Note: Descriptions are shown in the official language in which they were submitted.
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COMBINED RADIOT~RAPY AND IMhu~ KAPY TO TREAT CANCER
R~rRGROUND OF T~ INv~ ON
FIETn OF T~ INVENTION
The present invention relates generally to
cancer therapy and, more particularly, to compositions
and methods for increasing the susceptibility of a tumor
in a m~mm~l ian subject to the e~fects of radiation
therapy.
R~CKGROUln~ INFOR~TION
Improved methods and novel agents for treating
cancer have resulted in increased survival time and
,~urvival rate for patients with various types of cancer.
For example, improved surgical and radiotherapeutic
procedures result in more effective removal of localized
tumors. Surgical methods, however, can be limited due,
:Eor example, to the location of a tumor or to
dissemination of metastatic tumor cells. Radiotherapy
also can be limited by these factors, which limits the
dose that can be administered. Tumors that are
relatively radioresistant will not be cured at such a
dose .
Immunotherapeutic methods also are being
~m; ned as a means to treat a cancer by stimulating the
patient's immune response against the cancer. In
particular, the role of cytokines, which are cellular
factors that can modulate an immune response, is an
important factor to consider when planning an
immunotherapeutic procedure. For example, expression of
a cytokine such as interleukin-2 (IL-2) can increase the
proliferation of T cells, which are involved in the
cellular immune response against a cancer.
SUBSTITUTE SHEET (RULE 263
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It is well known, however, that cytokine
- A~; ni stration frequently is associated with toxic
effects that limit the therapeutic value of these agents.
For example, severe hypotension and edema limit the dose
and efficacy of intravenous and intralymphatic IL-2
administration. In addition, flu-like symptoms or
fatigue often are associated with the administration of
various cytokines. The toxicity of systemically
administered lymphokines is not surprising as these
agents mediate local cellular interactions and normally
sre secreted only in very small quantities.
To circumvent the toxicity of systemic cytokine
~' ; n; stration, an alternative approach involving
cytokine gene transfer into tumor cells has produced
anti-tumor immune responses in several An; -1 tumor
models. In these studies, the expression of cytokines
following cytokine gene transfer into tumor cells
resulted in a reduction in tumorigenicity of the
cytokine-secreting tumor cells when implanted into
syngeneic hosts. Reduction in tumorigenicity has been
reported in studies using, for example, IL-2,
interferon-y or interleukin-4. In addition, the treated
~n; - -1 S o~ten developed systemic anti-tumor ;mmlln;ty and
were protected against subsequent tumor cell challenges
with n--~A; fied tumor cells.
Although a single treatment mntl;~.l; ty such as
radiation therapy, chemotherapy, surgery or immunotherapy
can result in improvement of a patient, superior results
can be achieved when such modalities are used in
com~ination. In particular, treatment with a com~ination
of radiotherapy, which can be directed to a localized
area cont~; n; ng a tumor, and chemotherapy or
immunotherapy, which provide a systemic mode of
treatment, can be useful where dissemination of the
disease has occurred or is likely to occur.
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Un~ortunately, the therapeutic usefulness of radiation
~ therapy can be limited where the tumor cells are
relatively radioresi~tant, since the radiation dose is
limited by the tolerance of normal tissue in the
radiation field. Thus, there exists a need to increase
the susceptibility a cancer to the effects of radiation
therapy so as to provide a more effective means to reduce
the severity of a tumor in a patient. The present
invention satisfies this need and provides related
advantages as well.
8UM~RY QF ~U~ lNV~r~ lON
The invention provides a method of increasing
the susceptibility of a tumor to the effects of radiation
therapy in a mammalian subject, by contacting the tumor
with a cytokine selected from the group consisting of
interleukin-3, granulocyte-macrophage colony stimulating
factor and granulocyte colony stimulating factor, wherein
said contacting stimulates an immune response against the
tumor. A method of the invention provides the additional
advantage that a systemic immune response against the
tumor can be stimulated in the subject.
The invention also provides a method of
increasing the susceptibility of a tumor in a m~ - l; An
subject to the effects of radiation therapy, by
contacting the tumor with a nucleic acid molecule
encoding a cytokine selected from the group consisting of
interleukin-3, granulocyte-macrophage colony stimulating
factor and granulocyte colony stimulating factor,
wherein, following said contacting, said cytokine is
expressed and stimulates an immune response against the
tumor. For example, the invention provides a method of
increasing the susceptibility of a tumor in a subject to
the effects of radiation therapy by A~mi ni stering, at a
site other than the tumor, tumor cells that have been
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genetically modified to express a c~tokine such as
interleukin-3.
The invention further provides a method of
reducing the severity of a cancer in a ~mmAliAn subject
by A~m; n; stering a cytokine to the subject and
~i n; stering a radiotherapeutic dose of radiation to the
tumor. For example, the invention provides a method of
~ nizing a subject with a cytokine and a tumor antigen,
which stimulates an immune response again~t tumor cells
expressing the antigen, and A~; n; stering a
radiotherapeutic dose of radiation to a tumor comprising
such tumor cells in the subject.
D~TT~ DESCRIPTION OF T~E lNV~ ON
The present invention provides a method of
increasing the susceptibility of a tu-m--or in a subject to
the effects of radiation therapy by contacting the tumor
with a cytokine such as interleukin-3 (IL-3~,
granulocyte-macrophage colony stimulating factor (GM-CSF)
or granulocyte colony stimulating factor (G-CSF). As
used herein, the term "contacting," when used in
reference to a cytokine and a tumor, means that the
cytokine is present in the location of the tumor,
particularly in the location of a localized tumor. A
tumor can be contacted with a cytokine, for example, by
injecting a solution cont~; n; ng the cytokine into the
region of the tumor, by A~m; n; stering a nucleic acid
molecule encoding a cytokine into the region of the
tumor, wherein the nucleic acid molecule is taken up by
cells present in or around the tumor such that the
cytokine is expressed and secreted, or by A~m; n; ~tering a
cell that has been genetically modified to express and
secrete the cytokine into the region of a tumor.
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An additional advantage of a method of the
~ invention is that a systemic immune response against the
cancer can occur in the subject. Thus r a composition
cont~; n; ng a cytokine or a nucleic acid molecule encoding
5 a cytokine is referred to herein generally as an
"immunizing composition." In general, an ;mmlln;zing
composition contains a cytokine that is in a form for
~ i n istration to a subject. In addition, an immunizing
composition can contain a tumor antigen or other
immunostimulatory agent as desired. The term
"im~unizing' is used generally to refer to the
~i n; stration of such an ; n; zing composition to a
subject.
Contact of a cyto~ine such as IL-3, GM-CSF or
G-CSF with a tumor antigen results in sti l~tion of an
immune response against a tumor cont~i n i n~ cells that
express the antigen. As used herein, the term "immune
response" includes the innate (nonspecific) immune
response, which is characterized, in part, by the
inflammatory response, and the acquired (specific~ immune
response, which is characterized, in part, by a B cell or
T cell response (see, for example, Kuby, Immunology 2d
ed. (W.H. Freeman and Co. 1994); see Chap. 1).
Cytokines such as IL-3, GM-CSF and G-CSF
2'; stimulate proliferation of bone marrow precursor cells
such as granulocyte and macrophage precursor cells.
These cells, which are effector cells of the innate
immune response, can be involved in rendering a method of
the invention effective because they can infiltrate a
tumor and mediate an inflammatory response and
phagocytosis of cellular debris in the tumor.
IL-3, GM-CSF and G-CSF do not appear to
directly stimulate the proliferation of B cell or T cell
precursors. However, the cells of the innate immune
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response have a central role in stimulating an acquired
~ immune respon~e. For example, macrophages are involved
in antigen presentation, which i8 a prere~uisite to
stimulation of an acquired immune response. In addition,
cytokine production at the site of an inflammatory
response contributes to the stimulation of the acquired
immune response. Thus, a method of the invention results
in stimulation of the innate and acquired immune
responses, which can increase the susceptibility of a
tumor to the effects of radiation therapy. The
stimulation of an acquired immune response provides the
additional advantage that the response is systemic and,
therefore, can be effective in killing metastatic lesions
that may be outside of the field of radiation therapy.
