously untreated mice,
the median survival was 17 days. By contrast, the mice initially treated with
IL-2 and rechallenged with tumor on day 70 had a significantly prolonged
survival where the median survival was 36 days, with 3 of 6 surviving 70
days (p =0.01 versus positive control, Mann-Whitney Test) . As expected,
mice rechallenged with media all lived an additional 70 days.
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These results demonstrate long-term memory in an immune response
directed against intracranial melanoma useful in preventing metastatic tumor
recurrence after resection or inhibiting growth of synchronous metastases by
delivery of IL-2 transduced cells.
Example 6: Local intracranial delivery of IL-2 transduced cells protects
against challenges outside the central nervous system
Twenty-four C57B 16 mice were injected intracranially with irradiated,
IL-2 secreting B16-F10 melanoma cells and 12 C57B16 mice were injected
with medium, as controls. A11 animals simultaneously received a co-injection
of wild type B 16-F 10 cells which were uniformly fatal to untreated mice.
Nine of the twenty-four IL-2 treated animals survived 75 days. No controls
survived more than 20 days. At day 75, the 9 surviving animals and 8 new
control animals were challenged in the flank with l0,000 wild type B16-F10
melanoma cells.
Results
Only 1 of 9 long-term survivors developed a flank tumor; in contrast,
6 of 8 control mice developed a flank tumor, (p=.024). The 8 (of 9) long-
term survivors who rejected the first flank challenge, along with 8 new
control animals, were challenged a second time in the flank with a larger
bolus of 50,000 wild type B16-F10 melanoma cells, 126 days after the first
challenge. Seventeen days later) 8 of 8 control mice had developed tumors
whereas only 2 of 8 of the long term survivors had a flank tumor (p=0.01).
This study demonstrates that localized IL-2 immunotherapy delivered
intracranially not only generates an immediate anti-tumor response within the
central nervous system, but also establishes long-term memory capable of
generating potent anti-tumor responses against multiple subsequent tumor
challenges outside the central nervous system.
Example 7: Local intracranial delivery of IL-4-transduced tumor cells
Poorly immunogenic B16-F10 melanoma cells were transfected with
the IL-4 gene to deliver IL-4 at the site of a tumor in the central nervous
system. The transfected B 16-F 10 cells were irradiated to prevent replication
and injected intracranially in eight C57BL/6 mice along with non-irradiated,
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non-transfected B16-F10 (wild type) melanoma cells. The expression of IL-4
receptors on B16-F10 melanoma cells was examined by means of
fluorescence activated cell sorting (FACS).
Results
While animals treated with IL-4 had a median survival of 31 days,
control animals that received only wild type cells had a median survival of 18
days (range 15 to 20 days; p=0.002).
FACS analysis of B16-F10 cells did not reveal the presence of the IL-
4 receptor, suggesting that IL-4 does not interact with its receptor on tumor
cells to exert the cytotoxic effect. The results obtained in these experiments
indicate that high levels of IL-4 expressed locally stimulate a strong
immunologic antitumor response which leads to significant prolongation of
survival in mice with central nervous system tumors.
Example 8: Combination therapy: Systemic vaccination with GM-CSF
transductants and local intracranial administration of IL-2
transductants
For combination experiments, all animals received a subcutaneous
flank vaccination and were challenged after two weeks with the standard
intracranial dose of 10z wild type melanoma cells co-injected with the
specific
intracranial treatment. The flank vaccination was with a single subcutaneous
injection of 106 irradiated GM-CSF-producing cells and the intracranial
treatment two weeks later was with 5 x 10~ irradiated IL-2 producing cells
injected into the brain. Given that a dose of 8.0 ng per day of IL-2 (l05
cells), yielded a 33 percent long-term survivor rate and approached doses at
which toxicity was observed, a lower dose of 4.0 ng per day of IL-2 (5 x 104
cells) was selected. Control animals received either the subcutaneous GM-
CSF vaccine and intracranial treatment with medium, or subcutaneous
vaccination with medium and intracranial treatment with 5 x l04 IL-2
producing cells. Additional controls received vaccinations of medium alone
followed by intracranial treatment with medium along with an intracranial
tumor challenge.
