Combination of temozolomide with immunocytokine F16-IL2 for the treatment of glioblastoma

Abstract

Background: Glioblastoma patients are still not cured by the treatments available at the moment. We investigated the therapeutic properties of temozolomide in combination with F16-IL2, a clinical-stage immunocytokine consisting of human interleukin (IL)-2 fused to the human antibody F16, specific to the A1 domain of tenascin-C.

Methods: We conducted three preclinical therapy studies, using subcutaneous and intracranial U87MG glioblastoma tumours xenografted in BALB/c nude mice. The same therapeutic schedule was used, consisting of five total administrations every third day, of 0.525 mg temozolomide, 20 microg F16-IL2, the combination, or the control solution.

Results: Immunohistochemical analysis of U87MG xenografts and of human glioblastoma specimens showed selective tumour staining of F16. A quantitative biodistribution confirmed the preferential tumour accumulation of radiolabelled F16-IL2. In the study with subcutaneous xenografts, the combination of F16-IL2 with temozolomide induced complete remission of the animals, which remained tumour free for over 160 days. The same treatment led to a consistent size reduction of intracranial xenografts and to a longer survival of animals. The immunocytokine promoted the recruitment of leukocytes into tumours of both models.

Conclusion: The combined use of temozolomide with F16-IL2 deserves clinical investigations, which will be facilitated by the excellent safety profile in cynomolgus monkeys, and by the fact that F16-IL2 is in clinical trials in patients with cancer.

Figure 1

Immunohistochemical analysis of U87MG human glioblastoma xenografts and of human glioblastoma surgical specimens using the F16 antibody, specific to the extradomain A1 of tenascin-C, and the L19 antibody, specific to the extradomain B of fibronectin (serial tissue sections). Both antibodies stained tumour perivascular structures considerably. In negative controls (NC), the primary antibody was omitted. Scale bars indicate 100 μm.

Figure 2

(A) Preclinical therapy study with subcutaneous U87MG human glioblastoma xenografts. The treatment regimen consisted of five total administrations, every third day, of temozolomide (0.525 mg, corresponding to 75 mg m⁻²) in a saline 10% dimethyl sulfoxide (DMSO) solution, F16–IL2 (20 μg) in phosphate-buffered saline, a combination of F16–IL2 and temozolomide (same doses), or saline 10% DMSO solution. The combination therapy group exhibited the highest therapeutic benefit with a complete remission of the animals, which remained tumour free for over 160 days. (B) Preclinical therapy study, using intracranial U87MG human glioblastoma xenografts. The same therapeutic schedule of the subcutaneous study was used. The combination of F16–IL2 with temozolomide exhibited the highest therapeutic benefit. Pairwise comparisons between the combination therapy group and temozolomide alone (P=0.009), F16–IL2 alone (P=0.001), and the control group (P<0.001) were calculated using the Student's t-test and showed significant results. (I) Photograph of a mouse hemisphere with tumour, imaged from two sides. (II) Tumour volumes at day 25 from the start of treatment (13 days after the last drug administration), expressed as average mean±s.d. (C) Survival study using intracranial U87MG human glioblastoma xenografts, with the same therapeutic schedule of the previous subcutaneous and intracranial studies. Results indicate a longer survival for the combination treatment group (combo vs TMZ: P<0.002; combo vs F16–IL2: P<0.002; combo vs control: P<0.0001).

Figure 3

Immunofluorescence analysis of tumour-infiltrating immune cells and of microvascular density in the subcutaneous (A) and intracranial (B) glioblastoma models, 24 h after the third injection of therapeutic agents. The F16–IL2+temozolomide treatment groups show the largest increase in the infiltration of leukocytes and in particular of natural killer cells and macrophages (serial tissue sections). Scale bars indicate 100 μm.

Figure 4

Immunofluorescence analysis of F16–IL2 fusion protein localisation in subcutaneous (A) and intracranial (B) glioblastoma xenografts, 24 h after the third injection of therapeutic agents (serial tissue sections). Scale bars indicate 100 μm.

Figure 5

Immunofluorescence analysis of apoptosis and proliferation in subcutaneous glioblastoma xenografts, 24 h after the third injection of therapeutic agents. Results show a clear increase in apoptosis and the complete suppression of proliferation in the combination F16–IL2+temozolomide treatment group. Scale bars indicate 50 μm.