The invention also provides a method of
increasing the susceptibility of a tumor in a subject to
the effects of radiation therapy by A~; n; stering, at a
site other than the tumor, an ;~ln;zing composition
comprising a cell genetically modified to express a
cytokine. For example, the invention provides a method
of increasing the susceptibility of a tumor in a subject
to the effects of radiation therapy by A~' ; n; stering to
the subject an ;~lln;zing composition comprising tumor
cells genetically modified to express a cytokine. Such
an ;~-lln;zing composition can be A~i ni stered at the site
of the tumor, in which case the cytokine is IL-3 or
GM-CSF or G-CSF, or can be ~i n; stered at a site other
than the tumor site, in which case the cytokine can be an
interleukin, an interferon, a tumor necrosis factor or a
colony stimulating factor.
In one embo~;m~t of the invention, an
~ n;zing composition, which contains a cytokine such as
I~-3 or GM-CSF or G-CSF, is ~m; ni stered at the site of a
tumor in a sub]ect. As a result, the microenvironment of
the tumor is altered such that a systemic immune response
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against the tumor cells in the tumor occurs, thereby
~ rendering the tumor more susceptible to the effects of
radiation therapy.
~,
In another embo~; -nt of the invention, an
;mml7n;zing composition, which contains a c~tokine and a
tumor antigen, i8 ~tl~;n; stered at a site other than the
tumor site in the subject. As a result of the
~m; n; stration of the immunizing composition at a site
other than a tumor site, a systemic immune response is
stimulated in the subject, thereby rendering the tumor
more suscepti~le to the ef~ects of radiation therapy.
An ;~ml-~;zing composition, which contains a
cytokine or a nucleic acid molecule encoding a cytokine,
can be A~mi n; stered to a subject having a cancer. As a
result, an im.mune response is stimulated in the subject
against the cancer, wherein the systemic immune response
or alterations induced by the immune response in the
tumor microenvironment can increase the susceptibility of
the subject~s cancer to the effects of radiation therapy.
Subsequent radiotherapy then can be used to treat the
tumor and the systemic immune response can destroy
r~; ni ng tumor cells, including any metastatic lesions.
Thus, in another embo~;m~nt, the invention provides a
method of reducing the severity of a cancer in a subject,
comprising immunizing the subject with an immunizing
composition and administering a dose of radiation to the
site of the cancer.
As disclosed herein, ;mmlln;zation of a subject
provides a means to increase the susceptibility of a
tumor such that it can be treated more effectively by
radiotherapy (see Example I~. As used herein, the term
"tumor' means a localized growth of cancer cells, which
can be a primary tumor located at the site where a cancer
originally formed or can be a metastatic lesion. The
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terms "tumor cell~ and "cancer cell" are used
~ interchangeably herein to mean a malignant cell.
A method of the invention is particularly
useful for treating a subject having metastatic lesions
that have disseminated from an original tumor site
because, in addition to increasing the susceptibility of
a primary tumor to radiation therapy, a method of the
invention also provides a systemic immune response, which
can kill disseminated cancer cells. Thus, the invention
is particularly useful for treating a subject with a
cancer such as a melanoma or any other cancer in which
the dissemination of metastatic lesions is common, in
that recurrence of a tumor due to growth of metastatic
lesions is less likely to occur.
A method of the invention increases the
susceptibility of a tumor in a ~ lian subject to the
effects of radiation therapy. As used herein, the phrase
"increasing the susceptibility of a tumor to the effects
of radiation therapy" means that a method of
20 A~mi n; stering a cytokine a~ disclosed results in a
specified radiation dose having a greater therapeutic
effect against the tumor than the specified dose would
have in the absence of cytokine ~i ni stration. Thus,
the term "increasing the susceptibility of a tumor to the
effects of radiation therapy" is used in a comparative
sense to indicate that the radiation dose to reduce the
severity of a cancer in a patient that has been immunized
as disclosed is lower than the radiation dose that would
have been required if the patient had not been i n; zed.
The term "radiosensitize" often is used in a
therapeutic setting to indicate that a treatment results
in increased susceptibility of a tumor to the effects of
radiation therapy (see, for example, ~nc. Res.
54:4266-4269, which is incorporated herein by reference).
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However, the term ~radiosensitize" also is used
~ specifically to indicate that a particular treatment such
as exposure to a drug results in a decrease in the amount
of radiation required to produce an effect such as cell
death as compared to the amount of radiation required in
the absence of the treatment. It should be recognized,
however, that the increased susceptibility o~ a tumor to
the effects of radiation that occurs due to practicing
the present invention is an effect measured at the level
of the whole tumor, not at the level of an individual
tumor cell. Nevertheless, the fact that the effect is
measured at the level of the tumor does not preclude that
the effect on the tumor can be due, at least in part, on
an effect at the cellular level.
As used herein, the term "effects of radiation
therapy~ refers to the well known clinic~l effect that
radiation has on reducing the size or growth rate of a
tumor. Although no mechanism is proposed to explain how
a method of the invention increases the susceptibility of
a tumor to the effects of radiation therapy,
~;n; stration of a cytokine such as IL-3, GM-CS~ or
G-CSF, which stimulates production of inflammatory cells
such as granulocytes and macrophages, can result in
infiltration of such cells into the tumor. Such cell
infiltration can result in an inflammatory response and
phagocytic activity that can render the tumor more
susceptible to the effects of radiation therapy. For
example, cells such as granulocytes produce active oxygen
species that can kill tumor cells.
In addition, phagocytic activity the by
infiltrating such cells can allow increased blood flow to
the tumor, which, in turn, can increase the well known
cytostatic and cytotoxic effects that radiation has on a
cell. For example, exposure of a cell to radiation can
inhibit progression of the cell through the cell cycle;
.
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can damage nucleic acids, proteins, or other
macromolecules in a cell; or can kill the cell by
inducing apoptosis. These effects of radiation are
interrelated and represent a continuum of effects, the
magnitude of which is dependent, in part, on the
radiation dose and on the relative radiosensitivity of
the target cell.
Furthermore, the induction of a systemic
acquired immune response due, for example, to
presentation of tumor antigens to immunoeffector cells by
macrophages that have infiltrated the tumor can
contribute to the effectiveness of a method of the
invention for increasing the susceptibility of a tumor to
the effects of radiation therapy. The effectiveness of a
method of the invention can be due to one or more of
these or other mechanisms acting in concert with the
radiation therapy to produce an effect on the tumor that
is greater than might otherwise have been expected by
combining radiation therapy and immunotherapy (see
Example I.C.).
The present invention provides a means to
increase the susceptibility of a tumor to the effects of
radiation therapy, including tumor cell killing. For
convenience, reference is made generally herein to tumor
cell "killing." It should be recognized, however, that
an increased susceptibility of a tumor to any of the
effects of radiation can provide a significant
therapeutic benefit to a cancer patient.
The effectiveness of a method of the invention
in treating a ~ -lian subject can be identified using
well known methods. For example, the effectiveness o~
treatment can be identified by detecting, in a subject
~ n;zed as disclosed herein, prolonged survival of the
subject, disappearance of the tumor, or a decreased rate
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of growth of an irradiated tumor as compared to the rate
~ of growth prior to irradiation. In human cancer
patients, such measurements generally are made using well
known imaging methods such as magnetic resonance imaging,
computerized axial tomography and X-rays. In addition,
determ;~tion of the level of a tumor marker such as the
detection of levels of circulating carcinoembryonic
antigen (CEA) or pro~tate specific antigen or the like
also can be used as an indication of the effectiveness o~
a treatment. Thus, the effectiveness of a method of the
invention can be deter~;ne~ by measuring a decrease in
the growth rate of a tumor or an appropriate change in
the level of a circulating marker, the presence or
relative level of which is indicative of cancer.
~5 Radiation therapy i8 a conventional method for
treating cancer. In particular, radiotherapy is useful
in cases where the tumor is relatively localized and not
excessively large, or where surgical excision of the
tumor is contrA;n~icated due, for example, to the
location of the tumor. Radiation therapy is a preferred
method of treating, for example, pro~tate cancer and
brain tumors. The skilled artisan would know the
appropriate dosages, treatment schedules and radiation
sources to use for treating a particular cancer.
2!; Various factors can limit the usefulness of
radiotherapy. Ultimately, however, the success of
radiotherapy is limited due to unacceptable patient
morbidity that occurs as a result of consequent
irradiation of normal tissue in the radiation field. In
particular, exposure of rapidly renewing tissues,
including, bone marrow, small intestine and skin, to
radiation can lead to unacceptable patient morbidity.
However, slowly proliferating tissues, including nervous
tissue, also can be damaged irreversibly i~ exposed to an
excessively high dose of radiation.
