posted on 2021-05-07, 18:11authored byPenny
J. Le, Shane Miersch, Matthew W. Forbes, Nick Jarvik, Anthony Ku, Sachdev S. Sidhu, Raymond M. Reilly, Mitchell A. Winnik
Metal-chelating polymer-based radioimmunoconjugates
(RICs) are
effective agents for radioimmunotherapy but are currently limited
by nonspecific binding and off-target organ uptake. Nonspecific binding
appears after conjugation of the polymer to the antibody and may be
related to random lysine conjugation since the polymers themselves
do not bind to cells. To investigate the role of conjugation sites
on nonspecific binding of polymer RICs, we developed a microbial transglutaminase
reaction to prepare site-specific antibody–polymer conjugates.
The reaction was enabled by introducing a Q-tag (i.e., 7M48) into
antibody (i.e., Fab) fragments and synthesizing a polyglutamide-based
metal-chelating polymer with a PEG amine block to yield substrates.
Mass spectrometric analyses confirmed that the microbial transglutaminase
conjugation reaction was site-specific. For comparison, random lysine
conjugation analogs with an average of one polymer per Fab were prepared
by bis-aryl hydrazone conjugation. Conjugates were prepared from an
anti-frizzled-2 Fab to target the Wnt pathway, along with a nonbinding
specificity control, anti-Luciferase Fab. Fabs were engineered from
a trastuzumab-based IgG1 framework and lack lysines in the antigen-binding
site. Conjugates were analyzed for thermal conformational stability
by differential scanning fluorimetry, which showed that the site-specific
conjugate had a similar melting temperature to the parent Fab. Binding
assays by biolayer interferometry showed that the site-specific anti-frizzled-2
conjugate maintained high affinity to the antigen, while the random
conjugate showed a 10-fold decrease in affinity, which was largely
due to changes in association rates. Radioligand cell-binding assays
on frizzled-2+ PANC-1 cells and frizzled-2− CHO cells showed
that the site-specific anti-frizzled-2 conjugate had ca. 4-fold lower
nonspecific binding compared to the random conjugate. Site-specific
conjugation appeared to reduce nonspecific binding associated with
random conjugation of the polymer in polymer RICs.