NOVEL APPLICATIONS OF BISPECIFIC DART PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 527

FIGURE 36.2 Application of U-DART 1 to screen DART 1 lead candidate among mAb panel.
(A) U-DART 1 screening of 34mAbs against A498 renal cancer target cells incubated with resting
human PBMC effector cells (effector to target ratio ¼ 25:1). Each fluorescein labeled mAb
(100 ng/mL) was mixed with (solid bar) and without (open bar) an antifluorescein (4420) TCR
DART 1 protein (200 ng/mL) and added to the same 96-well culture plate. Cell-mediated cytotoxicity
was quantified by measuring lactate dehydrogenase release. Error bars represent SD. mAbs 1-8 were
selected for further screening. (B) Schematic representation of U-DART 1 molecule. The 4420
TCR DART 1 consists of two covalently linked polypeptide chains that assemble to generate two
specificities, one against TCR b and the other against fluorescein. (C) U-DART 1 dose response
against renal cancer cells. A498 renal cancer target cells were incubated with resting human PBMC
effector cells at an effector to target ratio of 20:1. Each fluorescein labeled antibody (400 ng/mL) was
mixed with 4420 TCR DART 1 (800 ng/mL) and followed by 1:4 serial dilution and individual
assays were done in duplicate. Cytotoxicity was quantified by measuring lactate dehydrogenase
(LDH) release. Dose response curve of each antibody/U-DART 1 mixture was processed with
GraphPad Prism software. (D) T-cell mediated cytotoxicity of mAb6 TCR DART 1 . ADCC assays
were performed using A498 cells with PBMC at an effector to target ratio of 20:1. Individual assays
were done in duplicate and with a titration of antibodies: mAb6 TCR DART 1 and U-DART 1 .
Cytotoxicity was quantified by measuring LDH release.

the human BCR complex is an example of such approach
aimed at inhibiting B-cell activation [23]. We termed
such bispecific DARTs “immunomodulatory DARTs” or
I-DART 1 (Figure 36.3).
A U-DART format was developed for screening of I-
DART 1 candidates using an approach similar to that
employed for redirected effector cell killing. In this
U-DART 1 format, either component arm can be fixed
(i.e., the CD32B or the CD79B arms of a B-cell targeted
I-DART), and fluoresceinated mAb(s) representing candi-
dates for the other arm can be evaluated. A test of this
functional screening format was carried out using the
CD32B and CD79B specificities in reciprocal U-DART
formats. In the first format, the anti-CD79B Fv was used

as the fixed arm (4420 hCB3.1; Figure 36.4A—left panel)

in a U-DART that can be combined with a fluoresceinated
anti-CD32B mAb (CD32B mAb-FITC) to support bispecific
engagement and downregulation of B cells. Conversely, the
anti-CD32B Fv can be kept fixed resulting in a U-DART
(4420 h2B6; Figure 36.4A—center panel) that can be
combined with a fluoresceinated anti-BCR mAb to down-
regulate B-cell activation. The U-DART proteins were
expressed in eukaryotic cells (293-H or CHO-S), affinity
purified and further purified by SEC. Simultaneous binding
of both arms of 4420 hCB3.1 U-DARTwere demonstrated

by bispecific ELISA (Figure 36.4B). In this case, a soluble
single-chain form of the human CD79A/B ectodomain was
used to capture the DART 1 and detection was accomplished