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12
AATn;n; stration of radiotherapy as fractionated
~ doses over a period of time can provide advantages over
~; n; stration of a single large dose. In particular,
fractionated doses of radiation are useful if the cells
in the normal tiss~le in the radiation field can repair
radiation induced damage faster or more efficiently than
the tumor cells in the radiation field. In this case,
fractionated doses can be A~' ; n; stered at intervals that
preferentially allow repair of the normal cells as
compared to the tumor cells. In addition, tumors
generally have relatively hypoxic regions that are less
susceptible to radiation damage. Fractionated radiation
doses also can permit reoxygenation to occur in such
regions, due to sloughing off of tumor cells killed by
previous doses, thus improving the effectiveness of
subse~uent radiation doses.
Considerable research has been directed to the
identification of chemical agents that selectively
increase the radiosensitivity of tumor cells, but not of
normal cells. Such radiosensitizers can work, for
example, by effecting reoxygenation of a hypoxic region
of a tumor or by acting as an oxygen mimetic. Since
normal tissue is well oxygenated, such a radiosensitizer
can increase the sensitivity of the tumor cells, while
having relatively less effect on the normal cells, thus
effectively radiosensitizing the cancer cells.
Cytokines are a class of molecules that, in
some cases, also can act as radiosensitizing agents.
Cytokines constitute a family of polypeptides that are
produced by leukocytes and other cells and regulate the
immune and inflammatory responses in humans (Thomson, The
Cytokine Handbook (Academic Press; 1994), which is
incorporated herein by reference; see Chap. 1~.
Cytokines are polypeptides or glycoproteins, including
the heterodimeric I~-12, that bind to high affinity cell
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13
surface receptors. Numerous cytokines, including
interleukins 1-13 (IL-l, IL-2, I:1~-3, etc.), interferons
~, ~ and y (Ifn-~, Ifn-~ and Ifn-y), tumor necrosis
factors ~ and ~ (TNF-~ and TNF-~), colony stimulating
factors such as macrophage colony stimulating factor
(M-CSF), G-CSF and GM-CSF, and transforming growth
factor ~ (TGF~) and stem cell factor, are known in the
art (Thomson, supra, 1994).
Cytokines can increase or decrease the rate o~
cell proliferation and can affect the differentiation
state of a target cell, including cells involved in the
immune response. For example, IL-2, ~:L--4 and IL-7 are
T cell growth factors, which increase proliferation of
T cells; M-CSF, GM-CSF, G-CSF and IL-3 can induce bone
marrow cells to differentiate into macrophages or
granulocytes or both; and Ifn-y, IL 4, IL-7 and GM-CSF
can activate macrophages, increasing their tumoricidal
activity.
In addition, the expression of specific
combinations of cytokines can be particularly useful for
~timulating an immune response. For example, expression
of IL-l, IL-6 or a TNF can enhance IL-2 induced T cell
proliferation; of IL-6 can enhance IL-4 induced T cell
proliferation; and of Ifn-y, IL-2 and IL-12 can stimulate
T cells of the T helper-1 class, which are involved in
the cellular immune response. Thus, it can be useful to
express specific combinations of cytokines for the
purpose of stimulating an immune response.
It is recognized, however, that the expression
~O of other combinations of cytokines can inhibit an immune
response. For example, TGF-~ can inhibit I~-2 action and
IL-10 can inhibit cytokine production and antigen-
specific proliferation of T helper-1 cells. In addition,
various types of cancer cells can express receptors for a
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14
cytokine such as IL-2 or IL-1, such that expression of
~ the cytokine can induce proliferation of the tumor cells.
Thus, the selection of a cytokine or com~ination of
cytokines to be administered to a subject must be
considered carefully (~homson, supra, 1989; see
Chap. 25).
In some cases, cyto~ines can act to
radiosensitize cancer cells. For example, tumor cells
genetically modified to express IL-6 or I~-7 were more
sensitive to radiation induced killing in vitro than were
the corresponding unmodified tumor cells (McBride et al.,
~cta Oncol. 34:447-451 (1995), which is incorporated
herein by reference). Surprisingly, however, when
unmodified tumor cells or IL-7 expressing tumor cells
were injected into a mouse thigh, then treated with
radiotherapy, the IL-7 expressing tumor cells were more
radioresistant than the unmodified tumor cells.
In other studies, human tumor cells that were
exposed to TNF-~ or genetically modified to express TNF-
~
when irradiated, then xenografted into nude mice, weremore sensitive to radiation therapy than were the
corresponding lln~o~;fied tumor cells (Hallahan et al.,
Nat. Med. 1:786-791 (1995); Sersa et al., Int. J. ~An~.
42:129-134 (1988); each of which is incorporated herein
by reference; see also Weichselbaum et al., supra, 1994 ) .
TNF-~ also can act as a radioprotector, however,
decreasing the lethal effect of radiation in mice (Neta
et al., J. Exp. Med. 175:689-694 (1992)). IL-1 also has
been described as providing a radioprotective effect
(Neta et al., supra, 1992), although studies using
another syst~m found no such radioprotective effect for
IL-1, or for IL-2 or IL-3 (Gallicchio et al., J. Biol.
Re~p. ~od. 8:479-487 (1989)).
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A8 disclosed herein, cytokines ~3uch as IL-3,
~ GM-CSF and G-CSF can be useful to increase the
susceptibility of a tumor in a ~ n subject to the
- effects of radiation therapy, and have the additional
advantage that a systemic immune response can be
stimulated in the subject. It is well known, however,
that ~mi ni stration of a cytokine to a subject is limited
by the generalized toxicity induced when the agent is
~inistered syst~mic~lly. Thus, cytokines may be better
LO ~. ;nistered locally, for example, at the site of a
tumor.
A cytokine can be ~i ni stered by injection as
a solution into the site of a tumor. Furthermore, the
cytokine can be A~; ni stered as a cytokine polypeptide,
~5 or can be ~i ni stered in the form of a nucleic acid
molecule encoding the cytokine, wherein the nucleic acid
molecule is taken up by a cell in the tumor and the
cytokine is expressed therefrom. Where a nucleic
molecule encoding a cytokine is ~ in; ~tered to the site
of a tumor, the nucleic acid molecule generally is linked
to an appropriate regulatory such as a promotor that
provides selective expression of the cytokine at the site
of the tumor (see, for example, Seung et al., Canc. Res.
55:5561-~565 (1995~, which is incorporated herein by
reference). In addition, the nucleic acid molecule
encoding the cytokine can be l; nke~ to a vector such as a
retrovirus vector (see below) or the nucleic acid
molecule encoding the cytokine can be physically
associated with a formulation such as a liposome or an
inert particle such as gold.
Preferably, the cytokine is expressed from a
cell such as a tumor cell that has been genetically
modified, in vltro or in vlvo, to express the cytokine.
Expression of a cytokine from a genetically modified cell
provides the advantage that sustained, localized
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expression of the cytokine can occur, thus obviating the
need for repeated A~m; n; strations.
Various studies have been performed with tumor
cells genetically modified to express a cytokine. As
discussed above, for example, tumor cells that were
genetically modified to express TNF-~ and injected into
unmodified tumors growing in immunodeficient mice,
sensitized the tumor to radiation therapy more than did
the corresponding unmodified tumor cells (Weichselbaum et
0 al., supra, 1994; Hallahan et al., supra, 1995). In
addition, human renal carcinoma cells that were
genetically modified to express IL-2, Ifn-~, or both
cytokines, then irradiated with a dose of radiation that
inhibited the growth, but not cytokine expression, of the
genetically modified cells, lost their tumorigenicity as
determined following injection into T cell depleted mice
(Belldegrun et al., J. Natl. C~n~. Inst. 85:207-216
(1993), which is incorporated herein by reference3. In
addition, the genetically modified renal carcinoma cells
prevented the growth of unmodlfied renal carcinoma cells
when injected together, but not if the genetically
modified cells were injected at a different site from the
unmodified cells (Id.). These studies indicate that
tumor cells that are genetically modified to express a
cytokine can be used for local delivery of the cytokine
to a desired site such as a tumor.
In other studies, intraperitoneal immllnization
of mice with either ;~llnogenic or non-immunogenic tumor
cells, each of which was genetically modified to express
IL-3, protected the mice from later challenge with the
corresponding unmodified tumor cells (McBride et al.,
Folia Biolog. 40:62-73 ~1994), which is incorporated
herein by reference; see Example I). These results
demonstrate that ;mmllnization with IL-3 expressing tumor
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17
cells can stimulate a systemic immune response that
~ protects against a later challenge with tumor cells.
