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

LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin

Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2

LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin

Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2

Danger of frustrated sensors: Role of Toll-like receptors and NOD-like receptors in aseptic and septic inflammations around total hip replacements

The innate immune sensors, Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), can recognize not only exogenous pathogen-associated molecular patterns (PAMPs), but also endogenous molecules created upon tissue injury, sterile inflammation, and degeneration. Endogenous ligands are called damage-associated molecular patterns (DAMPs), and include endogenous molecules released from activated and necrotic cells as well as damaged extracellular matrix. TLRs and NLRs can interact with various ligands derived from PAMPs and DAMPs, leading to activation and/or modulation of intracellular signalling pathways. Intensive research on the innate immune sensors, TLRs and NLRs, has brought new insights into the pathogenesis of not only various infectious and rheumatic diseases, but also aseptic foreign body granuloma and septic inflammation of failed total hip replacements (THRs). In this review, recent knowledge is summarized on the innate immune system, including TLRs and NLRs and their danger signals, with special reference to their possible role in the adverse local host response to THRs. Translational potential of this article: A clear understanding of the roles of Toll-like receptors and NOD-like receptors in aseptic and septic loosening of joint replacements will facilitate potential strategies to mitigate these events, thereby extending the longevity of implants in humans

Binding assay of LpMab-12 against sialylated glycopeptide of hPDPN using ELISA.

<p>Strategy for the sialylated glycopeptide synthesis. SA, sialic acid; Gal, galacose; GalNAc, <i>N</i>-acetyl-_D-galactosamine.</p

Immunohistochemical analysis of the oral cancer and heart tissue samples using LpMab-12 and LpMab-7.

<p>Serial sections of the tissues with oral cancer were incubated with LpMab-12 (A-D) or LpMab-7 (E-H), followed by the development with the EnVision+ kit and counterstaining with hematoxylin, or the HE staining (I-L). Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cells. Scale bars: 100 μm. LpMab-12 stains lymphatic vessels with high efficiency, similarly to LpMab-7.</p

The reaction of LpMab-12 against glycopeptides of hPDPN.

<p>The reaction of LpMab-12 against glycopeptides of hPDPN.</p

Epitope mapping of LpMab-12 by Western blot analysis and flow cytometry.

<p>(A) CHO-K1 cells were transfected with a plasmid expressing wild-type hPDPN with the FLAG-tag added to the C-terminus (WT), or the FLAG-tag hPDPN containing a point mutation in the sequence E47A-E57A, as indicated in the figure. Total cell lysates from the transfected cell lines were analyzed by Western blot with LpMab-12 or LpMab-7, as a positive control for hPDPN expression. Immunoblot with anti-FLAG antibody was also used as well to establish the expression of exogenous hPDPN. Anti-IDH1 and anti-β-actin mAbs were used as internal controls to show that total proteins are equal protein load. Red arrow, 40-kDa; blue arrow, 30-kDa. (B) CHO-K1 cells transfected as in (A) were analyzed by flow cytometry using indirect immunolabeling with LpMab-12. Cells exposed to the secondary anti-mouse IgG only were used as a negative control (Control).</p