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.

Despite the well-established antitumor activity of CD1d-restricted invariant natural killer T lymphocytes (iNKT), their use for cancer therapy has remained challenging. This appears to be due to their strong but short-lived activation followed by long-term anergy after a single administration of the CD1d agonist ligand alpha-galactosylceramide (αGC). As a promising alternative, we obtained sustained mouse iNKT cell responses associated with prolonged antitumor effects through repeated administrations of tumor-targeted recombinant sCD1d-antitumor scFv fusion proteins loaded with αGC. Here, we demonstrate that CD1d fusion proteins bound to tumor cells via the antibody fragment specific for a tumor-associated antigen, efficiently activate human iNKT cell lines leading to potent tumor cell lysis. The importance of CD1d tumor targeting was confirmed in tumor-bearing mice in which only the specific tumor-targeted CD1d fusion protein resulted in tumor inhibition of well-established aggressive tumor grafts. The therapeutic efficacy correlated with the repeated activation of iNKT and natural killer cells marked by their release of TH1 cytokines, despite the up-regulation of the co-inhibitory receptor PD-1. Our results demonstrate the superiority of providing the superagonist αGC loaded on recombinant CD1d proteins and support the use of αGC/sCD1d-antitumor fusion proteins to secure a sustained human and mouse iNKT cell activation, while targeting their cytotoxic activity and cytokine release to the tumor site.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1381-7) contains supplementary material, which is available to authorized users.

Purpose:To redirect an ongoing antiviralT-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. Experimental Design: First, lymphochoriomeningitis virus (LCMV)^infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)^transfected MC38 colon carcinoma cells precoated with anti-CEA  H-2D b /GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab ¶) 2 fragments. Second, influenza virus^infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2  H-2D b /NP366 influenza peptide conjugates, or anti-HER2 F(ab ¶) 2 fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA  H-2D b conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA + cells, s.c. grafted in LCMV-infected mice.Results: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA + cells did not develop into tumors, whereas all grafts with F(ab ¶) 2 -precoated MC38-CEA + cells did so (P = 0.0022). In influenza virus^infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA  H-2D b /cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). Conclusion:The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody  MHC/viral peptide conjugates.In recent years, a major effort has been dedicated to the development of active vaccination protocols for immunotherapy of cancer patients, using newly discovered tumor-specific or differentiation antigens (1 -3). Successful induction of specific T-cell responses and tumor infiltration by T lymphocytes were well documented, but the number of tumor remissions remained low (4,5). It is hard to understand the reasons for the relatively poor antitumor activity of the induced specific T lymphocytes, given that such effector cells are known to be so efficient in the elimination of virus-infected cells and in allograft rejection. Three major explanations have been given for the inefficiency of the T-cell antitumor response: the poor antigenicity of autologous tumor antigens, the low expression or absence of MHC molecules on the tumor cell surface, or some functional defects in their antigen-processing machinery (6, 7).Here, we present a new immunotherapeutic strategy that has the potential to overcome these obstacles. We coupled an antitumor antibody fragment to an MHC molecule loaded with an immunodominant viral peptide and showed that tumor cells coated with the...