NOVEL APPLICATIONS OF BISPECIFIC DART PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 530

chCB3.1-FITC mAb with that mediated by the h2B6 shown). As expected no inhibition was observed in

RBL/CD32B-Y292A cells (Figure 36.5), although expression
of the CD32B on the mutant cell line was similar to that of the
wild-type transfectant (data not shown). In terms of specific
signaling, increased phosphorylation of the CD32B ITIM was
observed in those conditions that led to inhibition of hexam-
inidase release, consistent with its role in initiating the inhibi-
tory cascade (Figure 36.5A).
In conclusion, the use of the antifluorescein Fv from the
mAb 4-4-20 as one partner in several of the DART 1
molecules, in combination with novel or commercially
available fluoresceinated mAbs, allows for the flexibility
to interrogate these different modes of inhibition. The anti-
hapten Fv was linked either to the Fv specific for the
inhibitory receptor CD32B or to the Fv specific for the
activating receptor, in this case CD79B. Such a DART 1

hCB3.1 I-DART 1 (Figure 36.4D—lower panels).

36.6.1 Inhibition of Basophil Degranulation

To further demonstrate the versatility of the 4420 CD32B

U-DART, we also evaluated the ability of this DART 1 to
inhibit granule release from basophils. In this case, we used
RBL cells transfected with either wild-type human CD32B
(RBL/CD32B) or a version mutated within the inhibitory
ITIMmotif (RBL/CD32B-Y292A). RBL/CD32B cells were
activated using chimeric anti-DNP IgE and DNP-BSA or
DNP-BSA-FITC together with DART. Significant, dose-
dependent inhibitory activity was seen only when 4420

CD32B U-DART and DNP-BSA-FITC were used (Figure
36.5), but not with DNP-BSA (nonfluoresceinated) (data not

FIGURE 36.5 Receptor coligation activates the inhibitory signaling pathway. Rat basophilic
leukemia RBL-2H3 (RBL) cells, RBL/CD32B (RBL cells stably expressing human CD32B) cells,
and RBL/CD32B/Y292A (RBL cells stably expressing a nonsignaling form of human CD32B) were
cultured in RPMI complete media. (A) Western blot analysis of whole cell lysate (1 10 6 cells)
obtained from different types of purified RBL cells that were activated alone or in combination with
anti-DNP IgE, FITC-BSA-DNP (0.5 m g/mL), and 4420 2B6 U-DART 1 (1 m g/mL). The blots were
probed with anti-pCD32B. Equivalent loading was confirmed by stripping and reprobing with anti-
GAPDH. (B) 2 10 4 cells/well were grown for overnight in 96-well plates in the presence of anti-
DNP IgE. The following day all RBL/CD32B cells were activated with FITC-BSA-DNP in the
presence of either 4420 2B6 U-DART ( ) or 4420 chCB3.1 DART 1 ( & ) or CD16 CD32B
DART 1 ( ~ ). Finally supernatants were transferred to microtiter plates and b -hexosaminidase
release was measured. (C) DART 1 mediated inhibition of b -hexoaminidase release. After overnight
treatment with anti-DNP IgE, different RBL cell lines were activated with FITC-BSA-DNP in the
presence of 1 m g/mL of 4420 2B6 U-DART.