- As disclosed herein, the expression of a
cytokine such as IL-3 or GM-CSF or G-CSF at the site of a
tumor can render the tumor more susceptible to the
effects of radiation therapy and can stimulate systemic
immunity in the subject against the cancer. For example,
a non-immunogenic murine ~ibrosarcoma tumor cell line,
FSA~, or a moderately immunogenic murine fibrosarcoma
tumor cell line, FSA ~also called FSAR), was genetically
modified by transduction using a retroviral vector
cont~in;ng an expressible IL-3 encoding nucleic acid.
FSAN cells that were genetically modified to express IL-3
or genetically modified with a vector lacking the IL-3
coding sequence were injected into mice, then, after the
tumors attained a size of about 6 to 8 mm, the tumors
were irradiated with a single dose of 25 Gray (Gy), 40 Gy
or 55 Gy of X-rays (see Example I). In all cases, the
I~-3 expressing tumors completely regressed after
irradiation, whereas unirradiated IL-3 expressing tumors
or irradiated tumor genetically modified with the control
vector continued to grow. Furthermore, systemic immunity
was stimulated in the cured mice, such that no tumors
developed when the mice were subsequently challenged with
25 11 -.~; fied tumor cells.
The results of Example I demonstrate that
expression of I~-3 at the site of the tumor can increase
the susceptibility of a tumor in a ~ ~lian subject to
the effects of radiation therapy and can induce a
systemic immune response in the subject against the
cancer. In addition, where ~i n; stration of an
immunizing composition is at the site of tumor, a
cytokine such as GM-CSF or G-CSF also can be useful in
the present invention. For example, the heterodimeric
receptors for IL-3 and GM-CSF share a common chain
CA 02244841 1998-07-31
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(Thomson, supra, 1994; see Chaps. 5 and 19), indicating
~ that these cytokines can sct, in part, through a shared
pathway such as through the stimulation of dendritic
cells, which can increase tumor antigen presentation. In
addition, GM-CSF and G-CSF can stimulate the
proliferation of granulocyte and macrophage precursor
cells, which, as observed for IL-3, can result in
increase infiltration of these cells into the tumor (see
Example I). Thus ~ A~m; ni stration of IL-3 or GM-CSF or
G-CSF at the site of a tumor can increase the
susceptibility of the tumor to the effects of radiation
therapy and can stimulate a systemic immune response
against the cancer.
Where a tumor is treated by ~mi n; stering IL-3
or GM-CSF or G-CSF at the site of the tumor in a subject,
it is not necessary to include a tumor antigen in the
immunizing composition, since the tumor provides a source
of the antigen. However, a cytokine, including an
interleukin, interferon, tumor necrosis factor or colony
stimulating factor, also can be administered at site
other than the tumor site and can induce an immune
response that can increase the susceptibility of the
tumor to the effects of radiation therapy. In this
embodiment of the invention, the immunizing composition
also can contain a tumor antigen in addition to the
cytokine. If desired, an immunizing composition also can
contain an adjuvant such as BCG (see Harlow and Lane,
Ant;hodies: a laboratory manual (Cold Spring Harbor
Laboratory Press 1988); Mishell and Shiigi, Selected
Methods in Cellular Immunology (W.H. Freeman and Co.
(1980)), each of which is incorporated herein by
reference) or other adjuvant as commercially available
(Ribi Immunochem Res., Inc.; Hamilton MT).
When a cytokine is ~m; ni stered at a site other
than the tumor site, the cytokine is administered in a
CA 02244841 1998-07-31
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19
form that results in controlled release of desirably low
~ levels of the cytokine. Thus, an ;mmllnizing composition
can be formulated to contain a cytokine in com~ination
with a material such as DEPOFOAM, a wafer, an immunobead,
a micropump or other material that provides for
controlled slow release of the cytokine. Such controlled
release materials are well known in the art and available
from commercial sources (Alzs Corp., Palo Alto CA;
Depotech, La Jolla CA; see, also, Pardoll, Ann. Rev.
T~mllnol~ 13:399-415 (1995), which is incorporated herein
by reference).
In addition, a cytokine can be A~m; n; stered in
combination with a tumor antigen, which can be in the
form of a tumor cell, a tumor cell extract or a purified
tumor antigen. A tumor antigen can be obtained from the
subject or can be a known tumor antigen, including, for
example, epithelial cell mucin, which i5 encoded by the
MUC-1 gene, or the melanoma antigen, MZ2-E, which is
encoded by the MAGE-l gene, each of which is associated
with particular tumor cells (Finn, Curr. Opin. Immunol.
5:701-708 (1993), which is incorporated herein by
reference).
An i~m-ln;zing composition also can comprise a
tumor cell, which can be obtained from the subject to be
treated, that is genetically modified to express a
cytokine. It should be recognized that, while reference
is made to the "expression" of a cytokine by a cell, such
a cell that is useful in the invention also must secrete
the cytokine. The genetic modification of a subject's
tumor cells to express a cytokine provides the advantage
that, in addition to expression of the cytokine, the
tumor cell also presents a tumor antigen, against which
an active immune response can be generated.
CA 02244841 1998-07-31
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2~
If a subject's tumor cells are not readily
~ available, another type of cell can be genetically
modified to express a cytokine and, in addition, can be
genetically modified to express a tumor antigen, which
can be the same as the tumor antigen expressed on the
subject's cancer cells or can be a known tumor antigen as
disclosed above. Such genetically modified cells, which
express a tumor antigen and a cytokine, are referred to
herein as "carrier" cells (see PCT/US92/08999, filed
October 23, 1~92, which is incorporated herein by
reference).
Genetically modifying a cell to express a known
tumor antigen can be particularly useful when the tumor
cells to be genetically modified are not obtained from
the subject to be treated. For example, it may not be
possible to obtain a sufficient number of tumor cells
from a cancer patient or the patient's tumor cells may
not be adaptable to growth in culture. In this ca~e,
cells that do not express a particular tumor antigen that
2Q is expressed by the patient~s cancer cells can be
genetically modified to express the tumor antigen and, in
addition, can be genetically modified to express a
cytokine. Upon A~m; n; stration of such a genetically
modified carrier cell to the subject, the subject's
immune response against the cancer can be st;mlllAted and
the susceptibility of the tumor to the effects of
radiation therapy can be increased.
A cytokine also can be expressed from a
genetically modified cell such as a fibroblast or an
antigen presenting cell 6uch as a monocyte, a dendritic
cell or a lymphocyte. Such cells, which can be obtained
from the sub3ect or can be allogenic cells, are referred
to herein as "cytokine-expressing cells" or "CE cells"
snd can be prepared as disclosed herein or using methods
well }cnown in the art (see PCT/US92/08999, supra, 1992).
CA 02244841 1998-07-31
'WO 97/28251 PCT~US97/01465
21
Such CE cells can be administered at the site of a tumor
~ or, if ~; n; stered at a site other than a tumor site,
pre~erably, are At' ; ni stered in combination with a tumor
antigen.
- 5 A cell such as a tumor cell that is genetically
modified to express a cytokine can be further modified to
express an immunostimulatory agent such as the
costi~~ tory B7 molecules, B7.1 and B7.2, (Baskar et
al., Proc. Natl. Acad. Sci.. USA 90:5687-5690 (1993);
Townsend and Allison, Science 259:368-37Q (1993); Tan et
al., ~. Tmmllnol. 149:32217-3224 ~1992), each which is
incorporated herein by reference), autologous MHC class I
and class II molecules (Plautz et al., P~oc. Natl. ~cad.
Sci., USA 90:4645-4649 (1993); Hui et al., Fems
15 Microhiol. Tr~llnol. 2:215-221 (1990~; Ostrand-Rosenberg
et al., ~_Lmmunol. 144:4068-4071 tl990), each of which
i5 incorporated herein by reference), allogeneic
histocompatability antigens such as HLA-B7 (Nabel et al.,
Proc. Natl. Acad. Sc;., USA 90:11307-11311 (1993), which
is incorporated herein by reference) and known tumor
antigens (Finn, supra, 1993). For example, a subject's
cancer cells may not express an MHC class I or II
molecule and, as a result, would not induce an optimal
immune response. In such a case, expression of the
appropriate MHC molecule can be useful for stimulating an
immune response in the subject against the cancer.
