**1. Introduction**

High-grade gliomas (HGGs) carry a dismal prognosis despite current treatments [1–4]. Options are particularly limited for patients with recurrent HGGs so new therapies are needed. Cancer vaccines are promising in this regard, designed to induce systemic immunity against antigens overexpressed by tumor cells and other components in the tumor microenvironment. Pilot clinical trials by us and others have exhibited the safety and potential efficacy of cytotoxic T lymphocyte (CTL) epitope peptide-based vaccinations for patients with HGGs [5–11].

Although cancer vaccines have been anticipated as a promising modality to treat cancer, recent reports indicated several mechanisms in tumor tissues that protect cancer cells from immune attacks [12]. For example, the limitation of the antitumor effects of CTLs was explained by interand intra-tumoral heterogeneity; a subset of tumor cells revealed downregulation, or loss of expression of human leukocyte antigen (HLA), or targeted antigen proteins [13,14]. To overcome the suppression of CTL antitumor effects, which occur due to tumor cell heterogeneity, we previously focused on a peptide vaccine targeting the tumor vasculature in the tumor microenvironment and demonstrated the safety and immunogenicity of vaccination with synthetic peptides for vascular endothelial growth factor receptor (VEGFR) epitopes in recurrent HGG patients [11].

Targeting of multiple glioma antigen epitopes also helps to address the issue of inter- and intra-tumoral heterogeneity of glioma cells. Furthermore, "oncoantigens" are ideal targets for a cancer vaccine [15–21] as they are essential for cell growth, and the probability of immune escape of cancer cells by reducing or lacking these proteins is expected to be low [22]. Therefore, this clinical trial was based on the use of HLA-A2402–restricted CTL epitopes derived from four glioma oncoantigens (GOAs) that we and others observed to be highly expressed in HGGs [23–26]: Lymphocyte antigen 6 family member K (LY6K), DEP domain containing 1 (DEPDC1), kinesin family member 20A (KIF20A), and forkhead box M1 (FOXM1)—in addition to two glioma angiogenesis-associated antigen (GAAAs): VEGFR1 and VEGFR2 [27,28].

To the best of our knowledge, this is the first study to evaluate a glioma vaccine therapy targeting tumor vasculature, as well as tumor cells with multiple glioma antigen epitope peptides derived from glioma cell-expressed oncoantigens and glioma angiogenesis factors. The primary objectives were to assess the tolerability of this regimen and its ability to induce GOA/GAAA epitope-specific immune responses.

#### **2. Materials and Methods**

The study protocol was approved by the institutional ethics committee (#20130294).

### *2.1. Vaccine Therapy Design*

This study was a non-randomized, open label clinical trial with cocktail peptide vaccines for recurrent/progressive HGGs. The primary endpoint of this study was the safety of the peptide vaccine treatment. Secondary endpoints were the GOA/GAAA epitope–specific immune responses and the therapeutic outcome of patients treated with this vaccine.
