Figure 7.

Antagonist Anti-hCD40 mAbs Engage CRD2-4 Domains

(A) Purified hCD40Tg mouse splenic B cells were incubated with or without the indicated mAb, all of m1 isotype, followed by hexameric mCD40L-h1Fc. Bound CD40L was then detected with anti-human-FITC and flow cytometry.

(B) Structure superposition of CD40:Lob 7/4 Fab' complex (colored as in Figure 2A) with the CD40:CD40L complex (PDB: 3QD6). CD40L is shown as a trimer binding to the opposite side of CD40 than Lob 7/4 and to a different CRD (CD40L shown as white surface representation).

(C) Purified hCD40Tg mouse splenic B cells were incubated with CD40L alone (10 μg/mL) or together with the indicated mAbs (m1 at 1 μg/mL) for 20 hr. Activation was assessed by homotypic adhesion (top) or flow cytometry to assess upregulation of activation markers. Results from one of at least three experiments for each mAb are shown.

(D) Purified hCD40Tg mouse splenic B cells were incubated with the indicated mAbs (m1 at 1 μg/mL) under control conditions (left panel) or in the presence of CD40L (10 μg/mL, right panel). Proliferation was assessed by [³H]thymidine incorporation as in Figure 1A and plotted as means ± SEM.

(E) As in (D), except that h2 mAbs were used. Means ± SEM of triplicate samples representing results from two or three experiments per mAb.

^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 using the unpaired Student's t test, left hand plots for indicated groups, right hand plots versus CD40L alone.