Engineering Antibody Reactivity for Efficient Derivatization to Generate Na_V1.7 Inhibitory GpTx-1 Peptide–Antibody Conjugates

Abstract

The voltage-gated sodium channel Na1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional Na1.7 targeting antibodies are not known. In this report, we describe Na1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified Na1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the Na1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked Na1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not Na1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit Na1.7 in engineered and neuronal cells.

The NaV1.7 Channel Subtype as an Antinociceptive Target for Spider Toxins in Adult Dorsal Root Ganglia Neurons

“…The results obtained are controversial and need to be further confirmed ( Lee et al, 2014 ; Liu et al, 2016 ). More recently, another approach was reported, using an antibody-drug conjugated: a potent Na V 1.7 toxin inhibitor (a GpTx-1 analog), connected by a PEG-linker to an antibody, showed greater stability in plasma and a biodistribution restricted to the regions expressing Na V 1.7 subtype, decreasing thus possible side-effect occurrence ( Biswas et al, 2017 ).…”

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V

“…Previously, we reported the identification, optimization, and derivatization of the 34 amino acid Na V 1.7 inhibitory peptide GpTx-1, from the Chilean tarantula Grammostola porteri , discovering analogs with single-digit nM Na V 1.7 IC 50 values [ 21 – 24 ]. To identify peptides with improved Na V 1.7 inhibitory potency compared to GpTx-1 analogs, we embarked on another fractionated tarantula venom electrophysiology screen and identified the 29-residue inhibitory cystine knot (ICK) peptide JzTx-V (β/κ-theraphotoxin-Cg2a also known as β-theraphotoxin-Cj2a) with sub-nM Na V 1.7 IC 50 from the Chinese earth tiger tarantula Chilobrachys jingzhao .…”

Use of Cryopreserved Hepatocytes as Part of an Integrated Strategy to Characterize In Vivo Clearance for Peptide-Antibody Conjugate Inhibitors of Nav1.7 in Preclinical Species

“…Previous reports have described internal efforts to identify potent inhibitory peptides of Nav1.7 based on GpTx-1 and JzTx-V peptide scaffolds that have been identified and isolated from tarantula spider venom (Biswas et al, 2017;Moyer et al, 2018;Wu et al, 2018). Because many therapeutic peptides suffer from poor pharmacokinetic profiles due to proteolysis and/or renal filtration (Diao and Meibohm, Fig.…”

“…activity have been reported in the literature. Recently, we described the engineering of two series of inhibitory Nav1.7 peptide-antibody conjugates, linking peptides based on the GpTx-1 or JzTx-V toxins found in tarantula venom to nontargeting antibodies to increase the halflife and stability of the inhibitory peptides (Biswas et al, 2017;Moyer et al, 2018;Wu et al, 2018;Murray et al, 2019).…”