Data presented in this article help to elucidate possible mechanisms of enhanced antitumor effects of localized IC therapy and assist in distinguishing the IT-IC antitumor effect from the i.v.-IC antitumor effect. In this study, we show that IT-IC treatment induces improved inhibition of tumor growth and augmented survival compared with no treatment or treatment with IT PBS. IHC and flow cytometric analyses show an increased percentage of NK and T cells in IT-IC–treated tumors. Depletion studies show that T cells and NK cells are involved in the antitumor effects of IT-IC on directly injected local tumors and on noninjected distant tumors in those same animals. Depletion of either immune cell population substantially attenuates the observed antitumor effects on both the local and distant tumors. These data suggest that both T cells and NK cells are necessary for the local and distant antitumor effects observed under these conditions. Although NK cells may exert a more direct and immediate effect (through Ab-dependent cellular cytotoxicity), and T cells may exert a more delayed effect through an adaptive immune response, we are not able to clarify these mechanisms from the data presented in this study. Furthermore, other possible mechanisms include direct induction of tumor apoptosis or disruption of the tumor microenvironment or tumor vascular system.
We also show that IT-IC treatment causes better tumor growth inhibition and survival than does i.v.-IC treatment, which is consistent with flow cytometry analyses demonstrating that IT-IC results in a lower percentage of living tumors cells compared with i.v.-IC treatment. Furthermore, flow cytometry analyses show that IT-IC treatment is characterized by a higher percentage of NKG2A/C/E+ cells. IT and i.v. treatment with IC increases the fraction of NK cells and CD8 cells that express NKG2A/C/E, whereas IT-IC increases expression levels of the NKG2D effector receptor on NK and CD8 cells to a greater degree than does i.v.-IC. Finally, flow cytometry analyses were used to show that IT-IC treatment results in augmented delivery and retention of IC to tumor compared with i.v.-IC treatment.
Although immunocytokines allow the targeting of immune-stimulating cytokines directly to the tumor microenvironment, systemic administration is still limited by dose-limiting toxicities (7
). These dose-limiting toxicities are due to the systemic effects of the IL-2 component of the immunocytokine (i.e., fever, capillary leakage, and secondary effects of capillary leakage, such as hypotension) and effects from the mAb component of the immunocytokine; for IC, these include neuropathic pain due to mAb recognition of selective GD2-expressing peripheral nerves. Finding a strategy of IC administration that maximizes the direct delivery of IC to the tumor site and potentially decreases dose-limiting systemic toxicities would be clinically beneficial. Using the NXS2 model, we demonstrate a greater antitumor response with localized IC administration compared with an equivalent systemic IC dose in the treatment of established s.c. tumors. Importantly, IT-IC treatment increased delivery of IC to the tumor site; substantially higher levels of IC were found at the tumor site for several hours following IT-IC compared with i.v.-IC.
IT-IC resulted in complete resolution of both the directly treated local and noninjected distant tumors in several mice. Several mechanisms may result in the antitumor effects of both local and distant tumors: IT-IC may be circulating and having a systemic effect, IT-IC may be inducing a systemic immune response by the host’s immune system, or a combination of both. We acknowledge that our experimental design may play some role in our observation that NK depletion (which begins on day 4, 4 d after implantation of the primary tumor and just prior to implantation of the secondary tumor) more potently interferes with the IC effects against the distant tumor than the primary one. We also know from previous studies that T cells from treated mice can respond to NXS2 in an Ag-dependent manner (18
). Our results indicate that IT-IC induces a systemic immune response. T cells and NK cells were required for rejection of both primary and distant tumors, and 90% of mice that became tumor-free following IT-IC treatment were able to subsequently reject rechallenge with 2 × 106
The IL-2 component of the IC augments the effects of the mAb and was shown to increase the number and activation state of NK cells, as well as to stimulate tumor cell killing by Ag-specific T cells (20
). The IL-2 component can stimulate both NK and T cells via IL-2R, independent of Fc or TCR binding, respectively (1
). Using a metastatic model of NXS2 neuroblastoma metastasizing to bone marrow in A/J mice, i.v.-IC therapy was previously shown to be exclusively mediated by NK cells (2
). In contrast, our model investigates possible immune effector cells within well-established s.c. tumors and shows a necessary role for both NK and T cells in response to localized IT-IC therapy. There is an increased percentage of NKG2A+
T cells, as well as increased NKG2D expression on CD8a+
T cells in tumors versus spleens within similarly treated mice (). This may suggest at least some inherent importance of T cells in combating tumor cells in this model because NKG2A/C/E+
cells were shown to be necessary for self recognition (19
). Furthermore, the statistically significant increase in NKG2D expression on NK cells after IT-IC versus i.v.-IC treatment () may reflect a mechanism that plays some part in the enhanced antitumor effect of IT versus i.v. IC treatment. In addition, the increased expression of NKG2D might be considered a marker of activation and suggests that other pathways (not assayed for in this study) might be further activated by IT-IC compared with i.v.-IC.
