While ICs enable targeting of immunomodulatory cytokines to the tumor microenvironment, systemic administration is still limited by dose-limiting toxicities [21
]. In this study, we show enhanced antitumor effects and increased tumor localization of IC following IT administration. Using the B16-KSA melanoma and NXS2 NB models, we demonstrated a greater antitumor response with IT IC compared to an equivalent systemic IC dose in the treatment of localized palpable s.c. tumors. Interestingly, IT hu14.18-IL2 IC delivery resulted in resolution of both the directly treated primary NXS2 tumor, as well as the non-locally treated distant NXS2 tumor in some animals. Evidence of a tumor-specific memory response was seen after primary NXS2 tumor regression. In the B16-KSA model, we show that the antitumor effect of IT IC was antigen-specific, dose-dependent, and greater than IL2 alone. The enhanced antitumor effect of IT IC compared to recombinant IL2 was also shown in the NXS2 model. These preclinical data suggest that the antitumor activities of huKS-IL2 and hu14.18-IL2, two ICs already undergoing clinical testing via systemic delivery, may result in enhanced tumor control when administered directly into the tumor microenvironment.
The advantage of local administration of IC compared to IV IC has been described previously using SCID mice bearing human tumor xenografts and infused with HLA identical human peripheral blood lymphocytes as effector cells [6
]. In that study, ICs consisting of anti-EGFR mAb linked to IL2 or TNF showed greater in vivo
antitumor effects when administered IT compared to IV. Furthermore, IT injection of intact IC resulted in a survival advantage compared to IT injection of the mAb and IL2 as separate IT injections. In another model, IT injection of huKS-IL12/IL2, where huKS1/4 mAb was linked to the synergizing cytokine combination of interleukin-2 and interleukin-12, resulted in complete resolution of s.c. Lewis lung carcinomas transfected with EpCAM [10
]. Our studies confirm and extend this previous work by evaluating IT administration of IC in several unique systems, including immunodeficient mice (). HuKS-IL2 IC delivered IT resulted in significant antitumor effects in both immunocompetent and T-cell deficient SCID mice bearing syngeneic B16-KSA tumors. Hu14.18-IL2 IC was also effective against human M21 melanoma xenograft tumors in nude mice. These data provide further insight into the mechanism of IC-mediated tumor cell destruction, suggesting that T cells are not the only potential effectors of antitumor activity in IT IC immunotherapy. Indeed, our studies show that IT IC induced antitumor effects in a T-cell independent manner. Based on previous studies that have shown a role for NK cells in the antitumor effects of IC, these innate immune cells are likely to be playing a significant role in the antitumor effects of IT IC [18
]. Since SCID and nude mice have expanded NK cell populations, the specific contribution of NK cells needs to be further evaluated through the use of depletion studies in immunocompetent animals to specifically characterize the immune effector cells involved in IT IC therapy.
The preclinical data presented in this report demonstrate several unique antitumor effects of IT IC compared to equivalent doses of systemic IC. In the NXS2 model, IC administration resulted in significantly more tumor resolutions when given IT. Complete tumor regressions are not typical at these doses when given systemically [30
]. Interestingly, IT IC also induced a tumor-specific memory response in tumor-free mice as evidenced by multiple rejections of NXS2 tumor rechallenges. These data indicate the likely development of a T cell memory response following initial tumor eradication, similar to what we have previously shown in an NXS2 model where T cells were involved in the resolution of tumors due to IV IC supplemented with systemic IL2 [30
]. The exact mechanism of this specific immune response in our IT IC model remains to be determined. While we observed complete resolution of some NXS2 tumors with IT IC therapy, we have not been able to demonstrate this curative effect against B16-KSA melanoma at the doses tested. This may be reflective of the quickly replicating, aggressive nature of the B16 cell line compared to the much slower growth pattern of the NXS2 NB cell line, as well as to differences in effector cell functions between the A/J and C57BL/6 mouse strains. In the same way that combination with chemotherapy seems to augment the efficacy of IV IC [16
], we hypothesize that combination therapy using IT IC will be more effective than IT IC alone against aggressive or more established tumors. On-going murine studies are attempting to augment the antitumor effect of IT IC with chemotherapeutic agents or antitumor immune stimulants such as αCD40 or CpG [2
]. In addition, since IV IC has been shown to have significant antimetastatic effects, combinatorial IT and IV IC therapy is a clinical possibility.
Also in the NXS2 model, we showed IT hu14.18-IL2 IC had significant antitumor effects on both the primary abdominal tumor treated directly with IT IC as well as a distant s.c. NXS2 tumor. Although the distant tumor was smaller at the initiation of treatment to the primary tumor, our data support a significant antitumor effect due to IT IC that is evident soon after IT IC administration. Here again, IT IC therapy induced tumor resolution in some animals. Additionally, we found that the effects at the distant tumor required IC delivery into the tumor microenvironment. Only IT IC, but not s.c. IC injected at a tumor-free site, resulted in the antitumor effect seen in the non-injected distant NXS2 flank tumors. These data suggest that the local effects of IC at the primary tumor site may induce systemic immunologic activity that provides a significant antitumor effect at the doses tested. Another model of intralesional immunomodulatory therapy, involving the chemotactic CC chemokine CCL16, also showed both local antitumor effects as well as reduced mortality due to distant metastatic disease [13
]. In this model, IT immunotherapy caused attraction and activation of immune effector cells in the tumor environment that led to significant antitumor effects. Local administration has been tested with other anticancer therapeutics as well. Altenschmidt, et al. used the antibody toxin scFV(FRP6)-ETA, consisting of a mAb against erbB2-receptor tyrosine kinase fused to Pseudomonas
endotoxin A, against established schwannomas in a preclinical murine model. They similarly demonstrated greater antitumor effect with IT administration compared to IV injection [1
]. Administration of the non-specific immune stimulant CpG directly into s.c. CT-26 colon adenocarcinoma and B16 melanoma has also been shown to suppress tumor growth and increase survival in mice via CD8+
T effector cells [35
]. Finally, IT injection of the chemotherapeutic agent doxorubicin in liposomes into Meth-A tumors in mice showed greater suppression of tumor growth when compared to the same agent administered IV [17
]. In the future, these individual agents may be combined with IC to enhance the therapeutic efficacy of each agent, with the ultimate goal of both local and systemic tumor eradication.
