The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity

Podoplanin (PDPN/Aggrus/T1α) binds to C-type lectin-like receptor-2 (CLEC-2) and induces platelet aggregation. PDPN is associated with malignant progression, tumor metastasis, and poor prognosis in several types of cancer. Although many anti-human PDPN (hPDPN) monoclonal antibodies (mAbs), such as D2-40 and NZ-1, have been established, these epitopes are limited to the platelet aggregation-stimulating (PLAG) domain (amino acids 29-54) of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-7, which is more sensitive than D2-40 and NZ-1, using the Cancer-specific mAb (CasMab) method. The epitope of LpMab-7 was shown to be entirely different from that of NZ-1, a neutralizing mAb against the PLAG domain according to an inhibition assay and lectin microarray analysis. In the present study, we produced a mouse-human chimeric anti-hPDPN mAb, chLpMab-7. ChLpMab-7 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, chLpMab-7 inhibited the growth of hPDPN-expressing tumors in vivo. Although chLpMab-7 recognizes a non-PLAG domain of hPDPN, it suppressed the hematogenous metastasis of hPDPN-expressing tumors. These results indicated that chLpMab-7 suppressed tumor development and hematogenous metastasis in a neutralization-independent manner. In conclusion, hPDPN shows promise as a target in the development of a novel antibody-based therapy

Chimeric Anti-PDPN Antibody ChLpMab-2

Human podoplanin (hPDPN ), a platelet aggregation‐inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C‐type lectin‐like receptor 2 (CLEC ‐2). The overexpression of hPDPN is involved in invasion and metastasis. Anti‐hPDPN monoclonal antibodies (mAbs) such as NZ ‐1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation‐stimulating (PLAG ) domain of hPDPN . Recently, we developed a novel mouse anti‐hPDPN mAb, LpMab‐2, using the cancer‐specific mAb (CasMab) technology. In this study we developed chLpMab‐2, a human–mouse chimeric anti‐hPDPN antibody, derived from LpMab‐2. chLpMab‐2 was produced using fucosyltransferase 8‐knockout (KO ) Chinese hamster ovary (CHO )‐S cell lines. By flow cytometry, chLpMab‐2 reacted with hPDPN ‐expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab‐2 exhibited high antibody‐dependent cellular cytotoxicity (ADCC ) against PDPN ‐expressing cells, despite its low complement‐dependent cytotoxicity. Furthermore, treatment with chLpMab‐2 abolished tumor growth in xenograft models of CHO /hPDPN , indicating that chLpMab‐2 suppressed tumor development via ADCC . In conclusion, chLpMab‐2 could be useful as a novel antibody‐based therapy against hPDPN ‐expressing tumors

Podoplanin Is Regulated by AP-1 and Promotes Platelet Aggregation and Cell Migration in Osteosarcoma

Podoplanin is a type-I transmembrane sialomucin-like protein, which is expressed in a wide range of cell types and is involved in platelet aggregation and tumor metastasis. Here, we investigated the function, regulation, and expression of podoplanin in osteosarcoma. Podoplanin expression was observed in three osteosarcoma cell lines (MG-63, HOS, and U-2 OS) with platelet aggregation–inducing ability, which was blocked by podoplanin small-interfering RNA or a neutralizing antibody. Overexpression of podoplanin in nonmetastatic Dunn osteosarcoma cells promoted cell migration without attenuating cell proliferation. Both podoplanin and TGF-β1 were up-regulated by c-Fos induction in MC3T3-E1 osteoblastic cells, and were highly expressed in c-Fos transgenic mouse osteosarcomas and c-Fos–transformed osteosarcoma cell lines. Immunohistochemistry of human osteosarcoma tissue microarrays (n = 133) showed staining of tumor cells embedded in an excess of irregular neoplastic bone matrix in 100% of tumors undergoing so-called “normalization/maturation.” Podoplanin was also expressed in osteosarcoma subtypes, with 65% of osteoblastic, 100% of chondroblastic, and 79% of fibroblastic tumors. CD44 and pERM immunohistochemistry showed coexpression with podoplanin in both mouse and human osteosarcoma. Podoplanin expression was significantly higher in metastatic osteosarcomas (n = 6) than in primary osteosarcomas (n = 10). Our data suggest that podoplanin, which is not expressed in normal osteoblasts but in osteocytes, is aberrantly expressed in transformed osteoblasts and in osteosarcoma, and is under AP-1 transcriptional control. Thus podoplanin is a candidate molecule for therapeutic targeting