MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 1, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0014
Open camera or QR reader andscan code to access this article
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Establishment of Anti-Dog Programmed Cell Death Ligand 1
Monoclonal Antibodies for Immunohistochemistry

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Tsunenori Ouchida,* Hiroyuki Suzuki,* Tomohiro Tanaka, Mika K. Kaneko, and Yukinari Kato

Immune checkpoint blockade therapy has shown successful clinical outcomes in multiple human cancers. In
dogs, several types of tumors resemble human tumors in many respects. Therefore, several groups havedeveloped the anti-dog programmed cell death ligand 1 (dPD-L1) monoclonal antibodies (mAbs) and showed
efficacy in several canine tumors. To examine the abundance of dPD-L1 in canine tumors, anti-dPD-L1
diagnostic mAbs for immunohistochemistry are required. In this study, we immunized the peptide in the dPDL1 intracellular domain, and established anti-dPD-L1 mAbs, L1Mab-352 (mouse IgG1, kappa), and L1Mab-354(mouse IgG1, kappa). In enzyme-linked immunosorbent assay, L1Mab-352 and L1Mab-354 showed highbinding affinity to the dPD-L1 peptide, and the dissociation constants (KD) were determined as 6.9 · 10-10 M
and 7.2 · 10-10 M, respectively. Furthermore, L1Mab-352 and L1Mab-354 were applicable for the detection ofdPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-overexpressed cells. These results
indicated that L1Mab-352 and L1Mab-354 are useful for detecting dPD-L1 in immunohistochemical analysis.
Keywords: dPD-L1, monoclonal antibody, peptide immunization, immunohistochemistry

antitumor immunity. CTLs are activated by recognizing
presented antigens by T cell receptor (TCR)-CD3 complex.8
After the recognition, zeta-chain associated protein kinase-70(ZAP-70) is recruited to the TCR-CD3 complex and phosphorylated by lymphocyte protein tyrosine kinase, whichtransduces the downstream signaling.9,10 In contrast, SH2containing protein tyrosine phosphatase-2 (SHP-2) is recruited
to PD-1 stimulated with PD-L1.11 Since SHP-2 dephosphorylates ZAP-70, PD-1 can inhibit the activation signals in
CTLs.
With the increase in life span in both humans and dogs, thecancer incidence has increased as well.12 Several naturally
occurring tumors in dogs resemble human tumors in many
respects. Therefore, the research on canine tumor therapy cangenerate knowledge that informs and prioritizes new tumor
therapy in humans.
The development of dog PD-L1 (dPD-L1) mAbs has beenreported.13–17 Using a canine chimeric mAb targeting dPDL1, tumor regression of undifferentiated sarcoma and oral
melanoma was achieved.14,16,17 On the other hand, tumorsthat did not respond to the treatment also existed.

Introduction

I

mmune checkpoint blockade therapy has recently
revolutionized the treatment and clinical outcome ofseveral cancer types.1 The therapies targeting programmed
cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1),
and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)
have become the standard therapy for several human tumors.
The therapeutic monoclonal antibodies (mAbs) against PD-1
(nivolumab and pembrolizumab), PD-L1 (atezolizumab,
durvalumab, and avelumab), and CTLA-4 (tremelimumab
and ipilimumab) were approved by U.S. Food and Drug
Administration (FDA).2 These mAbs have been used in either
monotherapy or combinatorial therapy with chemotherapy,
radiation therapy, or other modalities.1 These antibodies
improve the response rates, progression-free survival, andoverall survival in patients with various types of cancers.3–5
PD-L1 (CD274) is a type I membrane protein expressed on
nonlymphoid cells and a ligand of PD-1.6,7 PD-1 plays acritical role in the negative regulation of immune cells including cytotoxic T lymphocyte (CTL) and is important for

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
*These authors contributed equally to this work.

17

18

Furthermore, the combination therapy of anti-dPD-L1 mAbs
with hypofractionated radiotherapy is more effective toprolong overall survival.18 Therefore, the development ofanti-dPD-L1 mAbs for diagnostic use is essential for the
improvement of efficacy.
In this study, we established dPD-L1 mAbs (L1Mab-352
and L1Mab-354) by peptide immunization and showed theusefulness of the mAbs for immunohistochemical analysis inparaffin-embedded dPD-L1-positive cells.
Materials and Methods

