CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an anti-CD44 monoclonal antibody (mAb), C 44 Mab-5 (IgG 1 , kappa) was established by immunizing mice with CD44-overexpressing Chinese hamster ovary (CHO)-K1 cells. C 44 Mab-5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG 1 subclass of C 44 Mab-5 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), the antitumor activity of C 44 Mab-5 could not be determined. In the present study, we converted the mouse IgG 1 subclass antibody C 44 Mab-5 into an IgG 2a subclass antibody, 5-mG 2a , and further produced a defucosylated version, 5-mG 2a-f, using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 5-mG 2a-f was confirmed using fucose-binding lectins, such as AAL and PhoSL. The dissociation constants (K D) for 5-mG 2a-f against SAS and HSC-2 oral cancer cells were determined through flow cytometry to be 2.8x10-10 M and 2.6x10-9 M, respectively, indicating that 5-mG 2a-f possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5-mG 2a-f specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5-mG 2a-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 5-mG 2a-f significantly reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. Collectively, these results suggest that treatment with 5-mG 2a-f may represent a useful therapy for patients with CD44-expressing oral cancers.

CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo−resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti−CD44 monoclonal antibodies (mAbs) by immunizing mice with a CD44 variant (CD44v3−10) ectodomain and screening using enzyme−linked immunosorbent assay. We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established clones (C44Mab−46; IgG1, kappa) reacted with CD44 standard isoform (CD44s)−overexpressed Chinese hamster ovary−K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The apparent KD of C44Mab−46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1 × 10−8 M, 4.9 × 10−8 M, and 4.1 × 10−8 M, respectively. C44Mab−46 detected CD44s of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore, C44Mab−46 strongly stained the formalin−fixed paraffin−embedded ESCC tissues in immunohistochemistry. Collectively, C44Mab−46 is very useful for detecting CD44 in various applications.

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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.

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