STRAND EXCHANGE ENGINEERED DOMAIN (SEED): A NOVEL PLATFORM DESIGNED TO GENERATE MONO AND MULTISPECIFIC PROTEIN THERAPEUTICS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 539

FIGURE 37.5 Biological function testing of SEED molecules. Biological properties of SEED
proteins, C225-SEED (bivalent), C225-AG/GA (monovalent), C225-GA/AG (monovalent), were
tested for in vitro cell binding and ADCC activity and for in vivo PK properties compared to the
clinically validated antibody C225-Fc, and for CDC activity in comparison to hu14.18-Fc antibody.
(A) Ligand competition assays to measure binding to EGFR. A431 cell membranes were incubated
with 125 I-EGF (0.1 nM) and each antibody as indicated above. Incubations were performed in binding
buffer for 1.5 h at 37 C. Plates were washed and bound radioactivity measured. Competition curves
through the data points and K i values were determined using GraphPad Prism software. (B) ADCC
activity of human peripheral blood mononuclear cells against A431 target cells (100:1 effector:target
ratio) was measured by standard 51 Cr-release assay and plotted as percent target cell specific lysis
versus the concentration of each antibody as indicated above. Each data point is the mean SD of
triplicates. (C) CDC activity induced against M21 target cells by the indicated proteins was measured
by standard 51 Cr-release assay as described above. (D) Mouse PK study of Fc, SEED scaffold, C225-
Fc, C225-SEED, and C225-AG/GA proteins. Plasma concentration profiles after single IV adminis-
tration of 100 m g(

SD from three mice.
(Source: Reproduced from Reference [37] by permission of Oxford University Press.)

3.5mg/kg) into nude mice. Each data point is the mean

addressed by modulating a single target, (2) antibody bind-
ing to a single epitope of even a key target protein may be
insufficient by itself to modulate all of the biological activi-
ties of that protein, or (3) the bivalent nature of antibodies
can lead to crosslinking and subsequent activation of target
proteins. One potential solution to these obstacles is the
creation of a highly flexible biotherapeutic platform that can

be used to create antibody-like molecules in which the
specificities and valencies can be tuned to meet a specific
application.
Effectively treating cancer with single target-modifying
drugs such as antibodies has been particularly difficult
because of the high complexity of the oncogenic process
that often involves mutations or other genetic abnormalities