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Preparation of cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA).
The synthesized DNA (Eurofins Genomics KK,Tokyo, Japan) encoding signal sequence of N-terminus(1-MRMFSVFTFMAYCHLLKA-18)-deleted dPD-L1 (Accession No.: NM_001291972) was subsequently subcloned
into a pCAGzeo_ssPA16 vector (IL2-signal sequence andPA16 tag were added to N-terminus of construct). The aminoacid sequence of the PA16 tag was GLEGGVAMPGAEDDVV.19–22 The PA16 tag can be detected by an antihuman podoplanin mAb (clone NZ-1).19–35 The dPD-L1
plasmid was transfected into CHO-K1 cells, using a Neontransfection system (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were established through cellsorting using a cell sorter (SH800; Sony Corp., Tokyo, Japan), after which cultivation in a medium, containing
0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA) was
conducted.
CHO-K1, PA16-tagged dPD-L1-overexpressed CHO-K1(CHO/dPD-L1), and P3U1 cells were cultured in a Roswell
Park Memorial Institute (RPMI)-1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan), with 10% heat-inactivated fetal
bovine serum (FBS; Thermo Fisher Scientific. Inc.),
100 U/mL of penicillin, 100 lg/mL of streptomycin, and0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.). All
cells were grown in a humidified incubator at 37C, in an
atmosphere of 5% CO2 and 95% air.
Production of hybridomas

A 5-week-old BALB/c mouse was purchased from CLEA
Japan (Tokyo, Japan). The animal was housed under specificpathogen-free conditions. All animal experiments were approved by the Animal Care and Use Committee of Tohoku
University (Permit number: 2022MdA-001). The dPD-L1
peptide (260-KKHGRMMDVEKC-271) and keyhole limpet
hemocyanin (KLH)-conjugated dPD-L1 peptide (dPD-L1
peptide-KLH) were purchased from Eurofins Japan.
To develop mAbs against dPD-L1, we intraperitoneallyimmunized one mouse with the dPD-L1 peptide-KLH(100 lg) plus Imject Alum (Thermo Fisher Scientific, Inc.).
The procedure included three additional injections every
week (100 lg), which were followed by a final booster intraperitoneal injection (100 lg), 2 days before harvesting
splenocytes. The harvested splenocytes were subsequently
fused with P3U1 cells, using PEG1500 (Roche Diagnostics,
Indianapolis, IN). For the hybridoma selection, cells were
cultured in the RPMI-1640 medium with 10% FBS, 100 U/mL

OUCHIDA ET AL.

of penicillin, 100 lg/mL of streptomycin, 0.25 lg/mL ofamphotericin B, 5 lg/mL of Plasmocin, 5% Briclone (NICB,
Dublin, Ireland), and hypoxanthine, aminopterin, and thymidine (Thermo Fisher Scientific, Inc.). The supernatants were
subsequently screened using enzyme-linked immunosorbent
assay (ELISA) using the dPD-L1 peptide.
Purification of mAbs

The cultured supernatants of L1Mab-352 and L1Mab-354producing hybridomas were filtrated with Steritop (0.22 lm;
Merck KGaA, Darmstadt, Germany). The filtered supernatants were subsequently applied to 1 mL of Ab-Capcher
ExTra (ProteNova, Inc., Kagawa, Japan). After washing withphosphate-buffered saline (PBS), bound antibodies were
eluted with an IgG elution buffer (Thermo Fisher Scientific,Inc.), followed by immediate neutralization of eluates, using
1 M Tris-HCl (pH 8.0). Finally, the eluates were concentrated, after which buffer was replaced with PBS using
Amicon Ultra (Merck KGaA).
ELISA

The dPD-L1 peptide was immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific, Inc.) at 1 lg/mL
for 30 minutes at 37C. After washing with PBS containing 0.05%
Tween20 (PBST; NacalaiTesque, Inc.), wells were blocked with
1% bovine serum albumin (BSA) in PBST for 30 minutes at 37C.
Then, plates were incubated at 1 lg/mL of L1Mab-352 andL1Mab-354, followed by peroxidase-conjugated anti-mouse
immunoglobulins (1:1000; Agilent Technologies, Inc., Santa
Clara, CA). Finally, enzymatic reactions were conductedusing the ELISA POD substrate TMB kit (Nacalai Tesque,Inc.) and stopped by the addition of 1 M H2SO4. The absorbance at 450 nm was measured by using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
To determine the dissociation constant (KD), L1Mab-352
and L1Mab-354 were serially diluted from 40 mg/mL to
2.4 ng/mL. The KD was calculated by fitting saturation
binding curves to the built-in; one-site binding models in
GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA).
Flow cytometric analysis

CHO-K1 and CHO/dPD-L1 cells were harvested after a brief
exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells were subsequently washed with 0.1% BSA in PBS and treated with
1 lg/mL NZ-1 for 30 minutes at 4C. NZ-1 recognizes PA16
tag at the N-terminus of dPD-L1. The cells were treated with
2 lg/mL Alexa Fluor 488-conjugated anti-rat IgG (Cell Signaling Technology, Inc., Danvers, MA). The fluorescence data
were collected using the EC800 Cell Analyzer (Sony Corp.).
Immunohistochemical analysis
of paraffin-embedded CHO/dPD-L1