Localization studies using radiolabeled 111
In-GcT84.66-IL2 IC demonstrated that IT delivery resulted in more than an order of magnitude greater concentration of IC in the tumor microenvironment compared to systemic IC administration (). Our results are consistent with a previous report that showed approximately 5% of the ID of systemically injected IC was detectable in s.c. tumors eight hours later [25
]. We found that over 50% of the ID was detectable at the earliest time point after IT injection, and that radiolabeled-IC remained detectable at later time points than after IV IC, representing persistence of the drug in the tumor microenvironment. These analyses suggest that the enhanced efficacy of IT IC compared to IV IC may be due to higher and sustained concentrations of IC at the tumor site. We hypothesize that most of this radioactivity is retained at the tumor by cells which have bound the IC through specific Fab-mediated recognition of tumor antigen by the antibody component of the IC, thereby representing delivered drug. As non-specific IC (which is unable to recognize tumor antigen with its Fab component) also provides some antitumor effect when given IT (), further studies are needed to determine how each component of the IC molecule (mAb, FcR binding capability, IL2, or the larger complete protein) may be additionally contributing to the overall retention of the IC in the tumor microenvironment and the antitumor efficacy after IT delivery. Finally, it should be noted that IL2-containing IC are only one method of providing enhanced IL2-related effects. IT injection of liposomal IL2 or polyethylene glycol-modified recombinant IL2 have both been shown to induce significant antitumor effects against both primary and distant non-injected tumors in animal models [27
]. Comparison of IL2-containing IC to other methods of increasing the delivery of IL2 are indicated to understand the relative efficacy and toxicities related to these modalities. Additionally, comparisons of the dose-response curves of IT soluble IL2, formulations of IL2 with longer half-lives, and IL2-containing IC (with specific vs. non-specific antibody components) are necessary to further characterize the antitumor effects related to the IL2 component of the IC.
In our studies, the antitumor effect of IT huKS-IL2 was found to be significantly greater than that of IT administration of either IL2 alone or the non-specific IC hu14.18-IL2 against B16-KSA melanoma. Similarly, specific IC hu14.18-IL2 was also found to be more effective than IL2 alone in the treatment of NXS2 tumors. The mechanisms of IC-mediated antitumor activity are hypothesized to involve IC binding to specific antigens expressed on the tumor cell surface, followed by ADCC or complement mediated cytotoxicity (CMC). The studies performed in this study confirm that IC can localize to tumors after IV and IT administration. Tumor cell killing after antibody binding involves activation of FcR+
cells such as macrophages, granulocytes, monocytes, and NK cells, as well as CMC [38
]. Since the non-specific IC resulted in a limited but significant antitumor effect, it is likely that the localization of IL2 and/or non-specific activation through the Fc-component of the IC contributed to this effect. In this regard, a previous study involving genetically-modified ICs such that FcR-binding capability was eliminated showed that IC-mediated antitumor effects could be obtained without FcR-binding [11
]. MAb therapy has been used effectively in clinical trials as single agents, including the anti- GD2
Ab 3F8 and precursors to the hu14.18-IL2 IC including the 14.G2a murine mAb and ch14.18 chimeric mAb [4
]. The IL2 component of the IC augments the effects of mAb, and has been shown to increase the number and activation state of NK cells, as well as to stimulate tumor cell killing by antigen-specific T-cells [37
]. In addition, the IL2 component can stimulate both NK and T-cells via the IL2 receptor, independent of Fc or T-cell receptor binding, respectively [12
]. Taken together, our studies suggest that the proposed mechanisms of action for systemic IC also occur when IC therapy is administered directly to the tumor microenvironment. Currently, we are attempting to further characterize the immune effector cells within the tumor microenvironment and their activation following IT IC through immunohistochemistry and flow cytometry studies.
In summary, this study demonstrates that IT administration of IC is more effective than IV injection. Furthermore, the effect is greater than IT injection of IL2 alone or nonspecific IC. Complete tumor eradication of treated and untreated tumors, with development of a memory response, was demonstrated in immunocompetent mice. Furthermore, in immunocompetent mice, IT administration of IC induced an antitumor effect at a distant site that far exceeded the antitumor effect of s.c. injection of IC into a distant site of normal skin. Further characterization of the effector cell populations involved in the local antitumor effect at the IT site and at the distant, non-injected tumor site following this immunotherapeutic strategy may help determine the local and systemic mechanisms involved in immunocompetent mice, and enable development of novel strategies to simultaneously activate multiple arms of the host antitumor immune response. Further testing is required to determine how to potentially incorporate IT administration of IC into a regimen with IV IC in order to optimize local and systemic antitumor effects, and in combination with other antitumor therapies.