(2023) 54:82
Okagawa et al. Veterinary Researchhttps://doi.org/10.1186/s13567-023-01213-6

RESEARCH ARTICLE

Veterinary Research

Open Access

Development of a high‑affinity anti‑bovine
PD‑1 rabbit–bovine chimeric antibody using
an efficient selection and large production
systemTomohiro Okagawa1, Satoru Konnai1,2,3* , Shinya Goto2, Yamato Sajiki2, Otgontuya Ganbaatar2, Kei Watari2,
Hayato Nakamura2, Cai‑Xia Wang4, Taro Tachibana4,5, Yukinari Kato6,7, Yayoi Kameda8, Junko Kohara9,
Nobuhiro Terasaki10, Manabu Kubota10, Akira Takeda10, Hirofumi Takahashi10, Yasuhiko Suzuki1,3,8,11,
Naoya Maekawa1, Shiro Murata1,2 and Kazuhiko Ohashi1,2,12Abstract
Immune checkpoint molecules PD-1/PD-L1 cause T-cell exhaustion and contribute to disease progression in chronic
infections of cattle. We established monoclonal antibodies (mAbs) that specifically inhibit the binding of bovine PD-1/
PD-L1; however, conventional anti-PD-1 mAbs are not suitable as therapeutic agents because of their low binding
affinity to antigen. In addition, their sensitivity for the detection of bovine PD-1 is low and their use for immunostain‑
ing PD-1 is limited. To address these issues, we established two anti-bovine PD-1 rabbit mAbs (1F10F1 and 4F5F2)
and its chimeric form using bovine IgG1 (Boch1D10F1), which exhibit high binding affinity. One of the rabbit mAb
1D10F1 binds more strongly to bovine PD-1 compared with a conventional anti-PD-1 mAb (5D2) and exhibitsmarked inhibitory activity on the PD-1/PD-L1 interaction. In addition, PD-1 expression in bovine T cells could be
detected with higher sensitivity by flow cytometry using 1D10F1. Furthermore, we established higher-producing cellsof Boch1D10F1 and succeeded in the mass production of Boch1D10F1. Boch1D10F1 exhibited a similar binding affin‑
ity to bovine PD-1 and the inhibitory activity on PD-1/PD-L1 binding compared with 1D10F1. The immune activation
by Boch1D10F1 was also confirmed by the enhancement of IFN-γ production. Finally, Boch1D10F1 was administered
to bovine leukemia virus-infected cows to determine its antiviral effect. In conclusion, the high-affinity anti-PD-1 anti‑
body developed in this study represents a powerful tool for detecting and inhibiting bovine PD-1 and is a candidate
for PD-1-targeted immunotherapy in cattle.
Keywords Monoclonal antibodies, programmed death-1, cattle, antibody production

Handling editor: Tom McNeilly*Correspondence:
Satoru Konnai
konnai@vetmed.hokudai.ac.jp
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Okagawa et al. Veterinary Research(2023) 54:82

Introduction
Programmed death-1 (PD-1) is an immune checkpoint
molecule that interacts with its ligand PD-ligand 1(PD-L1) to cause the functional exhaustion of T cells,
which is evident through the loss of effector function by
inhibiting T-cell receptor (TCR) signaling [1–3]. During chronic infection, PD-1 is upregulated on T cells asa result of persistent stimulation via TCR, co-receptors,
and cytokines [4]. In cattle, the functional exhaustionof T cells is caused by the upregulation of the PD-1/
PD-L1 pathway and results in disease progression during chronic infections, such as bovine leukemia virus(BLV) infection [5–8]. BLV is a retrovirus which infects
bovine B cells and develops into a lifelong infection in
cattle. It can also result in an aggressive lymphoma,
known as enzootic bovine leukosis (EBL), in 1–5% of
the infected cattle after a long latent period [9]. In previous studies, we developed anti-bovine PD-1 and antibovine PD-L1 rat-bovine chimeric antibodies (chAbs)
in a mammalian expression system and tested theirantiviral effects in clinical studies using BLV-infected
cattle [10–12]. Blockade of the binding of PD-1/PD-L1
by these chAbs restored T-cell function and exerted
antiviral effects in the BLV-infected cattle [11, 12].
Although the antiviral effect was observed bytreatment with anti-bovine PD-1 rat-bovine chAb
(Boch5D2), the binding affinity of Boch5D2 to bovine
PD-1 was relatively low compared with that of commercial anti-human PD-1 blocking antibodies for immunotherapy [13, 14]. Recently, novel therapeutic antibodies
with higher binding affinities to PD-1 have been developed to enhance the clinical benefit of immunotherapy in humans [13, 14]. In addition, the application of
immunostaining for flow cytometry using anti-bovine
PD-1 rat mAb (5D2) established in a previous study [7]was also limited because of its low binding affinity [10].
Indeed, PD-1 expression was detectable by flow cytometry using 5D2 only in T cells of the cattle experiencing
a robust stimulation by antigens during chronic infection [8, 15–17]. To address the mechanism of T-cell
exhaustion mediated by PD-1 in various bovine diseases, a novel mAb capable of detecting bovine PD-1
with high sensitivity in the flow cytometric analysis isrequired.
Mice and rats are widely used for immunization to
generate mAbs; however, the use of rabbits for the mAbdevelopment has attracting interest because of theirunique and highly distinctive antibody repertoire, which
exhibits high-affinity, specificity, and diversity [18].
Indeed, previous studies have been succeeded in developing high-affinity mAbs to several antigens using rabbits[19–21]. These findings prompted us to establish a rabbit
mAb against bovine PD-1.

