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Communication

Establishment of Anti-Dog PD-L1 Monoclonal
Antibodies for Immunohistochemistry
Tsunenori Ouchida 1, Hiroyuki Suzuki 1,2,*, Tomohiro Tanaka 2, Mika K. Kaneko 1,2
and Yukinari Kato 1,2,*
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan; tsunenori.ouchida.d5@tohoku.ac.jp (T.O.);
k.mika@med.tohoku.ac.jp (M.K.K.)
2 Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan; tomohiro.tanaka.b5@tohoku.ac.jp* Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K);
Tel.: +81-22-717-8207 (H.S.&Y.K)

1

Abstract: 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 have developed the anti-dog programmed cell death ligand 1 (dPD-L1)
monoclonal antibodies (mAbs) and showed efficacy in several canine tumors. To examine theabundance of dPD-L1 in canine tumors, anti-dPD-L1 diagnostic mAbs for immunohistochemistry
are required. In this study, we immunized the peptide in the dPD-L1 intracellular domain, andestablished 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 high binding
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 thedetection of dPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-expressed
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

1. Introduction
Immune checkpoint blockade therapy has recently revolutionized the treatment and clinicaloutcome of several 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 mAbs against PD-1
receptor (nivolumab and pembrolizumab), PD-L1 (atezolizumab, durvalumab, and avelumab),
CTLA-4 (tremelimumab and ipilimumab) were approved by 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, progressionfree survival, and overall survival in patients with various types of cancers [3–5].
PD-L1 (CD274) is a type I membrane protein expressed on non-lymphoid cells and a ligand of PD-1
[6,7]. PD-1 plays a critical role in the negative regulation of immune cells including cytotoxic T lymphocyte(CTL) and is important for 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 (ZAP70) is recruited to the TCR-CD3 complex and phosphorylated by lymphocyte protein tyrosine kinase,which transduces the downstream signaling [9,10]. In contrast, SH2-containing protein tyrosine
phosphatase-2 (SHP-2) is recruited to PD-1 stimulated with PD-L1 [11]. Since SHP-2 dephosphorylates
phosphorylated ZAP-70, PD-1 can inhibit the activation signals in CTLs.

© 2023 by the author(s). Distributed under a Creative Commons CC BY license.

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With the increase in life span in both humans and dogs, the cancer incidence has increased aswell [12]. Several naturally occurring tumors in dogs resemble human tumors in many respects.
Therefore, the research on canine tumor therapy can generate knowledge that informs and prioritizes
new tumor therapy in humans.
The development of dog PD-L1 (dPD-L1) mAbs has been reported [13–17]. Using a canine
chimeric mAb targeting PD-L1, tumor regression of undifferentiated sarcoma and oral melanoma
was achieved [14,16,17]. On the other hand, tumors that did not respond to the treatment also existed.
Furthermore, the combination therapy of anti-dPD-L1 mAbs with hypofractionated radiotherapy ismore effective to prolong overall survival [18]. Therefore, the development of anti-dPD-L1 mAbs fordiagnostic use is essential for the improvement of efficacy.
In this study, we established anti-dog PD-L1 (dPD-L1) mAbs (L1Mab-352 and L1Mab-354) bypeptide immunization and showed the usefulness of the mAbs for immunohistochemical analysis inparaffin-embedded PD-L1-positive cells.
2. Materials and Methods
2.1. Preparation of cell lines
Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1 (P3U1) cells were obtained from theAmerican Type Culture Collection (Manassas, VA).
The synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding signal sequence of Nterminus (1-MRMFSVFTFMAYCHLLKA-18) deleted dPD-L1 (dPD-L1, Accession No.:
NM_001291972) was subsequently subcloned into a pCAGzeo_ssPA16 vector (IL2-signal sequenceand PA16 tag added to N-terminus of construct). The amino acid sequence of the tag system was asfollows: PA16 tag [19–22], sixteen amino acids (GLEGGVAMPGAEDDVV). The PA16 tag can bedetected by an anti-human podoplanin mAb (clone NZ-1) [19–35]. The dPD-L1 plasmid wastransfected into CHO-K1 cells, using a Neon transfection system (Thermo Fisher Scientific Inc.,
Waltham, MA). Stable transfectants were established through cell sorting 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-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 units/mL of
penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B (Nacalai Tesque, Inc.). All
cells were grown in a humidified incubator at 37oC, in an atmosphere of 5% CO2 and 95% air.
2.2. Production of hybridomas
A five-week-old BALB/c mouse was purchased from CLEA Japan (Tokyo, Japan). The animal
was housed under specific pathogen-free conditions. All animal experiments were approved by the
Animal Care and Use Committee of Tohoku University (Permit number: 2022MdA-001). The dPDL1 peptide (260-KKHGRMMDVEKC-271) and keyhole limpet hemocyanin-conjugated dPD-L1 peptide(dPD-L1 peptide-KLH) were purchased from Eurofins Japan.
To develop mAbs against dPD-L1, we intraperitoneally immunized one mouse with the dPDL1 peptide-KLH (100 μg) plus Imject Alum (Thermo Fisher Scientific, Inc.). The procedure included
three additional injections every week (100 μg), which were followed by a final booster
intraperitoneal injection (100 μg), two 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 units/mLof penicillin, 100 μg/mL of streptomycin, 0.25 μg/mL of amphotericin B, 5 μg/mL of Plasmocin, 5%
Briclone (NICB, Dublin, Ireland), and hypoxanthine, aminopterin and thymidine (HAT; Thermo
Fisher Scientific, Inc.). The supernatants were subsequently screened using enzyme-linked
immunosorbent assay (ELISA) using the dPD-L1 peptide.

