Figure 1
Characterization of JTA009, a novel anti-human ICOS mAb. JTA009, a fully human anti-human ICOS mAb, has greater avidity than SA12. (a) Avidity of anti-human ICOS antibodies was evaluated by direct ELISA using ICOS-Fc (as described in Materials and method). JTA009 (open circles) exhibited stronger binding to ICOS-Fc than did SA12 (closed circle), a previously reported anti-human ICOS mAb. (b) Peripheral blood T cells from normal control individuals were stimulated with anti-CD3 mAb (0.1 μg/ml) plus anti-CD28 mAb (2 μg/ml) for 72 hours. These cells were biotinylated and cell lysates were prepared. ICOS molecules in these lysates were immunoprecipitated, separated on SDS-PAGE gel, transferred to nitrocellulose membrane, and visualized using streptavidin-peroxidase and chemiluminescent substrate. A single band about 29 kDa was immunoprecipitated with JTA009 but not with JMAb23, the control antibody. The thin lower band corresponded to the position of the front dye of the gel. Human ICOS expressing (c) CHO-K1 and (d) its parental cell line CHO-K1 were stained with biotinylated JTA009 (thick line), biotinylated SA12 (broken line), or biotinylated JMAb23 (human IgG2; thin line) and streptavidin-FITC, and then analyzed using flow cytometry. (e) Human ICOS expressing CHO-K1 cells were stained biotinylated SA12 (6.25 μg/test) and streptavidin-FITC in the presence of various amounts of nonbiotinylated JTA009 (thick line: 0 μg/test; thin line: 5 μg/test; thick broken line: 10 μg/test; thin broken line: 25 μg/test). JTA009 dose dependently decreased the binding of SA12 to the ICOS expressing CHO-K1 cells. FITC, fluorescein isothiocyanate; ICOS, inducible co-stimulator; mAb, monoclonal antibody.