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Nanolipoprotein Particles as a Delivery Platform for Fab Based Therapeutics
journal contribution
posted on 2020-08-05, 19:08 authored by Martine Darwish, Whitney Shatz, Brandon Leonard, Kelly Loyet, Kathy Barrett, Janice L. Wong, Hong Li, Ryan Abraham, May Lin, Yvonne Franke, Christine Tam, Kyle Mortara, Inna Zilberleyb, Craig BlanchetteNanolipoprotein
particles (NLPs), a lipid bilayer-based nanoparticle
platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but
their potential to deliver Fabs with valencies that exceed those of
current multivalent formats has not yet been evaluated. Here we describe
the development, optimization, and characterization of Fab-NLP conjugates.
NLPs were generated with maleimide reactive lipids for conjugation
to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids
were shown to conjugate to the apolipoprotein when the NLPs were assembled
at pH 7.4. However, this undesirable reaction was not observed when
assembled at pH 6. Site-specific Fab conjugation conditions were then
optimized, and conjugation of up to 30 Fab per NLP was demonstrated.
Interestingly, although conjugation of higher numbers of Fabs had
a significant impact on NLP molecular weight, only a minimal impact
on NLP hydrodynamic radius was observed, indicating that particle
size is largely dictated by the discoidal shape of the NLP. Fab-NLP
viscosity and its stability upon lyophilization were also evaluated
as an assessment of the manufacturability of the Fab-NLP. Significantly
higher Fab concentrations were achieved with the Fab-NLP conjugates
relative to another multivalent format (Fab-PEG conjugates). Fab conjugation
to the NLP was also not found to have an impact on Fab activity in
both an inhibitory and agonist setting. Finally, the stability of
the Fab-NLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated
increased stability, with >63% of Fab-NLP remaining intact after
24
h at Fab per particle ratios of 7 or greater. Our findings suggest
Fab-NLPs are a promising platform for the targeted delivery of Fabs
in a multivalent format and are compatible with established manufacturing
processes.