In this study, we revealed that anti-CD137 switch antibody binds to target cells differently in tumor and spleen in vivo by using two-photon microscopy. We first examined the CD137 expression levels of tumor and spleen in tumor-bearing hCD137 KI mice. Conventional anti-CD137 antibody was then administered to tumor-bearing hCD137 KI mice to determine the distribution of the antibody to the tumor and spleen. Finally, two-photon microscopy was used to detect the distribution of anti-CD137 switch antibody to tumor and spleen in vivo.
To confirm the different binding ability of STA551 between tissues in vivo, we needed to detect the distribution of antibodies in a non-invasive way to minimize ATP-concentration changes in tissues. Therefore, we used two-photon microscopy to detect antibodies intravitally. In experiments using two-photon microscopy, it can be time-consuming to prepare animals, optimize imaging, and monitor animals. On the other hand, in ex vivo analysis using flow cytometry, several experimental conditions, such as dosing regimen, can be verified with better thruput. In addition, ex vivo studies can detect the expression of target molecules, the binding of antibodies, and identify the types of cells. Establishing optimal imaging conditions for intravital imaging by using ex vivo flow cytometric analysis would be useful in studying antibody distribution in vivo.
In this study, in vivo imaging revealed that Sta-MB distributed and bound to the cells in tumor but much less so in spleen. It has been suggested that the toxicity induced by anti-CD137 antibody is CD137 dependent, and that T-cell and macrophage infiltration and secreted cytokines by the cells are involved in the pathogenesis20–22. Ure-MB was highly distributed to not only tumor but also spleen, and it seemed to lead to systemic toxicity by inducing CD137 signaling to normal tissues. Our findings suggest that the tumor-selective target-binding ability of STA551 avoids the systemic reaction caused by conventional anti-CD137 antibodies.
Ure-MB, a conventional anti-CD137 antibody, bound to cells in tumor and spleen. Ure-MB mainly bound to T cells and NK cells, consistent with cell populations in which CD137 expression was observed Fig. 2B or previously reported23. Thus, this suggests that Ure-MB bound to the cells in a CD137 dependent manner. There were two possible reasons that the binding of Ure-MB was greater than the expression of CD137. The first reason is that the administration of antibody increased the expression of CD137 molecules. CD137 agonist signals are known to activate T cells and other immune cells, leading to increased expression of CD13724. Ure-MB administered to mice might bind to CD137, introduce CD137 agonist signals in tumor and spleen and induce increased expression of CD137 in tissues. The second reason is the Fc region-mediated binding of Ure-MB. Ure-MB, isotype control antibody and Sta-MB have engineered Fc regions that bind to murine FcγRs, particularly FcγRII6, and FcγRII is predominantly expressed in the myeloid lineage cells25. Isotype control antibody bound to CD45+ cells, especially CD11b+ cells, suggesting that the antibody bound to the cell via Fc region. However, the binding of Ure-MB to CD11b+ cells was comparable with that of isotype control antibody in either tumor or spleen, suggesting that Fab-mediated binding is more prevalent. In addition, since the main Ure-MB binding cells were confirmed to be CD137 expressing cells, such as T cells and NK cells, binding of Ure-MB to cells was thought to be primarily Fab-mediated binding.
In the imaging experiment, fluorescence from all antibodies were detected in tumor. Table1 and suppl Fig. 1 show the concentration of Ure-MB, Sta-MB, and isotype control antibody in plasma, spleen, and tumor. The concentration of isotype control antibody in each tissue, especially in tumor, was higher than the other two antibodies. In image analysis, the difference could not be distinguished between the antibodies which were binding to target molecule and non-binding antibodies and/or bound nonspecifically in tissues. Isotype control antibody which was highly distributed to tumor might be detected as fluorescent signal in the imaging experiment.
STA551 is designed to bind to CD137 strongly in the presence of 100 µM ATP but not in the absence of ATP6. Murine ATP levels have been reported to be approximately 100 µM of extracellular ATP in tumor and 10-100 nM in normal tissues7,8,26. However, it is difficult to measure the exact ATP concentration in physiological conditions because ATP concentration changes depending on the sampling and measurement conditions due to degradation of ATP and release of intracellular ATP. This study revealed that Sta-MB showed binding in tumor and little binding to spleen. This data suggested that, in physiological conditions, tumor had ATP levels of 100 µM or higher and normal tissue had lower ATP levels. The present imaging results may be useful for estimating ATP levels of tissues under physiological conditions. In addition, human ATP levels have been reported to be 10-100 nM in normal tissues26 and more than 10 µM in tumor, and there is around 1,000-fold difference in ATP concentration between tumor and normal tissues, which is similar in mice27. Considering the similarity in ATP distributions in human and mouse tissues, it is anticipated that STA551 will also exhibit tumor-selective binding in humans. Thus, STA551 is expected to exert anti-tumor efficacy with tumor selective CD137 signals, while reducing systemic reaction, even in human patients.
In conclusion, we showed that STA551 distributes in tumor but little in spleen. Such STA551 distribution demonstrated more clearly the reason why STA551 work in tumor but not in normal tissues. Because of less distribution in normal tissues, STA551 could be a promising therapeutic antibody for patients with currently difficult-to-treat cancers.