Takuro Nakamura

Isocitrate dehydrogenase mutation is frequently observed in giant cell tumor of bone

Giant cell tumors of bone (GCTB) are benign and locally destructive tumors that include osteoclast‐type multinuclear giant cells. No available treatment is definitively effective in curing GCTB, especially in surgically unresectable cases. Isocitrate dehydrogenase (IDH) mutations have been reported not only in gliomas and acute myeloid leukemias, but also in cartilaginous tumors and osteosarcomas. However, IDH mutations in GCTB have not been investigated. The IDH mutations are remarkably specific to arginine 132 (R132) in IDH1 and arginine 172 (R172) or arginine 140 (R140) in IDH2; IDH1/2 mutations are known to convert α‐ketoglutarate to oncometabolite R(‐)‐2‐hydroxyglutarate. We recently reported that the most frequent IDH mutation in osteosarcomas is IDH2‐R172S, which was detected by MsMab‐1, a multispecific anti‐IDH1/2 mAb. Herein, we newly report the IDH mutations in GCTB, which were stained by MsMab‐1 in immunohistochemistry. DNA direct sequencing and subcloning identified IDH mutations of GCTB as IDH2‐R172S (16 of 20; 80%). This is the first report to describe IDH mutations in GCTB, and MsMab‐1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation‐bearing GCTB.

Development of Monoclonal Antibody LpMab-10 Recognizing Non-glycosylated PLAG1/2 Domain Including Thr34 of Human Podoplanin

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein that possesses a platelet aggregation-stimulating (PLAG) domain in the N-terminus. PLAG domain includes three tandem repeats of eight amino acids: PLAG1, PLAG2, and PLAG3. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. In contrast, the glycosylation of Thr34 of PLAG1 of human PDPN remains to be clarified. Herein, we developed and characterized a novel anti-PDPN monoclonal antibody, LpMab-10, which targets PLAG1/2 domain. LpMab-10 detects endogenous PDPN of cancer cells and normal cells independently of glycosylation. The minimum epitope of LpMab-10 was identified as Glu33-Gly45 of PDPN using Western blot and flow cytometry. The Thr34 of PLAG1 is critical for LpMab-10 recognition, and O-glycan is not included in LpMab-10 epitope, indicating that Thr34 of PLAG1 is not O-glycosylated. In immunocytochemical and immunohistochemical analyses, LpMab-10 strongly detected PDPN-expressing tumor cells. By using monoclonal antibodies against different Ser/Thr, including epitopes of PDPN, it becomes possible to determine whether Ser/Thr residues of PDPN are O-glycosylated.

Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

Development and characterization of anti‐glycopeptide monoclonal antibodies against human podoplanin, using glycan‐deficient cell lines generated by CRISPR/Cas9 and TALEN

Human podoplanin (hPDPN), which binds to C‐type lectin‐like receptor‐2 (CLEC‐2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer‐associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung‐type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti‐hPDPN mAb, LpMab‐21. To characterize the hPDPN epitope recognized by the LpMab‐21, we established glycan‐deficient CHO‐S and HEK‐293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab‐21 is Thr76–Arg79. LpMab‐21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab‐21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell‐type‐specific. LpMab‐21 combined with other anti‐hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN.

Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity

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.

Establishment of Novel Monoclonal Antibody PMab-32 Against Rabbit Podoplanin.

Podoplanin (PDPN) is a type I transmembrane O-glycoprotein, which is known as a specific lymphatic marker. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. PDPN is also expressed in several normal tissues, including podocytes and type I alveolar cells. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN), mouse PDPN (mPDPN), and rat PDPN (rPDPN) have been established, useful antibodies against rabbit PDPN (rabPDPN) have not been developed. In this study, we immunized mice with the recombinant proteins of rabPDPN, and developed a novel anti-rabPDPN MAb, named PMab-32. PMab-32 could detect endogenous and exogenous rabPDPN in flow cytometry and Western blot analysis. The KD of PMab-32 was determined to be 6.2 × 10(-8) M by flow cytometry. Immunohistochemical analysis showed that PMab-32 is useful for detecting podocytes, type I alveolar cells, and lymphatic endothelial cells in normal rabbit tissues. PMab-32 is expected to be useful for various rabbit experiments.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375–394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP−386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1–3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.

Establishment of P38Bf, a Core-Fucose-Deficient Mouse-Canine Chimeric Antibody Against Dog Podoplanin

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. The overexpression of PDPN in cancers is associated with hematogenous metastasis by interactions with the C-type lectin-like receptor 2 (CLEC-2). We have previously reported the development of a mouse monoclonal antibody (mAb) clone, PMab-38 (IgG1, kappa), against dog PDPN (dPDPN). PMab-38 reacted strongly with canine squamous cell carcinomas and melanomas, but not with lymphatic endothelial cells, indicating its cancer specificity. In this study, we developed and produced several mouse-canine chimeric antibodies originating from PMab-38. A mouse-canine chimeric antibody of subclass A (P38A) and a mouse-canine chimeric antibody of subclass B (P38B) were transiently produced using ExpiCHO-S cells. Core-fucose-deficient P38B (P38Bf) was developed using FUT8 knockout ExpiCHO-S cells. We compared the binding affinities, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of P38A, P38B, and P38Bf against Chinese hamster ovary (CHO)/dPDPN cells. Flow cytometry analysis showed that the KD of P38A, P38B, and P38Bf were 1.9 × 10−7, 5.2 × 10−9, and 6.5 × 10−9, respectively. Both P38B and P38Bf revealed high ADCC activities against CHO/dPDPN cells; P38Bf demonstrated significantly higher ADCC compared with P38B, especially at low concentrations. P38B and P38Bf exhibited higher CDC activities against CHO/dPDPN cells. Conversely, P38A did not exhibit any ADCC or CDC activity. In summary, P38Bf is a good candidate for antibody therapy against dPDPN-expressing canine cancers.

Establishment of Monoclonal Antibody PMab-202 Against Horse Podoplanin

Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs. One of the established mAbs, PMab-202 (IgG1, kappa), was specifically able to detect horse PDPN in Chinese hamster ovary/horse PDPN (CHO/horPDPN) cells in flow cytometry experiments. PMab-202 was also able to detect endogenous horse PDPN expressed in and a horse kidney cell line, FHK-Tcl3.1, in flow cytometry and Western blot analyses. PMab-202 is expected to prove useful in investigating the function of horse PDPN.