Biomedical Engineering Reference
In-Depth Information
pairing, which has allowed the production of bispecific
antibodies. Although this technology has led to the genera-
tion of the clinically approved bispecific antibody Catumax-
omab, its reliance on the use of nonhuman heavy and light
chains increases the risk of immunogenicity that may ulti-
mately limit the general utility of this approach in the clinic.
An alternative approach to address the heavy and light chain
pairing issue is the use of a single common light chain that
can associate with different variable heavy chain domains
[30]. This can be accomplished using antibody Fab fragment
libraries containing broad heavy chain diversity but only a
single light chain. While there is evidence that this approach
is effective, it likely requires that the light chain contributes
minimally to the overall binding affinity. The removal of the
light chain entirely, though engineering of human heavy
chains [31] or use of naturally occurring light chain-free
camelid antibodies [32], eliminates the potential for random
chain pairing. Although such an approach would negate any
potential contribution to the overall affinity that light chains
normally provide, some heavy chain only-based binders
have been reported to attain affinities up to 100 pM [32].
All of the aforementioned binder technologies have been
used as building blocks for the creation of multispecific
antibodies through the genetic fusion to the C- and/or N-
termini of conventional antibodies of differing specificity.
For example, fusions of scFv moieties to the termini of
conventional antibodies have been reported [33], as have
fusions of additional VH and VL domains, creating dual
variable domain immunoglogulins [34]. When fused to a
conventional homodimeric Fc-based antibody, such mole-
cules typically have four or more binding sites and a
significantly larger size than traditional antibodies. The
increased overall size of these molecules has the potential
to impact production, stability, pharmacokinetics, tissue
distribution, and tumor penetration [6]. To create a platform
for bispecific antibodies possessing the biophysical charac-
teristics, size, pharmacokinetic profile, and ease of manu-
facture as conventional antibodies, investigators have
examined methods for selectively heterodimerizing heavy
chains of immunoglobulins having different specificities.
Such investigations have resulted in different solutions to
preferentially drive this heavy chain heterodimerization. In
the “knobs-into-holes” technology first described over a
decade ago [18,30], heavy chains engineered with amino
acid protuberances were coexpressed with heavy chains
engineered to have corresponding concavities such that
the maintenance of van der Waals contacts through high
surface complementarity favored heterodimerization rather
than homodimerization. In combination with a single com-
mon light chain, this technology proved successful in creat-
ing bivalent and bispecific antibodies [30]. Although this
technology has been somewhat limited by the presence of
homodimer contaminants during large-scale expression in
mammalian cells, a recent report demonstrates that process
improvements allow good production of bispecific antibod-
ies in a bacterial expression system [35].
Another recent approach to preferentially drive heavy
chain heterodimerization relies upon the introduction of
oppositely charged amino acids in opposing heavy chains
[36]. Results clearly demonstrate that this approach can be
used to generate heterodimeric heavy chain pairs. Moreover,
by fusing scFv domains with different specificities to each
heavy chain, the resulting molecules could demonstrate true
bispecific targeting and be readily expressed in mammalian
cells.
The novel method for creating a heterodimerizing plat-
form described within this chapter differs from the two
technologies summarized above in that the SEEDs were
designed by incorporating complementary amino acid
sequence stretches from human IgA and IgG within the
Fc CH3 domain of a normal antibody [19]. Results demon-
strate the strong heterodimerizing propensity of the engi-
neered heavy chains. Notably, the SEED-based antibodies
retain the biophysical, biological, and pharmacokinetic
properties of conventional antibodies [37]. The SEED plat-
form has proven to be highly flexible when coupled with
different binding domains, such as Fabs, scFv, and heavy
chain only binders. Unlike either conventional antibodies or
dual variable domain immunoglobulins, the heterodimeriz-
ing heavy chain-based platforms can be readily used to
create monovalent antibodies [36-38]. Such molecules
may have significant advantages over multivalent antibodies
for antagonizing targets that can become activated upon
crosslinking. One such target of significant therapeutic
relevance is cMet. This target has proven difficult to func-
tionally inhibit with conventional antibodies because of the
propensity of bivalent binding to dimerize and activate the
receptors, even when the ligand is completely blocked.
However monovalent Fab fragments of these same activat-
ing antibodies can effectively antagonize ligand-mediated
activation and lead to increased antitumor activity in vivo
[39]. This observation was substantiated when the “knobs-
into-holes”-based monovalent antibody MetMab, which
includes a Fab as the single binding arm, was demonstrated
to fully antagonize cMet activation, resulting in potent
inhibition of c-Met dependent tumor cell growth in pre-
clinical models [40]. Notably, MetMab has sufficient drug-
like characteristics to be progressed into clinical trials. The
SEED-based monovalent antibodies retain similar bio-
physical properties and expression/purification character-
istics as conventional antibodies.
While monovalent antibodies generated from platforms
such as knobs-into-holes and SEED can specifically bind
targets with high affinity, the lack of a second arm may
reduce overall avidity that, in certain circumstances, could
impact efficacy. Use of a heterodimerizing platform pro-
vides the potential to create biparatopic molecules having
two binding arms that
recognize two nonoverlapping
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