Methods for det~ ;ning whether a tumor cell expresses a
particular immunost;mnl~tory agent are known in the art
and can be used to determine whether the tumor cell
should be genetically modified to express the
immunostimulatory agent.
In some aspects of the invention, the
A~min;stration of viable tumor cells is required.
However, administration of viable tumor cells to a
subject requires that the tumor cells be inactivated so
CA 02244841 1998-07-31
W O97/282Sl PCTrUS97/01465
22
they do no~ grow in the subject. Inactivation can be
accomplished by any of various methods, including, for
example, by irradiation, which is ~i n; stered at a dose
that inhibits the ability of the cells to replicate but
5 does not immediately kill the tumor cells. Where the
irradiated tumor cell i8 a carrier cell that has been
genetically modified to express a cytokine, it is known
that such irradiation does not substantially affect the
expre~sion of the cytokine (Belldegrun et al., supra ,
10 1993). Treatment of tumor cells with a cytostatic agent
or with a low dose of a cytotoxic agent also can render
the cells reproductively inactive. Such viable tumor
cells can present tumor antigens to the subject's immune
system but cannot multiply and form new tumors.
It is further recognized that, in some cases, a
tumor cell can express an immunosuppressive agent such as
a TGF~. Thus, if it is desirable to use such a tumor
cell as an antigen or as a carrier cell in the present
invention, the tumor cell can be genetically modified to
20 reduce the expression of the immunosuppressive factor.
Tumor cells that produce immunosuppressive factors are
known in the art and are present, for example, in
carc; nn~s~ sarcomas, gliomas, mel~nn ~s, lymphomas and
leukemias (Sulitzeanu, Adv. C~nr. Res. 60:247-267 (1993~,
25 which is incorporated herein by reference). Whether a
cancer cell is producing an immunosuppressive agent can
be readily determined using methods as disclosed herein
or otherwise known in the a~t.
Immunosuppressive agents are known in the art
30 and include, for example, TGF~, lymphocyte blastogenesis
inhibitory factor, the retroviral pl5E protein,
suppressive E-receptor (see Sulitzeanu, sup~a, 1993) anc~
extracellular matrix molecules such as fibronectin and
tenascin ~Olt et al., Cancer 70:2137-2142 (1992);
Hemasath et al., ~. Immunol. 152:5199-52Q7 (1994), each
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23
of which is incorporated herein by reference). It i~
~ recognized, for example, that various isoforms of TGF~
such as TGF~l, TGFB2, TGF~3, TGFA4 and TGFB5 exist (see,
for example, Roszman et al., Immllnol. To~y, 12:3~Q-274
(1991); Constam et al., J. Immunol., 148;1404-1410
- (1992); Elliot et al., J. Neuro-Oncolo~y, 14:1-7 (1992~,
each of which is incorporated herein by reference) and
that the immunosuppressive effect of one or more of these
isoforms of TGFB depends, for example, on the target
cell. The term "TGF~" is used generally herein to mean
any isoform of TGF~, provided the isoform has
immunosuppressive activity.
The present invention provides a method of
reducing the severity of a cancer in a subject ~y
15 A~mi n; stering to the subject an i ni zing composition,
which stimulates an immune response that increases the
susceptibility of a tumor in the subject to the effects
of radiation therapy, then administering a
radiotherapeutic dose of radiation to the tumor. As used
herein, the term "reducing the severity of a cancer"
means that the clinical signs or symptoms of the cancer
in a subject are indicative of a beneficial effect to the
subject due to treatment using a method of the invention.
Although complete remission is the optimal
result, it is recognized that any decrease in the rate of
progression of the cancer can provide a palliative effect
in the subject, thus improving the subject~s quality of
life. Methods for dete~;n; ng whether a treatment is
reducing the severity of a cancer are well known in the
art and include, for example, imaging methods such as
magnetic resonance imaging, computerized axial tomography
and X-rays, and tumor marker assays such as the detection
of levels of circulating carcinoembryonic antigen (CBA)
or prostate specific antigen or the like or by detecting
~5 the activation of immunoeffector functions in a subject
CA 02244841 1998-07-31
W O 97/28251 PCT~US97/01465 24
such as the activation of tumor cytolytic immunoeffector
~ cells.
A method of the invention can reduce the
severity of a cancer in a subject, by increasing the
susceptibility of a tumor in the subject to the effects
of radiation therapy. In addition, a method of the
invention can provide systemic i n i ty against the
subject's cancer, such that cancer cells that are not
killed by the radiotherapy are killed by the subject's
immune response. The presence of such an immune response
can be identified by comparing the immune functions of a
subject prior to A~3Tn; ni stration of an ir~ n; zing
composition with the immune functions following
administration. Such immune functions can be determined
using methods well known in the art for measuring a
humoral or cellular immune response (see, for example,
~arlow and Lane, supra, 1988; see, also, Example I.B.).
Genetic modification of a tumor cell or a
fibroblast or antigen presenting cell for use in the
present invention is advantageous because the genetically
modified cell provides sustained expression of the
cytokine. Viral vectors such as retrovirus, adenovirus
or adenovirus-associated vectors can be particularly
useful for genetically modifying a cell (see, for
example, Flotte, J. Bioenerg. Bi~m~mh.~ 25:37-42 (1993)
and Kirshenbaum et al., J. Clin. Invest, 92:381-387
(1993), each of which is incorporated herein by
reference; see, also, Hallahan et al., supra, 1995).
Such vectors are particularly useful when the vector
contains a promoter sequence, which can provide
constitutive or, if desired, inducible or tumor selective
expression of a cloned nucleic acid sequence. Such
vectors are well known in the art (see, for example,
~eth. Enzymol. t Vol. 185, D.V. Goeddel, ed. (A~A-1~m~C
Press, Inc., 1990), which is incorporated herein by
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W O 97/28251 PCTrUS97/01465
reference) and commercially available (Promega; Madison,
~ WI~. In particular, a vector contA;n;ng a radiation
inducible promotor can be useful in the present invention
(see Hallahan et al., supra , 1995).
~ ~ Vectors can be introduced into a cell or into
cells within a tumor by any o~ a variety of methods known
in the art (see, for example, Sambrook et al., Molecular
Cloning: A l~horatory manll~l (Cold Spring Harbor
La~oratory Press 1989); and Ausubel et al., Current
ProtocolF~ ;n Molecular R; ology, John Wiley and Son~,
Baltimore, MD (1994), each of which is incorporated
herein by reference). Such methods include, for example,
transfection, lipofection, electroporation and infection
with recombinant vectors or the use of liposomes.
]5 Introduction of nucleic acids by infection
(transduction) using a viral vector is particularly
advantageous in that it can be effective in vitro or in
vivo. Higher efficiency can also be obtAine~ due to the
infectious nature of a viral vector. Moreover, viruses
are very specialized and typically in~ect and propagate
in specific cell types. Thus, their natural specificity
can be used to target the vectors to specific tumor cell
types in a biopsy culture, which may be cont~ in~ted with
other cell types. Viral or non-viral vectors can also be
modified with specific receptors or ligands to alter
target specificity through receptor mediated events.
A nucleic acid molecule also can be introduced
into a cell using methods that do not require the initial
introduction of the nucleic acid sequence into a vector.
For example, a nucleic acid sequence encoding a cytokine
can be introduced into a cell using a cationic liposome
preparation (Morishita et al., J. Clin. Invest., 91:2580-
2585 (1993), which is incorporated herein by reference;
~ee, also, Nabel et al., s~pra, 1993)). In addition, a
CA 02244841 1998-07-31
W O 97/28251 PCTrUS97101465
26
nucleic acid seq~ence can be introduced into a cell
~ using, for example, adenovirus-polylysine DNA complexes
(see, for e~ample, Michael et al., J. Riol. Chem.,
268:6866-6869 (1993), which is incorporated herein by
reference). Other methods of introducing a nucleic acid
sequence are well known (see Goeddel, supra, 1990).
Nucleic acid sequences encoding various
cytokines have been cloned and are available for use
(Ge~R~nk; Thomson, supra, 1994). Nucleic acid sequences
encoding, for example, cytokines such as various
interleukins, including IL-3, interferons and colony
stimulating factors, including GM-CSF and G-CSF, are
available from the American Type Culture Collection (see
ATCC/NIH Repository Catalogue of Human and Mouse DNA
1~ Probes and Libraries, 6th ed., 1992) or are available
commercially (Amgen, Thousand Oaks, CA; see, also,
Patchen et al., Ey~tl. ~ematol., 21:338-344 (1993);
Broudy et al., Bloo~, 82:436-444 (1993), each of which is
incorporated herein by reference).
