NOVEL APPLICATIONS OF BISPECIFIC DART PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 529

FIGURE 36.4 CD32B CD79b cross-linking by dual affinity retargeting molecules activates the
inhibitory pathway in human B cells. (A) Schematic representation of DART 1 molecules. (B) Dual-
affinity ELISA assay demonstrates that for typical DART, both arms can simultaneously engage their
respective targets. ELISA plates were coated with soluble human CD79a/b ectodomain followed by
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hCB3.1 or 4420 h2B6 U-DART 1 protein. (C) B-cell proliferation in the presence of

hCB3.1 U-DART. Purified human B cells activated with polyclonal F(ab) 0 2 fragment of goat
anti-human IgM antibody (50 m g/mL) were treated with varying concentrations of 4420 hCB3.1
DART 1 in the presence of a fixed concentration (3 m g/mL) of fluorescein-labeled anti-CD32B (2B6),
anti-CD22 (HIB22), and isotype control mAbs or with unlabeled anti-CD32B and anti-CD22 mAbs.
Following 48 h of culture, cell proliferation was assessed by incorporation of radiolabeled thymidine
(1 m Ci/well) added during the last 16 h of culture. (D) B-cell proliferation and Ig secretion mediated
by 4420 hCB3.1 U-DART 1 compared to h2B6 hCB3.1 DART. Upper panels: activated human B
cells were treated with varying concentration of 4420 2B6 U-DART 1 in combination with
indicated mAbs (left panel) or to h2B6 chCB3.1 DART, chCB3.1(N297Q) and h2B6(N297Q)
mAbs alone (right panel) with cell proliferation assessed by thymidine incorporation following 48 h
culture. Lower panels: activated human B cells were treated with 20 nM of 4420 2B6 U-DART in
the presence of anti-CD79B Mab, anti-CD79b-FITC Mab, or an irrelevant FITC-labeled antibody of
the same isotype (left panel) or to 20 nM of h2B6 chCB3.1 DART 1 , anti-human CD79b mAb, or
anti-human CD32B mAb (right panel). Supernatants were tested in ELISA using plates coated with
polyclonal anti-human Ig Ab to capture secreted immunoglobulins by activated B cells. Bound
human immunoglobulins were detected using a polyclonal anti-human k -chain HRP-conjugated
antibody.

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