1 research outputs found
Development of Copper-Catalyzed Azide–Alkyne Cycloaddition for Increased in Vivo Efficacy of Interferon β‑1b by Site-Specific PEGylation
The development of protein conjugate therapeutics requires
control
over the site of modification to allow for reproducible generation
of a product with the desired potency, pharmacokinetic, and safety
profile. Placement of a single nonnatural amino acid at the desired
modification site of a recombinant protein, followed by a bioorthogonal
reaction, can provide complete control. To this end, we describe the
development of copper-catalyzed azide–alkyne cycloaddition
(CuAAC, a click chemistry reaction) for site-specific PEGylation of
interferon β-1b (IFNb) containing azidohomoalanine (Aha) at
the N-terminus. Reaction conditions were optimized using various propargyl-activated
PEGs, trisÂ(benzyltriazolylmethyl)Âamine (TBTA), copper sulfate, and
dithiothreitol (DTT) in the presence of SDS. The requirement for air
in order to advance the redox potential of the reaction was investigated.
The addition of unreactive PEG diol reduced the required molar ratio
to 2:1 PEG–alkyne to IFNb. The resultant method produced high
conversion of Aha-containing IFNb to the single desired product. PEG–IFNbs
with 10, 20, 30, and 40 kDa linear or 40 kDa branched PEGs were produced
with these methods and compared. Increasing PEG size yielded decreasing
in vitro antiviral activities along with concomitant increases in
elimination half-life, AUC, and bioavailability when administered
in rats or monkeys. A Daudi tumor xenograft model provided comparative
evaluation of these combined effects, wherein a 40 kDa branched PEG–IFNb
was much more effective than conjugates with smaller PEGs or unPEGylated
IFNb at preventing tumor growth in spite of dosing with fewer units
and lesser frequency. The results demonstrate the capability of site-specific
nonnatural amino acid incorporation to generate novel biomolecule
conjugates with increased in vivo efficacy