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Gram-scale quantities of antibody are required forclinical trials of therapeutic antibodies in adult cattle. We have established an expression system for large
amounts of antibody using a mammalian cell line andan engineered expression plasmid with a DHFR marker
for efficient selection of transfected cells and a ubiquitous chromatin opening element (UCOE) to reduce
DNA methylation in the transgene promoter [22, 23]. We
applied this system for the large production of chAb for aclinical trial in cattle.
In this study, we generated an anti-bovine PD-1 rabbitmAb (1D10F1) and rabbit–bovine chAb (Boch1D10F1)
which exhibit high-affinity to bovine PD-1. The biochemical properties of these antibodies were compared with
anti-bovine PD-1 rat mAb (5D2) and chAb (Boch5D2)
which were established previously [7, 10]. Accordingly,
mass production of Boch1D10F1 was achieved andadministration of Boch1D10F1 to BLV-infected cattle
was conducted to evaluate the antiviral effects of thishigh-affinity antibody.

Materials and methods
Generation of anti‑bovine PD‑1 rabbit mAbs

A rabbit was immunized with recombinant bovine PD1-Ig fusion protein [24] in TiterMax Gold Adjuvant(Sigma–Aldrich, St. Louis, MO, USA). Lymphocytes
were collected from the immunized rabbit 3 weeks later,
were fused with SP2 myeloma cells. Culture supernatantsfrom heterohybridomas [25, 26] were screened using
ELISA with recombinant polyhistidine-tagged bovine
PD-1 protein (BoPD-1-His) [10] and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (H + L) antibody(Jackson ImmunoResearch, West Grove, PA, USA). The
hybridomas that tested positive in ELISA were cloned
by limiting dilution. Culture supernatant of cultivatedclones were tested again by ELISA against BoPD-1-His
and total RNA was isolated from the positive clones ofhybridomas using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. cDNAs were synthesized from the
RNA samples with SuperScript III One-Step RT-PCR
System with Platinum Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific). The gene encoding variable regions of heavy and light chains of antibody (VH
and VL) were amplified using KOD -Plus- Neo DNA Polymerase (Toyobo, Osaka, Japan) and gene specific primers. The amplicons were cloned into pCAGGS [27] with
the nucleotides encoding constant regions of heavy andlight chains (CH and CL) of rabbit IgG and sequenced by
Eurofins Genomics (Tokyo, Japan). HEK293T cells weretransfected with the expression plasmids and grown infetal bovine serum (FBS)-free D-MEM medium (Fujifilm Wako Pure Chemical, Osaka, Japan). The cultureOkagawa et al. Veterinary Research(2023) 54:82

supernatants were tested by ELISA against bovine PD-1
as shown above. The supernatants from two clones(1D10F1 and 4F5F2) were positive in ELISA and further tested in the following screening test using purified
antibodies.
Expression and purification of anti‑bovine PD‑1 rabbit
mAbs

Transient cell lines expressing anti-bovine PD-1 rabbit mAbs were established using the Expi293 Expression System (Thermo Fisher Scientific). Briefly, Expi293F
cells were transfected with the expression plasmids,
pCAGGS-1D10F1-VHCH and pCAGGS-1D10F1-VLCL
or pCAGGS-4F5F2-VHCH and pCAGGS-4F5F2-VLCL,
using Expifectamine (Thermo Fisher Scientific) and cultured with shaking in Expi293 Medium (Thermo Fisher
Scientific) at 37 °C and 125 rpm with 8% C
­ O2 for 7 days.
For negative control antibodies, cells were transfectedwith a combination of pCAGGS-1D10F1-VHCH andpCAGGS-4F5F2-VLCL (1H4L) or the other combination(4H1L). Antibodies were purified from the culture supernatants by affinity chromatography with an Ab-Capcher
ExTra (ProteNova) and the buffer was exchanged withphosphate-buffered saline (PBS) by size exclusion chromatography using PD-10 Desalting Column (GE Healthcare). The concentration of Boch5D2 was measured byultraviolet (UV) absorbance at 280 nm with a NanoDrop
8000 spectrophotometer (Thermo Fisher Scientific).
Expression and purification of anti‑bovine PD‑1
bovine‑rabbit chAb

The nucleotide sequences of the VH and VL genes of
1D10F1 were combined with the constant regions ofbovine ­IgG1 (GenBank accession number X62916) with
reduced Fc-mediated effector functions (­IgG1 ADCC −)[10] and bovine Ig lambda (GenBank accession number X62917), respectively. In selecting the subclasses of
the constant region, the dominant subclasses in bovine
immunoglobulins (IgG1 and Igλ) were chosen [28, 29].
The resulting sequences were modified according to the
optimal codon usage of the Chinese hamster, synthesized(GenScript), and cloned into the expression vectors,
pDC62c5-U533, pDC61, and pNC32c5-U533 [23].
CHO DG44 cells (a dhfr-deficient CHO cell line) weretransfected with the expression vectors and cloned bylimiting dilution in CD OptiCHO medium (Thermo
Fisher Scientific) supplemented with 2 mM GlutaMAXI (Thermo Fisher Scientific). For the selection of cells
transfected with pNC32c5-U533, 800 μg/mL of G418
sulfate (Enzo Life Sciences) were added to the medium.
After 3 weeks, the cloned cells were screened for theirability to produce Boch1D10F1 using an enzymelinked immunosorbent assay (ELISA) with horseradish

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peroxidase (HRP)-conjugated anti-bovine IgG Fc rabbit
polyclonal antibody (Rockland Immunochemicals) as
previously described [24]. Boch1D10F1 was producedby shaking the established cell lines in Dynamis Medium(Thermo Fisher Scientific) with EfficientFeed B + AGT
Supplement (Thermo Fisher Scientific) at 37 °C with 5%­CO2 for 14 days. Live and dead cells were counted witha Countess Automated Cell Counter (Thermo Fisher Scientific). The concentration of Boch1D10F1 in the culture
supernatant was determined using a bovine IgG ELISA as
described above.
Purification of Boch1D10F1 from the culture supernatant was performed by affinity chromatography with
an Ab-Capcher ExTra (ProteNova) and PD-10 Desalting
Column (GE Healthcare) as described above. The concentration of the purified Boch1D10F1 was measured by
UV absorbance at 280 nm with a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific). The purity of
Boch1D10F1 was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under
reducing or nonreducing conditions on a SuperSep Ace
5%–20% gradient polyacrylamide gel (Fujifilm Wako Pure
Chemical) with 2 × Laemmli Sample Buffer (Bio-Rad,
Hercules, CA, USA). The Precision Plus Protein Dual
Standard (Bio-Rad) was used as a molecular weight size
marker and the proteins were visualized with Quick-CBB(Fujifilm Wako Pure Chemical).
Binding assay of anti‑bovine PD‑1 antibodies

