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Experimental Therapeutics |
Departments of Immunology [N. K. E., R. B. B.] and Surgery [M. S. S.], Roswell Park Cancer Institute, Buffalo, New York 14263, and Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912 [Y. S. J., J. S. J., E. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The attraction of gene-modification lies mainly in the fact that the
cytokine of choice can be delivered to the tumor microenvironment in a
paracrine manner, circumventing the severe side effects associated with
systemic cytokine immunotherapy (1, 2, 6)
. Local and
sustained delivery of therapeutic agents can also be achieved with
biodegradable controlled-release polymers (7)
.
Biodegradable polymer microspheres have been used in humans for
in vivo drug delivery (7)
, cancer chemotherapy
(8)
, and vaccination with antigenic peptides
(9)
. Although these delivery systems were initially
developed for the sustained delivery of low-molecular-weight
therapeutics (7, 8)
, recent advances in encapsulation
technologies and protein stabilization have led to the successful
encapsulation of a number of bioactive macromolecules including
immunostimulatory cytokines (10)
. In two in
vivo murine studies, stimulation of antitumor responses with
GM-CSF3
and IL-1
-loaded microsphere formulations have been reported (11, 12)
.
Recently, we described a novel technology (PIN) for highly efficient encapsulation of biologically active molecules into polymer microspheres (13) . This spontaneous process does not require vigorous stirring/sonication during the formation of emulsions, and labile proteins are efficiently encapsulated without denaturation or losses to aqueous nonsolvent baths. In preliminary studies, we demonstrated that recombinant human IL-2-loaded PLA microspheres, prepared by PIN, release physiologically relevant quantities of bioactive IL-2 for extended periods and that the in vivo release of IL-2 from the PLA microspheres provokes a mouse NK cell-mediated suppression of human tumor xenografts in SCID mice (14) . On the basis of these initial observations, we have further investigated the clinical potential of PIN/PLA microspheres loaded with three different cytokines for cancer immunotherapy in a weakly immunogenic syngeneic murine tumor model. We report here that intratumoral injection of IL-12-loaded PIN/PLA microspheres, but not IL-2 nor GM-CSF-loaded microspheres, induces the regression of established tumors, prevents spontaneous metastasis and promotes the development of tumor-specific immunity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytokines.
Recombinant human PEG-IL-2 (6 x 106 units/mg) was a gift from Chiron, Inc.
(Emeryville, CA). Recombinant murine IL-12 (2.7 x 106 units/mg) was donated by Genetics Institute,
Inc. (Andover, MA) and recombinant murine GM-CSF (7.2 x 107 units/mg) was donated by Immunex, Inc.
(Seattle, WA).
Microspheres.
A PIN technique was used for encapsulation of cytokines as described
previously (13)
. Briefly, BSA (RIA grade, Sigma Chemical
Co., St. Louis, MO), PLA (Mr
24,000 and Mr 2,000; 1:1 (w/w)],
Birmingham Polymers, Inc, Birmingham, AL), and recombinant cytokine in
methylene chloride (Fisher, Pittsburgh, PA) was rapidly poured into
petroleum ether (Fisher) for formation of microspheres. Microspheres
were filtered and lyophilized overnight for complete removal of
solvent. Four formulations containing 1% BSA (w/w) were produced:
(a) control (no cytokines); (b) human PEG-IL-2
[
10 µg (60,000 IU)/mg PLA); (c) murine IL-12 [10
µg (270,000 units)/mg PLA]; and (d) murine GM-CSF [10
µg (7.2 x 105 units)/mg PLA).
Scanning electron micrographs demonstrated that the microspheres were
1–5 µm in diameter and were easily injectable with a 28.5-gauge
needle. The encapsulation efficiencies for the cytokines were
extrapolated from the measurements of total protein encapsulated into
the microspheres as described (16)
.
Cytokine Release and Bioactivity Assays.
The assay for the quantitation of in vitro cytokine release
from the microspheres has been described previously (14)
.
