RESURGENCE OF BISPECIFIC ANTIBODIES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 514

Chemical cross-linking or dimerization domains have been
employed for joining two different Fab fragments, or, their
recombinant fusion with scFvs. A bispecific construct called
MDX-210 is based on two Fab fragments chemically cross-
linked at the natural hinge region of IgG. The antibody
construct has been in clinical development since the early
1990s [5]. It targets the tumor-asscociated tyrosine kinase
receptor HER2 with one arm, and the high-affinity Fc g
receptor I (CD64) on macrophages with the second arm
(Figure 35.5A). MDX-210 was designed to bind CD64 in a
way not impacted by excess serum IgG and to cause
redirected lysis of cancer cells by CD64-expressing cyto-
toxic macrophages.
Genentech/Roche revived chemical cross-linking for
development of bispecific antibodies called “dual action
Fab” (DAF) or “bis-Fab” (Figure 35.5B) [53]. In order to
obtain a site-specific linkage, free cysteine residues were
introduced in an accessible position on the surface of Fab
fragments. The separately in E. coli produced and purified
“thiobodies” are sequentially reacted with a chemical cross-
linker to form the desired bispecific variant at high yield. By
use of a special cross-linker containing polyethylene glycol,
a means is given to prolong the serum half-life of the
otherwise short-lived bispecific constructs. A bispecific
antibody in this format is being developed for dual EGFR
and HER3 receptor inhibition.
ImmunoMedics has worked out a very unique approach
of connecting various Fab fragments with each other or with
full IgG molecules [54,55]. For their “dock-and-lock”
(DNL) platform, small dimerization domains derived
from protein kinase A and its scaffold protein AKAP are
employed. The C-terminally fused AD2 sequence is used to
combine two Fab fragments of the same specificity (Figure
35.5C). This domain is then recognized by a DDD2
sequence, which is C-terminally fused to a second kind
of Fab fragment. The resulting AD2/DDD2 homo- and
heterodimerization complex ultimately combines three
Fab fragments and is covalently stabilized by disulfide
bridges. ImmunoMedics has published bispecific variants
that combine CD20 and CD22-specific Fab fragments [54],
and DNL antibodies for pre-targeting cancer cells for radio-
immunotherapy [55].
The Portuguese Biotech Company Biotecnol has devel-
oped a bivalent bispecific antibody for T-cell engagement
where two scFv are C-terminally fused to a Fab fragment.
This format goes back to one developed by Schoonjans et al.
[56]. Because the Fab fragment represents a heterodimeri-
zation domain, fusion of different scFvs to C-termini of CH1
and C k / l domains will lead to homogenous bi- or tri-specific
constructs. In the example shown in Figure 35.5D, the Fab-
scFv construct recognizes CD3 on T cells and the BCL1
antigen on lymphoma cells. Both the Fab fragment and one
of the two-fused scFvs are specific for BCL1 providing for
bivalent target binding to lymphoma cells. A format called

“heterominibody” has been developed by Micromet [57]. It
just uses the heterodimerizing CH1/C k domain of the Fab
fragment for fusion of scFvs and proinflammatory cytokines.
UCB-Celltech presented a novel bispecific format with
extended serum half-life called “Fab-Fv” [58]. As shown in
Figure 35.5E, a variable vH/vL (Fv) domain is C-terminally
fused to a Fab fragment. By binding to serum albumin, the
Fv domain confers a long serum half-life to the fusion
protein.
Fab fragments provide attractive features as building
blocks for bispecific antibodies. They already contain one
specific binding site; they represent a heterodimerization
element and allow fusion of additional V H /V L domains or
single-chain antibodies to their N- or C-termini.

35.7 BISPECIFIC CONSTRUCTS BASED ON
DIABODIES OR SINGLE-CHAIN ANTIBODIES

Since decades, bispecific antibodies are being developed
that use diabodies or scFvs as sole structural basis [59].
Initial issues with the stability of small antibody fragments
[60] can today be overcome by screening for fragments with
high thermal stability, which typically translates into attract-
ive biophysical features. Both scFvs and diabodies result
from recombinantly fusing V H and V L domains with non-
immunogenic peptide linker sequences [61,62]. If the pep-
tide linker is short (between 5 and 9 amino acid residues) a
diabody will result, which is composed of two polypeptide
chains. For making a bispecific diabody, V H and V L domains
from two different antibodies need to be fused with each
other. If the linker between V H and V L domains is greater
than 12 residues in length (usually between 15 and 20
residues), a scFv will result, which can bind to antigen as
a single polypeptide chain. For construction of a bispecific
antibody, at least two scFvs need to be fused in tandem by
the help of a third peptide linker. Examples of bispecific
antibody formats based on scFvs and diabodies are shown in
Figure 35.6.
Bispecific T-cell engagers (BiTE), as developed by
Amgen, consist of tandemly arranged scFvs
(Figure 35.6A) that have been designed and optimized for
redirected lysis of target cells with the help of cytotoxic
T cells [63,64]. In its newest version, a primate cross-
reactive, anti-CD3 human scFv is fused with a second
human or humanized scFv specific for a tumor-associated
antigen. Figure 35.6a lists several target antigens where the
corresponding BiTE antibodies have been characterized in
clinical and nonclinical studies (reviewed in Reference [64]).
Properties of BiTE antibodies include (i) monovalent binding
to CD3 and target antigen, (ii) activation of unstimulated
T cells in a strictly target cell-dependent manner, (iii) support
of serial lysis by activated T cells, (iv) extreme potency of
redirected lysis at sub-picomolar concentrations, and (v) an