To confirm the binding activity of the anti-bovine PD-1
rabbit mAbs and chAb (Boch1D10F1) to bovine PD-1
protein, flow cytometry was performed using myc-tagged
bovine PD-1-expressing CHO DG44 cells (BoPD-1-myc
cells) [7]. Briefly, BoPD-1-myc cells were incubated withthe purified mAbs (1D10F1, 4F5F2, 1H4L, or 4H1L), antibovine PD-1 rat mAb (5D2) [7], rat I­ gG2a isotype control(R35-95, BD Biosciences), Boch1D10F1, or bovine IgG
control (Sigma–Aldrich) at room temperature for 30
min. The cells were then washed with PBS supplemented
with 1% bovine serum albumin (BSA; Sigma–Aldrich)and labeled with Alexa Flour 647-conjugated anti-rabbit
IgG (H + L) goat F(ab’)2 (Thermo Fisher Scientific), APCconjugated anti-rat immunoglobulin antibody (Southern Biotech) or APC-conjugated anti-bovine IgG Fc goat
antibody (Jackson ImmunoResearch) at room temperature for 30 min. Finally, the cells were washed and analyzed immediately using FACS Verse (BD Biosciences).
To evaluate the detection sensitivity and specificity of anti-bovine PD-1 rabbit mAbs to PD-1 protein inbovine lymphocytes, multicolor flow cytometric analysis was performed using bovine leukocytes isolated
from fresh blood samples. To prevent nonspecific reactions of staining antibodies, leukocytes were incubatedOkagawa et al. Veterinary Research(2023) 54:82

in PBS containing 10% goat serum (Sigma–Aldrich) at
room temperature for 15 min. The cells were stainedwith anti-bovine PD-1 rabbit mAb (1D10F1 or 4F5F2),
rabbit IgG controls (1H4L), anti-bovine PD-1 rat mAb(5D2) [7], or rat ­IgG2a isotype control (R35-95, BD Biosciences) for 30 min at room temperature. After washing with 1% BSA-PBS, the cells were labeled with Alexa
Flour 647-conjugated anti-rabbit IgG (H + L) goat F(ab’)2(Thermo Fisher Scientific) or APC-conjugated anti-rat
immunoglobulin antibody (Southern Biotech) for 30 min
at room temperature with a cocktail of staining antibodies, including PerCp/Cy5.5-conjugated anti-bovine CD3
mAb (MM1A; Washington State University Monoclonal Antibody Center), FITC-conjugated anti-bovine
CD4 mAb (CC8; Bio-Rad), PE-conjugated anti-bovine
CD8 mAb (CC63, Bio-Rad), APC/Cy7-conjugated antiTCR1-N24 mAb (GB21A; Washington State University
Monoclonal Antibody Center), and PE/Cy7-conjugatedanti-bovine IgM mAb (IL-A30; Bio-Rad). The mAbs
MM1A, GB21A, and IL-A30 were conjugated with eachfluorochrome using Lightning-Link Conjugation Kits
(Innova Biosciences). Finally, the cells were washed andanalyzed by FACS Verse (BD Biosciences).
Surface plasmon resonance analysis

To assess the binding affinity of 1D10F1, 4F5F2, andBoch1D10F1 to bovine PD-1, surface plasmon resonance(SPR) analysis was performed using the Biacore System(GE Healthcare) with BoPD-1-His as described previously [10]. SPR measurements were performed usinga CM5 sensor chip (GE Healthcare) immobilized with
BoPD-1-His and 1D10F1, 4F5F2, 5D2, and Boch1D10F1
on a Biacore X100 or Biacore 3000 instrument (GEHealthcare) at 25 °C. The kinetic constant of each antibody was determined by fitting with a 1:1 kinetic binding
model.
Blockade assay of PD‑1/PD‑L1 binding

To confirm the ability of 1D10F1, 4F5F2, andBoch1D10F1 to block PD-1/PD-L1 binding, biotinylated
BoPD-1-Ig (5 μg/mL) [24] was incubated with various
concentrations (0.39 to 50 μg/mL) of 1D10F1, 4F5F2, andBoch1D10F1 at 37 °C for 30 min. The incubated BoPD1-Ig proteins were incubated with bovine PD-L1-EGFPexpressing CHO DG44 cells (BoPD-L1-EGFP cells) [24]at 37 °C for 30 min. BoPD-1-Ig bound to BoPD-L1-EGFP
cells was labeled with APC-conjugated streptavidin (BioLegend) at room temperature for 30 min, washed with
PBS, and analyzed by FACS Verse (BD Biosciences). Rat­IgG2a isotype control (R35-95, BD Biosciences), rabbit
IgG controls (1H4L and 4H1L), and bovine ­IgG1 control(Bethyl Laboratories) were used as negative controls.

