Figure 3.

Epitope Mapping of Anti-hCD40 mAbs

(A) C-terminally His-tagged CD40 proteins consisting of 4, 3, or 2 CRD domains (4 = full-length; 3 = CRD1 deleted; 2 = CRD1 and CRD2 deleted) were analyzed by western blotting with the mAbs indicated above each panel used for detection. A composite image from multiple blots with different antibodies is shown.

(B) hCD0Tg mouse B cells were incubated with FITC-labeled Lob 7/4 or Lob 7/6, as indicated, in the absence or presence of a 10-fold excess of competitor (comp) mAbs indicated above each plot. Cell labeling was assessed by flow cytometry; filled histogram, unlabeled cells; light gray, FITC-labeled mAbs alone; black line, FITC-labeled mAbs + competitor.

(C) Surface plasmon resonance analysis of mAb competition for CD40 binding. The mAbs indicated above each plot were flowed over immobilized hCD40 ECD for 800 s to allow saturation of hCD40 binding sites, followed by the addition of individual competitor mAb for 350 s. Non-competitive mAbs (i.e., mAbs without overlapping epitope) give a response greater than 500 response units (RU).

(D) Untransfected 293 cells (none), or cells transfected with full-length (WT) human CD40 or CD40 in which N-terminal amino acids 23–37 had been deleted, were incubated with the indicated anti-hCD40 mAbs (h2 isotype). Bound mAbs were detected with anti-human Fc-FITC. The percentage of cells in the boxed region, denoting positive staining, is shown for each plot. Results from one of two experiments shown.

(E). Schematic of the approximate locations of epitopes for nine human CD40 mAbs analyzed. See also Figure S1.