Cell blocks were produced using iPGell (Genostaff Co.,
Ltd., Tokyo, Japan) and 4% paraformaldehyde (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan). To deparaffinize, rehydrate, and retrieve antigen, the sections fromparaffin-embedded cell blocks were autoclaved in EnVision
FLEX Target Retrieval Solution, High pH (Agilent Technologies, Inc.) at 121C for 20 minutes. Then, sections wereESTABLISHMENT OF ANTI-DOG PD-L1 MAB

blocked using the Super Block T20 (PBS) Blocking Buffer(Thermo Fisher Scientific, Inc.), incubated with 50 lg/mL
L1Mab-352 and L1Mab-354 for 1 hour at room temperature,and treated with the Envision + Kit for mouse (Agilent
Technologies, Inc.) for 30 minutes. Finally, color was developed using 3,3¢-diaminobenzidine tetrahydrochloride(Agilent Technologies, Inc.) for 5 minutes, and counterstaining was performed using hematoxylin (FUJIFILM Wako
Pure Chemical Corporation).
To inhibit L1Mab-352 and L1Mab-354 binding, dPD-L1
peptide (final concentration: 20 lg/mL) was mixed with
L1Mab-352 and L1Mab-354 (final concentration: 50 lg/mL).
After incubation for 1.5 hour at room temperature, immunohistochemical analysis was performed.
Results

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Establishment of novel anti-dPD-L1 mAbs

For this study, we immunized a mouse with a KLHconjugated synthetic peptide corresponding to an intracellularregion of dPD-L1. To produce hybridomas, the splenocytes
from the mouse were fused with P3U1 cells by using polyethylene glycol. The wells which reacted with the synthetic peptide
were selected by ELISA. After limiting dilution and additional
screening, two clones L1Mab-352 (mouse IgG1, kappa) andL1Mab-354 (IgG1, kappa) were finally established (Fig. 1).
Binding affinity analysis of L1Mab-352 and L1Mab-354
against the dPD-L1 peptide

To determine the KD of L1Mab-352 and L1Mab-354 withthe dPD-L1 peptide, we conducted the binding affinity
analysis using ELISA. After immobilization of the dPD-L1

19

peptide on immunoplates, serially diluted L1Mab-352 andL1Mab-354 were added. The mean of the absorbance at
450 nm was plotted versus the concentrations of L1Mab-352
and L1Mab-354. The KD values of L1Mab-352 and L1Mab354 for the dPD-L1 peptide were calculated as 6.9 · 10-10 M
and 7.2 · 10-10 M, respectively (Fig. 2).

Immunohistochemical analysis of L1Mab-352
and L1Mab-354 using paraffin-embedded CHO/
dPD-L1

We next performed immunohistochemical analysis against
paraffin-embedded CHO/dPD-L1 cells (Fig. 3A) using
L1Mab-352 and L1Mab-354. We first confirmed the cellsurface expression of dPD-L1 using flow cytometry(Fig. 3B). Then, we prepared the paraffin-embedded CHOK1 and CHO/dPD-L1 cells and stained the section using
L1Mab-352 and L1Mab-354. As shown in Figure 3C and D,
L1Mab-352 and L1Mab-354 stained the section of CHO/dPDL1, but not that of CHO-K1. These results indicated that
L1Mab-352 and L1Mab-354 can stain dPD-L1 in paraffinembedded samples.

Peptide blocking of L1Mab-352 and L1Mab-354
in immunohistochemical analysis

To confirm the specificity, we performed peptide blocking
in the immunohistochemical analysis. As shown in Figure 4,
the reactivity of L1Mab-352 and L1Mab-354 was blocked in
the presence of dPD-L1 peptide. This result indicated that thesignals of staining with L1Mab-352 and L1Mab-354 arecaused by the specific binding to dPD-L1.

FIG. 1. Establishment of novel anti-dPD-L1 mAbs, L1Mab-352, and L1Mab-354. (A) The workflow of the establishmentof L1Mab-352 and L1Mab-354. First, dPD-L1 peptide conjugated with KLH was immunized into a mouse. The splenocytes
were then fused with P3U1 cells. After 6 days, hybridoma supernatants were screened by ELISA. (B) The dPD-L1 peptide(1 mg/mL) was immobilized on immunoplates for 30 minutes at 37C. The plates were incubated with 1 mg/mL of L1Mab352 and L1Mab-354, followed by the treatment of peroxidase-conjugated anti-mouse immunoglobulins. OD450 was
measured using a microplate reader. Error bars represent means – SDs (n = 3). dPD-L1, dog programmed cell death ligand 1;
ELISA, enzyme-linked immunosorbent assay; KLH, keyhole limpet hemocyanin; mAbs, monoclonal antibodies; OD450,
optical density was measured at 450 nm; SD, standard deviation.

