RESURGENCE OF BISPECIFIC ANTIBODIES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 516

Although Tandabs have a size in excess of 100 kDa, the
serum half-life of the CD19/CD3 bispecific Tandab in mice
was surprisingly short with a t1/2 b of 2.8 h [73]. In animal
models, the CD19/CD3-bispecific Tandab required costimu-
lation of human T cells with anti-CD28 antibody [71]. This
may indicate that this bispecific format did not allow for
single-agent activation of unstimulated T cells in vivo.
Another kind of diabody-based bispecific antibody con-
structs is based on MacroGenic's DART proteins
(Figure 35.6C) of which at least two are in formal preclinical
development. One is designed to engage NK cells via
binding to their Fc g receptor CD16A for lysis of CD32B-
expressing lymphoma cells [74]. The other is cross-linking
the B cell receptor subunit CD79B with the inhibitory Fc g
receptor CD32B on the surface of B cells for the treatment of
rheumatoid arthritis [75]. In order to stabilize the diabodies
used in DART constructs, extra cysteine residues for making
an additional disulfide bond have been introduced. This
technology will allow use of otherwise instable diabodies
and prevent their aggregation. Diabodies with high thermo-
dynamic stability can be also be obtained by other methods
[76]. MacroGenics is developing bispecific DART antibody
constructs together with Pfizer and Boehringer Ingelheim,
and is about to commence a clinical study with their
CD32B/CD16A-bispecific DART.
By introducing a peptide linker, Kontermann and col-
leagues have developed an alternative to disulfide-based
diabody stabilization as used by DART by introduction of
a peptide linker fusing the two chains of a diabody to form a
“single-chain diabody” (Figure 35.6D). T cell-engaging
single-chain diabodies with specificity for carcinoma
embryonal antigen (CEA) or fibroblast-activating protein
alpha and CD3 on T cells have shown high activity in cell-
based assays, and human serum albumin (HSA) binding
versions of these proteins a much prolonged serum half-life
in mice [77].
Bispecific tandem scFv-based constructs for T-cell
engagement are being developed by two more companies
(Figure 35.6E). Applied Immune Technologies (AIT) and
ReceptorLogics are using single-chain antibodies derived
from “T-cell receptor mimic (TCRm) mAbs” for targeting
tumor cells expressing a certain MHC class I antigen/peptide
antigen complex [78]. With their TCR-like specificity, these
antibody constructs can only bind target cells expressing the
proper MHC class I/peptide complex and will thereby face
the same limitations as the format shown in Figure 35.7C,
which is discussed.
By fusing scFvs to both N- and C-terminus of HSA, a
bispecific antibody was engineered by Merrimack that
confers the long serum half-life of HSA to the fusion protein
(Figure 35.6F) [79]. Similar to IgG, HSA is retained by the
FcRn system [48]. A dual receptor-inhibiting antibody
construct of Merrimack's called MM-111 is bispecific for
HER2 and HER3 and commencing Phase II clinical

FIGURE 35.7 Bifunctional fusions of antibodies or fragments
with unrelated proteins. Disclosed binding specificities and partic-
ular structural features are labeled. Single-chain antibodies (scFvs)
show a peptide linker between variable domains.

development for the treatment of breast cancer patients.
Several more multispecific antibodies in this format are in
preclinical development.
Epigen Biotech has crafted a multivalent T-cell-engaging
bispecific construct by fusing a single domain antibody
specific for HLA-A2 with a scFv specific for CD3 using
a coiled-coiled peptide domain from human cartilage matrix
protein (COMP) 48 domain (Figure 35.6G) [80]. This
construct is called a “COMBODY.”

35.8 BIFUNCTIONAL FUSIONS OF ANTIBODIES
OR FRAGMENTS WITH OTHER PROTEINS

While Figures 35.1-35.6 have described bispecific antibod-
ies entirely based on immunoglobulin sequences (with
exception of examples in Figures 35.6F and 35.6G), we
will, for the sake of completeness, also discuss a selection of
bifunctional proteins that combine immunoglobulin
sequences with those of unrelated proteins (Figure 35.7).
Because the second, immunoglobulin-unrelated protein is
likewise binding to a protein target—be it a cytokine recep-
tor or the TCR—such fusion proteins should also be con-
sidered bispecific antibodies.
A number of so-called immunocytokines are in pre-
clinical and clinical development by several companies
including EMD Serono, ImmunoMedics, Philogen, Bayer