Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity

Introduction

Podoplanin (PDPN) is expressed in many tumors, such as brain tumors, esophageal cancers, lung cancers, malignant mesotheliomas, osteosarcomas, fibrosarcomas, testicular cancers, and bladder cancers.^(1–15) Importantly, the expression of human podoplanin in cancer-associated fibroblasts contributes to poor prognosis.^(16–21) C-type lectin-like receptor 2 (CLEC-2) is an endogenous receptor of human podoplanin.^(22,23) It binds to human podoplanin through residues Glu47 and Asp48 within its platelet aggregation-stimulating domain, and it also binds to the α-2,6-linked sialic acid linked to Thr52.⁽²⁴⁾

Although many antihuman podoplanin monoclonal antibodies (mAbs) are commercially available, almost all the mAbs react with the N-terminus of human podoplanin.^(6,25–29) In contrast, we have used the cancer-specific mAb (CasMab) technology to produce antiglycopeptide mAbs (GpMabs) against human podoplanin.^(30–39) Recently, we have successfully developed a novel antihuman podoplanin mAb, LpMab-21, which recognizes a sialylated glycopeptide epitope.⁽⁽⁴⁰⁾ LpMab-21 is one of the GpMabs, but not a CasMab. Importantly, LpMab-21 is the first mouse antihuman podoplanin mAb of the IgG_2a subclass, which was generated using the CasMab technology.^{(30–36,38,39)}

Results and Discussion

We have produced several antihuman podoplanin mAbs using the CasMab technology; however, the isotypes of the mAbs are IgG₁ (seven clones) and IgG₃ (one clone), which do not induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The applications of mouse IgG₃ mAbs are limited because they often aggregate.⁽⁽⁴¹⁾ In this case, we have had to convert them to human IgG₁ to investigate whether these mAbs cause ADCC and CDC.⁽⁽³³⁾ In contrast, the mouse IgG_2a subclass can induce ADCC and CDC; therefore, we investigated whether LpMab-21 can induce ADCC and CDC against human podoplanin-expressing cancer cell lines. As shown in Figure 1A, LpMab-21 demonstrated ADCC against LN319, the Chinese hamster ovary (CHO)/human podoplanin, and PC-10 cell lines, whereas it did not induce ADCC against human podoplanin-negative parental CHO cells. Similarly, LpMab-21 induced CDC against LN319 and PC-10 cell lines (Fig. 1B). We previously demonstrated that LN319 expresses human podoplanin at a higher level than does PC-10⁽⁽³⁰⁾; therefore, ADCC and CDC might depend on the human podoplanin expression levels in these cell lines.

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FIG. 1.

Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of LpMab-21 against human podoplanin-expressing cell lines. (A) ADCC activity of LpMab-21. Mouse splenocytes were harvested from severe combined immunodeficiency mouse spleens. Spleens were homogenized in RPMI 1640 and centrifuged. To deplete red blood cells, the cell pellet was suspended in red blood cell lysis buffer (Sigma–Aldrich). After washing and resuspension in cRPMI1640, splenocytes were used as effector cells. ADCC was determined using the ⁵¹Cr release assay. Target cells were incubated with 0.1 μCi of [⁵¹Cr]sodium chromate at 37°C for 1 hour. After washing with cRPMI1640 three times, ⁵¹Cr-labeled target cells were placed in 96-well plates in triplicate. Effector cells and LpMab-21 or control mouse IgG were added to the plates. After 6 hours of incubation, ⁵¹Cr release was measured in the supernatant (100 μL) from each well using a gamma counter (PerkinElmer). The percentage of cytotoxicity was calculated using the following formula: specific lysis (%) = (E − S)/(M − S) × 100, where E is the ⁵¹Cr release in the test sample, S is the spontaneous release, and M is the maximum release. Statistical significance of the differences in in vitro data was analyzed by the standard Student's t-test. In this study, p values of <0.05 were considered statistically significant in all experiments. **p < 0.01 (B) CDC was evaluated with the ⁵¹Cr release assay. Target cells were incubated with [⁵¹Cr]sodium chromate (0.1 μCi) for 1 hour at 37°C. The cells were then washed with cRPMI1640. The ⁵¹Cr-labeled cells were incubated with a baby rabbit complement (Cedarlane) at a dilution of 1:32 (Chinese hamster ovary [CHO] and CHO/human podoplanin) or 1:4 (LN319 and PC-10) in the presence of LpMab-21 (1 μg/mL) or control mouse IgG (1 μg/mL) for 3 hours (CHO and CHO/human podoplanin) or 6 hours (LN319 and PC-10) in 96-well plates. After the incubation, the ⁵¹Cr radioactivity was measured in the supernatants using a gamma counter. The percentage of cytotoxicity was calculated as described previously. **p < 0.01.

To investigate the antitumor activity of LpMab-21 on primary tumor growth in vivo, CHO/human podoplanin cells were subcutaneously implanted into flanks of nude mice. LpMab-21 or control mouse IgG (clone PMab-2)⁽⁽⁴²⁾ was injected into the peritoneal cavity of the mice once weekly for 4 weeks (n = 6 each). PMab-2 was raised against rat podoplanin and was shown to not cross-react with human podoplanin.⁽⁽⁴²⁾ Tumor formation was observed in five mice from the control group (tumor incidence on day 35: 83.3%, 5/6; Supplementary Fig. S1). In contrast, LpMab-21 dramatically reduced the tumor development (tumor incidence on day 35: 50%, 3/6; Supplementary Fig. S1). The tumor volume was significantly reduced by LpMab-21 treatment (Fig. 2). These results indicate that administration of LpMab-21 inhibited the primary tumor growth of CHO/human podoplanin cells. In our previous study, only CDC of a human–mouse chimeric anti-PDPN mAb could show antitumor activity because human NK cell was not added in this xenograft model.⁽⁽⁴³⁾ In contrast, we also showed that ADCC of a human–mouse chimeric anti-PDPN mAb is more important than CDC in another study.⁽⁽⁹⁾ Therefore, we think that both ADCC and CDC are important to induce antitumor activity in the xenograft model.

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FIG. 2.

Antitumor effects of LpMab-21 on primary tumor development. CHO/human podoplanin cells (3 × 10⁶ cells/100 μL) were subcutaneously inoculated into BALB/c nude mice. After 1 day, 100 μg of LpMab-21 or control mouse IgG (clone PMab-2) was injected into the peritoneal cavity of the mice. The antibodies were injected once weekly for 4 weeks (control group: n = 6; LpMab-21 group: n = 6). The tumor diameter was measured at intervals of 3 to 4 days and was calculated using the following formula: tumor volume = W² × L/2, where W is short diameter and L is long diameter. *p < 0.05; **p < 0.01 with two-way analysis of variance.

Taken together, LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers. Our developed CasMabs as well as LpMab-21 could be applied to the novel antitumor reagents, including T cells and viruses,⁽⁽⁴⁴⁾ to give strict specificity against tumor cells.