The lack of an effective, translatable strategy for the paracrine delivery of cytokines, including IL-12, has limited the clinical potential of cytokine-based immunotherapies. Cell-based, virus-based, and plasmid-based IL-12 delivery strategies have all shown promise preclinically, but each faces unique obstacles. Recombinant IL-12 protein-based delivery strategies are the most direct and quantifiable approaches in terms of ensuring the accuracy and reproducibility of locally delivered IL-12. Several protein-based delivery strategies, such as IL-12 encapsulation in polymeric microspheres and IL-12 incorporation into gels9,10
are designed to maximize IL-12 levels in the tumor microenvironment while limiting systemic exposure. Most notably, several studies by Egilmez et al have demonstrated that encapsulation of IL-12 in polylactic acid (PLA) microspheres and polycaprolactone:PLA microspheres can control murine28
and human tumors29,30
following i.t. immunotherapy. Specifically, IL-12-loaded PLA microspheres were found to eradicate 70% of Line-1 tumors and 80% of CT26 tumors.30
However, others have found that the same immunotherapy regimen could not prevent the growth of B16 melanoma31
or MT-901 mammary carcinomas.32
It is not known whether these differences in efficacy are due to methodological variations, differences in tumor models, or inherent limitations of IL-12-loaded microspheres. Another drawback of IL-12-loaded microspheres is the need to use organic solvents during formulation, which can denature IL-12 immediately or adversely affect long-term storage if the solvents are not completely removed. In fact, over 80% of the bioactivity of IL-12 was lost when PLA/IL-12 microspheres were stored for 3 weeks.33
When loaded into liposomes, IL-12 has shown a sustained release and antitumor activity against tumor xenografts.11
In addition, previous studies have shown that IL-12 can be encapsulated in liposomes for potential use as a vaccine adjuvant.12,34
IL-12 has also been incorporated into biocompatible gels for either slow systemic release10
or paracrine enhancement of cancer vaccines.9
To our knowledge, none of these protein-based delivery platforms has been evaluated in clinical studies.
Here, we utilize a straightforward technology whereby recombinant IL-12 is admixed with chitosan under mild, aqueous conditions. Chitosan/IL-12 coformulations offer several advantages. Chitosan is inexpensive and can be reproducibly manufactured under cGMP conditions. Preparation of chitosan/IL-12 requires neither harsh organic solvents nor sonication that could denature IL-12. Chitosan and IL-12 can be admixed at bedside immediately prior to administration, eliminating the need for long-term storage. Finally, chitosan has an excellent safety record in humans and is readily digested by lysozyme, released by polymorphonuclear neutrophils and macrophages, to yield glucosamine.
This is the first study to demonstrate enhanced retention of IL-12 in the tumor microenvironment following i.t. administration with a delivery system. Chitosan increases the retention of IL-12 in the tumor from 1 to 2 days to 5 to 6 days (). This is probably an underestimate of IL-12 retention since the lower limit of detection—between 16 and 31 ng (see Materials and Methods)—is far greater than the limit of IL-12 bioactivity. Furthermore, chitosan increases total IL-12 exposure in the tumor microenvironment by approximately 3-fold, as determined by integrating the area under the curve (). Once again, this is likely an underestimate, as the imaging system becomes saturated at high levels (≥ 1 µg).
Interestingly, despite the enhanced local retention of IL-12, our recently published paper demonstrated that s.c. injections of IL-12 and chitosan/IL-12 resulted in similar serum levels of IL-12 and IFN-γ.21
Taken together, these findings imply that the majority of IL-12 is released from chitosan in < 24 h, while a significant and biologically relevant concentration of IL-12 remains at the injection site for at least 6 days. IL-12 in circulation may complement local IL-12 by expanding CD8+
T-cell and NK-cell populations35,36
and by enhancing the trafficking and migration of NK37
In preliminary studies, we found that i.t. administration of chitosan/IL-12 may activate splenic NK and CD8+
cells, as demonstrated by increases in size (forward scatter) and granularity (side scatter) during flow cytometry analyses (unpublished data).