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Immune activation assay by Boch1D10F1

To analyze immune activation by Boch1D10F1, peripheral blood mononuclear cell (PBMC) cultivation assay
was conducted as previously described with some modifications [8]. PBMCs were isolated from the peripheral
blood of BLV-infected cattle by density gradient centrifugation using Percoll (GE Healthcare), washed threetimes with PBS, and suspended in RPMI1640 Medium(Sigma–Aldrich) supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific), 200 IU/mL of penicillin, 200 μg/mL of streptomycin, and 0.01% L-glutamine(Thermo Fisher Scientific). PBMCs were cultured with
20 μg/mL of Boch1D10F1, anti-bovine PD-1 rat-bovine
chAb (Boch5D2) [10], or bovine IgG control (Sigma–
Aldrich) in the presence of 2% heat-inactivated supernatant from BLV-infected fetal lamb kidney cells (FLK-BLV)
at 37 °C in 5% ­CO2 for six days. Culture supernatants
were then harvested and IFN-γ concentrations in the
supernatants were determined using a bovine IFN-γELISA (Mabtech) performed in duplicate according to
the manufacturer’s protocol.
Evaluation of the antiviral effect of Boch1D10F1 treatment
in BLV‑infected cattle

To confirm the antiviral effects of Boch1D10F1 in cattle, the administration of Boch1D10F1 was performedin cattle naturally infected with BLV (n = 6, Holstein,female; Additional file 1) in animal facilities at the Animal Research Center, Agricultural Research Department,
Hokkaido Research Organization (Shintoku, Hokkaido,
Japan) and Faculty of Veterinary Medicine, Hokkaido
University (Sapporo, Hokkaido, Japan). BLV infection
was confirmed by the detection of anti-BLV antibodies
in plasma using a commercial ELISA kit (JNC, Tokyo,
Japan) and by the detection of BLV provirus in DNA
using real-time PCR as described previously [10]. The
number of lymphocytes in blood samples was counted
using an automated hematology analyzer (Celltac α;
Nihon Kohden, Tokyo, Japan). BLV-infected cattle wereclassified as aleukemic (AL) or persistent lymphocytosis (PL) based on the lymphocyte counts as follows:
AL < 8000 cells/μL; PL > 8000 cells/μL.
Four BLV-infected cattle (AL2, AL3, PL2, and PL3)
were intravenously administered 1 mg/kg (AL2 andAL3) or 2 mg/kg (PL2 and PL3) of purified Boch1D10F1
diluted in saline (Additional file 1). Two of the animals(AL3 and PL3) were also administrated 0.5 mg/kg of
meloxicam (Metacam; Boehringer Ingelheim, Ingelheim, Germany) subcutaneously three times at sevendays intervals (Additional file 1). The other two animals(AL1 and PL1) did not receive any treatment andwere used as negative controls (Additional file 1). This

Okagawa et al. Veterinary Research(2023) 54:82

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animal experiment was approved by the Ethics Committee of the Animal Research Center, Agricultural
Research Department, Hokkaido Research Organization (approval #1703) and the Ethics Committee of theFaculty of Veterinary Medicine, Hokkaido University(Approval #17-0024).
To determine proviral loads in the peripheral blood,
the BLV tax gene was measured in genomic DNA by
quantitative real-time PCR using the Cycleave PCR
Reaction Mix (Takara Bio) and a Probe/Primer Mix
for BLV (Takara Bio) with a LightCycler 480 System II(Roche Diagnostics) as described previously [10]. EachDNA sample was tested in triplicate. Genomic DNA
was extracted from PBMCs with the Wizard Genomic
DNA Purification Kit (Promega) and the concentrationof DNA was measured by UV absorbance at 260 nmwith a NanoDrop 8000 spectrophotometer (Thermo
Fisher Scientific).
Statistical analysis

All statistical tests were performed with GraphPad
Prism 6 software (GraphPad Software Inc.). Differenceswere considered statistically significant at p < 0.05.

Results
Establishment and characterization of anti‑bovine PD‑1
rabbit mAbs

Supernatants containing rabbit mAbs from two hybridoma clones were established using lymphocytes from a
rabbit immunized BoPD-1-Ig and showed specific binding to BoPD-1-His protein in ELISA. These anti-bovine
PD-1 rabbit mAbs were purified and tested for theirbinding affinity to bovine PD-1 protein by SPR analysis
using a Biacore instrument. 1D10F1 and 4F5F2 showed
higher affinities for PD-1 compared with 5D2, with ­KDvalues of 1.11 ± 0.07, 2.85 ± 0.50, and 0.12 ± 0.04 nM,
respectively (Table 1). In particular, 1D10F1 exhibitedthe highest association constant (ka) compared with theother mAbs (Table 1). The binding ability and specificity

Table 1 Binding affinity of anti-PD-1 mAbs to BoPD-1-His
proteinAnti-PD-1
mAbka (1/Ms)kd (1/s)

KD (M)

5D2**

1.84 × ­104 ± 0.27

2.15 × ­10–4 ± 0.44

1D10F16–3

1.63 × ­10 ± 0.16* 1.80 × ­10 ± 0.06* 1.11 × ­10–9 ± 0.07*

4F5F2

7.39 × ­105 ± 0.90* 2.08 × ­10–3 ± 0.28* 2.85 × ­10–9 ± 0.50*

These values were measured on a Biacore X100 instrument*p < 0.05 (vs. 5D2; Tukey’s test)**

The values for 5D2 were shown in a previous paper [10]1.22 × ­10–8 ± 0.39

to bovine PD-1-expressing cells (BoPD-1-myc cells) werethen tested by flow cytometry. Both anti-PD-1 rabbit
mAbs (1D10F1 and 4F5F2) bound to BoPD-1-myc cells
in a dose-dependent manner (Figure 1A) but not to mockcells (Additional file 2). Negative controls of rabbit mAbs
(1H4L and 4H1L) did not bind to BoPD-1-myc cells at
any tested concentrations (Figure 1A). 1D10F1 exhibiteda stronger fluorescent intensity compared with 4F5F2
and anti-PD-1 rat mAb (5D2), which was established in
our previous study (Figure 1A).
To determine the reactivity of anti-PD-1 rabbit mAb(1D10F1) to PD-1 naturally expressed on bovine T cells,
the surface expression of PD-1 was analyzed in T-cell
subsets in freshly isolated bovine leukocytes by flow
cytometry. The 1D10F1 mAb recognized PD-1 expressedon ­CD3+, ­CD3+CD4+, ­CD3+CD8+, and ­CD3+γδTCR​+
T cells, whereas 4F5F2 did not (Figure 1B). The detection sensitivity of PD-1 on the T-cell subsets by 1D10F1
was higher compared with the previously establishedmAb 5D2 (Figures 1B and C). Therefore, 1D10F1 has
high sensitivity for the detection of bovine PD-1 in flow
cytometry.
To analyze the inhibitory activity of 1D10F1 and 4F5F2,
the effect of the mAbs in a binding assay with BoPD-1-Ig
to BoPD-L1-EGFP expressing cells was determined byflow cytometry compared with 5D2. 1D10F1, 4F5F2 and
5D2 inhibited PD-1/PD-L1 binding in a dose-dependent
manner (Figure 1D). At 50 μg/mL, the 1D10F1, 4F5F2,
and 5D2 mAbs inhibited 79.2%, 54.6%, and 44.4% of
PD-1/PD-L1 binding, respectively (Figure 1D). Basedon these results, the mAb 1D10F1 was considered as
the best blocking antibody among the established antibovine PD-1 mAbs.
Establishment of an anti‑bovine PD‑1 rabbit–bovine chAb

