Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain

Cell lines

CHO-K1 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). LN319 and D397 were provided by Dr. Kazuhiko Mishima (Saitama Medical University, Saitama, Japan) and Dr. Darell D. Bigner (Duke University Medical Center, Durham, NC), respectively. PC-10 cells were purchased from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan). CHO-K1 and PC-10 were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and LN319 and D397 were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Antibodies

A rat anti-human podoplanin mAb (NZ-1) and a human–rat chimeric anti-human podoplanin antibody (NZ-8) were developed as described previously.^(5,27,29,30) For the generation of human–rat chimera anti-human podoplanin (NZ-12), the appropriate V_H of a rat NZ-1 antibody and C_H of human IgG₁ were subcloned into the pCAG-Neo (Wako Pure Chemical Industries Ltd., Osaka, Japan), and V_L of a rat NZ-1 antibody and C_L of human lambda chain were subcloned into pCAG-Ble vectors (Wako Pure Chemical Industries Ltd.). Antibody expression vectors were transfected into CHO-K1 cells using Lipofectamin LTX (Thermo Fisher Scientific, Inc.). Stable transfectants of CHO/NZ-12 were selected by cultivating the transfectants in medium containing 1 mg/mL G418 (Wako Pure Chemical Industries Ltd.) and 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA). CHO/NZ-12 cells were cultivated in CHO-S-SFM II medium (Thermo Fisher Scientific, Inc.). NZ-12 was purified using Protein G-Sepharose (GE healthcare Bio-Sciences, Pittsburgh, PA). Human IgG was purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Determination of the binding affinity by flow cytometry

LN319 cells (2 × 10⁵ cells) were resuspended with 100 μL of serially diluted antibodies (NZ-1, NZ-8, or NZ-12; 0.02–100 μg/mL) followed by FITC-labeled anti-human IgG or Oregon green-labeled anti-rat IgG (Thermo Fisher Scientific, Inc.). Fluorescence data were collected using a cell analyzer (EC800; Sony Corp.). The dissociation constants (K_D) were obtained by fitting the binding isotherms using the built-in one-site binding models in Prism software.

Preparation of effector cells

The preparation methods of effector cells have been described previously.⁽³¹⁾ Human peripheral blood mononuculear cells (MNCs) were obtained from leukocytes, which were separated from peripheral blood of healthy donors. The human study was approved by the Ethics Committee of Tokushima University.

Antibody-dependent cellular cytotoxicity

ADCC was determined with ⁵¹Cr release assay.^(31,32) Target cells were incubated with 0.1 μCi of ⁵¹Cr-sodium chromate at 37°C for 1 hour. After washing with CRPMI1640 (RPMI1640 medium including 10% FBS) three times, ⁵¹Cr-labeled target cells were placed in 96-well plates in triplicate. Effector cells and antibodies were added to the plates. After 6 hours incubation, ⁵¹Cr release of the supernatant from each well (100 μL) was measured using a gamma counter (PerkinElmer, Waltham, MA). The percentage of cytotoxicity was calculated using the following formula: % Specific lysis =(E−S)/(M−S) × 100, where E is the release in the test sample, S is the spontaneous release, and M is the maximum release.

Complement-dependent cytotoxicity

CDC was evaluated by ⁵¹Cr release assay as described previously.^(31,32) Target cells were incubated with ⁵¹Cr-sodium chromate (0.1 μCi) for 1 hour at 37°C. After that, the cells were washed with CRPMI1640. ⁵¹Cr-labeled cells were incubated with baby rabbit complement (Cedarlane, Ontario, Canada) at a dilution of 1:4 and antibodies (1 μg/mL) for 6 hours in 96-well plates. After incubation, the supernatant including ⁵¹Cr was measured using a gamma counter. The percentage of cytotoxicity was calculated as described above.

Statistical analyses

The statistical significance of differences in in vitro data was analyzed by standard Student's t-test. In this study, p-values less than 0.05 were considered significant in all experiments.

Development of a novel human–rat chimeric anti-podoplanin antibody (NZ-12)

The sensitivity of NZ-8 is lower than that of NZ-1.⁽²⁹⁾ NZ-8 was generated by fusing the V_H of rat antibody (NZ-1) with C_H of human IgG₁ and fusing V_L regions of NZ-1 with C_L of human kappa because almost all chimeric antibodies and humanized antibodies are comprised of kappa light chains.^(33,34) In contrast, NZ-1 possesses lambda light chain, the rare isotype found among rat IgGs (<5%).⁽³⁵⁾ Therefore, we subsequently generated the human–rat chimeric antibody (NZ-12) by fusing the V_H of rat antibody (NZ-1) with C_H of human IgG₁ and fusing V_L regions of NZ-1 with C_L of human lambda.

NZ-12 demonstrated high sensitivity against podoplanin-expressing cancer cell lines in flow cytometry, and its reactivity was shown to be much higher than that of NZ-8 (data not shown). K_D of NZ-1 was determined to be 1.2 × 10⁻⁹ M by flow cytometry (Fig. 1). Using the same methods, K_D of NZ-8 was determined to be 7.3 × 10⁻⁷ M (Fig. 1), indicating that the binding affinity of NZ-8 was diminished following conversion from rat mAb to a human–rat chimeric antibody. In contrast, K_D of NZ-12 was determined to be 8.5 × 10⁻⁹ M by flow cytometry (Fig. 1), indicating that the binding affinity against a podoplanin-expressing cell line (LN319) became much higher following conversion from a human kappa light chain (NZ-8) to a human lambda light chain (NZ-12).

OPEN IN VIEWER

FIG. 1.

Determination of binding-affinity using flow cytometry. LN319 (2 × 10⁵ cells) was resuspended at 100 μL of serially diluted NZ-1, NZ-8, or NZ-12 (0.02–100 μg/mL).

ADCC and CDC mediated by anti-podoplanin antibodies

To apply targeted therapy to podoplanin, we further assessed whether anti-podoplanin antibodies could induce ADCC against podoplanin-expressing cell lines mediated by human MNC as effector cells. We compared the ADCC and CDC activities against NZ-1, NZ-8, and NZ-12 using LN319, D397, and PC-10 cell lines. ADCC against podoplanin-expressing cell lines was not exhibited by NZ-1 using human MNC, whereas NZ-8 and NZ-12 showed the induction of significant level of ADCC mediated by human MNCs against LN319 and D397 glioblastoma cells and PC-10 lung cancer cells (Fig. 2A). Furthermore, ADCC by NZ-12 was higher compared with NZ-8 particularly against LN319 and D397 glioblastoma cells. Interestingly, CDC was induced by NZ-12 against all podoplanin-expressing cell lines, and this was significantly higher than that of NZ-8 (Fig. 2B).

OPEN IN VIEWER

FIG. 2.

ADCC and CDC activities of anti-podoplanin antibodies. (A) ADCC activities induced by human MNC against podoplanin-expressing cell lines LN319, D397, and PC-10 were determined with 6 hours ⁵¹Cr release assay at the E/T ratio of 100 in the presence of 1 μg/mL of NZ-1, NZ-8, NZ-12, and human IgG. (B) CDC activities against podoplanin-expressing cell lines LN319, D397, and PC-10 were determined by ⁵¹Cr release assay. **p < 0.01, *p < 0.05 versus control (values are means ± S.E.). ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity; MNC, mononuculear cell.

Taken together, NZ-12 could be useful for antibody therapy against human podoplanin-expressing cancers. In the future, NZ-1/NZ-12 could be further applied to the novel anti-tumor reagents, including T cells and viruses.^(36,37)