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Results
When animals were treated with the GM-CSF-secreting cell vaccine in
combination with intracranial IL-2-producing cells, a greatly enhanced
response was achieved. The results are shown in Figure 4. Median survival
was not yet reached at l00 days, with 58% of the mice still alive after
combination therapy. This represents a significant advantage in survival over
flank vaccination with GM-CSF-producing cells alone (median survival, 27
days, p=0.018), over local intracranial administration of IL-2-producing cells
alone (median survival, 35 days, p=0.01), and over control (median
survival, 17.5 days, p < 0.001). The rate of 58 % of long term survivors,
those surviving more than 100 days, in the combination group exceeds the
percentage in the GM-CSF flank vaccine group (16%, p=0.08), the
intracranial IL-2 transductant group (8.3 %, p=0.03)) or the sum of the two,
providing a greatly enhanced response. Both the mice receiving the GM-CSF
vaccine alone and those treated with intracranial IL-2-secreting cells alone
showed prolongation of survival compared to controls (IL-2, p =0.0006; GM-
CSF) p < 0.001 ), confirming the efficacy of single cytokine therapy in this
model.
These results indicate that the GM-CSF vaccine generates a cohort of
CD4 + and CD8 + T-cells specific for a tumor antigen, which activity of the
cells is enhanced by the subsequent local IL-2 therapy at the tumor site. The
results correspond with the theory that while the blood-brain barrier poses an
obstacle to delivery of chemotherapeutic agents to the brain, it functions as
an avenue of entry into the central nervous system for immunologically active
cells. Wekerly, H., et al., TINS, 27:1-7 (1986).
The degree of inflammatory i~ltrate in the brain parenchyma of
animals treated with these cytokines was small, regardless of the mode of
delivery. These findings are in contrast to studies of cytokine enhanced
antitumor immune responses done in flank models, where a marked local
inflammatory infiltrate was apparent. These results indicate that intrinsic
immune response cells within the brain such as microglia may also be
involved in the protective immune response.
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Example 9: Systemic vaccination with GM-CSF transductants and local
intracranial administration of IL-2 transductants produces
long term memory protection against re-challenges
Genetically engineered non-replicating B16-F10 melanoma cells were
used for systemic delivery of GM-CSF or intracranial delivery of IL-2.
Forty-nine C57B16 mice were vaccinated in the flank with either GM-CSF
producing B 16-F I O cells or medium followed by intracranial delivery of IL-2
or medium 2 weeks later. Every animal received an intracranial co-injection
of wild-type melanoma at a dose uniformly fatal to untreated animals. Initial
survivors and a new control group were rechallenged with a second
intracranial dose of wild-type B16-F10 melanoma 134 days after their first
challenge.
Results
Animals receiving both flank GM-CSF and intracranial IL-2 had the
longest survival, with median survival not yet reached at l30 days (7 of 13
alive). This is a significant improvement over either flank GM-CSF alone
(median survival 27 days, p=0.018)) intracranial IL-2 alone (32 days,
p=0.01) or control (17.5 days) p<0.00l). GM-CSF alone and IL-2 alone
were significantly more effective than the controls, (p < 0.006).
Seven animals who received combination therapy with GM-CSF and
IL-2 survived more than 130 days. Five of the 7 mice rechallenged were
alive 28 days later. All 8 controls died by day 21 (p=0.0l8). Furthermore,
the antitumor effect persisted for greater than 4 months. These results
demonstrate that use of cytokines can lead to immunologic memory in the
brain.
Example 10: Interstitial delivery of IL-2 to the brain
Methods
An alternative approach to the control of metastatic brain tumors by
using local immunotherapy with interstitial delivery of interleukin-2 (IL-2)
as
30. both a primary treatment for establishing tumors and a vaccine against
subsequent recurrence is described. IL-2 was incorporated into biodegradable
sustained release microspheres (average diameter 4 wm) which were
stereotactically injected into the brains of mice to deliver 100 ng IL-2 over
a
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7 day period. Safety testing showed no evidence of clinical or histologic
toxicity from this dose over a 120 day period. To evaluate efficacy as a
primary treatment, a lethal challenge of B16 melanoma was stereotactically
co-injected into the left parietal lobe of 25 mice with either: saline
(controls), empty (no IL-2) microspheres, or the IL-2 containing
microspheres.
Results
75 % of the IL-2 treated animals (6I8) were alive with no sign of
tumor 90 days after initial treatment while median survival of untreated
controls was 18 days with no animal surviving more than 21 days. The
empty microspheres had no effect on survival. In a similar mouse melanoma
test, no impact was seen on survival from either bolus injection of IL-2 to
the
tumor site or systemic IL-2 administration, suggesting that the sustained
local
delivery of IL-2 in the brain provided by the microspheres is important for
the continued stimulation of immune cells attracted to the tumor bed.
