Development of Core-Fucose-Deficient Humanized and Chimeric Anti-Human Podoplanin Antibodies

Abstract

Podoplanin (PDPN), a 36-kDa type I transmembrane O-glycoprotein, is expressed in normal cells, including renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, and in cancer cells, including brain tumors and squamous cell lung carcinomas. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and PDPN/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced an anti-human PDPN monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda) and its rat-human chimeric mAbs (NZ-8/NZ-12), which neutralize PDPN/CLEC-2 interactions and inhibit platelet aggregation and cancer metastasis. In this study, we first developed a humanized anti-human PDPN mAb, named as NZ-27. We further produced a core-fucose-deficient version of NZ-27, named as P1027 and a corefucose-deficient version of NZ-12, named as NZ-12f. We investigated the binding affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of P1027 and NZ-12f. We demonstrated that the binding affinities of P1027 and NZ-12f against LN319 (a human glioblastoma cell line) are 1.1 • 10 -8 and 3.9 • 10 -9 M, respectively. ADCC reporter assays demonstrated that NZ-12f shows 1.5 times higher luminescence than P1027. Furthermore, NZ-12f showed 2.2 times higher ADCC than P1027, whereas both P1027 and NZ-12f showed high CDC activities against LN319 cells. Using LN319 xenograft models, P1027 and NZ-12f significantly reduced tumor development in an LN319 xenograft model compared with control human IgG. Treatment with P1027 and NZ-12f may be a useful therapy for patients with PDPN-expressing cancers.

Improved anti‐solid tumor response by humanized anti‐podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model

“…The antibodies used for staining are listed in Table S2. NZ‐1 and NZ‐27 scFvFc antibodies were developed as described previously (Chandramohan et al, 2013; Kaneko et al, 2020).…”

Defucosylated mouse‑dog chimeric anti‑HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors

“…Canine mononuclear cells (MNCs) obtained from Yamaguchi University were resuspended in DMEM (Nacalai Tesque, Inc.) with 10% FBS and were used as effector cells (37,38,42). Target cells (CHO-K1, CHO/dHER2, and SNP) were labeled with 10 µg/ml Calcein AM (Thermo Fisher Scientific, Inc.) (31,(39)(40)(41)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53). The target cells (2x10 4 cells) were plated in 96-well plates and mixed with effector canine MNCs (effector/target cells ratio, 50), 100 µg/ml of H77Bf or control dog IgG.…”

“…CDC of H77Bf. Target cells (CHO-K1, CHO/dHER2, and SNP) were labeled with 10 µg/ml Calcein AM (31,(39)(40)(41)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53). The target cells (2x10 4 cells) were plated in 96-well plates and mixed with rabbit complement (final dilution 1:10; Low-Tox-M Rabbit Complement; Cedarlane Laboratories,) and 100 µg/ml of control dog IgG or H77Bf.…”

Defucosylated Mouse–Dog Chimeric Anti-EGFR Antibody Exerts Antitumor Activities in Mouse Xenograft Models of Canine Tumors

“…ADCC assay was performed as previously described [ 16 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. Briefly, canine mononuclear cells derived from dog blood were obtained from Yamaguchi University and resuspended in DMEM with 10% FBS to be used as effector cells.…”