Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice
Kathrin Stirnemann, Jackeline F. Romero, Lucia Baldi, Bruno Robert, Valérie Cesson, Gurdyal S. Besra, Maurice Zauderer, Florian Wurm, Giampietro Corradin, Jean-Pierre Mach, H. Robson MacDonald, Alena Donda
Kathrin Stirnemann, Jackeline F. Romero, Lucia Baldi, Bruno Robert, Valérie Cesson, Gurdyal S. Besra, Maurice Zauderer, Florian Wurm, Giampietro Corradin, Jean-Pierre Mach, H. Robson MacDonald, Alena Donda
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Research Article Oncology

Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice

Abstract

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand α-galactosylceramide (αGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when αGalCer was loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-γ secretion as well as DC maturation in mice. Most importantly, when αGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2–7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free αGalCer at this time had no effect. The antitumor activity of the CD1d–anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy.

Authors

Kathrin Stirnemann, Jackeline F. Romero, Lucia Baldi, Bruno Robert, Valérie Cesson, Gurdyal S. Besra, Maurice Zauderer, Florian Wurm, Giampietro Corradin, Jean-Pierre Mach, H. Robson MacDonald, Alena Donda

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Figure 4

In vivo antitumor activity in systemic treatments.

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In vivo antitumor activity in systemic treatments. (A) Mice were grafted...
(A) Mice were grafted i.v. with 700,000 B16-HER2 cells and i.v. treatment was started 48 hours later. Mice were injected 5 times i.v. every 3–4 days (arrows) with either PBS (control), equimolar amounts of αGalCer (0.4 μg), or αGalCer-loaded sCD1d–anti-HER2 fusion (40 μg). Mice were analyzed after 3 weeks and results are shown as pictures of tumors-invaded lungs (1 representative lung per group; original magnification, ×6.3), and in the graph expressed as percent of lung surface invaded by melanin-loaded tumor nodules. Results represent the mean ± SD of 5 mice per group of 2 independent experiments. **P < 0.005 versus control; *P < 0.04 versus αGalCer. (B) Mice were grafted as above, and treatment was started 6 days after with the same protocol as in A including treatment with sCD1d (25 μg). Lung nodules were analyzed after 2 weeks. Results represent the mean ± SD of 6 mice per group of 2 independent experiments. ***P = 0.0006 versus control; **P < 0.004 versus αGalCer; *P < 0.02 versus sCD1d. (C) Mice were grafted s.c. on the right flank with 700,000 B16-HER2 cells and i.v. treatment as in B was started 7 days later, when all tumors were palpable. Additional groups treated with 4D5 alone (80 μg) or the combination of 4D5 + sCD1d (80 + 25 μg) were included. Results expressed as the mean tumor size in mm3 ± SD measured at the end of the treatment (day 18) of 4 mice per group. Statistical significance of αGalCer/sCD1d–anti-HER2–treated group was of *P < 0.05 versus all other groups.