Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates

ACS Chem Biol. 2017 Sep 15;12(9):2427-2435. doi: 10.1021/acschembio.7b00542. Epub 2017 Aug 28.

Abstract

The voltage-gated sodium channel NaV1.7 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 NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 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 NaV1.7 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 NaV1.7 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 NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.

MeSH terms

  • Animals
  • HEK293 Cells
  • Humans
  • Immunoconjugates / chemistry
  • Immunoconjugates / pharmacokinetics
  • Immunoconjugates / pharmacology*
  • Male
  • Mice
  • Models, Molecular
  • NAV1.7 Voltage-Gated Sodium Channel / metabolism*
  • Peptides / chemistry
  • Peptides / pharmacokinetics
  • Peptides / pharmacology*
  • Tissue Distribution
  • Voltage-Gated Sodium Channel Blockers / chemistry
  • Voltage-Gated Sodium Channel Blockers / pharmacokinetics
  • Voltage-Gated Sodium Channel Blockers / pharmacology*

Substances

  • Immunoconjugates
  • NAV1.7 Voltage-Gated Sodium Channel
  • Peptides
  • Voltage-Gated Sodium Channel Blockers