Epitope Mapping of a Cancer-Specific Anti-Podocalyxin Monoclonal Antibody (PcMab-60) Using Enzyme-Linked Immunosorbent Assay and Surface Plasmon Resonance

Cell lines

Chinese hamster ovary (CHO)-K1 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). PODXL mutant plasmids were transfected into cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Stable transfectants were sorted using a cell sorter (SH800; Sony Corp., Tokyo, Japan). Cells were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂. Transfectants were cultivated in a medium containing 0.5 mg/mL G418 (Nacalai Tesque, Inc.).

Plasmid preparation

cDNA encoding the full-length open reading frame of PODXL was obtained through polymerase chain reaction (PCR) using cDNA derived from the LN229 cell line (ATCC) as a template. We produced PODXL deletion mutants (dN60, dN100, dN140, dN200, dN300, and dN400) using HotStar HiFidelity polymerase kit (Qiagen Inc., Hilden, Germany) with appropriate oligonucleotides. In-Fusion PCR cloning kit (Takara Bio, Inc., Shiga, Japan) was used to subclone PCR products into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical Industries Ltd., Osaka, Japan) containing a signal sequence and PA tag (GVAMPGAEDDVV),⁽²¹⁾ which is recognized by an anti-PA tag mAb (NZ-1).⁽²²⁾ All amino acid numbers were consistent with the NCBI reference sequence (NP_005388.2).

Flow cytometry

Cells were harvested by briefly exposing them to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). After washing the cells with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), they were treated with PcMab-60 (10 μg/mL) or NZ-1 (1 μg/mL) for 30 min at 4°C and then with Alexa Fluor 488-conjugated antimouse IgG or antirat IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluorescence data were collected using BD FACSLyric system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

PODXL peptides

PODXL (Accession No. NP_005388.2) peptides, including 6 deletion mutants and 20 alanine-substituted peptides (Tables 1–3), were synthesized using PEPScreen (Sigma-Aldrich Corp., St. Louis, MO, USA).

Enzyme-linked immunosorbent assay

Synthesized PODXL peptides were immobilized in Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at a concentration of 10 μg/mL for 30 min at 37°C. After washing with PBS containing 0.05% Tween 20 (PBST) (Nacalai Tesque, Inc.), wells were blocked with 1% BSA-containing PBST for 30 min at 37°C. The plates were incubated with PcMab-60 (20 μg/mL), followed by a peroxidase-conjugated antimouse immunoglobulins (1:2000 diluted; Agilent Technologies, Inc., Santa Clara, CA, USA). Enzymatic reactions were conducted using ELISA POD Substrate TMB kit (Nacalai Tesque, Inc.). Optical density was measured at 655 nm using iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).

Measurement of dissociation constants (K_D) between PcMab-60 and alanine-substituted peptides using SPR

PcMab-60 was immobilized on the sensor chip CM5 according to the protocol described by Cytiva (Marlborough, MA, USA). In brief, PcMab-60 was diluted to 10 μg/mL using acetate buffer (pH 4.0: Cytiva) and immobilized using an amine-coupling reaction. The surface of the flow cell 2 of the sensor chip CM5 was treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (NHS), followed by injecting PcMab-60. Unreacted NHS-ester was blocked with ethanolamine after PcMab-60 immobilization. K_D values between PcMab-60 and alanine-substituted peptides given in Table 2 were measured using Biacore X100 (Cytiva) at 25°C. The buffer used was PBS containing 0.05% (v/v) Tween 20 and 1.4% (v/v) dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation). A single cycle kinetics method was used to measure binding signals. We conducted an equilibrium analysis to analyze the data and determine K_D using BIAevaluation software (Cytiva).

Determination of the PcMab-60 epitope using PODXL deletion mutants and synthesized peptides

To determine the PcMab-60 epitope, we constructed PODXL N-terminal deletion mutants (dN60, dN100, dN140, dN200, dN300, and dN400) with the PA tag at their N-terminus and expressed these mutants in CHO-K1 cells. Reactions between PcMab-60 and deletion mutants and between anti-PA tag mAb (NZ-1) and deletion mutants were characterized using flow cytometry. The results of flow cytometry showed that PcMab-60 reacted with dN60 and dN100, but did not react with dN140, dN200, dN300, and dN400 (Fig. 1A). All deletion mutants were detected by NZ-1 (Fig. 1B). Both PcMab-60 and NZ-1 did not recognize parental CHO-K1 cells (Fig. 1A, B). These results indicated that the N-terminus of PcMab-60 epitope existed between the 100–140 amino acids (aa) of PODXL (Fig. 1C).

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FIG. 1.

Epitope mapping of PcMab-60 using deletion mutants. (A, B) PODXL deletion mutants were expressed in CHO-K1 cells, incubated with PcMab-60 (A) or anti-PA tag mAb (NZ-1) (B) for 30 min at 4°C, and analyzed using flow cytometry, followed by corresponding secondary antibody treatment. Lines, with first antibody; shadowed, without first antibody in blocking buffer (negative control). (C) Schematic illustration of PcMab-60 epitope mapping with PODXL deletion mutants (dN60, dN100, dN140, dN200, dN300, and dN400) with a PA tag at N-terminus, and synthesized peptides corresponding to 70–89, 80–99, 90–109, 100–119, 110–129, and 120–139 amino acids. Black bars: positive reaction to PcMab-60. White bars: negative reaction to PcMab-60. IC, intracellular domain; PA, PA tag; PODXL, podocalyxin; TM, transmembrane domain.

Next, we prepared six peptides of 20 aa each (70–89, 80–99, 90–109, 100–119, 110–129, and 120–139 of PODXL; Table 1) and performed ELISA. PcMab-60 reacted with the 100–119 peptide (₁₀₀-SGPVNTTVARGGGSGNPTTT-₁₁₉), but did not react with other peptides (Table 1). This result indicated that the PcMab-60 epitope exists between the 100–119 aa of PODXL (Fig. 1C).

Epitope mapping using SPR

Next, we synthesized wild-type and 20 alanine-substituted PODXL peptides of 100–119 aa and determined the K_D values using SPR. The K_D values between 11 peptides (S100A, G101A, P102A, V103A, N104A, T106A, V107A, A108G, T118A, T119A, and wild-type 100–119) and PcMab-60 were determined to be ∼300–600 nM. However, the K_D values between 10 peptides (T105A, R109A, G110A, G111A, G112A, S113A, G114A, N115A, P116A, and T117A) and PcMab-60 were not determined. The K_D values for each peptide are given in Table 2. These results indicated that Thr105, Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, Pro116, and Thr117 are the critical epitope of PcMab-60 (Fig. 2A).

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FIG. 2.

Schematic illustration of PcMab-60 epitope mapping using SPR (A) and ELISA (B). Critical epitope residues are underlined. ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; SS, signal sequence.

Epitope mapping using ELISA

We further evaluated the reactivity of PcMab-60 to the alanine-substituted PODXL peptides using ELISA. PcMab-60 reacted with S100A, G101A, P102A, V103A, N104A, T105A, T106A, V107A, A108G, T117A, T118A, and T119A, but not with R109A, G110A, G111A, G112A, S113A, G114A, N115A, and P116A (Table 3). The results of ELISA indicated that Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, and Pro116 are the critical epitope of PcMab-60 (Fig. 2B).