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.
Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG
1
, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.
†A complete list of the members of the International Breast Cancer Study Group (IBCSG), a division of the ETOP IBCSG Partners Foundation, and lists of the study groups, steering committee members, and investigators participating in the POSITIVE trial are provided in the Supplementary Appendix, available at NEJM.org.
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