Selectable marker genes encoding, for example,
a polypeptide conferring neomycin (neo) resistance also
are readily available and, when linked to a nucleic acid
sequence or incorporated into a vector, allow for the
selection of cells that have successfully incorporated
the desired nucleic acid sequence. A "suicide" gene also
can be incorporated into a vector so as to allow for
selective inducible killing of a cell, particularly of a
genetically modified tumor cell, when the treatment is
completed or otherwise term;n~ted. A gene such as the
herpes simplex virus thymidine kinase gene (TK) can be
used as a suicide gene to provide a means of inducible
destruction of a ce~l. For example, when a cell such as
a tumor cell cont~in;ng such a TK gene no longer is
useful in the subject, a drug such as acyclovir or
gancyclovir can be ~m; n; stered to the subject. Either
CA 02244841 1998-07-31
W O97/282~1 PCTAUS97/0146S 27
of these drugs selectively kills cells expressing the
~ viral TK, thus elimin~ting the genetically modified
cell~. Additionally, a suicide gene can encode a
non-secreted cytotoxic polypeptide and can be l; nke~ to
an inducible promotor. If destruction of the cells i5
desired, the appropriate inducer of the promotor is
~i n i stered so that the cytotoxic polypeptide is
expressed.
Numerous methods are available for transferring
nucleic acid sequences into cultured cells, including the
methods described above. In addition, a useful method
can be similar to that employed in previous human gene
transfer studies, where tumor infiltrating lymphocytes
(TILs) were modified by retroviral gene transduction and
~m;n; stered to cancer subjects ~Rosenberg et al., New
~ngl. J. Me~. 323:570-578 (1990); see, also, U.S. Patent
No.: 5,460,959, issued October 24, 1995, U.S. Patent
5,399,346, issued March 21, 1995; each of which is
incorporated herein by reference~. In the Phase I safety
study of retroviral ~-~;Ated gene transfer, TILs were
genetically modified to express the neomycin resistance
gene. Following intravenous infusion, polymerase chain
reaction analyses consistently found genetically modified
cells in the circulation for as long as two months after
A~; n; stration. No infectious retroviruses were
identified in these subjects and no side effects due to
gene transfer were noted in any subjects. These
retroviral vectors have been altered to prevent viral
replication ~y the deletion of viral gag, pol and env
genes.
When retroviruses are used for gene transfer,
replication competent retroviruses theoretically can
develop due to recombination of retroviral vector and
viral gene sequences in the packaging cell line utilized
3'; to produce the retroviral vector. Packaging cell lines
CA 02244841 1998-07-31
W O97t28251 PCT~US97/01465
28
in which the production of replication competent virus by
recombination has been reduced or eliminated can be used
to m; n; m; ze the likelihood that a replication competent
retrovirus will be produced. Hence, all retroviral
vector supernatants used to infect subject cells wi~l be
screened for replication competent virus by st~n~d
assays such as PCR and reverse transcriptase assays.
As discussed above, a cancer cell can express
an immunosuppressive agent such as an immunosuppressive
isoform of TGF~. Such cancer cells should be genetically
modified to reduce or inhibit the expression of the
immunosuppressive agent if the cells are to be used as
carrier cells or are to be ~; n; stered in com~ination
with an immunostimulatory agent such as a cytokine,
CE cells or the like. Reduction or inhibition of
expression of an immunosuppressive agent that is
expressed by a tumor cell can be accomplished using known
methods of genetic modification. For example, a tumor
cell expressing an immunosuppressive agent such as an
immunosuppressive isoform of TGF~ can be genetically
modified such that the expression of the TGFB is reduced
or inhibited using a homologous recombination gene
"knock-out method (see, for example, Capecchi, Nature,
344:~05 (1990) and references cited therein; Koller et
al., Science, 248:1227-1230 (1990); Zijlstra et al.,
N~tllre, 342:435-438 (1989), each of which is incorporated
herein by reference; see, also, Sena and Zarling, ~
Genet., 3:365-372 (1993), which i8 incorporated herein by
reference). The homologous recombination gene knock-out
method provides several advantages. For example,
expression of a gene encoding an immunosuppressive agent
such as a TGF~ gene in a tumor cell can be inhibited
completely if both alleles of the target gene are
inactivated. In addition to providing complete
inhibition of the immunosuppressive agent, the method of
CA 02244841 1998-07-31
W 0 97/28251 PCTAUS97/01465
29
homologous recombination gene knock-out is essentially
~ permanent.
The expression of an ; -nosuppressive agent ~y
a tumor cell also can be reduced or inhi~ited by
providing in the tumor cell an antisense nucleic acid
sequence, which is complementary to a nucleic acid
sequence or a portion of a nucleic acid sequence encoding
an immunosuppressive agent such as an immunosuppressive
isoform of TGFB. Methods for using an antisense nucleic
acid sequence to inhibit the expression of a nucleic acid
sequence are known in the art and described, for example,
by Godson et al., J. ~iol. Chem., 268:11946-11950 (1993),
which is incorporated herein by reference. Expression of
an immunosuppressive agent by a tumor cell also can be
reduced or inhibited by providing in the tumor cell a
nucleic acid sequence encoding a ribozyme, which can be
designed to recognize and inactivate a specific mRNA such
as a mRNA encoding an i~lnosuppressive isoform of TGF~
(see, for example, McCall et al., Proc. Natl. Acad. Sci.,
USA, 89:5710-5714 (1992~; Cremisi et al., Proc. Natl.
Acad. Sci.. USA, 89:1651-1655 (1992); Williams et al.,
Proc. Natl. Acad. Sc;. US~, 89:918-921 (1992); Neckers
and Whitesell, Amer. J. Physiol. 265:L1-12 (1993);
Tropsha et al., J. Mol. Recog. 5:43-54 (1992), each of
which is incorporated herein by reference).
Various assays to determine whether a subject-s
cancer cells express an immunosuppressive agent such as
an immunosuppressive isoform of TGFB are available and
known to those skilled in the art. For example, a
radioimmunoassay or enzyme linked immunosorbent assay can
be used to detect a specific immunosuppressive agent in a
serum or urine sample obtained from a subject. In
addition, an assay such as the mink lung epithelial cell
assay can be used, for example, to identify TGFB2
activity (Ogawa and Seyedin, ~eth. Enzymol. 198:317-327
CA 02244841 1998-07-31
W O 97128251 PCTrUS97/01465
(1991), which is incorporated herein by reference). A
biopsy of the tumor also can be e~ ined~ for example,
immunohistochemically for the expression of an
immunosuppressive agent. In addition, the tumor cells
can be evaluated by northern blot analysis, reverse
transcriptase-polymerase chain reaction or other known
methods (see, for example, Erlich, PCR Technolo~y:
Principles ~n~ ~ppl ications for DNA ~m~l;fic~t;on
(Stockton Press 1989); Sambrook et al., Molecular
Clo~; ng: ~ 1 ~horatory manllAl (Cold Spring Harbor
Laboratory Press 1989), each of which is incorporated
herein by reference).
It is recognized that, in order to increase the
susceptibility of a subject's cancer to radiotherapy, the
cytokine must be expressed in an effective amount. As
used herein, the term "effective amount" means an amount
of a cytokine that can stimulate a systemic immune
response in the sub]ect or alter the tumor
microenvironment, such that a tumor in the subject is
radiosensitized. An effective amount can be determined,
for example, by detecting a stimulation of the subject's
immune response or by detecting a reduction in the
severity of the subject's cancer following radiotherapy.
Such an effective amount can be determined using assays
for det~rmi n; ng the activity of immunoeffector cells
following ~ ; n; stration of an ; n; zing composition to
the subject or by monitoring the effectiveness of the
radiotherapy using well known imaging methods.
Where an ;~lnizing composition includes a
genetically modified cell such as a tumor cell, carrier
cell or a CE cell, the number of cells to be A~inistered
depends, in part, on the amount of cytokine secreted by
the cells. Methods for determi n ing the level of a
cytokine expressed by a genetically modified cell are
3~ disclosed herein or otherwise known in the art (see, for
CA 02244841 1998-07-31
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31
example, ~homson, supra, 199 4; Chap. 25). For example,
~ the IL-3 expressing cells generated as described in
Example I produced 50 ng bioactive IL-3/ml medium/48 hr/
1 x 106 cells. In general, about 1 x 105 to about 1 x 107
cell8 i5 required for immlln;zation~ depending, for
ex_mple, on the nu-mber of times the composition is to be
~-' ; n; stered, a8 well as the amount of a particular
cytokine secreted.