Example 11: Intracranial injections of IL-2 into the brain
lVlethods
To evaluate the potential brain vaccine effect of this therapy) a group
of non tumor-bearing mice were co-injected intracranially with IL-2
microspheres and melanoma cells (see example 10) which had been irradiated
to prevent replication. 30 days later these animals and a group of control
mice (no previous treatment) were challenged intracranially with the lethal
dose of live melanoma with no additional IL-2 therapy.
Results
Previous IL-2 vaccination extended median survival by 45 % and
generated 2I5 animals alive at l60 days after tumor challenge with all
controls dead by 23 days. These results suggest that brain interstitial
immunotherapy with IL-2 delivered by microspheres is an effective and safe
treatment against brain metastases and may prevent tumor recurrence.
_ _ _.~ . . . ,.
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Example 12: Combination therapy with locally delivered IL-2 and
chemotherapeutic agents
Methods
A combination therapy consisting of immunotherapy, using non-
replicating genetically engineered tumor cells that produce IL-2, and local
delivery of chemotherapy was tested to determine if the component therapies
acted synergistically against an intracranial tumor challenge in a rnurine
brain
tumor model. B16-F10 melanoma cells were transduced with murine IL-2
using a replication-defective MFG retroviral vector. Chemotherapeutic
agents, BCNU and carbopiatin, were incorporated into controlled-release
polymers. C571B 16 mice received intracranial challenge of wild-type tumor
with and without non-replicating IL-2 producing tumor cells. On day 5,
animals received polymer implants containing 4.0 % BCNU, 1 % carboplatin,
or placebo.
Results
Animals receiving IL-2 and 1 % carboplatin (n=10, median survival
greater than 100 days, p < 0.01 ) had significantly improved survival over
animals receiving IL-2 (n=10, median survival 38 days) or 1 % carboplatin
alone (n=, median survival 23 days). Animals receiving IL-2 and 4.0%
BCNU (n=10, median survival 44 days) had improved survival over animals
receiving 4.0% BCNU (n=10, median survival 24 days) or IL-2 alone
(n=10, median survival 38 days). The median survival for the control group
(n=10) was 19 days. This example demonstrates that combination
intracranial immunotherapy with IL-2 and local delivery of chemotherapy
improves survival over either antitumor therapy alone. This effect was
obtained even when utilizing low doses of chemotherapy ( 1 % carboplatin and
4.0 % BCNU) .
Example 13: IL-2 inhibited tumor growth in murine model of hepatic
melanoma metastasis
Methods
Useful immunotherapeutic strategies must demonstrate success in
tumors within the liver, an organ with unique immunologic properties and a
common site of metastases in humans. The anti-tumor effects of local or
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paracrine IL-2 delivery was evaluated using the non-immunogenic B 16-F 10
melanoma implanted into the liver of C57B116 mice. Irradiated autologous
tumor cells transfected with the IL-2 gene was used as the source of IL-2.
Varying numbers of these cells (5 x 105 to 2 x 106) were admixed with 1 x
10' wild type B 16-F 10 melanoma cells and injected intrahepatically, and
compared to cells alone, cells coinjected with non-transfected cells, or with
IL-2 cells injected at a distant site (flank) (n=7lgroup). Presence or absence
of tumor within the liver and tumor volumes were measured on day 21.
Results
Inhibition of tumor growth was seen in mice receiving locally
delivered IL-2 at all doses. The number of mice without any tumor was
significantly less in those receiving the highest IL-2 dose (p < 0.05) and, in
those livers in which tumor was present, the mean tumor volume was
markedly reduced (2 mm' versus 884 mm', p < 0.05). This effect was not
seen when IL-2 was administered at a distant site nor in those mice receiving
irradiated wild type controls. The effect was dose-dependent, with maximum
inhibition seen at the highest IL-2 dose. Histologic examination revealed a
marked infiltrate at the site within the livers with IL-2 secreting cells,
comprised of both lymphocytes and Kupffer cells. In summary, these results
demonstrate that local delivery of IL-2 inhibited tumor growth in this marine
model of hepatic melanoma metastasis. This strategy has potential
application for the development of immunotherapy of liver tumors.
Those skilled in the art will recognize, or be able to ascertain using
no more than routine experimentation, many equivalents to the specific
embodiments of the invention described herein. Such equivalents are
intended to be encompassed by the following claims.
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