The rabbit mAb 1D10F1 was engineered into a rabbit–
bovine chAb, Boch1D10F1. To obtain a large amountof Boch1D10F1 for further in vitro and in vivo characterization, Boch1D10F1 was stably expressed and produced using the CHO DG44 cell expression system.
Three plasmids were evaluated as an expression vector
for Boch1D10F1. UCOE reduces DNA methylation in atransgene promoter and contributes to the maintenanceof the expression of recombinant proteins in CHO cells[22]. pDC62c5-U533 was designed with UCOEs at the
5’ and 3’ ends of the expression cassettes of VL + CL and
VH + CH (Figure 2A). This plasmid was modified using
the pDC61 expression vector for the selection of highprotein expressing cell lines using thymidine-hyposanthin regulated by the selection marker DHFR (Figure 2A),
which was established previously [11, 23]. In addition, theother vector (pNC32c-U533) with UCOEs with a neomycin resistance (npt) selection marker was used to evaluate

Okagawa et al. Veterinary Research(2023) 54:82

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Figure 1 Generation and characterization of anti-bovine PD-1 rabbit mAbs. A Flow cytometric analysis using anti-bovine PD-1 rabbit mAbs
(1D10F1 and 4F5F2) and anti-bovine PD-1 rat mAb (5D2). BoPD-1-myc cells were stained with the mAbs using serial dilutions (10 μg/mL to 100 pg/mL). Rat ­IgG2a isotype control and rabbit IgG controls (1H4L and 4H1L) were used as negative controls. B, C Flow cytometric analysis of PD-1
expression in T-cell subsets of cattle (n = 3). Freshly isolated bovine PBMCs were stained with anti-bovine PD-1 rabbit mAbs (1D10F1 and 4F5F2)
and anti-bovine PD-1 rat mAb (5D2). Rat ­IgG2a isotype control and rabbit IgG control (1H4L) were used as negative controls. B Representative
histograms of staining with anti-PD-1 mAbs (orange, green, or blue histograms) and matched negative controls (gray histograms) are shown. The
gating strategy of this assay was provided as Additional file 3. C Percentages of PD-1+ cells in each T-cell subset in leukocytes from healthy cattle(n = 3). Bars indicate the group mean percentage. Significant differences between each of the two groups were determined using a Student’s
t test. *p < 0.05. D BoPD-1-Ig was preincubated with anti-bovine PD-1 rabbit mAbs (1D10F1 and 4F5F2) and anti-bovine PD-1 rat mAb (5D2),
and then reacted with BoPD-L1-EGFP cells. BoPD-1-Ig bindings were evaluated by flow cytometry. Rat ­IgG2a and rabbit IgG controls (1H4L
and 4H1L) were used as negative controls. Each curve represents the relative binding of BoPD-1-Ig preincubated with each antibody comparedwith the no-antibody control.

Okagawa et al. Veterinary Research(2023) 54:82

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the performance of the combination of UCOEs and the
DHFR selection marker (Figure 2A) [10, 23]. More than
100 clones were randomly selected from the cloned CHO
DG44 cells transfected with each plasmid and examined for their Boch1D10F1 production by ELISA. Cells
transfected with pDC62c5-U533-Boch1D10F1, pDC61Boch1D10F1, and pNC32c-U533-Boch1D10F1 stably
produced 62.2, 41.8, and 4.10 μg/mL of Boch1D10F1
after three days of shaking culture (Figure 2B). In particular, 22.6% (24/106 clones), 1.8% (2/112 clones), and
0% (0/105 clones), respectively, of the established celllines transfected with pDC62c5-U533-Boch1D10F1,
pDC61-Boch1D10F1, and pNC32c-U533-Boch1D10F1
were among the higher-producing cell lines producing > 20 μg/mL protein (Figure 2B). Thus, pDC62c5-U533
was selected for the production of recombinant antibody because high-expressing cells could be obtained
efficiently.
To produce large amounts of Boch1D10F1, one of
the higher Boch1D10F1-producing cell lines was cultured in 2 L of shaking culture using Dynamis Medium.
Boch1D10F1 was present in the supernatants at 623.5
mg/L after 14 days (Figure 2C) and successfully purified
using Protein A resin (Figure 2D). The heavy and lightchains of Boch1D101 were detected at approximately 50
and 25 kDa under reducing conditions, respectively (Figure 2B). Under nonreducing conditions, Boch1D10F1
was detected as a pure, single band at > 150 kDa, whichsuggests that the antibody consists of two heavy and twolight chains. Thus, mass production of Boch1D10F1 wasachieved using a mammalian expression system.

Thus, Boch1D10F1 is capable of robustly inhibiting thebinding of bovine PD-1/PD-L1.
The T-cell response of BLV-infected cattle is impairedby the interaction of PD-1/PD-L1 [6–8, 11]. To determine
whether the treatment of Boch1D10F1 reactivates the
T-cell response of BLV-infected cattle, IFN-γ production
was analyzed in a cultivation assay containing PBMCs
from BLV-infected cattle in the presence of Boch1D10F1
and viral antigen stimulation. The Boch1D10F1 blockade
significantly enhanced IFN-γ production by PBMCs (Figure 3C), even though Boch5D2 also activated a significant IFN-γ response (Figure 3D). These results suggestthat Boch1D10F1 has the potential to enhance the T-cell
response in cattle.