Briefly, 3 mg of particles in 200 µl of tissue culture medium
(DMEM + 10% FCS) were incubated in the wells of a 96-well
culture plate in triplicate at 37°C. The medium was changed daily for
12–16 consecutive days, and the aliquots were stored at 4°C. The
quantity of cytokine in the medium was determined either by ELISA (R&D
Systems, Minneapolis, MN), or in the case of PEG-IL-2, by a bioactivity
assay using an IL-2-dependent murine T-cell line proliferation assay
(14)
. The bioactivity assay for recombinant murine IL-12
was performed using a murine splenocyte proliferation assay as
described previously (17)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The initial release of cytokines is followed by a rapid decline with
an eventual stabilization of the release kinetics after day 7. Both
PEG-IL-2 and IL-12, which were released from the cytokines were shown
to be bioactive in vitro (Fig. 1)
. The results indicate that
significant quantities of cytokine can be released from the
microspheres for at least 12 days, but that the absolute quantities and
the release rates vary, depending on the particular cytokine that is
encapsulated.
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. At the tumor cell dose used, all of the mice in the
control group (BSA microspheres) developed palpable tumors by day 3,
with tumors reaching a diameter of 5 mm within 7–8 days. In
contrast, all of the mice that were treated with the IL-12-loaded
microspheres remained tumor-free for at least 6 weeks. Mice that were
treated with GM-CSF- or PEG-IL-2-loaded microspheres experienced a
significant, albeit less dramatic, inhibition or delay in tumor growth.
Two of five mice that received the GM-CSF microspheres remained
tumor-free for 6 weeks, whereas all of the mice that were treated with
PEG-IL-2-loaded microspheres developed tumors, although tumor growth in
these mice was delayed compared with the controls. The antitumor effect
observed with GM-CSF was surprising. This cytokine induces potent
antitumor immunity when used in a prophylactic vaccine setting;
however, it has not been shown to suppress tumor growth directly
(1)
. Interestingly, IL-2, which has been shown to induce
tumor suppression in numerous murine tumor models, had only a weak
antitumor effect here. The observed effects (or lack thereof) could be
related to the dose and the release pattern of the particular cytokine
delivered by the microspheres. Regardless of the relative antitumor
efficacy of individual cytokines, the above results establish that the
cytokines released from the microspheres are biologically active
in vivo, and that tumor growth can be completely arrested
when IL-12-loaded microspheres are injected at the same time that
tumors are inoculated into mice.
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4 mm in diameter
prior to treatment. These tumors were then injected with
cytokine-loaded microspheres, and tumor growth was monitored weekly. In
these experiments, the dose of microspheres was increased significantly
as compared with that used in the coengraftment studies (2 mg as
opposed to 50 µg per injection) because the number of tumor cells
within the established tumors is greater and established tumors are
more difficult to suppress and eradicate. The results are shown in Fig. 2B
. There was no significant difference between the growth
patterns of tumors treated with control (BSA-loaded) microspheres and
PEG-IL-2 or GM-CSF-loaded microspheres, with which tumors grew
progressively. However, a single intratumoral injection of IL-12-loaded
microspheres promoted complete tumor regression in 7 of 10 mice, and
tumor growth was suppressed in the 3 remaining mice. These results
demonstrate that the sustained release of IL-12 from the microspheres
can induce potent antitumor activity in a clinically relevant setting.
Tumor Regression Is Accompanied by the Development of
Protective Antitumor Immunity, the Potency of Which Is Dependent on the
Method of Vaccination.
The ultimate goal of immunotherapy is to promote the development of
long-term systemic antitumor immunity to prevent recurrence of
tumors, which cannot be achieved with conventional treatments such as
chemotherapy and radiation. Cytokine gene-modified tumor cell vaccines,
especially those involving the cytokines IL-12 and GM-CSF, have been
shown to provoke the development of effective antitumor immunity in
mice (19, 20)
. To test whether IL-12 delivered by
microspheres directly into existing tumors is able to promote similar
protective antitumor immunity, mice that were able to reject
established s.c. tumors after treatment with IL-12-loaded microspheres
were challenged with live tumor cells at a different site 5–6 weeks
after the original tumor had completely regressed. The results of this
experiment are shown in Table 1
. Of the 15 vaccinated mice that were challenged, 12 (80%)
rejected the tumor, which suggests the development of potent protective
antitumor immunity in these mice.
