MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 2, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0004

Monoclonal Antibody L1Mab-13 Detected
Human PD-L1 in Lung Cancers

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Shinji Yamada,1 Shunsuke Itai,1 Takuro Nakamura,1 Miyuki Yanaka,1
Yao-Wen Chang,1 Hiroyoshi Suzuki,2 Mika K. Kaneko,1 and Yukinari Kato1,3

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting
cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has
been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer
patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L1Mab-13 (IgG1, kappa) was produced using acell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using
flow cytometry, Western blot, and immunohistochemical analyses. L1Mab-13 specifically reacted hPD-L1 ofhPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (humanfibroblast) in flow cytometry and Western blot. Furthermore, L1Mab-13 reacted with lung cancer cell lines (EBC1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L1Mab-13, is very useful for
detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.
Keywords: programmed cell death-ligand 1, monoclonal antibody, lung cancer

IntroductionL

ung cancer is one of the commonest fatal malignant
tumors and is a leading global cause of cancer-related
deaths.(1) There exist two subtypes of lung cancers: smallcell lung cancer and nonsmall cell lung cancer. The former
includes squamous carcinoma, adenocarcinoma (AC), andlarge cell carcinoma.(2) Despite development in therapeutic
treatments for this disease, chemotherapy and radiotherapy
still demonstrate low efficacy because of its 5-year survival
rate being *15%.(3) Recently, some molecular targeteddrugs were shown to be effective for lung cancer under specificconditions and were consequently approved for clinicaluse.(4–7) In addition, several immune checkpoint inhibitors for lung cancer treatment have also been permitted.
These antibodies include nivolumab (fully human IgG4
monoclonal antibody [mAb] against programmed cell
death 1 [PD-1](8)), pembrolizumab (mouse–human IgG4chimeric antibody against PD-1(9,10)), and atezolizumab(mouse–human IgG1 chimeric antibody against programmed
cell death ligand-1 [PD-L1](11)).
PD-L1 is a type I transmembrane glycoprotein expressedin several cancers including breast, gastric, ovarian, pan1
2
3creatic as well as in renal cell carcinoma, melanoma,
and glioblastoma.(12–18) It is also an immune regulatory
molecule that limits T cell effector function.(19) Binding of
PD-L1 to its receptor PD-1 inhibits activated T cell proliferation and allows cancer cells evade host immune surveillance.(20,21) Furthermore, several studies have revealeda strong correlation between PD-L1/L2 expression on tumor cells and the poor prognosis in some cancers.(22–24) To
use nivolumab or pembrolizumab for lung cancer treatment, information about its effectiveness in detecting PDL1 expression in immunohistochemistry is necessary.(25)
Therefore, more sensitive and specific mAbs for detecting
PD-L1 are required. In this study, we generated a novelantihuman PD-L1 (hPD-L1) antibody, L1Mab-13 (mouse
IgG1, kappa).
Materials and Methods
Cell lines

LN229, CHO-K1, and P3U1 cell lines were obtained fromthe American Type Culture Collection (ATCC, Manassas,
VA). KMST-6, EBC-1, LK-2, Lu65, and Lu99 were obtained
from the Cell Resource Center for Biomedical Research

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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ANTI-PD-L1 MAB FOR LUNG CANCER

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FIG. 1. Development of anti-hPD-L1 mAbs. (A) Flow cytometry. Cells were treated with 1 mg/mL L1Mab-13 (red line)
and negative control (black line) followed by treatment with Alexa Fluor 488-conjugated antimouse IgG. (B) Western blot
analysis. Cell lysates (10 mg) were electrophoresed and proteins were transferred onto PVDF membranes. After blocking,membranes were incubated with 10 mg/mL of L1Mab-13 or 1 mg/mL of anti-b-actin (clone: AC-15) followed by peroxidaseconjugated antimouse IgG. mAb, monoclonal antibody; hPD-L1, human programmed cell death ligand-1.

Institute of Development, Aging and Cancer Tohoku University (Miyagi, Japan). PC-10 was purchased from ImmunoBiological Laboratories Co., Ltd. (Gunma, Japan). LN229/
hPD-L1 was produced by transfecting pCAG/PA-hPDL1-RAP-MAP into LN229 cells using a Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories, Inc., Berkeley,CA). CHO/hPD-L1 was produced by transfecting pCAG/hPDL1-RAP-MAP into CHO-K1 cells using Lipofectamine LTX
Reagent (Thermo Fisher Scientific, Inc., Waltham, MA).
The stable transfectants of LN229/hPD-L1 and CHO/hPD-L1
were established by limiting dilution. PA tag system(GVAMPGAEDDVV vs. clone: NZ-1), RAP tag system(DMVNPGLEDRIE vs. clone: PMab-2), and MAP tag
system (GDGMVPPGIEDK vs. clone: PMab-1) have beenpreviously established in Tohoku University Graduate
School of Medicine and described in detail.(26–28)
CHO-K1, CHO/hPD-L1, P3U1, KMST-6, EBC-1, LK-2,
Lu65, Lu99, and PC-10 cell lines were cultured in RPMI 1640
medium (Nacalai Tesque, Inc., Kyoto, Japan), and LN229 and
LN229/hPD-L1 were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere containing 5% CO2 and 95% air, supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc.). The culture medium was supplemented
with penicillin (100 units/mL), streptomycin (100 mg/mL), andamphotericin B (25 mg/mL) (all from Nacalai Tesque, Inc.).
Zeocin (0.5 mg/mL; InvivoGen, San Diego, CA) was added tothe culture medium of LN229/hPD-L1 and CHO-K1/hPD-L1.