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FIG. 2. The determination of the binding affinity of L1Mab-352 and L1Mab-354 by ELISA. The dPD-L1 peptide(1 mg/mL) was immobilized on immunoplates for 30 minutes at 37C. The plates were incubated with L1Mab-352 (A) andL1Mab-354 (B) at 2.4 ng/mL–40 mg/mL, followed by the treatment of peroxidase-conjugated anti-mouse immunoglobulins.
OD450 was measured using a microplate reader. The KD was calculated using GraphPad PRISM 6. Error bars representmeans – SDs (n = 3). KD, dissociation constant.

FIG. 3. Immunohistochemical staining of paraffin-embedded CHO/dPD-L1 with L1Mab-352 and L1Mab-354. (A) The
construction of dPD-L1. PA16 tag, the epitope of NZ-1, was tagged to the N-terminus of dPD-L1. The sequence of dPD-L1
peptide is located in the intracellular domain. (B) The cell surface expression of dPD-L1 was confirmed by flow cytometry
using NZ-1. Red line: NZ-1. Black line: negative control. (C, D) Immunohistochemical analysis of paraffin-embedded CHO/
dPD-L1 (C) and CHO-K1 (D) using 50 mg/mL of L1Mab-352 and L1Mab-354. Scale bar = 100 mm. CHO, Chinese hamster
ovary. NC, negative control.
20

ESTABLISHMENT OF ANTI-DOG PD-L1 MAB

21
Author Disclosure Statement

No competing financial interests exist.
Funding Information

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This research was supported in part by the Japan Agency
for Medical Research and Development (AMED) under
grant nos. JP23ama121008 (to Y.K.), JP23am0401013(to Y.K.), JP23bm1123027 (to Y.K., JP22ck0106730(to Y.K.), and JP21am0101078 (to Y.K.), and by the Japan
Society for the Promotion of Science ( JSPS) Grants-inAid for Scientific Research (KA-KENHI), grant nos.
21K20789 (to T.T.), 22K06995 (to H.S.), 21K07168(to M.K.K.), 23K19494 (to T.O.), and 22K07224(to Y.K.).

FIG. 4. The blockade of L1Mab-352 and L1Mab-354 reactivity by dPD-L1 peptide. Immunohistochemical stainingof paraffin-embedded CHO/dPD-L1 with L1Mab-352(50 mg/mL) (A), L1Mab-352 (50 mg/mL) plus dPD-L1 peptide (20 mg/mL) (B), L1Mab-354 (50 mg/mL) (C), and
L1Mab-354 plus dPD-L1 peptide (20 mg/mL) (D). Scalebar = 100 mm.
Discussion

Immune checkpoint blockade therapies have improved thepatients’ outcomes with various types of tumors.36 However,
only 30% of patients receive the benefit from the therapies.37
Immunohistochemical analyses to determine the PD-L1positive tumor cells are widely validated and used as predictive
biomarkers to select the patients for immune checkpoint blockade therapy.38 However, different diagnostic anti-PD-L1 mAbclones were approved for specific therapeutic ones by FDA. For
instance, clone 22C3 has been utilized as a predictive biomarker
for pembrolizumab in several cancers. A clone 28-8 has been
approved as a complementary assay for nivolumab. Each clone
has a different cut-off point and cancer-specific scoring algorithm.2 Both 22C3 and 28-8 recognize the extracellular domain
of PD-L1 and exhibit similar subcellular patterns of PD-L1 expression in tumor and immune cells.2 However, different staining patterns by 22C3 and 28-8 are also reported.39
In this study, we developed novel anti-dPD-L1 mAbs
(L1Mab-352 and L1Mab-354), using peptide immunization of
the intracellular domain and showed the usefulness for immunohistochemical analysis in paraffin-embedded dPD-L1positive cells (Figs. 3 and 4). Since the paraffin-embedded
CHO/dPD-L1 cells were sliced during the preparation of samples, L1Mab-352 and L1Mab-354 could access the intracellulardomain of dPD-L1. Further studies are necessary to showwhether L1Mab-352 and L1Mab-354 apply to formalin-fixedparaffin-embedded canine tumors. An anti-dPD-L1 mAb(6C11-3A11) which recognizes the extracellular domain ofdPD-L1 was reported to apply to immunohistochemistry.17 The
comparison of the staining pattern of mAbs targeting dPD-L1
extracellular and intracellular domains could provide supportive
information in human cancer diagnosis.
Currently, several anti-dPD-L1 mAbs have been developed for canine tumor therapy.16,17,40 Like human tumors,
standardization of PD-L1 immunohistochemistry will be required in the future.

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23

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: August 22, 2023
Accepted: September 29, 2023