P3X63Ag8U.1 (P3U1), Chinese hamster ovary (CHO)-K1, Lec1, Lec2, Lec8, and LN229 cells were obtained from the American Type Culture Collection. Raji and BALL-1 were obtained from the Cell Resource Center for Biomedical Research (Institute of Development, Aging and Cancer, Tohoku University, Miyagi, Japan). DNA encoding the CD20 gene (IRAL012D02) was provided by the RIKEN BRC through the National BioResource Project of MEXT, Japan. The open reading frame of CD20 plus an N-terminal PA tag was subcloned into a pCAG-Neo or pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation). CHO/CD20 and CHO/mock were produced by transfecting pCAG-Neo/CD20 and pCAG-Neo into CHO-K1 cells, respectively, using a Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc.). LN229/CD20 and LN229/mock were produced by transfecting pCAG-Ble/CD20 and pCAG-Ble into LN229 cells, respectively, using a Neon transfection system (Thermo Fisher Scientific, Inc.). Lec1/CD20 (N-glycan-deficient), Lec2/CD20 (sialic acid-deficient), and Lec8/CD20 (galactose-deficient) were produced by transfecting pCAG-Ble/CD20 into Lec1, Lec2, and Lec8 cells, respectively, using a Neon transfection system. Lec1/mock, Lec2/mock, and Lec8/mock were produced by transfecting pCAG-Ble into Lec1, Lec2, and Lec8 cells, respectively, using a Neon transfection system. The cell line BALL-1/CD20-KO (BINDS-24) was generated by transfecting CRISPR/Cas9 plasmids for CD20 (Thermo Fisher Scientific, Inc.) using a Neon transfection system. Stable transfectants were established using SH800 (Sony Corp.).

P3U1, CHO-K1, CHO/CD20, CHO/mock, Lec1, Lec1/CD20, Lec1/mock, Lec2, Lec2/CD20, Lec2/mock, Lec8, Lec8/CD20, Lec8/mock, Raji, BALL-1, and BINDS-24 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.). LN229, LN229/CD20, and LN229/mock were cultured using Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.). The media were supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 100 units/ml of penicillin, 100 µg/ml of streptomycin, and 25 µg/ml of amphotericin B (Nacalai Tesque, Inc.). L-proline (0.04 mg/ml; MP Biomedicals, LLC) was added to Lec1, Lec2, and Lec8. The cells were grown in an incubator at 37°C with humidity and 5% CO₂ and 95% air atmosphere.

Total RNAs were prepared from cell lines using an RNeasy mini prep kit (Qiagen Inc.). The initial cDNA strand was synthesized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Inc.) by priming nine random oligomers and an oligo(dT) primer according to the manufacturer's instructions. We performed 35 cycles of PCR for amplification using HotStarTaq DNA Polymerase (Qiagen Inc.) with 0.2 mM of primer sets: CD20 sense (5′-ATGACAACACCCAGAAATTC-3′), CD20 antisense (5′-TTAAGGAGAGCTGTCATTTTC-3′), GAPDH sense (5′-CAATGACCCCTTCATTGACC-3′), and GAPDH antisense (5′-GTCTTCTGGGTGGCAGTGAT-3′).

All animal experiments were performed in accordance with relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments described in the hybridoma production were approved by the Animal Care and Use Committee of Tohoku University (Permit no: 2016MdA-153). Mice were monitored for health every day. The duration of the experiment was four weeks. A body weight loss exceeding 25% of total body weight were defined as a humane endpoint. Mice were euthanized by cervical dislocation, and the death was verified by respiratory arrest and cardiac arrest.

Two female BALB/c mice (6 weeks old) were purchased from CLEA Japan. Animals were housed under specific pathogen-free conditions. Animal experiments described in the hybridoma production were approved by the Animal Care and Use Committee of Tohoku University (Permit no: 2016MdA-153). Briefly, LN229/CD20 cells (1×10⁸ cells) were immunized into two BALB/c mice using intraperitoneal injection together with Imject Alum (Thermo Fisher Scientific, Inc.). After three additional immunizations, a booster injection was administered two days before harvesting spleen cells. These spleen cells were fused with P3U1 cells using polyethylene glycol 1500 (Roche Diagnostics), and then hybridomas were grown in an RPMI medium supplemented with sodium hypoxanthine, aminopterin, and thymidine (Thermo Fisher Scientific, Inc.). The culture supernatants were used for hybridoma screening by flow cytometry.

Cells were harvested by brief exposure to 0.25% trypsin with 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing with phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), the cells were treated with 10 µg/ml of C₂₀Mab-11 for 30 min at 4°C, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (1:2,000; Cell Signaling Technology, Inc.). Fluorescence data were collected using Spectral Cell Analyzer SA3800 (Sony Corp.).