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FIG. 2. Flow cytometry. Cells were treated with 1 mg/mL 29E.2A3 (positive control; blue line) and L1Mab-13 (red line)
followed by treatment with Alexa Fluor 488-conjugated antimouse IgG (negative control; black line).
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ANTI-PD-L1 MAB FOR LUNG CANCER

FIG. 3. Immunohistochemical analysis by L1Mab-13 against lung AC. (A, B) After antigen retrieval using citrate buffer(pH 6), sections were incubated with 2 mg/mL of Abcam28-8 for 1 hour at room temperature followed by treatment withEnvision+ antirabbit kit for 30 minutes. (C, D) After antigen retrieval using citrate buffer (pH 6), sections were incubatedwith 10 mg/mL of L1Mab-13 with Envision+ antimouse kit. Color was developed using DAB for 2 minutes, and sections
were then counterstained with hematoxylin. (E, F) Negative control, (G, H) HE; scale bar = 100 mm. DAB, 3,3diaminobenzidine tetrahydrochloride; HE, hematoxylin and eosin staining.
Human tissues

One lung AC cancer patient who underwent surgery at the
Sendai Medical Center was recruited for analysis. The study wasapproved by the ethical committee of Sendai Medical Center.
Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/
1-mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin in
phosphate-buffered saline, the cells were treated eitherwith 1 mg/mL of anti-hPD-L1 mAbs clone 29E.2A3 (BioLegend, Inc., San Diego, CA) or L1Mab-13 for 30 minutes
at 4C. The antibodies were subsequently observed using
Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA). Fluorescencedata were acquired using EC800 Cell Analyzers (SonyCorp., Tokyo, Japan).

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Western blot analysis

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.) and proteins werethen electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Industries., Ltd., Osaka, Japan)
and were transferred onto PVDF membranes (Merck KGaA,
Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), membranes were incubated with
10 mg/mL of anti-hPD-L1 (clone L1Mab-13) and 1 mg/mL ofanti-b-actin (clone AC-15; Sigma-Aldrich Corp., St. Louis,MO), and then with peroxidase-conjugated antimouse IgG(1:1000 diluted; Agilent Technologies, Inc., Santa Clara,
CA), and were finally developed using ImmunoStar LD(FUJIFILM Wako Pure Chemical Industries Ltd.) using a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

YAMADA ET AL.

In conclusion, L1Mab-13 is very useful for detecting hPDL1 using flow cytometry, Western blot, and immunohistochemical analysis of lung cancers. Studies investigating
L1Mab-13 reactivity for its applicability in other humancancers are warranted.
Acknowledgments

We thank Noriko Saidoh, Saori Handa, and Yoshimi
Nakamura for their excellent technical assistance. This
research was supported by AMED under Grant Numbers:
JP17am0301010 (Y.K.), JP17am0101078 (Y.K.), andJP17ae0101028 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.

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Immunohistochemical analyses

Histological sections of 4 mm thickness were autoclavedin citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo,
Japan) for 20 minutes. The sections were then incubatedwith 10 mg/mL of L1Mab-13 and 2 mg/mL of 28-8 (Agilent
Technologies, Inc.) for 1 hour at room temperature, andtreated using an Envision+ kit (antimouse; Agilent Technologies, Inc.) and Envision+ kit (antirabbit; Agilent Technologies, Inc.) for 30 minutes, respectively. Color wasdeveloped using 3,3-diaminobenzidine tetrahydrochloride(Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin (FUJIFILM Wako
Pure Chemical Industries., Ltd.).
Results and Discussion

We immunized one mouse with LN229/hPD-L1 cells andperformed flow cytometric screening. We previously named
this cell-based strategy as the cell-based immunization andscreening (CBIS) method.(29,30) Culture supernatants of 672
wells were mixed with LN229/hPD-L1 and LN229 cells, and27 wells (27/672; 4.0%), showing a stronger reaction against
LN229/hPD-L1 cells than LN229 cells, were selected. After
limiting dilution, 10 clones were established, of which 7 wereclassified as IgG1 subclass and 3 as IgG2b subclass. Only one
clone, L1Mab-13 (IgG1, kappa), reacted with lung AC tissue,
which was previously diagnosed as hPD-L1-positive in an
immunohistochemical analysis. In this study, we characterized L1Mab-13.
L1Mab-13 reacted with CHO/hPD-L1, but did not reactwith CHO-K1 (Fig. 1A). L1Mab-13 also detected endogenous hPD-L1 of human fibroblast cells (KMST-6), indicating
L1Mab-13 is useful for flow cytometry. Furthermore, L1Mab13 showed specific bands of hPD-L1 of CHO/hPD-L1 andKMST-6 in Western blot (Fig. 1B), indicating that L1Mab-13
is also useful for detecting hPD-L1 in Western blot.
L1Mab-13 recognized endogenous hPD-L1 in lung cancer
cell lines, such as EBC-1, Lu65, and Lu99 in a similar patternof clone 29E.2A3 using flow cytometry (Fig. 2). To further
investigate the utility of L1Mab-13 in human lung cancers,
we performed immunohistochemical analyses. Figure 3
shows that both 28-8 (positive control) and L1Mab-13 stained
lung cancer cells in a membrane-staining pattern, demonstrating the utility of L1Mab-13 in immunohistochemical
analysis in lung cancers.

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-Machi
Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinari-k@bea.hi-ho.ne.jp
Received: January 15, 2018
Accepted: March 7, 2018