Delivery of NKG2D Ligand Using an Anti-HER2 Antibody-NKG2D Ligand Fusion Protein Results in an Enhanced Innate and Adaptive Anti-Tumor Response.
Authors: Shin S. , Cho H. , Tolba K. , Shin S. , Shin D. , Calfa C. , Zhang Y. , Rosenblatt J. 
Published: December 2010

Background: NKG2D ligands activate via the NKG2D receptor expressed on effector cells belonging to both the innate (NK) and adaptive immune systems (CD8+ T cells). Over-expression of NKG2D ligand on the surface of tumor cells leads to tumor regression in several murine models. We reasoned that antibody targeting of NKG2D ligand to tumor cells could also be used to evoke an enhanced anti-tumor immune response.Methods: We fused the murine NKG2D ligand, Rae-1β, to the 3' end of an anti-HER2 IgG3 containing an intact Fc domain (anti-HER2 IgG3-Rae-1β) to target NKG2D ligand to HER2+ breast tumor cells. The cytotoxicity of the fusion proteins was tested in vitro in direct and indirect assays with the murine NK cell line, KY-2. Anti-tumor efficacy was examined in a murine mammary tumor model.Results: Anti-HER2 IgG3-Rae-1β fusion protein bound to both the HER2 antigen, and to NKG2D as determined by flow cytometry. Anti-HER2 IgG3-Rae-1β fusion induced enhanced cytotoxicity of NK effectors against HER2 expressing tumor cells in vitro. EMT6-HER2 bearing BALB/c mice treated with anti-HER2 IgG3-Rae-1β showed markedly decreased tumor growth relative to anti-HER2 IgG3 or PBS injected control mice. Treatment of mice that were implanted on one flank with EMT6 and contralaterally implanted with EMT6-HER2, resulted in prompt regression of EMT6-HER2, and delayed regression of EMT6, suggesting “epitope spreading.” To examine the involvement of NK, CD4, or CD8 cells in the growth inhibition of the Rae-1β fusion protein targeted tumors, we performed lymphocyte subset depletion studies. Mice depleted of either NK cells or CD8+ T cells showed decreased inhibition of tumor growth following treatment with anti-HER2 IgG3-Rae-1β, compared to control mice. These results suggest that NK cells and CD8+ cells play an important role in the anti-tumor activity of anti-HER2 antibody-Rae-1β fusion. Anti-HER2 IgG3-Rae-1β also inhibited growth of EMT6-HER2 in FcγR-/- mice, indicating activity of the fusion in the absence of FcR interaction. No anti-tumor efficacy of anti-HER2 IgG3-Rae-1β was observed in IFNγ-/- mice.To determine if anti-HER2 antibody-Rae-1β fusion led to priming of an adaptive response, twelve mice that had previously rejected EMT6-HER2 tumors following treatment with anti-HER2 IgG3-Rae-1γ, were rechallenged with either EMT6-HER2, or with parental EMT6. Five of six mice rechallenged with EMT6-HER2 and five of six mice rechallenged with EMT6 showed no evidence of tumor growth at day 25, while both EMT6 and EMT6-HER2 grew rapidly in six of six control mice, respectively.Conclusion: Antibody-NKG2D ligand fusion proteins administration leads to an enhanced anti-tumor innate and adaptive immune response. The immune response evoked by targeting of the fusion to HER2, a single surface antigen, encompassed additional non-targeted antigens. Antibody-NKG2D ligand fusion proteins could potentially be used to treat other malignancies by substituting alternate tumor specificities. Murine NKG2D ligand in the antibody fusion molecules could be replaced with human NKG2D ligands for testing in man.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6102.