Cell lysates were prepared by 1% Triton X-100 and cell debris was removed by centrifugation. Cell lysates were boiled in sodium dodecyl sulfate sample buffer with a reducing reagent (Nacalai Tesque, Inc.). These proteins (10 µg) were electrophoresed on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubated with 10 µg/ml of C₂₀Mab-11, 1 µg/ml of NZ-1 (anti-PA tag) or 1 µg/ml of anti-β-actin for control (clone AC-15; Sigma-Aldrich Corp.), followed by incubation with secondary antibody peroxidase-conjugated anti-mouse immunoglobulin (1:1,000; Agilent Technologies Inc., Santa Clara, CA, USA) or anti-rat IgG (1:10,000; Sigma-Aldrich Corp.), respectively. Finally, proteins were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co. Ltd.).

One formalin-fixed paraffin-embedded (FFPE) tissue sample from an oropharyngeal squamous cell carcinoma patient who underwent surgery at Sendai Medical Center was used for this study (13). Written informed consent was obtained from the patient for sample procurement and subsequent data analyses. Tissue microarray (CC00-10-001) including lymphomas, normal lymph node, and normal thyroid was purchased from Cybrdi, Inc.

The 4-µm thick paraffin-embedded tissue sections were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After blocking with the SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.), tissue sections were incubated with C₂₀Mab-11 (5 µg/ml) for 1 h at room temperature and treated with the Envision+ Kit for mouse (Agilent Technologies, Inc.) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Statistical analysis was conducted using t-test with GraphPad Prism 6 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference. All data are expressed as the mean ± SEM.

In this study, we employed the CBIS method (Fig. 1). First, we immunized two mice with LN229/CD20 cells and their spleen cells were harvested and grown as hybridomas in cell culture. Next, supernatants from cultured hybridomas positive for CHO/CD20 and negative for CHO-K1, were selected by flow cytometry. We detected strong signals from CHO/CD20 and weak or no signals from CHO-K1 in 14 of the 960 wells (1.5%). Further screening using western blotting and immunohistochemistry techniques led to the establishment of C₂₀Mab-11 (IgM, kappa).

CD20 expression was investigated by RT-PCR. CD20 was not detected in CHO-K1/mock, LN229/mock, Lec1/mock, Lec2/mock, and Lec8/mock (Fig. 2A). Then, overexpression of CD20 was confirmed in CHO/CD20, LN229/CD20, Lec1/CD20, and Lec2/CD20, Lec8/CD20. We used these stable transfectants of CD20 in flow cytometry and western blot analyses to characterize C₂₀Mab-11.

Using flow cytometry analysis, we found that C₂₀Mab-11 reacted with CHO/CD20 cells, but not with CHO-K1 cells (Fig. 2B). Similarly, C₂₀Mab-11 reacted with LN229/CD20 cells, but not with LN229 cells. Additionally, we performed flow cytometry using CD20-stable transfectants of glycan-deficient CHO cell lines (Lec1, Lec2, and Lec8), although it was previously reported that CD20 is not glycosylated (18). C₂₀Mab-11 reacted with Lec1/CD20 (N-glycan-deficient), Lec2/CD20 (sialic acid-deficient), and Lec8/CD20 (galactose-deficient) cells, but not with Lec1, Lec2, and Lec8 cells, indicating that the binding epitope of C₂₀Mab-11 is independent of glycans. These results indicate that C₂₀Mab-11 is specific for CD20. Fluorescence intensity was quantitatively analyzed (Fig. 2C).

Next, we performed western blot using C₂₀Mab-11. C₂₀Mab-11 detected CD20 with a 45-kDa band in CHO/CD20 and a 37-kDa band in BALL-1 and Raji cells; it did not detect CD20 in CHO-K1 cells and CD20-knockout BALL-1 (BIND-24) cells (Fig. 3), indicating that C₂₀Mab-11 is specific for CD20. An anti-PA tag mAb (NZ-1) detected CD20 at a 45-kDa band without detecting any bands in CHO-K1, BALL-1, BINDS-24, and Raji cells. The difference of molecular size between CHO/CD20 and BALL-1 cells might be due to N-terminal PA-tag of CD20. Although additional bands of 30 and 20 kDa were detected by C₂₀Mab-11 and an anti-PA tag mAb (NZ-1), respectively, in CHO/CD20 cells, we postulated that they might be degraded fragments of the CD20 protein.

We further investigated the immunohistochemical utility of C₂₀Mab-11 in a lymph node of oropharyngeal squamous cell carcinoma patient. C₂₀Mab-11 strongly stained the lymph follicle and weakly stained the cortex (Fig. 4A and B). No staining was observed without the primary antibody (Fig. 4C and D). Hematoxylin and eosin (HE) staining was also performed using consecutive oropharyngeal squamous cell carcinoma tissues (Fig. 4E and F).

C₂₀Mab-11 weakly stained B cells of normal lymph node (Fig. 4G and H) and B cell-lymphomas (Fig. 5A and B). No staining was observed in B cell-lymphomas without the primary antibody (Fig. 5C and D). C₂₀Mab-11 (Fig. 5E and F) and control (Fig. 5G and H) did not stain normal thyroid tissues. These results indicate that C₂₀Mab-11 is useful for detecting B cells in immunohistochemical analyses using FFPE tissues.