2 research outputs found
High-Affinity VEGF Antagonists by Oligomerization of a Minimal Sequence VEGF-Binding Domain
Vascular endothelial growth factor (VEGF) neutralizing
antagonists
including antibodies or receptor extracellular domain Fc fusions have
been applied clinically to control angiogenesis in cancer, wet age-related
macular degeneration, and edema. We report here the generation of
high-affinity VEGF-binding domains by chemical linkage of the second
domain of the VEGF receptor Flt-1 (D2) in several configurations.
Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including
a C-terminal cysteine to enable its dimerization by disulfide bond
formation or by attachment to divalent PEGs and oligomerization by
coupling to multivalent PEGs. Disulfide-linked dimers produced by
Cu<sup>2+</sup> oxidation of the free-thiol form of the protein demonstrated
picomolar affinity for VEGF in solution, comparable to that of a D2-Fc
fusion (sFLT01) and ∼50-fold higher than monomeric D2, suggesting
the 26 a.a. tag length between the two D2 domains permits simultaneous
interaction of both faces of the VEGF homodimer. Extending the separation
between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced
a modest ∼2-fold increase in affinity over the disulfide, thus
defining the optimal distance between the two D2 domains for maximum
affinity. By surface plasmon resonance (SPR), a larger (∼5-fold)
increase in affinity was observed by conjugation of the D2 monomer
to the termini of 4-arm PEG, and yielding a product with a larger
hydrodynamic radius than sFLT01. The higher affinity displayed by
these D2 PEG tetramers than either D2 dimer or sFLT01 was largely
a consequence of a slower rate of dissociation, suggesting the simultaneous
binding by these tetramers to neighboring surface-bound VEGF. Finally,
disulfide-linked D2 dimers showed a greater resistance to autocatalytic
fragmentation than sFLT01 under elevated temperature stress, indicating
such minimum-sequence constructs may be better suited for sustained-release
formulations. Therefore, these constructs represent novel Fc-independent
VEGF antagonists with ultrahigh affinity, high stability, and a range
of hydrodynamic radii for application to multiple therapeutic targets
Site-Specific Antibody–Drug Conjugation through Glycoengineering
Antibody–drug
conjugates (ADCs) have been proven clinically
to be more effective anti-cancer agents than native antibodies. However,
the classical conjugation chemistries to prepare ADCs by targeting
primary amines or hinge disulfides have a number of shortcomings including
heterogeneous product profiles and linkage instability. We have developed
a novel site-specific conjugation method by targeting the native glycosylation
site on antibodies as an approach to address these limitations. The
native glycans on Asn-297 of antibodies were enzymatically remodeled <i>in vitro</i> using galactosyl and sialyltransferases to introduce
terminal sialic acids. Periodate oxidation of these sialic acids yielded
aldehyde groups which were subsequently used to conjugate aminooxy
functionalized cytotoxic agents via oxime ligation. The process has
been successfully demonstrated with three antibodies including trastuzumab
and two cytotoxic agents. Hydrophobic interaction chromatography and
LC-MS analyses revealed the incorporation of ∼1.6 cytotoxic
agents per antibody molecule, approximating the number of sialic acid
residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative
activity toward antigen-positive tumor cells and significantly greater
antitumor efficacy than naked antibody in a Her2-positive tumor xenograft
model. These findings suggest that enzymatic remodeling combined with
oxime ligation of the native glycans of antibodies offers an attractive
approach to generate ADCs with well-defined product profiles. The
site-specific conjugation approach presented here provides a viable
alternative to other methods, which involve a need to either re-engineer
the antibody sequence or develop a highly controlled chemical process
to ensure reproducible drug loading