Epitope Mapping Using the Cell-Based 2 × Alanine Substitution Method About the Anti-mouse CXCR6 Monoclonal Antibody,Cx 6 Mab-1

Cell lines

Chinese hamster ovary-K1 (CHO-K1) cells were purchased from the American Type Culture Collection (Manassas, VA). The mCXCR6 mutation plasmids, pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), containing an N-terminal PA tag,^27–32 were transfected into CHO-K1 cells using a Neon Transfection System (Thermo Fisher Scientific, Inc., Waltham, MA). The PA tag comprises 12 amino acids, GVAMPGAEDDVV, and is recognized by NZ-1 mAb.^27,33

Cells transiently transfected with wild type (WT) or two alanine (or glycine) residues-substituted mCXCR6 mutants were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.), and 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA). Then, cells were grown in a humidified incubator, which was supplied with 5% CO₂ and 95% air at 37°C.

Production of mCXCR6 2 × alanine mutants

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding mCXCR6 (Accession No. NM_030712) with N-terminal PA tag was subcloned into a pCAG-Ble vector. Plasmids were transfected using a neon transfection system. Substitutions of consecutive amino acids with two alanine (or glycine) in the mCXCR6 sequence were produced by QuikChange Lightning Site Directed Mutagenesis Kits (Agilent Technologies, Inc., Santa Clara, CA). Polymerase chain reaction fragments-bearing desired mutations were inserted into the pCAG-Ble vector using KAPA HiFi HotStart ReadyMix (NIPPON Genetics Co., Ltd., Tokyo, Japan).

Flow cytometry

Transiently transfected CHO-K1 cells were detached by 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.) and collected using 10% FBS containing RPMI 1640 medium. After washing with 0.1% bovine serum albumin (BSA; Nacalai Tesque, Inc.) in phosphate-buffered saline (PBS; Nacalai Tesque, Inc.), cells were treated with anti-mCXCR6 mAb (Cx₆Mab-1; 1 μg/mL) or NZ-1 (anti-PA tag; 1 μg/mL) for 30 minutes at 4°C, followed by incubation with Alexa Fluor 488-conjugated anti-rat IgG (1:1000; product no. 4416; Cell Signaling Technology, Inc.). Fluorescence data were collected using the SA3800 Cell Analyzer (Sony Biotechnology Corp., Tokyo, Japan).

Epitope mapping of Cx₆Mab-1 with alanine-substituted mCXCR6 in CHO-K1

We previously developed an anti-mCXCR6 mAb (Cx₆Mab-1) by N-terminal peptide immunization of mCXCR6. ²² To identify the binding epitope of Cx₆Mab-1, we produced 10 mutants of mCXCR6, in which sequential two amino acids were replaced to two alanine (or glycine) residues. This method was named as “2 × Ala-scan method.”

Based on past ELISA analysis, we could not determine the epitope with 1 × Ala-scan method ²⁶ ; therefore, we introduced 2 × Ala-scan method using cells in this study. For instance, a mutant (D2A-D3A) indicates the alanine substitution of second Asp and third Asp of mCXCR6 protein. As shown in Figure 1A, all 2 × Ala mutants and WT mCXCR6 containing the N-terminal PA tag were recognized by NZ-1 (an anti-PA tag mAb), indicating that the expression level of each construct was sufficient. Cx₆Mab-1 recognized D2A-D3A, G4A-H5A, Q6A-E7A, E16A-G17A, D18A-F19A, and W20A-L21A, but not S8A-A9G, L10A-Y11A, D12A-G13A, and H14A-Y15A (Fig. 1B).

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FIG. 1.

Epitope determination of Cx₆Mab-1 using 2 × Ala (or Gly) substituted mutants by flow cytometry. (A) CHO-K1 cells, which transiently overexpressed WT or two amino acids-substituted mutants (D2A-D3A, G4A-H5A, Q6A-E7A, S8A-A9G, L10A-Y11A, D12A-G13A, H14A-Y15A, E16A-G17A, D18A-F19A, and W20A-L21A) of mCXCR6, were incubated with NZ-1 (anti-PA tag mAb; red line), or buffer control (black line) for 30 minutes at 4°C, followed by treatment with secondary antibody. (B) CHO-K1 cells, which transiently expressed WT or two amino acids-substituted mutants (D2A-D3A, G4A-H5A, Q6A-E7A, S8A-A9G, L10A-Y11A, D12A-G13A, H14A-Y15A, E16A-G17A, D18A-F19A, and W20A-L21A) of mCXCR6, were incubated with Cx₆Mab-1 (anti-mCXCR6 mAb; red line), or buffer control (black line) for 30 minutes at 4°C, followed by treatment with secondary antibody. (C) Schematic illustration of mCXCR6 and the Cx₆Mab-1 epitope. The Cx₆Mab-1 epitope involves Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 of mCXCR6, shown underline. mCXCR6, mouse CXC chemokine receptor 6; WT, wild type.

These results suggest that Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 are included in the critical epitope of Cx₆Mab-1. The results are shown as a schematic illustration in Figure 1C. Previous results of ELISA experiments indicated that Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 were the critical epitopes of Cx₆Mab-1, we speculate that this discrepancy is due to the structural differences between peptides and proteins. This 2 × Ala-scanning strategy could be another option to identify the epitope of mAbs even if the conventional alanine scanning is not effective.

Several GPCRs, including CCR2, CCR3, CCR5, CCR9, and CXCR1, interact with ligands at the N-terminal region.^34,35 A molecular dynamics simulation about docking of CXCL16–CXCR6 was reported. ³⁶ Cx₆Mab-1 may block CXCL16 binding, although further approach is needed. CXCL16 is highly expressed by tumor cells, including pancreatic cancer, and tumor-infiltrating immune cells. Since CXCR6-armed T cells enhance the recognition and lysis of tumor, ³⁷ Cx₆Mab-1 is expected to be one of the immunotherapy strategies by applying modalities such as bispecific antibodies against tumor antigen.