Characterization of anti‑bovine PD‑1 rabbit–bovine chAb

Discussion
In this study, two high-affinity rabbit mAbs (1D10F1
and 4F5F2) against bovine PD-1 were successfully
generated. The mAb 1D10F1 was very useful for thedetection of bovine PD-1 in immunostaining for flow
cytometry and exhibited strong inhibition of the PD-1/
PD-L1 interaction, compared with the rat mAb 5D2. In
contrast, 4F5F2 was also expected to be a good detection antibody because of its high binding affinity to
the extracellular region of the PD-1 soluble protein(BoPD-1-His) in the Biacore assay (Table 1), but flow
cytometric assays with this mAb detected a smaller
percentage of full-length PD-1 protein in BoPD-1myc overexpressing cells (Figure 1A) and none at all inbovine T cells (Figure 1B). This is presumably because
the epitope to which 4F5F2 binds could be a motif not
exposed on the surface of full-length PD-1 protein
expressed at plasma membrane. More validation isneeded to explain well why the seemingly contradictory
results were obtained for 4F5F2.

The binding ability of Boch1D10F1 was determinedby flow cytometry using bovine PD-1-expressing cells(BoPD-1-myc cells). Boch1D10F1 was bound to BoPD1-myc cells in a dose-dependent manner (Figure 3A).
To confirm the binding affinity of Boch1D10F1 tobovine PD-1 protein, SPR analysis was performed usinga Biacore 3000 instrument with BoPD-1-His protein.
Boch1D10F1 and the original 1D10F1 rabbit mAb had asimilar affinity for PD-1, with ­KD values of 1.23 ± 0.05 and1.37 ± 0.09 nM, respectively (Table 2). Therefore, the chimerization of anti-PD-1 antibody did not alter its binding
affinity to bovine PD-1 protein.
To analyze the blocking activity of Boch1D10F1, thebinding of BoPD-1-Ig to BoPD-L1-EGFP-expressing cells
was determined in the presence of Boch1D10F1, 1D10F1,
Boch5D2, and 5D2 by flow cytometry. Boch1D10F1
inhibited the binding of PD-1/PD-L1 in a dose-dependent manner at a level similar to that of 1D10F1 (Figure 3B). In contrast, Boch5D2 and 5D2 exhibited a partial
blockade of the PD-1/PD-L1 interaction (Figure 3B).

Reduction of proviral load by the treatment of BLV‑infected
cattle with Boch1D10F1

To evaluate the antiviral effect of Boch1D10F1 in vivo,
BLV-infected cattle were inoculated with anti-PD-1
chAb (Boch1D10F1). AL2 and PL2 were treated with
Boch1D10F1 intravenously at a dose of 1 or 2 mg/kg,
respectively. AL3 and PL3 also received an intravenous
dose of Boch1D10F1 (1 or 2 mg/kg) on day 0 as well asa subcutaneous injection of 0.5 mg/kg meloxicam (Metacam) on day 0, 7, and 14, because the combination of
PD-1/PD-L1 blockade and COX-2 inhibition represents
a promising strategy to synergistically enhance the antiviral T-cell response during BLV infection [12, 30]. Amongthe treated animals, AL2 and PL3 exhibited a sustained
decrease in BLV proviral load in PBMCs following
Boch1D10F1 administration (Figure 4).

Okagawa et al. Veterinary Research(2023) 54:82

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Figure 2 Production of anti-PD-1 chimeric antibody, Boch1D10F1. A Schematic structures of plasmid vectors encoding Boch1D10F1(pDC62c5-U533-Boch1D10F1, pDC61-Boch1D10F1, and pNC32c-U533-Boch1D10F1). The light chain consisting of a variable region (VL)
and a constant region (CL). The heavy chain consisting of a variable region (VH) and a constant region (CH). mDHFR: modified dihydrofolate
reductase, UCOE: ubiquitous chromatin opening element (UCOE), npt: neomycin-resistant gene. B Selection of CHO DG44 cell clones
producing Boch1D10F1. The production capacity of Boch1D10F1 from each cell clone transfected with pDC62c5-U533-Boch1D10F1 (blue bars),
pDC61-Boch1D10F1 (red bars), and pNC32c-U533-Boch1D10F1 (green bars) are shown. The production capacity of the highest producing clones
for each vector are shown in the plot. C Expression of Boch1D10F1. Boch1D10F1 was expressed in 2L of shaking culture of a higher-producing
cell line. The antibody production (left axis: gray bar) and the density of live and dead cells (right axis: black and white circles) were measured
at 3- to 4-day intervals. D Purification of Boch1D10F1. Boch1D10F1 was purified from the supernatants of shaking cultures. Purified protein
was confirmed by reducing and nonreducing SDS-PAGE. An uncropped gel image generated during the current study is provided in Additional
file 4.

Okagawa et al. Veterinary Research(2023) 54:82

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Figure 3 Characterization of Boch1D10F1. A Reactivity of Boch1D10F1 with BoPD-1-myc cells. BoPD-1-myc cells were stained with Boch1D10F1
in serial dilutions (10 μg/mL to 100 pg/mL) and analyzed by flow cytometry. B Blockade of PD-1/PD-L1 binding by Boch1D10F1. BoPD-1-Ig
was preincubated with 5D2 and Boch5D2 and incubated with BoPD-L1-EGFP cells. BoPD-1-Ig bindings were evaluated by flow cytometry. Eachcurve represents the relative binding of BoPD-1-Ig preincubated with Boch1D10F1, Boch5D2, 1D10F1, and 5D2 compared with no-antibody
control. Bovine IgG (for Boch1D10F1 and Boch5D2), rabbit IgG (1H4L), and rat ­IgG2a controls (for 5D2) were used as negative controls. C, D Effectof PD-1/PD-L1 blockade on the IFN-γ response. PBMCs of BLV-infected cattle (n = 13) were cultured with Boch1D10F1 and Boch5D2 (10 μg/mL)or bovine IgG control in the presence of FLK-BLV antigen (2%) for 6 days. IFN-γ production for each animal was measured by ELISA in duplicate. Bars
indicate the group median response. Significant differences between each group were determined using Wilcoxon signed-rank test Friedman test.
**p < 0.01.