Depletion data from a two-tumor model showed that T cells, as well as NK cells, play a large role in the IT-IC antitumor effect compared with the previously shown NK-predominant cell-mediated i.v.-IC antitumor effect (3
). Perhaps having substantial IL-2 bound to the surface of tumor cells after IT-IC treatment () is directly or indirectly responsible for increasing these specific lymphocytes (NKG2A/C/E+
TIL) or altering their phenotype (increasing expression of NKG2D effector receptor) and enhancing the antitumor effect compared with i.v.-IC treatment. In vitro data suggest that immunocytokine on tumor cells enables cells with IL-2Rs to form more activated immune synapses with the tumor cells than they would using mAb in combination with IL-2 (24
). IT–IC-treated mice compared to i.v.–IC-treated mice show a small, but significant, increase in NKG2D expression on NKp46+
double-positive cells, as well as a small, but nonsignificant, increase in NKG2D expression on CD8a+
cells (, ). This may indicate a phenotypic advantage of resulting TILs when treating with IT-IC. Expression of NKG2D on activated CD8+
T cells was shown to account for TCR-independent cytotoxicity against malignant cells in vitro (26
). Its enhanced expression on CD8+
TILs, but not spleen cells, following i.v. or IT treatment with IC in our study demonstrates the localized activation of T cells, at the tumor site, induced by IC treatment. We examined expressivity of NKG2D ligands on NXS2 and found a large increase in RAE-1γ expression ex vivo (data not shown). Therefore, in addition to being a marker of activation, NKG2D may play a direct role in the enhanced antitumor effects.
Previous data showed therapeutic and safety benefits of localized versus systemic immunocytokine therapy in preclinical models (11
), as well as localized versus systemic immune therapy in melanoma patients (14
). Our studies confirm and extend this previous work by evaluating possible underlying mechanisms of IT administration of IC, as well as showing antitumor efficacy in an established measureable disease setting in a preclinical model. IHC infiltration data collected using the quantitative method that we developed are consistent with infiltration data collected by flow cytometry. Although the experimental design intended for each animal within a treatment group to be identical to one another, there is substantial heterogeneity within treatment groups, which is the topic of a separate article (R.K. Yang, N.A. Kalogriopoulos, A.L. Rakhmilevich, E.A. Ranheim, S. Seo, K. Kim, K.L. Alderson, J. Gan, R.A. Reisfeld, S.D. Gillies, J.A. Hank, and P.M. Sondel, manuscript in preparation). The preclinical data presented in this report demonstrate several unique antitumor effects of IT-IC compared with equivalent doses of systemic IC. Flow cytometry analyses of IC delivery and retention show a substantial difference in the amount of IC present at initial time points at the tumor site between IT and i.v. administration. This direct exposure to greater concentrations of IT-IC at the tumor site between IT-IC and i.v.-IC treatment may be responsible, at least in part, for the different tumor-response rates and the different infiltration and expression patterns of TILs between the two treatment groups. These mechanisms of enhanced activity by localized IT treatment provide further justification for proceeding with clinical testing of IT-IC, potentially testing IT delivery of IC in patients with GD2+
tumors, such as melanoma patients with metastatic disease having cutaneous, s.c., or readily injectable involved lymph nodes. Furthermore, the type of analyses presented in this preclinical report might also be conducted on biopsies of patient samples following IT-IC administration. Such immune monitoring by IHC and flow cytometry may become a potential means to assess the immunologic effects of immunocytokine treatment; if these parameters show correlation with antitumor effect, they may be considered indicators for prognosis of patients’ response to IT-IC.