Development and Characterization of Novel Monoclonal Antibodies Against Human DNAM-1

Abstract

DNAM-1 (CD226) is an activating immunoreceptor expressed on lymphocytes and myeloid cells. CD155 and CD112 are the ligands for DNAM-1. DNAM-1 plays an important role in tumor immunity mediated by CD8 T cells and NK cells. Moreover, the interaction of DNAM-1 with the ligands contributed to the development of acute graft versus host disease (GVHD) and treatment with anti-DNAM-1 monoclonal antibodies (mAb) dramatically improved acute GVHD in a mouse model, suggesting that DNAM-1 may be a good molecular target for therapy to acute GVHD in human. In this study, we generated and characterized five novel clones of anti-human DNAM-1 mAbs, named TX94, TX95, TX96, TX107, and TX108. Among these mAbs, TX94 is a unique neutralizing mAb that most efficiently blocked the interaction between DNAM-1 and CD155. Furthermore, TX94 inhibited NK cell-mediated cytotoxicity against a tumor cell line and suppressed CD8 T cell proliferation mediated by allogeneic mixed lymphocyte reaction. Thus, TX94 may be useful for molecular therapy targeting DNAM-1.

G307S DNAM-1 mutation exacerbates autoimmune encephalomyelitis via enhancing CD4+ T cell activation

“…To analyze tyrosine phosphorylation of DNAM-1, FLAG-DNAM-1-expressing Jurkat transfectants were incubated with 10 µg/mL anti-human DNAM-1 mAb (clone TX94) (Okumura et al, 2017) on ice for 30 min, followed by 15 µg/mL rabbit polyclonal antibody against mouse IgG (Bethyl Laboratories, Montgomery, TX) for 0, 3, 6, or 10 min at 37 °C. The cells were then lysed with 1% NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0) supplemented with protease inhibitors (1 mM PMSF, 20 U/mL aprotinin) and phosphatase inhibitors (1 mM EGTA, 10 mM NaF, 1 mM Na 4 PO 7 , 0.1 mM β-glycerophosphate, 1 mM Na 3 VO 4 ).…”

“…To generate Jurkat transfectants, culture of Jurkat T cells was treated with culture supernatants containing retroviruses encoding FLAG-WT DNAM-1, FLAG-G307S DNAM-1, FLAG-Y322F DNAM-1, FLAG-G307S-Y322F DNAM-1, FLAG-WT DNAM-1-BirA*, or FLAG-G307S DNAM-1-BirA* in the presence of 10 μg/mL protamine sulfate (Sigma-Aldrich) and centrifuged at 1000g at 32 °C for 60 min. FLAG + cells were sorted by flow cytometry, and the equivalent expressions of FLAG and DNAM-1 were confirmed.ImmunoblottingTo analyze tyrosine phosphorylation of DNAM-1, FLAG-DNAM-1-expressing Jurkat transfectants were incubated with 10 µg/mL anti-human DNAM-1 mAb (clone TX94)(Okumura et al, 2017) on ice for 30 min, followed by 15 µg/mL rabbit polyclonal antibody against mouse IgG (Bethyl Laboratories, Montgomery, TX) for 0, 3, 6, or 10 min at 37 °C. The cells were then lysed with 1% NP-40 lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0) supplemented with protease inhibitors (1 mM PMSF, 20 U/mL aprotinin) and phosphatase inhibitors (1 mM EGTA, 10 mM NaF, 1 mM Na4PO7, 0.1 mM β-glycerophosphate, 1 mM Na3VO4).…”

Suppression of Th1 and Th17 Proinflammatory Cytokines and Upregulation of FOXP3 Expression by a Humanized Anti-DNAM-1 Monoclonal Antibody

“…Furthermore, TX94 inhibited NK cell-mediated cytotoxicity against a tumor cell line and suppressed CD8 + T cell proliferation mediated by allogeneic mixed lymphocyte reaction (MLR). (18) These functional characteristics of TX94 mAb suggest that a humanized TX94 mAb may be useful for the clinical application to immunotherapy for inflammatory diseases, in which DNAM-1 is involved in the pathogenesis. In this study, we generated a humanized TX94 mAb, named TNAX101A, which contains an engineered Fc portion of human IgG1 to reduce Fc-mediated effector functions, and show the functional characteristics of TNAX101A.…”

“…We previously generated several mouse antihuman DNAM-1 mAbs. (18) Among these mAbs, TX94 clone is a unique mAb; it most efficiently interfered the binding of DNAM-1 to CD155. Furthermore, TX94 inhibited NK cell-mediated cytotoxicity against a tumor cell line and suppressed CD8 + T cell proliferation mediated by allogeneic mixed lymphocyte reaction (MLR).…”