Okagawa et al. Veterinary Research(2023) 54:82

Page 10 of 14

Table 2 Binding affinity of anti-PD-1 rabbit mAb and chAb
to BoPD-1-His protein
Anti-PD-1
antibodyka (1/Ms)*kd (1/s)*

KD (M)*

1D10F15.00 × ­105 ± 0.29 6.17 × ­10–4 ± 0.38 1.23 × ­10–9 ± 0.05

Boch1D10F1

4.37 × ­105 ± 0.09 6.13 × ­10–4 ± 0.61 1.37 × ­10–9 ± 0.09

These values were measured on a Biacore 3000 instrument*

The values of 1D10F1 and Boch1D10F1 are not significantly different (p > 0.05)

We have been working on the establishment andcharacterization of bovine chAb [10, 11, 31], and thisstudy represents our first attempt at establishing a rabbit–bovine chAb to achieve increased binding affinity to an antigen. The anti-PD-1 rabbit–bovine chAb
(Boch1D10F1) was generated in a high-producing cellline selected from more than 300 cell clones. As reported
previously, the addition of UCOE into the expression plasmid also increased the expression level of thetransgene compared with conventional plasmids in our
experiments. Although we have not examined the mechanism of this increase, UCOE is known to inhibit DNA
methylation in the transgene promoter and maintain theexpression level of the transgene after many passagesof the established cell line [22, 23]. In the present study,
Boch1D10F1 was prepared for animal experiments using
adult cattle in a flask-shaking culture system. We established as system to develop a higher antibody producing
cell line, which will be very useful for in vivo antibody
research using large animal models, such as cattle, dogs,
pigs, horses, and monkeys.
Although Boch1D10F1 exhibited a higher binding
affinity and stronger blocking activity to bovine PD-1
compared with Boch5D2 [10], immune activating efficacy was observed in PBMC cultivation assays using
these chAbs. IFN-γ was measured as a representative cytokine as well as a key regulator of Th1 response
based on our previous studies [7, 8, 10]. We attempted tomeasure other cytokines, such as IL-2 and TNF-α using
ELISA, but they were not detectable in the culture supernatant. To determine the blockade effect in EBL animals,
other methods to detect cytokine production from BLVspecific T cells are needed, such as the Enzyme-Linked
ImmunoSpot Assay or Luminex Assay System withimmunogenic peptides or proteins [32–34].
With the spread of BLV on farms throughout
Japan [35], the incidence of EBL has increased in recent
years, causing significant economic losses for dairy andbeef farmers. Because of the high prevalence in Japan,
it has been extremely difficult to implement cleanup
through the detection and culling of infected cattle. Furthermore, no effective treatment or vaccine has becomecommercially available at this time. Therefore, a novel
control strategy against BLV infection is required.
In the animal experiment of Boch1D10F1 administration, PD-1 blockade reduced the BLV proviral load in
one of the aleukemic (AL) cattle (AL2), which is consistent with a previous observation in a pilot clinical
trial of Boch5D2 [10]. The antiviral effect in this animal
was observed throughout the experiment. In contrast,
a continuous antiviral effect was not observed in PL2,
which is also consistent with our previous study demonstrating that PD-1/PD-L1 and COX-2/prostaglandin E2 ­(PGE2) pathways cooperatively suppresses the
T-cell response in BLV-infected cattle with progressive
disease, such as persistent lymphocytosis (PL) animals[12]. Consistently, the combination of Boch1D10F1
and meloxicam induced an antiviral effect in a PL cow(PL3); however, the viral load was reversed at day 49
post-administration. The mechanism of this increase
may be a decreased blocking effect resulting from antibody degradation or compensation by other immunosuppressive factors, such as the immune checkpoint
molecule, cytotoxic T-lymphocyte antigen 4 (CTLA-4),
or regulator T-cell subsets [36–38]. These possibilities
should be tested in subsequent clinical studies throughrepeated administration of chAb or a combination
with other blocking antibodies, such as anti-CTLA-4
chAb [31]. In addition, phenotyping T-cell subsets
in these animals will be important to understand thevarying efficacy of PD-1 blockade observed in eachindividual.
We demonstrated that treatment with anti-PD-L1
chAb activated the T-cell response in cattle infectedwith Mycobacterium avium subsp. paratuberculosis
and Mycoplasma bovis [39–41]. The clinical benefitof Boch1D10F1 treatment may also be determined incattle with paratuberculosis and mycoplasmosis anaplasmosis to determine whether targeting PD-1/PD-L1
pathway can be applied as broad-spectrum immunotherapy against chronic infectious diseases in cattle.
In addition, functional role of PD-1/PD-L1 pathway inT-cell response of other bovine infections should beclarified, such as bovine tuberculosis, brucellosis, babesiosis, and theileriosis to consider the spectrum of thisstrategy. Furthermore, bovinized antibodies, in which
the frameworks of the variable region are replacedwith bovine antibody, are expected to improve stability in vivo and further enhance clinical efficacy andcould be the next option of the strategy for bovine
immunotherapy.
To develop new control methods for chronic infectious
diseases, it is necessary to further analyze the immunosuppressive mechanisms of each disease and to consider

Okagawa et al. Veterinary Research(2023) 54:82

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Figure 4 Effect on proviral loads on BLV-infected cattle administered Boch1D10F1. Provirus copy number per 50 ng DNA of PBMCs
from BLV-infected cattle administrated Boch1D10F1 with or without meloxicam (Metacam). Proviral loads of BLV were quantified in PBMCs at eachtime point by real-time genomic PCR targeting the BLV tax gene. Each dot represents the mean of three independent experiments. Significant
differences were determined by Dunnett’s multiple-comparison test across the time points. *p < 0.05 versus pre-administration. AL: aleukemic, PL:persistent lymphocytosis.