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, vaccination of mice
with mixtures of IL-12 microspheres and live Line-1 cells provided less
protection from a subsequent tumor challenge than did in
situ vaccination (57 versus 80%). Only 10% of the
mice were protected from tumor challenge with an irradiated-cell/IL-12
microsphere vaccine, which was identical to that obtained with
irradiated cells alone. In the control nonvaccinated group, none of the
mice were able to reject tumor challenge.
To determine whether the immunity provoked by the cytokine-loaded
microspheres was tumor-specific, mice that rejected s.c. Line-1 tumors
after vaccination in situ were challenged either with Line-1
or Colon 26 (an unrelated colon tumor cell line derived from BALB/c
mice) cells and tumor growth was monitored. Whereas six of six mice
vaccinated with Line-1 rejected the Line-1challenge, only one of six
vaccinated mice rejected a challenge with Colon 26 tumor cells (Table 2
). Nonvaccinated control mice did not reject challenges
with either tumor cell line. These results demonstrate that the
systemic antitumor immunity induced by the IL-12-loaded microspheres
was tumor-specific.
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-dependent mechanism
(21, 22)
. To determine whether the microsphere-mediated
tumor regression observed here was induced by a similar mechanism,
microsphere vaccination experiments were repeated in CB.17 SCID mice,
which lack functional B- and T-lymphocytes. Mice with established s.c.
tumors were treated with intratumoral injections of IL-12-loaded
microspheres, and tumor growth was monitored. The results are shown in
Fig. 3
. Treatment with IL-12-loaded microspheres delayed tumor
growth by 1 week in the CB.17 SCID mice but failed to promote tumor
regression. The limited antitumor response observed in the CB.17 SCID
mice was shown to be NK-cell-dependent because the depletion of the
mouse NK cells with the monoclonal antibody TMß1 resulted in the loss
of the tumor-suppressive activity. In contrast, a significant tumor
suppression was observed in the immunocompetent BALB/c mice with tumors
regressing completely in three of five mice.
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. In this experiment, 53% of the tumors regressed
completely after intratumoral delivery, whereas none of the tumors
regressed when the microspheres were injected on the contralateral
flank of tumor-bearing mice. Moreover, a single intratumoral injection
of free IL-12 at a dose equal to that delivered by the microspheres
resulted in the regression of tumors in only 20% of the animals;
whereas i.p. delivery of free IL-12 did not promote any tumor
regression. These results demonstrate that local and sustained delivery
of IL-12 to tumors is superior to local or systemic bolus delivery.
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7–8 mm) s.c. tumors were treated with IL-12-loaded
microspheres, and their lungs were analyzed 2 weeks after treatment.
The results are shown in Fig. 4
. Treatment with IL-12-loaded microspheres induced
significant suppression of tumor growth compared with treatment with
BSA-loaded microspheres (Fig. 4A
). Although the primary s.c.
tumors were suppressed, treatment here did not result in complete
regression because of the larger tumor inoculum and the greater size of
the tumors at the time of treatment compared with previous experiments.
More interestingly however, the examination of the lungs 2 weeks after
treatment revealed significant suppression of lung metastasis in the
IL-12-treated animals as compared with the controls (Fig. 4B
). These results demonstrate that the local treatment of
primary tumors with IL-12-loaded microspheres can suppress both the
growth of the primary tumor and metastasis to distant sites. Whether
the antimetastatic effect observed here was attributable to the
systemic presence of the cytokine released by the microspheres or to
the development of systemic antitumor immunity that resulted from a
release of the cytokine into the tumor microenvironment was not
determined. The results shown in Tables 1 and 2
establish that
intratumoral delivery of IL-12 microspheres induces the development of
a potent tumor-specific systemic immunity. Moreover, the results
summarized in Table 3
demonstrate that when the IL-12 microspheres are
injected contralateral to tumors, tumor regression is not induced.