Prior to A~m; n; stration, genetically modified
lD cells can be mixed with an appropriate adjuvant or with a
phA -cologically acceptable solution such as
physiological saline or the like for A~m; ni stration,
which can be accomplished by any of various methods such
as subcutaneous or intramuscular injection or any manner
1!5 acceptable for i~l7nization. Pharmacologically
acceptable solutions useful for administration to a
subject are known in the art (see, for example, Khan et
al.~, supra, 1994; Audibert and Lise, supra, 1993; Mishell
and Shiigi, supra, 1980). In addition, various methods
of A~m; n; ~tration can be used and are known to those
skilled in the art. ~m; ni stration can be at a body
location other than an active tumor site or, if desired,
at the site of a tumor in a cancer subject.
One skilled in the art would know that the
effectiveness of therapy can be determined by monitoring
a subject-s immunoresponsiveness. For example, the
cytolytic activity of immune effector cells against the
subject's cancer cells can be assayed using well known
methods. In addition, the size or growth rate of a tumor
can be monitored in vivo using methods of diagnostic
imaging. Also, biopsy samples of the tumor can be
exAm;ned histologically for infiltration, for example, of
granulocytes, macrophages or lymphocytes.
CA 02244841 1998-07-31
W O 97/28251 PCTrUS97/01465
3~
It is understood that modifications that do not
~ su~stantially affect the em~odiments of this invention
al~o are included within the invention provided herein.
Accordingly, the following examples are intended to
illustrate but not limit the present invention.
EXAMPLE I
RESSION OF IL-3 AT THE SITE OF A TUMOR
TN~EASES T~F SUSCEPT~RILITY OF T~E TUMOR
TO T~ ~FECTS OF RADIATION THE~APY
This example demonstrates that a tumor formed
by tumor cells genetically modified to express IL-3 is
more more susceptible to the effects of radiation than is
a tumor formed by lln~o~ified tumor cells.
~ 3 transduction:
The Jzen.1 retroviral vector (Laker et al.,
Proc. NAtl. Acad. Sci.r USA 84:8458-8462 (1987), which is
incorporated herein by reference~ was used to introduce
the full length cDNA encoding murine IL-3 into cultured
non-immunogenic (FSAN) or moderately immunogenic (FSA)
murine fibrosarcoma cells as previously described
(McBride et alO, supra, 1992; McBride et al., supra,
1994; see, also, McBride and Howie, Rr. ~. Canc. 53:707-
71~ (1986), which is incorporated herein by reference).
Briefly, the IL-3 cDNA was inserted downstream
of the 5~-LTR promoter and upstream of a neo gene driven
by an internal TK promotor pre~ent in the vector. The
IL-3-contA;n;ng vector was transfected using calcium
phosphate precipitation into the ecotropic packaging cell
line GP+env-86 (Markowitz et al., J. Virol. 62:1120-1124
(1988), which is incorporated herein by reference).
Cloned cell lines produced viral titers in excess of
1 x 106 pfu/ml.
CA 02244841 1998-07-31
'WO 97/28251 PCT~US97/01465
33
FSA and FSAN tumor cells were infected with the
~ IL-3-cont~i n; ng vector (Jzen-IL-3) or with the parental
vector lacking the IL-3 cDNA insert (Jzen) and infected
- cells were selected in 0.6 mg G418/ml medium. Sequential
daily supernatant infections with the retroviral vectors
~ over 3 days transduced about 50% of the cells.
Expression of IL-3 mRNA by the Jzen--IL-3
transfected cell lines was confirmed by northern ~lot
analysis using the murine IL-3 cDNA as a probe. ~he IL-3
transduced cells expressed the appropriate 4 kb RNA. In
addition, IL-3 protein expression was confirmed by
3H-thymidine incorporation into the IL-3-dependent B~SUtA
murine mast cell line (McBride et al., supra, 1994 ) .
IL-3 transduced cells produced approximately 50 ng
bioactive IL-3/ml medium/1 x 106 cells in a 48 hr period.
B. I~-3 expressing tllm~r cells are immunogen;c:
Su~cutaneous injection of ~2.5-3.0) x 10 FSA or
FSAN cells result in 50% incidence of tumors (TD-50) in
the C3Hf/Sed/Kam female mice (10-12 weeks old) used in
these studies. In comparison, IL-3 transduced FSA
(FSA-IL-3) or FSAN (FSAN-IL-3) cells had a TD-50 of
1.2 x 106 or 2.8 x 105 cells, respec~ively. The decreased
tumorigenicity is correlated with granulocyte
infiltration into the tumors (see McBride et al., supra,
1994). For all experiments described herein, appropriate
controls were run in parallel, including the comparison
of genetically modified cells with the corresponding
unmodified cells or cells modi~ied with the control
Jzen.l vector.
i
The ability of irradiated IL-3 expressing tumor
cells to induce immunity to a subsequent challenge of
nmo~;fied tumor cells was e~mined. Cells were
irradiated using a Gammacell 220 (Atomic Energy Limited;
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C'AnA~) with a cobalt source at a dose rate of 3. 3 Gy/min
~ to a total of 30 Gy. 1 x 106 irradiated (30 Gy) IL-3
transduced FS~ (FSA-IL-3) or FSAN (FSAN-IL-3) cells were
injected intraperitoneally into mice, then, 10 days
later, the mice were challenged with 1 x 106 unmodified
FSA or FSAN cells, respectively. The number of
~ nizing IL-3 transduced tumor cells was deliberately
chosen to be too low to protect against growth of the
moderately immunogenic FSA tumor cells in order allow the
detection of e~h~n~ed immunogenicity.
T~-lln;zation with the irradiated IL-3
transduced tumor cells, but not with Jzen.1 modified
tumor cells, induced specific ; n;ty against the
;~lln;zing cell type (i.e., FSA or FSAN), protecting the
mice from subsequent challenge with ~ ified tumor
cells (McBride et al., supra, 1994). Complete protection
occurred in 80~ to 90% of the immunized mice and no
cross-protection was observed against the antigenically
unrelated tumor.
The generation of immunologic memory in the
; ni zed mice was demonstrated ~y intravenously
in~ecting 2 x 10' spleen cells from ; n;zed mice or
control (not previously treated) mice into syngeneic C3H
SCID mice bearing previously established 4 day old
parental FSA or FSAN tumors. Regression of the
established tumors was o~served following adoptive
transfer of spleen cells from the ;~ml~n;zed mice but not
from the control mice. In addition, a moderate amount of
cross-protection was observed against the non-;m~lln;zing
tumor cells t~cBride et al., supra , 1994). These results
demonstrate that a specific, systemic immune response is
induced in mice ; n;zed with IL-3 expressing tumor
cells.
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C. Expression of ITI-3 incr~ses susceptibility of tumor
~ to the effects of r~ t;on:
- (1-5) x 10~ IL-3 transduced FSA or FSAN cells or
Jnez.1 transduced FSA or FSAN cells were injected
subcutaneously into the thigh muscles of mice (N = 5-10).
After about 16 to 20 days~ when the tumors were about
6 to 8 mm in diameter, the tumors were irradiated. For
irradiation, mice were placed in a full body shield, with
the tumor bearing thigh exposed. Tumors were irradiated
using a 250 kVp Phillip's X-ray source at a dose rate of
11.2 Gy/min. The tumors in mice bearing the moderately
immunogenic FSA tumors were irradiated with a single do~e
of 25 Gy or 40 Gy. The tumors in mice bearing the
non-immunogenic FSAN tumor~ were irradiated with a single
dose of 55 Gy.
IL-3 expressing FSA tumors regressed completely
following radiotherapy with 25 Gy or 40 Gy. In
comparison, unirradiated IL-3 expressing FSA tumors and
irradiated unmodified parental FSA tumors continued
growing during the course of the experiment, although
growth of the irradiated, lln -';fied FSA tumors was
slightly delayed following irradiation. Furthermore,
mice that were cured of the IL-3 expressing FSA tumors
were protected from challenge with 2 x 106-ln~o~;fied FSA
tumor cells, but not of FSAN tumor cells.