Okagawa et al. Veterinary Research(2023) 54:82

the most effective combination of therapies (therapeutictargets). In addition, the development and production
costs of antibody drugs must be fully considered. This
study provides a technological basis for producing highperformance antibodies in large quantities and at low
cost.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13567-​023-​01213-6.
Additional file 1. Animals used in the experiment of antibody admin‑
istration. Six BLV-infected cattle (Holstein, female) were used for theadministration experiments of Boch1D10F1 and meloxicam to evaluate
their antiviral effects in vivo.
Additional file 2. Flow cytometric analysis using anti-bovine PD-1
mAbs to mock cells. Flow cytometric analysis was performed using
anti-bovine PD-1 rabbit mAbs (1D10F1 and 4F5F2) and anti-bovine PD-1
rat mAb (5D2) and mock-transfected CHO DG44 cells. CHO DG44 cells
were transfected with pCI-neo (mock plasmid) and cloned by limiting
dilution. The mock cell line was stained with the mAbs (100 μg/mL) at
room temperature for 30 min. Rat I­gG2a isotype control and rabbit IgG
controls (1H4L and 4H1L) were used as negative controls. The cells werelabeled with Alexa Flour 647-conjugated anti-rabbit IgG (H + L) goat
F(ab’)2 (Thermo Fisher Scientific), APC-conjugated anti-rat immunoglobu‑
lin antibody (Southern Biotech) at room temperature for 30 min. Finally,
the cells were washed and analyzed immediately using FACS Verse (BD
Biosciences).
Additional file 3. Gating strategy of T-cell subsets in leukocytes fromhealthy cattle. ­CD4+, ­CD8+, or γδTCR​+ T cells were gated in C
­ D3+IgM−
lymphocytes and then analyzed for expression of PD-1 in each T cellsubset as shown in Figure 1B.
Additional file 4. An uncropped gel image for Figure 2 D. Purified
anti-PD-1 chAb (Boch1D10F1) was analyzed by reducing and nonreducing
SDS-PAGE.
Acknowledgements
We are grateful to Dr Hideyuki Takahashi, Dr Yasuyuki Mori, and Dr Tomio
Ibayashi for their valuable advice and discussions, and the technical staffs of
the animal facilities for the excellent care and handling of animals. We thankEnago for the English language review.
Authors’ contributions
Conceptualization: TO, SK, YSu, KO; Investigation: TO, SK, SG, YSa, OG, KW, HN,
CXW, YuK, YaK, JK, NT, HT; Analysis and interpretation of data: TO, SK, SG, TT,
YuK, JK, MK, AT, YSu, NM, SM; Methodology and resources: YuK, JK, TT, MK, YSu,
NM; Supervision: SK, SM, KO; Writing original draft: TO, SK; Review and editing:
SG, YaS, CXW, TT, YuK, JK, NT, MK, AT, HT, YSu, NM, SM, KO. All authors read andapproved the final manuscript.
Funding
This work was supported by JSPS KAKENHI grant number 19KK0172,
22H02503, 22K19232 [to S.K.], 19K15993, and 22K15005 [to T.O.], grants fromthe Project of the NARO, Bio-oriented Technology Research Advancement
Institution (Research Program on Development of Innovative Technology
26058 BC [to S.K.] and Special Scheme Project on Regional Developing Strat‑
egy, Grant 16817557 [to S.K.]), grants from Ito Memorial Foundation [to S.K.]
and Regulatory Research Projects for Food Safety, Animal Health and Plant
Protection (JPJ008617.17935709 [to S.K.]) and supported by the Japan Agency
for Medical Research and Development (AMED) under Grants JP223fa627005[to S.K.], JP21am0101078 [to Y.K.] and JP22ama121008 [to Y.K.]. The funders had
no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.

Page 12 of 14

Availability of data and materials
The datasets used during the current study are available from the correspond‑
ing author on reasonable request.

Declarations
Ethics approval and consent to participate
This animal experiment was approved by the Ethics Committee of the Animal
Research Center, Agricultural Research Department, Hokkaido Research
Organization (approval # 1703) and the Ethics Committee of the Faculty of
Veterinary Medicine, Hokkaido University (Approval #17-0024).
Competing interests
YSu, YK, SK, TO, NM, SG, YSa, KO, and SM have a patent pending for materials
and techniques described in this paper (Japanese patent, Application number
2018–99704).
Author details
1
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine,
Hokkaido University, Sapporo, Japan. 2 Department of Disease Control, Facultyof Veterinary Medicine, Hokkaido University, Sapporo, Japan. 3 Institute for Vac‑cine Research and Development (HU‑IVReD), Hokkaido University, Sapporo,
Japan. 4 Department of Bioengineering, Graduate School of Engineering,Osaka City University, Osaka, Japan. 5 Department of Bioengineering, Graduate
School of Engineering, Osaka Metropolitan University, Osaka, Japan. 6 Depart‑
ment of Antibody Drug Development, Tohoku University Graduate Schoolof Medicine, Sendai, Japan. 7 Department of Molecular Pharmacology, Tohoku
University Graduate School of Medicine, Sendai, Japan. 8 Division of Biore‑
sources, International Institute for Zoonosis Control, Hokkaido University, Sap‑poro, Japan. 9 Animal Research Center, Agriculture Research Department, Hok‑kaido Research Organization, Shintoku, Japan. 10 Hokkaido Agricultural Mutual
Aid Association, Shibecha, Japan. 11 Global Station for Zoonosis Control, Global
Institution for Collaborative Research and Education (GI‑CoRE), Hokkaido
University, Sapporo, Japan. 12 International Affairs Office, Faculty of Veterinary
Medicine, Hokkaido University, Sapporo, Japan.
Received: 12 December 2022 Accepted: 2 September 2023

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