Together, these data support the notion that the suppression of lung
metastasis observed here is most likely mediated by the development of
a systemic antitumor immunity and is not simply attributable to
systemic release of IL-12 from the microspheres.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our studies extend and confirm previous reports demonstrating the ability of IL-12 to induce tumor regression and the development of potent antitumor immunity in preclinical murine models (20–24) . Additional studies have established that the potent antitumor effects of IL-12 are tempered by the dose- and schedule-dependent toxicity in mice (25) and in humans (26, 27) when administered systemically. Recent studies demonstrated that systemic administration of IL-12 also induces a transient generalized immunosuppression in mice (28, 29) . The severe toxicity associated with systemic infusion of IL-12 in early clinical trials was partially alleviated by altering the schedule and dose of treatment, but the lack of significant antitumor efficacy in these trials has been disappointing (27, 30) . Ineffectiveness of systemic IL-12 therapy in the clinic could be attributable to the inability of the cytokine to reach effective local concentrations in the tumor bed at maximum tolerated dose and/or the induction of a generalized suppression of T-cell responses.
The risk of toxicity and generalized immunosuppression is obviated when therapy is restricted to the intralesional treatment of a single tumor nodule (even in a patient with multiple tumor foci and/or distant metastases) rather than attempting to treat cancer patients systemically to deliver the cytokine to all of the tumor sites. The rationale for such an approach is clearly supported by the findings reported here that low doses of IL-12, released locally from the microspheres at a single tumor site in a sustained fashion, have a significant antitumor effect resulting in the disappearance of the primary tumor, reduction in distant metastases, and the development of systemic antitumor immunity. Although local and sustained delivery of cytokines to tumors can also be achieved by ex vivo or in situ cytokine gene-modification strategies resulting in a similar induction of antitumor immunity in mice (1–3, 20) , these approaches involve complex and expensive protocols coupled with low and/or variable gene transfer efficiency that present difficulties in the clinical setting (1, 2, 6) . In contrast, the approach described here with the PIN/PLA microspheres represents a simple, universal, and much less expensive alternative to gene therapy protocols for delivering low doses of cytokines to the tumor microenvironment in a sustained fashion.
Furthermore, our data establish that vaccination of tumor-bearing mice with IL-12-loaded microspheres in situ is superior to vaccination of mice with mixtures of live tumor cells and IL-12 microspheres, which in turn is more effective than irradiated tumor cell/microsphere mixtures in inducing protective antitumor immunity. This finding has obvious potential clinical relevance with respect to ultimately designing vaccination strategies for cancer patients. Others have shown that vaccination with live cytokine gene-modified tumor cells is more effective than vaccination with irradiated cytokine gene-modified tumor cells, and antigen dose has been suggested as a critical factor to explain these observations (6, 20) . Regardless of the mechanism, these data are consistent with the notion that in situ tumor vaccination strategies should be preferred in the clinic when feasible, and microsphere-mediated delivery of therapeutics to tumors represents one such approach. The intralesional inoculation of a patient’s tumor nodule with cytokine-loaded microspheres before surgical resection and/or chemotherapy would also allow for maximal stimulation of antitumor immunity without interfering with standard therapy. Accessibility of tumor would not be a major concern because stereotactic injections could be used for a large variety of lesions that are not directly accessible.
The ease of preparation, consistency, and long-term stability of cytokine-loaded microspheres (31) are also conducive to further manipulation of the vaccination protocols in the clinical setting to improve efficacy. Repeated booster vaccinations with mixtures of microspheres and irradiated tumor cell suspensions obtained from postsurgical specimens can be used to augment antitumor immunity after the initial vaccination and standard therapy. Other potential applications include more complex strategies involving multiple cytokines and/or combination therapy with other therapeutic agents that have been shown to be more effective than single cytokine approaches (32–34) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by USPHS Grants
CA54491, CA75235, and CA79879; Institutional Core Grant CA16056 (to
R. B. B.); and Grant GM55245–01 (to E. M.). 
2 To whom requests for reprints should be
addressed, at Roswell Park Cancer Institute, Department of Immunology,
Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-8540;
E-mail: egilmez{at}sc3102.med.buffalo.edu
3 The abbreviations used are: GM-CSF,
granulocyte-macrophage colony-stimulating factor; IL, interleukin; PIN,
phase inversion nanoencapsulation; PLA, polylactic acid; NK, natural
killer; SCID, severe combined immunodeficient; PEG, polyethylene
glycol.
4 N. K. Egilmez and R. B. Bankert, unpublished
results.
Received 12/29/99. Accepted 5/15/00.
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REFERENCES |
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induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect. J. Exp. Med., 188: 1603–1610, 1998.
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