Intratumoral hu14.18-IL-2 (IC) induces local and systemic antitumor effects that involve both activated T and NK cells as well as enhanced IC retention

Abstract

hu14.18-IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18-IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18-IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E⁺ NKp46⁺ NK cells and NKG2A/C/E⁺ CD8⁺ T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.

Figures

FIGURE 1.

IT-IC shows enhanced antitumor effects compared with control treatments. Tumors were established according to the well-established tumor model. (A) Tumor growth curves of IT-IC–treated (n = 12), IT PBS-treated (n = 12), and untreated (n = 8) s.c. NXS2 tumor in A/J mice. Data were obtained from two independent experiments. (B) Kaplan–Meier survival curves of IT-IC–treated (n = 13), IT PBS-treated (n = 6), and untreated (n = 5) mice. Data represent three independent experiments. (C) H&E stain of IT-IC–treated NXS2 tumor 4 d posttreatment initiation. Original magnification ×40. (D) H&E stain of untreated NXS2 tumor. Original magnification ×40.

FIGURE 2.

IT-IC is distinguished by increased TILs. Representative IHC stains of s.c. NXS2 tumor frozen sections were photographed. Sections were stained with either anti-CD8a (A, B) or anti-NKG2A/C/E (C, D) Abs. Sections from either IT-IC–treated tumors (A, C) or untreated tumors (B, D) are shown. Original magnification ×40. (E) Quantification of IHC photographs using the manual counting method. IT-IC–treated (n = 20) tumors are characterized by higher tumor leukocyte infiltration percentages (CD45+, CD3+, CD8a+, NKG2A+, F4/80+) than are IT PBS-treated (n = 10) and untreated (n = 15) tumors. Data were obtained from three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 3.

T cell and NK cell depletion reduces IT-IC–induced antitumor effects. Tumors were established according to the early-established tumor model. Local tumor (A) and distant tumor (B) growth curves of IT-IC–treated T cell-depleted mice (▴, n = 8), IT-IC–treated NK cell-depleted mice (▼, n = 16), IT-IC–treated mice without depletion (▪, n = 16), and IT PBS-treated mice (●, n = 16) bearing s.c. NXS2 tumors. (C) Kaplan–Meier survival curves of mice with the early established two-tumor NXS2 model, shown in (A) and (B) for groups receiving IT-IC treatment, with and without NK or T cell depletion. Data were obtained from two independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

IT-IC is more effective than i.v.-IC in slowing tumor growth and prolonging survival. Tumors were established according to the well-established tumor model. Mice given IT-IC, i.v.-IC, or no treatment were followed. Tumor growth curves (n = 30/group, four independent experiments) (A) and Kaplan–Meier survival curves (n = 13/group, three independent experiments) (B) are plotted. Mice in these treatment groups were also sacrificed for characterization of the TILs, using both manual blinded counting of IHC photographs (n = 15/group, three independent experiments) (C), as well as flow cytometric analyses (D). (E) Populations of TILs as a percentage of total leukocytes within a tumor (CD45+). (F) Populations of live NXS2 tumor cells as a percentage of TLNCs within a tumor. (D–F: n = 11/group, three independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

Treatment with IC increases NKG2A/C/E+ NK and T cells in tumor and spleen and increases NKG2D expression within tumors. Flow cytometric analyses show percentages of NKG2A/C/E+ cells within the NKp46+ NK cell populations (A) and the CD8a+ cytotoxic T cell populations (B) within tumors and spleens. Flow cytometric analyses of NKG2D expression on NKp46+/NKG2A/C/E+ NK cell populations (C) and the CD8a+/NKG2A/C/E+ cytotoxic T cell populations (D) within tumors and spleens. All data represent three independent experiments (n = 11 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6.

IT-IC treatment results in augmented IC localization and increased IC retention compared with i.v.-IC. Tumor-bearing mice given IC IT or i.v. were sacrificed at various times, and their tumors were disaggregated. (A) Flow cytometric measurements of levels of human IgG Fcγ Ab fragment on NXS2 tumor cells ex vivo at various times posttreatment. (B) Flow cytometric measurements of levels of human IL-2 on NXS2 tumor cells ex vivo at various times posttreatment. All MFI values are normalized to an IT nonspecific control immunocytokine (IT-KS–IL-2). Data represent three independent experiments with approximately six mice/group/time point. **p < 0.01, ***p < 0.001, ****p < 0.0001.