**4. Discussion**

This is the first clinical evaluation of peptide-based vaccine therapy, targeting glioma cells as well as glioma neovascular endothelial cells, using multiple GOA/GAAA-derived epitopes for recurrent/progressive HGG. Our findings demonstrate tolerability and immunoreactivity to GOAs/GAAAs, as well as the preliminary efficacy of this treatment.

The population was very small and not homogeneous in this study. However, this was a pilot study to assess safety and immunoreactivity to the antigens, which allowed us to assess the tolerability and immune response regardless of patient characteristics.

The peptide epitopes included in this vaccine treatment were derived from six proteins known as GOAs or GAAAs [23–28]. ELISPOT data demonstrated that all evaluable vaccinated patients mounted an immune response against at least three of the target antigens, supporting the use of such epitopes in glioma vaccine regimens. ELISPOT data also showed that CTLs specific for three oncoantigens, DEPDC1, FOXM1, and LY6K were frequently observed in peripheral blood mononuclear cells from the vaccinated patients—indicating that these oncoantigens are highly immunogenic in advanced HGG patients. To evaluate if the induced CTLs contributed to reduction of tumor cells or tumor vascular endothelial cells in the microenvironment, further immunohistochemical analyses of tumor tissues obtained from vaccinated patients or blood flow analyses that can detect hypoperfusion peri-/intra-tumorally are warranted.

Although this was a pilot study focusing on safety and immunoreactivity to the antigens, we also evaluated treatment response in the vaccinated patients. In this study, the mOS in all patients and GB patients was 9.2 months and 9.1 months, respectively. Our median survival results are comparable to, but do not exceed those reported in the literature by previous clinical studies of glioma vaccines [9,10] and various combination regimens of chemotherapy and/or radiotherapy for recurrent GB patients [36–39]. This may be reflected in immune tolerance or a hostile immune status mediated by regulatory T-cell populations or tumor-secreted immunosuppressive factors in immunocompromised patients with recurrent HGG. Previous studies have demonstrated that anti-VEGF agents, such as bevacizumab, inhibit proliferation of immunosuppressive cells, such as regulatory T-cells and myeloid derived suppressor cells [40–42]—suggesting that VEGF-VEGFR pathway blockade could restore and improve antitumor immune responses. Nevertheless, the HGG patients who did not receive bevacizumab had a longer survival period than the patients treated with bevacizumab following a combination of chemotherapy and/or radiotherapy, although the sample size was relatively small (Figure 3). These results suggest that such approaches may be most effective if applied early in treatment, particularly in patients likely to have a robust immunity, such as the patients who have not yet received any chemotherapy or radiation therapy. In fact, cancer vaccines often need more time to elicit beneficial immune responses that demonstrate biological activity, which is shown by the occurrence of delayed vaccine effects.

**Figure 3.** Overall survival of glioblastoma patients with or without bevacizumab. The median overall survival time (mOS) was 23.6 months in three patients that did not receive bevacizumab before enrollment (solid line). The mOS was 8.6 months in four patients treated with bevacizumab before enrollment (dotted line).

One HGG patient (Patient 6) experienced objective clinical tumor regression (response rate of this vaccine treatment was 10%). Furthermore, it is noteworthy that this patient exhibited PR at week six and CR at week nine. CTLs specific for all six antigens were strongly induced in the patients, suggesting that this CTL response might contribute to the observable effect.

As biological features of HGGs in children are different from those that arise in adults [43,44], it is necessary to discuss the cases of children specifically. Our heterogenous patient cohort included a 17 year-old patient, whose OS was 8.9 months after enrollment. This vaccine therapy could not extend OS significantly, but could safely induce CTLs specific for three oncoantigens in this patient, suggesting that this vaccine therapy theoretically has the potential to exert an antitumor effect for pediatric HGGs expressing target antigens. Therefore, immunoreactivity to antigens and clinical efficacy of this regimen for children with HGGs will be assessed in a future study.

In summary, we performed a pilot study for HLA-A2402+ patients with recurrent/progressive HGGs to assess the safety, feasibility, and immunoreactivity of the peptide-based vaccine targeting GOAs and GAAAs. The safety and immunogenicity of this vaccine therapy was verified. The data suggest that this vaccine treatment may show preliminary evidence of clinical responses. However, a future study of this vaccine in combination with standard treatment for newly-diagnosed HGGs as well as immune-checkpoint blockade therapies, is required to improve the efficacy of glioma vaccine therapy.

**Author Contributions:** Conceptualization, R.U. and M.T.; formal analysis, R.K. and S.N.; investigation, R.K., K.S., S.S., H.N., R.T., Y.M., H.S., and M.T.; resources, S.N., Y.K., and K.Y.; data curation, R.K.; writing—original draft preparation, R.K.; writing—review and editing, R.U. and M.T.; visualization, R.K.; supervision, Y.K. and K.Y.; project administration, M.T.; funding acquisition, R.K., R.U., and M.T.

**Funding:** This research was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B), Grant Number 17K16661.

**Acknowledgments:** We thank Yuko Aikawa and Tomoko Muraki (Department of Neurosurgery, Keio University School of Medicine) for their technical assistance. We also thank Gillian Campbell, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
