NOVEL APPLICATIONS OF BISPECIFIC DART PROTEINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 524

the proinflammatory cytokines interleukin 1 a and 1 b (IL-
1 a , IL-1 b ) in a mouse collagen-induced arthritis (CIA)
model of rheumatoid arthritis yielded positive results using
a bispecific tetravalent IgG approach [21]. An alternative to
dual mediator blockade is interference with signaling path-
ways by engaging negative regulatory loops. One such
approach employs DART 1 molecules to interfere with B-
cell activation by inducing inhibitory signaling via the Fc
receptor, Fc g RIIB (CD32B) [23].
The physiological role of inhibitory receptors such as
CD32B is to dampen or raise the threshold for cellular
activation. For example, CD32B engagement downmodu-
lates activation initiated through the B-cell receptor complex
(BCR) on B cells; through Fc j RI on mast cells; and through
FcRI, FcRIIA, or FcRIIIA on macrophages. In each case, the
antigen itself serves as a scaffold for the formation of an
immune complex linking the stimulatory and inhibitory
receptors. When this occurs, the receptor tyrosine-based
inhibitory motif (ITIM) of CD32B is phosphorylated by
kinase(s) associated with the stimulatory receptor, initiating
the inhibitory cascade, and abrogating activation and down-
stream effects. We used this well-established paradigm to
demonstrate the applicability of the DART 1 platform to
coligate the inhibitory and stimulatory receptors in the
absence of antigen on human B cells.
CD32B is a component of an inhibitory receptor loop
triggered when coligated with the BCR complex. A DART 1
protein designed to modulate B-cell function was con-
structed by pairing an Fv specific for CD32B with an Fv
specific for the Ig- b component (CD79B) of the human BCR
complex [23]. Two different DART 1 proteins that recognize
different epitopes on CD32B and have the same specificity
for CD79B were expressed in Chinese hamster ovary (CHO)
cells and purified to homogeneity using antigen-affinity and
size-exclusion chromatography (SEC). Bispecific enzyme-
linked immunosorbent assays (ELISA) and surface plasmon
resonance (SPR) analyses demonstrated that both of the
arms could bind to both target antigens simultaneously and
retained the affinity of the parent Fab fragments.
As reported [23], both DART 1 proteins specific for
CD32B and CD79B demonstrated preferential simultaneous
binding to both the activating and inhibitory receptors on the
surface of the same B cells as evidenced by fluorescence-
activated cell sorting (FACS) studies. The coligation of
receptors decreased BCR-induced calcium mobilization
and phosphorylation of selected signaling molecules, which
in turn inhibited both B-cell proliferation and IgG secretion
in vitro. Consistent with this mechanism of action, a
DART 1 protein specific for the mouse orthologs of
CD32B and CD79B reduced disease severity in a mouse
model of collagen-induced inflammatory arthritis. This
novel approach to B-cell function modulation forms the
basis for translating inhibitory signaling into therapeutic
tools for treating autoimmune and inflammatory diseases.

36.4 APPLICATION OF BISPECIFIC ANTIBODIES
IN ONCOLOGY

Thedevelopmentofbispecificantibodies for recruitingimmune
effector cells to kill tumor cells has long been explored as a
potential therapeutic agent. However, inefficient production
and poor stability have prevented wide use. An early example is
the use of humanized Fab anti-CD64

humanized Fab anti-
EGFR to engage cytotoxic immune cells other thanT cells. This
bispecific antibody was intended for CD64-directed immuno-
therapy in cancer patients, but failed in clinical trials [28].
Greater success hasbeenachievedusingbispecific antibodies to
CD16 on NK and macrophages and CD3 on T cells [29-31].
More recently, blinatumomab (MT103), a bispecific T-cell
engager (BiTE 1 ) specific for CD19 and CD3, has successfully
been used to treat patients with non-Hodgkin's lymphoma by
redirecting cytotoxic T cells to kill tumor cells [32].

36.4.1 CD16-Based DART 1 Proteins
We recently reported on the application of DART 1 proteins
for redirected killing, whereby an Fv specific for human CD16
(Fc g RIII) on effector cells (e.g., NK cells) was coupled to an
Fv specific for human CD32B, used here as a target on B cells
[24]. DART 1 proteins were produced at high levels in mam-
malian cells and retained the binding activity of the respective
parental Fv domains. Simultaneous binding of both antigens
was evident from dual-affinity ELISA and SPR analysis.
These proteins showed good stability upon extended storage
of about 9months in phosphate-buffered saline at 4 C and also
in human serum at 37 C for up to 8 weeks.
Functionally, theDART 1 proteins demonstrated extremely
potent, dose-dependent cytotoxicity in retargeting human
peripheral blood mononuclear cells (PBMC) against
CD32B-positive B-lymphoma cell lines. When cultured with
human PBMC these proteins also mediated autologous B-cell
depletion,measuredas the residual fractionofCD20-positiveB
cells. Thepotencyof theseDART 1 molecules inADCCassays
was

100-fold greater than that of the corresponding IgG
bearing an Fc that was optimized for enhanced CD16 binding.
In vivo, the DART 1 proteins effectively depleted B cells
in mice transgenic for both CD16A on the effector cells and
CD32B on the B-cell targets. No depletion was observed in
either hCD16A or hCD32B single transgenic animals, indi-
cating that the presence of both receptors was required for
the observed effect. Furthermore, DART 1 proteins showed
potent in vivo antitumor activity in a mouse xenograft model
using Raji cells, a human Burkitt's lymphoma B-cell line
expressing CD32B antigen.

36.4.2 CD3/TCR-Based DART 1 Proteins

Given the potent activity of bispecific proteins that recruit T
cells as effector cells, we developed DART 1 proteins