Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4

Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L1Mab-4 (IgG2b, kappa), using cell-based immunization and screening (CBIS) method and investigated hPD-L1 expression in oral cancers. L1Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4) in flow cytometry and stained oral cancers in a membrane-staining pattern. L1Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of L1Mab-4. These results indicate that L1Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers.


Introduction
Oral cancer is the eleventh highest of all cancer types [1] and constitutes approximately 2% of all cancer cases worldwide [2]. In oral cancers, there are certain histological tumors, including squamous cell carcinoma (SCC), adenosquamous cell carcinoma, adenoid cystic carcinoma, osteosarcoma, and mucoepidermoid carcinoma. SCC is the most common type, accounting for > 90% of oral cancers [3]. Because of improvements and progression in therapeutic techniques such as surgery, chemotherapy, and radiotherapy, the 5-year survival rate has reached > 80% [4,5]. In contrast, it is sometimes difficult to provide sufficient therapeutic effects because of the risks of adverse events [6][7][8].
Recently, tumor immunotherapies, which is focused on several immune checkpoint molecules, such as programmed cell death-1 (PD-1), programmed cell death-ligand 1 (PD-L1), and cytotoxic T-lymphocyteassociated antigen 4 (CTLA-4), have emerged. Clinically, nivolumab, a complete human IgG 4 against PD-1, was firstly approved for the treatment of recurrent and/or metastatic head and neck cancer, which was previously treated with platinum-based chemotherapy [9]. Avelumab, a complete human IgG 1 against PD-L1, was recently approved for the treatment of metastatic Merkel cell carcinoma [10,11], which is detected in oral cavity [12].
PD-L1, also known as B7-H1 and CD274, is a type I transmembrane glycoprotein, which is expressed on antigen-presenting cells and some tumor cells, including melanoma, ovarian, and lung cancer cells [13][14][15]. PD-L1 is a ligand for PD-1 and is involved in inhibiting T-cell effector functions [16], leading to the escape of tumor cells from immune response. Recent studies have revealed a strong correlation between PD-L1/PD-L2 expression on tumor cells and negative prognosis in cancer patients [17][18][19].
Lin et al. have revealed a correlation between high PD-L1 expression and metastasis and poor prognosis in oral SCC [20]. Several reports have shown that PD-L1 could be an effective target for treatment [21][22][23][24][25][26]. However, PD-L1 expression in oral cancers has not been completely investigated. In this study, we established a novel anti-PD-L1 antibody and performed immunohistochemistry for oral cancers.

Immunohistochemical analyses
Histological sections of 4-μm thickness were deparaffinized in xylene and subsequently rehydrated and autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) or EnVision FLEX Target Retrieval Solution, High pH (Agilent Technologies Inc., Santa Clara, CA) for 20 min. Sections were then incubated with 10 μg/mL of L 1 Mab-4 for 1 h at room temperature, treated using an Envision+ kit (Agilent Technologies Inc.) for 30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and counterstained with hematoxylin (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Immunohistochemical analysis against oral cancer tissues
We further investigated the immunohistochemical utility of L 1 Mab-4 in human oral cancers. First, we performed immunohistochemical analysis using two different conditions of antigen retrieval. Fig. 3 shows that antigen retrieval using citrate buffer (pH 6) revealed a better staining pattern (Fig. 3A and B) than did EnVision FLEX Target Retrieval Solution, High pH ( Fig. 3C and D). Therefore, we employed citrate buffer (pH 6) for antigen retrieval in this study. L 1 Mab-4 stained oral cancer cells in a membrane-staining pattern, indicating that L 1 Mab-4 is very useful for immunohistochemical analysis against oral cancers.  4. Immunohistochemical analysis of L 1 Mab-4 against oral cancers (tissue microarray). After antigen retrieval, sections were incubated with 10 μg/mL of L 1 Mab-4 followed by treatment with Envision+ kit. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB), and sections were counterstained with hematoxylin. Scale bar = 100 µm.
In conclusion, we successfully produced L 1 Mab-4, a novel anti-hPD-L1 mAb, using the CBIS method. This method is very advantageous because it does not require the membrane protein to be purified in all steps of mAb production. L 1 Mab-4 is very useful in the detection of hPD-L1 using flow cytometry and immunohistochemical analysis. In the near future, studies investigating the reactivity of L 1 Mab-4 for other human cancers need to be conducted. Furthermore, we should consider the possibility that L 1 Mab-4 might cross-react with other membrane proteins or react with post-translational modification, including glycosylation, on PD-L1 because the reaction of L 1 Mab-4 is much higher than those of 29E.2A3 against LN229 and HO-1-u-1 (Figs. 1 and 2).