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2.3. Purification of mAbs
The cultured supernatants of L1Mab-352 and L1Mab-354-producing hybridomas were filtrated
with Steritop (0.22 μm, Merck KGaA, Darmstadt, Germany). The filtered supernatants were
subsequently applied to 1 mL of Ab-Capcher ExTra (ProteNova, Inc., Kagawa, Japan). After washingwith phosphate-buffer saline (PBS), bound antibodies were eluted with an IgG elution buffer(Thermo Fisher Scientific, Inc.), followed by immediate neutralization of eluates, using 1M Tris-HCl(pH 8.0). Finally, the eluates were concentrated, after which PBS was replaced with the elution bufferusing Amicon Ultra (Merck KGaA).
2.4. ELISA
The dPD-L1 peptide was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher
Scientific, Inc.) at 1 μg/mL for 30 minutes at 37°C. 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 37°C. Then, plates were incubated at 1 μg/mL of L1Mab-352 and L1Mab-354, followed byperoxidase-conjugated anti-mouse immunoglobulins (1:1000; Agilent Technologies, Inc., Santa Clara,
CA). Finally, enzymatic reactions were conducted using the ELISA POD substrate TMB kit (Nacalai
Tesque, Inc.) and stopped by the addition of 1M H2SO4. The absorbance at 450 nm was measured byusing 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 μg/mL to 2.4 ng/mL. The KD was calculated by fitting saturation binding curves to the builtin; one-site binding models in GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA).
2.5. 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 (EDTA, Nacalai Tesque, Inc.). The cells were subsequently
washed with 0.1% BSA in PBS and treated with 1 μg/mL NZ-1 for 30 min at 4°C. The cells weretreated with 2 μg/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.).
2.6. Immunohistochemical analysis of paraffin-embedded CHO/dPD-L1
Cell blocks were produced using iPGell (Genostaff Co., Ltd., Tokyo, Japan). To deparaffinize,
rehydrate and retrieve antigen, the sections were autoclaved in EnVision FLEX Target Retrieval
Solution, High pH (high pH; #DM828, Agilent Technologies, Inc.) at 121°C for 20 minutes. Then,
sections were blocked using the Super Block T20 (PBS) Blocking Buffer (Thermo Fisher Scientific,Inc.), incubated with 50 μg/mL L1Mab-352 or L1Mab-354 for 1 h at room temperature, and treatedwith the Envision + Kit for mouse (Agilent Technologies, Inc.) for 30 min. Finally, color wasdeveloped using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 5min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan).
To inhibit L1Mab-352 and L1Mab-354 binding, dPD-L1 peptide (final concentration: 20 μg/mL)
was mixed with L1Mab-352 or L1Mab-354 (final concentration: 50 μg/mL). After incubation for 1.5 h
at room temperature, immunohistochemical analysis was performed.
3. Results
3.1. Establishment of novel dPD-L1 mAs.
For this study, we immunized a mouse with a KLH-conjugated synthetic peptide corresponding
to an intracellular region of dPD-L1. To produce hybridomas, the splenocytes from the mouse werefused 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 L1Mab352 (mouse IgG1, kappa) and L1Mab-354 (IgG1, kappa) were finally established (Figure 1).

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Figure 1. Establishment of novel dPD-L1 mAbs, L1Mab-352 and L1Mab-354. (A) The workflow ofthe establishment of 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 six days, hybridoma
supernatants were selected by ELISA. (B) Synthesized peptides of dPD-L1 peptide (1 μg/mL) were
immobilized on immunoplates for 30 min at 37℃. The plates were incubated with 1 μg/mL of L1Mab352 and L1Mab-354, followed by the treatment of peroxidase-conjugated anti-mouse
immunoglobulins. Optical density was measured at 450 nm (OD450) using a microplate reader. Error
bars represent means ± SDs (n = 3).