Although a significant amount of IL-12 reaches the blood stream following i.t. chitosan/IL-12 administration, it is unlikely that the severe IL-12-related toxicities seen in early trials would be duplicated at the schedule reported here. In humans, IL-12-related toxicities have been associated more with daily administration than with a specific dose level. In fact, weekly or twice-weekly systemic administrations of IL-12 at the maximum tolerated dose or higher have been well tolerated.39,40
Therefore, it is reasonable to infer that weekly i.t. immunotherapy with chitosan/IL-12 would be well tolerated. It is important to note that no evidence of toxicity, such as ruffled fur, hunched habitus, or lethargy, was noted in any mouse receiving i.t. chitosan/IL-12. Additional toxicology studies to quantify organ weights, serum chemistry, and hematological parameters are planned.
Regarding antitumor activity, chitosan/IL-12 is at least as effective as any IL-12-based local immunotherapy published to date. When given alone, IL-12 had marginal impact against established, poorly immunogenic MC32a and Panc02 tumors (). However, coformulations of chitosan/IL-12 cured 80% to 100% of mice with established MC32a and Panc02 tumors. In fact, only 2 i.t. treatments with chitosan/IL-12 were required to achieve 100% eradication of the Panc02 line, which is less aggressive than the MC32a line. This complete tumor regression appears to be unique to chitosan/IL-12, as coformulations of chitosan with other cytokines such as GM-CSF and IFN-γ were totally ineffective ().
Perhaps more clinically significant than eradication of primary tumors is the finding that chitosan/IL-12 immunotherapy appears to protect cured mice from tumor recurrence, as determined in tumor rechallenge studies (). These findings agree with previous reports which show that IL-12-based therapies can generate long-term protection in numerous animal models.41,42
It should be noted that tumor rechallenge is only an approximation of the ability of an immunotherapy to protect from tumor recurrence or metastasis. demonstrates that manipulation of the timing and dose of tumor rechallenge can alter interpretations of the level of protection. Furthermore, an assault with hundreds of thousands of tumor cells is not representative of tumor recurrence in humans. Therefore, future studies of the ability of i.t. chitosan/IL-12 to confer protection from metastasis in the neoadjuvant setting must employ a more clinically relevant model.
The high frequency of tumor rejection in our rechallenge studies correlated with the robust CTL activity measured in cured mice (). Together, these data show that i.t. chitosan/IL-12 is capable of generating systemic, tumor-specific immunity in the absence of traditional vaccination. In vitro
-stimulated splenocytes from cured mice reacted strongly to an epitope of gp70, which is an endogenous murine retroviral envelope protein overexpressed on numerous murine tumors.27
This demonstrates that i.t. chitosan/IL-12 immunotherapy can make use of antigen from host tumor cells to create an adaptive immune response and that targeting specific tumor antigens may not be a prerequisite for effective immunotherapy.
cells and NK cells were revealed as critical immune cell subsets during chitosan/IL-12-mediated tumor regression (). The importance of these particular immune cell subsets in IL-12-based immunotherapies has also been shown by others.43
Our finding on the nonessential role of CD4+
cells is in agreement with some reports44
but not others.42,45
Given the ability of IL-12 to induce a strong TH
1 cytokine cascade, including the production of massive amounts of IFN-γ by NK and CD8+
cells, additional assistance from CD4+
helper cells does not appear to be necessary.
Finally, many cancer vaccines have been shown to induce robust tumor-specific T-cell responses. However, these T-cell responses are not always successful in controlling tumor growth because T cells have difficulty infiltrating the tumor or are inactivated by the immunosuppressive tumor microenvironment.46
I.t. injections of chitosan/IL-12 may disrupt the restrictive tumor architecture and encourage infiltration by both innate and adaptive immune cells. Furthermore, a high local concentration of IL-12 may reverse the action of, or eliminate, immunosuppressive cells such as tumor-associated macrophages47
and regulatory T cells.48
In sum, we have demonstrated that i.t. chitosan/IL-12 immunotherapy can (a) increase local retention of IL-12 in the tumor microenvironment, (b) eradicate aggressive murine tumors, and (c) generate systemic tumor-specific immunity capable of inhibiting tumor recurrence. These results, together with a favorable weekly administration schedule, as well as its simplicity of formulation, form the rationale for clinical investigation of i.t. chitosan/IL-12 immunotherapy for the management of solid tumors.