Intratumoral treatment of smaller mouse neuroblastoma tumors with a recombinant protein consisting of IL-2 linked to the hu14.18 antibody increases intratumoral CD8+ T and NK cells and improves survival

Abstract

Hu14.18-IL2 is an immunocytokine (IC) consisting of human IL-2 linked to hu14.18 mAb, which recognizes GD2 disialoganglioside. Phase II clinical trials of intravenous-hu14.18-IL2 (IV-IC) in neuroblastoma and melanoma are underway, and have already demonstrated activity in neuroblastoma. In our Phase II trial, lower neuroblastoma burden at the time of treatment was associated with a greater likelihood of clinical response to IV-IC. We have previously shown that intratumoral-hu14.18-IL2 (IT-IC) compared to IV-IC results in enhanced local and systemic antitumor activity in tumor-bearing mice. We utilized a mouse model to investigate the impact of tumor burden on hu14.18-IL2 treatment efficacy in IV- versus IT-treated animals. Studies presented here describe the analyses of tumor burden at the initiation of treatment and its effects on treatment efficacy, survival, and tumor-infiltrating leukocytes in A/J mice bearing subcutaneous NXS2 neuroblastoma. We show that smaller tumor burden at treatment initiation is associated with increased infiltration of NK and CD8+ T cells and increased overall survival. NXS2 tumor shrinkage shortly after completion of the 3 days of hu14.18-IL2 treatment is necessary for long-term survival. This model demonstrates that tumor size is a strong predictor of hu14.18-IL2-induced lymphocyte infiltration and treatment outcome.

Conflict of interest statement

Conflict of Interest:

The authors declare that they have no conflict of interest.

Figures

Figure 1. Initial Tumor Volume Inversely Correlates with Survival in hu14.18-IL2 Treated Mice

A/J mice bearing subcutaneous NXS2 tumors were treated with 3 daily doses of hu14.18-IL2 (either IV or IT) or received control treatment (IT-PBS or no treatment). (A) Contingency table showing the long-term outcomes of mice treated with IC (hu14.18-IL2 IV or IT) stratified by median initial tumor volume of 34 mm3 or control mice (IT-PBS or untreated). (B) Kaplan Meier curves showing number of survival days post-treatment initiation in IC treated or control groups stratified by median initial tumor volume of 34 mm3. Experiments represent data from 3 independent experiments with a total of 37 mice.

Figure 2. Hu14.18-IL2 Treatment of Mice with Smaller Initial Tumor Volume Leads to Increased Tumor NK Infiltration and NKG2D Expression on Tumor NK and CD8+ T cells

A/J mice bearing subcutaneous NXS2 tumors were treated with IT-IC, IV-IC or Untreated. Mice were sacrificed 4 days post-treatment initiation and tumors underwent flow cytometric analysis. Initial tumor volumes of all mice are stratified into groups greater or less than the median value of 101.6 mm3. (A): post-treatment tumor leukocytes (CD45+ cells) are presented as a percentage of Total Live Nucleated Cells (TNCs) within the tumor; (B): macrophages (F4/80+) are presented as a percentage of leukocytes (CD45+) within the tumor; (C): natural killer cells (NKp46+) are presented as a percent of mouse leukocytes (CD45+); (D): cytotoxic T cells (CD8+) are presented as a percent of mouse leukocytes (CD45+); (E): NKG2D expression levels are presented as MFI on NKp46+, NKG2A+ activated natural killer cells; and (F): NKG2D expression levels are presented as MFI on CD8+, NKG2A+ activated cells. Results represent data from 6 independent experiments with an average of 19 mice within each treatment group.

Figure 3. Tumor NK Cell and CD8+ T cell Infiltration Inversely Correlates with Tumor Growth Following IC Treatment

A/J mice bearing subcutaneous NXS2 tumors were treated with IT-IC, IV-IC or Control treatment. Mice were sacrificed 4 days post-treatment initiation and tumors underwent quantitative histological (A–B) or flow cytometric analyses (C–D). (A–B) After immunohistochemistry, 5 distinct microscopic fields were counted in a blinded fashion within each mouse’s tumor. NKG2A/C/E+ cells (A) and CD8a+ cytotoxic T cells (B) as a percentage of Total Nucleated Cells (TNCs) within the each mouse’s tumor are plotted against each mouse’s percent tumor growth. (A–B) represent data from 3 independent experiments, with an average of 18 mice total within each treatment group. Horizontal error bars represent standard error of the mean for subsamples within an individual mouse. (C) Post-treatment tumor leukocytes (CD45+ cells) as a percentage of Total Live Nucleated Cells (TNCs) are plotted against each mouse’s percent tumor growth. (D) Mice are stratified by treatment group and by tumor growth or shrinkage. Within each group, tumor immune cells (CD8+, NKp46+, F4/80+, CD4+) are shown as a percentage of tumor leukocytes (CD45+ cells). Asterisks in (D) compare tumor immune cell populations between IT-IC treated mice whose tumor grew vs. IT-IC treated mice whose tumor shrank. (C–D) represent data from 6 independent experiments with an average of 19 mice total within each treatment group. Note, the IT-IC treatment group consists of 6 mice that had tumors shrink and 15 mice that had tumors grow.

Figure 4. NKG2D Expression on NK and CD8+ T cells Correlates with Tumor Volume Reduction following Hu14.18-IL2 Treatment

A/J mice bearing subcutaneous NXS2 tumors were treated with IT-IC or IV-IC or received no treatment. Mice were sacrificed 4 days post-treatment initiation and tumors underwent flow cytometric analysis. Using flow cytometry, post-treatment tumor NKG2D expression (MFI) on activated natural killer cells (A) and cytotoxic T cells (B) are plotted against each mouse’s percent tumor growth. (C) shows bar graphs of the same data in (A–B). (D) shows, within each mouse’s tumor, the NKG2D expression (MFI) on double positive NKp46+, NKG2A/C/E+ NK cells vs that same animal’s NKG2D MFI on double positive CD8+, NKG2A/C/E+ T cells (x-axis). All panels represent data from 6 independent experiments with an average of 19 mice total within each treatment group.

Figure 5. Initial Tumor Load Shows an Inhibitory Interaction Effect on Hu14.18-IL2 Treatment and Correlates with Tumor Growth

Four days after randomization and treatment initiation, A/J mice bearing subcutaneous NXS2 were sacrificed for flow cytometric analysis of their tumors. (A) shows individual mouse tumor volumes at the day of randomization (Initial Tumor Volume (mm3)) plotted against Day 4 Tumor Volume. Linear regression models for each treatment group are shown. (B) shows Day 4 Tumor Volume data stratified into four quartiles based on initial tumor volume (separated by 33.9, 76.3, 130.9) as well as three treatment groups. All panels represent data from 11 independent experiments with an average of 48 mice total within each treatment group.