3.2. Kinetic analyses of L1Mab-352 and L1Mab-354 against the dPD-L1 peptide.
To determine the KD of L1Mab-352 and L1Mab-354 with the dPD-L1 peptide, we conducted the
kinetic analysis using ELISA. After immobilization of the dPD-L1 peptide on immunoplates, seriallydiluted L1Mab-352 and L1Mab-354 were added. The mean of the absorbance at 450 nm was plottedversus the concentrations of L1Mab-352 and L1Mab-354. The KD values of L1Mab-352 and L1Mab-354
for the dPD-L1 peptide were calculated as 6.9 ×10-10 M and 7.2 ×10-10 M, respectively (Figure 2).

Figure 2. The determination of the binding affinity of L1Mab-352 and L1Mab-354 by ELISA.
Synthesized peptides of dPD-L1 peptide (1 μg/mL) were immobilized on immunoplates for 30 min at
37℃. The plates were incubated with L1Mab-352 (A) and L1Mab-354 (B) at 2.4 ng/mL–40 μg/mL.,followed by the treatment of peroxidase-conjugated anti-mouse immunoglobulins. Optical density
was measured at 450 nm (OD450) using a microplate reader. The dissociation constant (KD) was
calculated using GraphPad PRISM 8. Error bars represent means ± SDs (n = 3).

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3.3. 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(Figure 3A) using L1Mab-352 and L1Mab-354. We first confirmed the cell surface expression of dPD-L1
using flow cytometry (Figure 3B). Then, we prepared the paraffin-embedded CHO-K1 and CHO/dPD-L1
cells, and stained the section using L1Mab-352 and L1Mab-354. As shown in Figure 3C,D, L1Mab-352 andL1Mab-354 stained to the section of CHO/dPD-L1, but not that of CHO-K1. These results indicated that
L1Mab-352 and L1Mab-354 can stain dPD-L1 in paraffin-embedded samples.

Figure 3. Immunohistochemical staining of paraffin-embedded CHO/dPD-L1 with L1Mab-352 andL1Mab-354. (A) The construction of dPD-L1. PA16 tag, the epitope of NZ-1, was tagged to the Nterminus of dPD-L1. The 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) andCHO-K1 (D) using 50 μg/mL of L1Mab-352 and L1Mab-354. Scale bar = 100 μm.

3.4. Peptide blocking of L1Mab-352 and L1Mab-354 in Immunohistochemical anayisis.
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 ofdPD-L1 peptide. This result indicated that the signals of staining with L1Mab-352 and L1Mab-354 arecaused by the specific binding to dPD-L1.

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Figure 4. The blockade of L1Mab-352 and L1Mab-354 reactivity by dPD-L1 peptide.
Immunohistochemical staining of paraffin-embedded CHO/dPD-L1 with L1Mab-352 (50 μg/mL) (A),
L1Mab-352 (50 μg/mL) plus dPD-L1 peptide (20 μg/mL) (B), L1Mab-354 (50 μg/mL) (C), and L1Mab354 plus dPD-L1 peptide (20 μg/mL) (D). Scale bar = 100 μm.

4. Discussion
Immune checkpoint blockade therapies have improved the patients’ outcomes with various types oftumors [36]. However, only 30% of patients receive the benefit from the therapy [37].
Immunohistochemistry analyses to determine the PD-L1-positive tumor cells are widely validated andused as predictive biomarkers to select the patients for immune checkpoint blockade therapy [38].
However, different diagnostic anti-PD-L1 mAb clones 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 adifferent cut-off point and cancer-specific scoring algorithm [2]. Both 22C3 and 28-8 recognize theextracellular domain of PD-L1, and exhibit similar subcellular patterns of PD-L1 expression in tumor andimmune cells [2]. However, different staining patterns by 22C3 and 28-8 are also reported [39].
In this study, we developed novel 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-L1-positive cells (Figures 3 and 4). Further studies are essential
to show whether L1Mab-352 and L1Mab-354 apply to formalin-fixed paraffin-embedded canine
tumors. An anti-dPD-L1 mAb (6C11-3A11) which recognizes the extracellular domain of dPD-L1 wasreported to apply to immunohistochemistry [17]. The comparison of the staining pattern of mAbs
targeting dPD-L1 extracellular and intracellular domains could provide supportive information inhuman 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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7Author Contributions: T.O. and T.T. performed the experiments. M.K.K. and Y.K. designed the experiments.
T.O., H.S., and Y.K. analyzed the data. T.O. and H.S. wrote the manuscript. All authors read and approved thefinal manuscript and agreed to be accountable for all aspects of the research in ensuring that the accuracy or
integrity of any part of the work is appropriately investigated and resolved. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was supported in part by the Japan Agency for Medical Research and Development(AMED) under grant nos. JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), 23bm1123027h0001 (to Y.K.),
JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.), and by the Japan Society for the Promotion of Science(JSPS) Grants-in-Aid for Scientific Research (KAKENHI), grant nos. 21K20789 (to T.T.), 22K06995 (to H.S.),
21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal Care and Use
Committee of Tohoku University (Permit number: 2022MdA-001) for studies involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: All related data and methods are presented in this paper. Additional inquiries
should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

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