Biomedical Engineering Reference
In-Depth Information
(A)
TNF-
α
biotin/Streptavidin-PE
(B)
Anti-hu lg kappa chain mAb-FITC
160
1200
140
1000
120
800
100
Rituximab
80
600
RX-TNF353-2
60
400
40
200
20
0
0
mAb (
μ
g/mL)
mAb (
μ
g/mL)
FIGURE 39.8
mAb
2
RX-TNF353-2 can interact simultaneously with two different antigens. The
human CD20 positive B-cell line CFB4.2 was incubated with increasing concentrations of mAb
2
or
rituximab. Cell-bound proteins were detected either with biotinylated TNF-
a
plus phycoerythrine
(PE) conjugated streptavidin (A) or with fluorescein-iso-thiocyanate (FITC) conjugated antihuman
kappa chain antibodies (B).
imagine the usefulness of a mAb
2
as antibody drug conju-
gate in which a toxic compound is specifically transported to
the diseased tissue. Finally, mAb
2
proteins could be used to
enhance immune responses or initiate alternative forms of
immune responses by using one antigen specificity for the
recruitment and activation of immune-effector cells, such as
cytotoxic T cells [12]. Such a concept is currently being
tested in cancer patients in the clinic.
Phase I and Phase II studies with their bispecific scFv-based
molecules. These compounds are biologically highly potent,
yet their intrinsically short in vivo half-life limits their poten-
tial, similar to all other fragment-based platforms. These
limitations can only be overcome by fusion of the antibody
fragments with molecules that can provide the missing prop-
erties, such as Fc fragment of IgG, HSA, or HSA-binding
moieties. Among the preclinical full-size IgG formats such as
the knobs-into-holes IgG or the SEEDbody, their monovalent
binding toward each of the recognized targets is considered to
be a major difference when compared to a normal IgG anti-
body, which is always bivalent. Compared to these platforms,
the mAb
2
format is the ideal solution, since it provides a
normal IgG with its full functionality in terms of effector
functions and in vivo half-life. Bivalent antigen binding via its
Fab arms is fully retained, and bispecificity is achieved by
engineering the C-terminal tip of the CH3 domain, which is
otherwise not known to have any biological function in
antibodies. Further validation of the mAb
2
concept in vivo
with clinically relevant mAb
2
proteins is in progress.
39.5 CONCLUSIONS AND FUTURE
PERSPECTIVES
During the last two decades, a number of different bispecific
antibody or antibody-fragment formats have been developed
(reviewed in [13,14]). They include bispecific IgGs and F(ab
0
)
2 fragments generated by variations of the hybridoma tech-
nology, by chemical cross-linking or by genetic engineering,
as well as recombinant bispecific formats based on smaller
fragments such as scFvs and single-domain antibodies. Fur-
thermore, recombinant formats utilizing various fusion part-
ners (HSA, Fc fragment, leucine zipper) to achieve
bispecificity have been described. Among all these, hetero-
dimeric IgG formats such as the knob-into-holes [15] or the
SEEDbody [16] constructs are the ones that are most similar
to normal, full-size IgG. Clinically, the most advanced full-
size IgG platform is being developed by Trion Pharma, and is
based on the ability of mouse IgG2a and rat IgG2b to
heterodimerize, leading to monovalent bispecific mouse/rat
IgG molecules [17]. However, immunogenicity of these
nonhuman antibodies remains one of the major drawbacks
of this platform as well as the demanding production process
which is based on Quadroma technology. Among the frag-
ment platforms, Micromet [18] and Merrimack have entered
ACKNOWLEDGMENTS
We thank Austria Wirtschaftsservice for funds provided
under its “Preseed” program, the Austrian Research Promo-
tion Agency (FFG) for funds provided under its “BRIDGE”
program (project 813002 “Modular Antibody Design Based
on Immunoglobulin Libraries”), and the Christian Doppler
Forschungsgesellschaft. f-star provided funds as the industry
partner in connection with the “BRIDGE” program and the
Christian Doppler Laboratory for Antibody Engineering. We
thank all colleagues from f-star GmbH for their valuable
contributions to this work.
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