Similarly, IL-3 expressing FSAN tumors
regressed completely following radiotherapy with 55 Gy,
whereas Jzen.1 modified FSAN tumors continued to grow
after a brief delay due to the irradiation. In addition,
31D mice cured of the IL-3 expressing FSAN tumors were
protected from challenge with unmodified FSAN cells.
These results demonstrate that the expression
of IL-3 at the site of tumor increases the susceptibility
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of the tumor to the effects of radiation therapy, thus
~ allowing complete regression of an otherwise progressive
tumor. In addition, a specific, systemic immune response
was generated such that no tumors formed upon further
challenge with tumor cells.
EXAMPLE II
~RN~AL CONSIDERATIONS FOR T~EAT~NT OF A SURIT~T
This example illustrates the general factors to
consider in treating a cancer patient with a method of
the invention.
Patient Selection:
Patients will have a histologically confirmed
diagnosis of cancer and can have metastatic lesions.
Patients with tumors that must be resected for
therapeutic purposes or with tumors readily accessible
for biopsy can be treated as disclosed herein.
Autologous fibroblasts and tumor cells can be cultured
using routine methods. However, where the autologous
tumor cells are not Am~nAhle to growth in culture, tumor
antigens can be provided by sources as disclosed herein.
Thus, allogeneic haplotype-matched genetically modified
tumor cells can be used provided such tumor cells are of
the same histologic origin as the patient's tumor.
Pretre~tment Evaluat;on:
StAn~Ard pretreatment evaluations are performed
as follows:
1) History and physical PX~m; n~tion including
a description and quantitation of disease activity and
tissue-typing of the patient.
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2) Performance Status Assessment
~ 0 = Normal, no symptoms
1 = Restricted, but ambulatory
2 - Up greater than 50~ of waking
hours, capable of self-care
3 = Greater than 50% o~ waking hours
confined to bed or chair,
limited self-care
4 = Bedridden
3) Pretreatment laboratory analysis,
including complete blood count, including differential
count, platelet count, PT, PTT, glucose, suN~ creatinine,
electrolytes, SGOT, SGPT, LDH, ~lk~;ne phosphatase,
bilirubin, uric acid, calcium and total protein albumin.
Other analyses are performed as deemed
appropriate, including urinalysis, serum complement
levels and immunophenotyping of peripheral blood B cell
and T cell subset~. In addition, pretreatment
evaluations can include chest X-ray and other diagnostic
studies including computerized tomography, magnetic
resonance imaging or radionuclide scans to document and
~uantify the extent of disease activity. Follow-up
evaluations of these assessments are performed at regular
intervals during the course of therapy (approximately
every 1 to 3 months) to monitor the subject's response to
therapy and to identify potential signs of toxicity, thus
permitting adjustments in the number and distribution of
;m~ln;zations. For example, routine histological methods
can be used to identify the presence of various cells
such as lymphocytes, granulocytes and macrophages in a
biopsy sample of a tumor.
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~estrictions on Concurrent Therapy:
For optimal effects of this treatment, patients
should receive no concurrent therapy that is known to
suppress the immune system.
Treatment Protocol:
Each patient will receive either intratumoral
or subcutaneous administrations an i n; zing
composition, which can be provided in the form of
autologous or allogeneic haplotype-matched carrier cells
or in the form of CE cells and a tumor antigen, if
desired. Tumor cells generally will be irradiated.
Prior to ~m; n; stration, tumor cells can be irradiated
with approximately 70 to 100 Gy of radiation, to render
the tumor cells incapable of proliferation in vivo.
When an immunizing composition is ~mi n; stered
to the site of a tumor, radiotherapy can begin one to
three days following the administration. When
; ln;zation i~ at a site other than the tumor,
~ nization generally will require two to four
A~mi ni strations of the immunizing composition at one to
four week intervals, with adjustments being made as
required. Radiotherapy can begin concurrently with the
immunization protocol, since the localized radiation will
not substantially affect the immunoresponsiveness of the
subject. Preferably, radiotherapy will begin after it
has been determined that the patient's immune response
has been sti~llAted by the i~-ln i zations.
Conventional methods of radiotherapy are
performed. The stimulation of a patient's immune
response will be determined by stAn~Ard methods,
including, for example, detecting the presence of
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activated immunoeffector cells either in vitro or by
~ detecting a delayed hypersensitivity-type reaction.
~ In general, a tumor biopsy is taken
approximately two months prior to the initiation of the
methods disclosed herein. If the tumor cells are
adaptable to tissue culture, they can be genetically
modified to expres~ a cytokine gene and used as carrier
cells. However, even if the tumor cells cannot be grown
in culture, they can be stored under appropriate
conditions and used as a source of tumor antigen, if
desired.
The ; n; zing composition is a~m; n i stered in a
form that provides controlled slow release of the
cytokine, particularly when administration is at a site
other than the tumor. In addition, if a tumor cell or a
carrier cell i8 not the source of the cytokine,
irradiated unmodified tumor cells also can be
A~mi n; stered as a source of tumor antigen, particularly
when administration is at a site other than the tumor.
Where A~;n;stration involves, for example, the
use of IL-3-expressing cells, the level of IL-3 secreted
at the site of ;~llnization can be escalated as reguired
during the immunization procedure. The number of
injected IL-3-expressing cells will remain relatively
constant at approximately 1 x 105 to 1 x 10' cells per
~m;n;stration site by adding an appropriate number of
irradiated llnmn~;fied cells. Multiple ;mmlln;zation sites
can be used if it is deemed desirable to increase the
dose of the cytokine to the sub~ect. The subject will be
3t) physically ~;ned on each of the three consecutive days
following A~; n;stration and physical and laboratory
evaluations will be made at weekly intervals.
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Dsse ~justments:
-
The immunoresponsiveness of the su~ject isdetermined using the assays described above, including,
for example, assays to determine changes in the activity
of the cellular immune response in the subject. So long
8S no toxicity is observed, subsequent Ar~mi ni strations
are administered at intervals of 1 to 4 weeks, as
desired. The results of the cellular and humoral
immunoresponsiveness assays and tumor monitoring studies
can be used to optimize the treatment protocol as
determined by one skilled in the art. Although toxic
side effects are not expected to result, potential side
effects can be treated using conventional methods.
Tr~tment of Pot~nt;~l Toxicity:
Unacceptable toxic side effects at the site of
A~min;stration are not expected to result during the
course of treatment. However, potential side effects can
be treated as required. For example, if massive tumor
cell lysis results, any resulting uric acid nephropathy,
adult respiratory distress syndrome, disseminated
intravascular coagulation or hyperkalemia will be treated
using stAn~A~d methods well known in the art. Local
toxicity at the sites of A~m; ni stration will be treated
with either topical steroids and, if necessary, surgical
excision of the in~ection site. Generalized
hypersensitivity reactions such as "the chills," fever or
rash will be treated symptomatically with antipyretics
and antihistamines. Patients should not be treated
prophylactically. Edema, arthralgia, lymphadenopathy or
renal dysfunction can be treated using corticosteroids
and/or antihistamines. Anaphylaxis will be treated by
stAn~A~d means such as A~m; ni stration of epinephrine,
fluids and steroids.
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41
Other Assays:
Provided that sufficient material is available
for evaluation, the following assays also are performed.
Standard immunofluorescence flow cytometry procedures are
useful to evaluate changes in the percentage of T cells,
natural killer cells and B cells associated with cytokine
gene therapy. Monoclonal antibodies specific for T cells
(CD2, CD3, CD4, CD8), natural killer cells (CD16, CD57,
CD58) and B cells (CDl9, CD20) can be used for these
studies.
Briefly, Ficoll-Hypaque purified mononuclear
cells are incubated with the primary antibody for 1 hr at
room temperature, washed, then incubated with
fluorochrome conjugated secondary antibody. The cells
are washed, fixed and the percentage of positive cells
are det~rmine~ using a Coulter Epics 4 flow cytometer.
Incubation of the cells with isotype-matched control
antibody instead of the primary antibody is useful as a
negative substitution control.
21~
St~n~rd immunohistological methods employing
monoclonal antibodies specific for the hematopoietic cell
subsets described above can be used to characterize the
immune effector cell infiltrates observed in delayed-type
hypersensitivity type skin test biopsy sites. Methods
for immunohistological evaluations of fresh frozen
cryostat tissue sections are well known in the art.
Although the invention has been described with
reference to the above examples, it is understood that
various modifications can be made without departing from
the spirit of the invention. Accordingly, the invention
is limited only by the following claims.
