Cyclophosphamide augments the efficacy of in situ vaccination in a mouse melanoma model

Abstract

Introduction: We have previously shown that an intratumoral (IT) injection of the hu14.18-IL2 immunocytokine (IC), an anti-GD2 antibody linked to interleukin 2, can serve as an in situ vaccine and synergize with local radiotherapy (RT) to induce T cell-mediated antitumor effects. We hypothesized that cyclophosphamide (CY), a chemotherapeutic agent capable of depleting T regulatory cells (Tregs), would augment in situ vaccination. GD2⁺ B78 mouse melanoma cells were injected intradermally in syngeneic C57BL/6 mice.

Methods: Treatments with RT (12Gy) and/or CY (100 mg/kg i.p.) started when tumors reached 100-300 mm³ (day 0 of treatment), followed by five daily injections of IT-IC (25 mcg) on days 5-9. Tumor growth and survival were followed. In addition, tumors were analyzed by flow cytometry.

Results: Similar to RT, CY enhanced the antitumor effect of IC. The strongest antitumor effect was achieved when CY, RT and IC were combined, as compared to combinations of IC+RT or IC+CY. Flow cytometric analyses showed that the combined treatment with CY, RT and IC decreased Tregs and increased the ratio of CD8+ cells/Tregs within the tumors. Moreover, in mice bearing two separate tumors, the combination of RT and IT-IC delivered to one tumor, together with systemic CY, led to a systemic antitumor effect detected as shrinkage of the tumor not treated directly with RT and IT-IC. Cured mice developed immunological memory as they were able to reject B78 tumor rechallenge.

Conclusion: Taken together, these preclinical results show that CY can augment the antitumor efficacy of IT- IC, given alone or in combination with local RT, suggesting potential benefit in clinical testing of these combinations.

Keywords: cyclophosphamide; immunocytokine; in situ vaccine.; melanoma; radiation.

Figure 1

CY and RT improve the antitumor efficacy of IC. (A) Schema of dosing schedule for RT, IC, and CY. Treatment began when the tumor reached a mean volume of ~100-300mm³. (B, C) compare IC, CY and IC+CY; (D, E) compare RT, IC and RT+IC. Mean +/- SEM of the tumor volumes (B, D) and mouse survival (C, E) are shown (n=5). The results for each pair of tumor growth and survival data (B–E) are representative of two independent experiments. *p≤ 0.05; **p≤ 0.01. p values comparing all groups to one another are included in Supplemental Table 1.

Figure 2

A combination of CY, RT and IT-IC results in enhanced antitumor efficacy against B78 melanoma. Mean +/- SEM of tumor volumes (A) and mouse survival until day 90 post RT (B) are shown. The percentages of tumor-free mice until day 175 post RT are shown. The ratio on each bar shows the number of tumor-free mice out of the total number of mice from four independent experiments (C). The results show the combined data of four independent experiments with p-values from proportion tests. *p≤ 0.05; **p≤ 0.01;***p ≤ 0.001; ****p ≤ 0.0001.

Figure 3

Treatments with CY in combination with IC or RT+IC induce reduction of Tregs. B78 tumor-bearing mice were treated with RT and/or CY on Day 0 and/or IC on Day 5-9 as shown in Figure 1A. Flow cytometry of tumor-infiltrating lymphocytes was performed on day 10. Percentages of CD4⁺ T cells (A), CD8⁺ T cells (B), Tregs (C) and ratio of CD8⁺ T cells/Tregs (D) within the population of CD45⁺ cells are shown and were compared using unpaired t-test. The combined data from two independent experiments are shown (n=9 mice per group).

Figure 4

Combination of CY, RT and IT-IC induces systemic immunity against B78 Melanoma. (A) Schema of RT + IT-IC + CY treatment regimen. C57BL/6 mice were injected with B78 tumor cells on both the right and left flank. Right flank (treated) tumors were given RT on day 0 once their right flank tumor reached ~100mm³. CY was injected on Day 0. IC was injected IT into right tumors on days 5-9. (B) Data shown are Means +/- SEM of tumor volumes, for the indicated treatment groups. (n=5 per group per experiment). Statistical comparisons of the response at the untreated tumor are shown, for the data from two independent experiments (labelled 1 and 2 in the statistical comparison schematic), except for RT + CY group which was done in one experiment. (C) Tumor volumes (Mean +/- SEM) of the untreated tumor from mice bearing two B78 tumors on day 61 following RT were compared using one-way ANOVA. (D) Combined survival of mice bearing two B78 tumors from two independent experiments [n=10, except RT+CY(n=5)] is shown. *p≤ 0.05; **p≤ 0.01. P-values from the statistical comparisons between groups are included in Supplementary Table 1.

Figure 5

Combination of CY, RT and IC enables immune memory against B78 Melanoma. After mice had been tumor-free for at least 60 days following treatment-induced B78 single flank tumor rejection, these mice and naïve mice were injected with 2x10⁶ B78 melanoma cells on the abdomen. (A) Individual mouse tumor growth curves for naïve mice, or mice previously cured by RT+ IC or RT+ IC+ CY following implantation with B78 tumor cells are shown. The ratio above each graph indicates the number of tumor-free mice out of the total number of implanted mice. (B) Percentages of tumor-free mice, calculated from the number of tumor-free mice out of total number of mice as indicated above the bars in (A), are shown.