In vivo imaging with two-photon microscopy to assess the tumor-selective binding of an anti-CD137 switch antibody.

STA551, a novel anti-CD137 switch antibody, binds to CD137 in an extracellular ATP concentration-dependent manner. Although STA551 is assumed to show higher target binding in tumor tissues than in normal tissues, quantitative detection of the target binding of the switch antibody in vivo is technically challenging. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy. Tumor-bearing human CD137 knock-in mice were intravenously administered fluorescently labeled antibodies. Flow cytometry analysis of antibody-binding cells and intravital imaging using two-photon microscopy were conducted. Higher CD137 expression in tumor than in spleen tissues was detected by flow cytometry analysis, and T cells and NK cells were the major CD137-expressing cells. In the intravital imaging experiment, conventional and switch anti-CD137 antibodies showed binding in tumors. However, in the spleen, the fluorescence of the switch antibody was much weaker than that of the conventional anti-CD137 antibody and comparable with that of the isotype control. In conclusion, we were able to assess switch antibody biodistribution in vivo through intravital imaging with two-photon microscopy. These results suggest that the tumor-selective binding of STA551 leads to a wide therapeutic window and potent antitumor efficacy without systemic immune activation.

Amino acid 159 of the envelope protein affects viral replication and T-cell infiltration by West Nile virus in intracranial infection.

West Nile virus (WNV) is an important cause of viral encephalitis in birds and animals, including humans. Amino acid 159 of the envelope (E) protein is reportedly implicated in the different levels of neurovirulence in mice infected with WNV NY99 or Eg101. We investigated the role of amino acid 159 of the E protein in the pathogenesis of WNV infection. We produced recombinant WNV with the structural proteins of the NY99 or Eg101 strain (NY-WT or EgCME-WT) and mutant viruses with substitutions of amino acid 159 of the E protein (NY-E-V159I or EgCME-E-I159V). The NY-WT and NY-E-V159I or EgCME-WT and EgCME-E-I159V titers in culture supernatant were similar. The mortality rate and viral titer in the brains of mice inoculated intraperitoneally with NY-WT or NY-E-V159I were also similar. In contrast, the mortality rate and viral titer in the brains of mice inoculated intracranially with EgCME-E-I159V were significantly higher than those of mice inoculated with EgCME-WT. The numbers of CD3-positive and CD8-positive T cells were greater in brains inoculated with EgCME-E-I159V than in those inoculated with EgCME-WT. Therefore, amino acid 159 of the E protein modulates the pathogenicity of WNV by affecting viral replication and T-cell infiltration in the brain.