Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A

Byeong Sung Lee,Yumi Lee,Bo Seok Jeong,Migyeong Jo,Sang Taek Jung,Jisoo Park,Tae Hyeon Yoo

doi:10.1186/s13036-019-0188-x

Byeong Sung Lee, Yumi Lee + Show 5 more

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https://doi.org/10.1186/s13036-019-0188-x

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Abstract

BackgroundImmunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. Immunotoxins based on full-length antibodies are less studied, even though the fragment crystallizable (Fc) domain plays an important role in regulating the concentration of immunoglobulin G (IgG) in the serum and in antibody-mediated immune responses against pathogens.ResultsWe devised a method to site-specifically conjugate IgG and another protein using a cysteine residue introduced into the IgG and a bio-orthogonally reactive unnatural amino acid incorporated into the other protein. The human epidermal growth factor receptor 2 (Her2)-targeting IgG, trastuzumab, was engineered to have an unpaired cysteine in the heavy chain, and an unnatural amino acid with the azido group was incorporated into an engineered Pseudomonas exotoxin A (PE24). The two protein molecules were conjugated site-specifically using a bifunctional linker having dibenzocyclooctyne and maleimide groups. Binding to Her2 and interaction with various Fc receptors of trastuzumab were not affected by the conjugation with PE24. The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines, which involved the inhibition of cellular protein synthesis due to the modification of elongation factor-2.ConclusionsWe constructed the site-specifically conjugated immunotoxin based on IgG and PE24, which induced target-specific cytotoxicity. To evaluate the molecule as a cancer therapeutic, animal studies are planned to assess tumor regression, half-life in blood, and in vivo immunogenicity. In addition, we expect that the site-specific conjugation method can be used to develop other antibody-protein conjugates for applications in therapeutics and diagnostics.

Highlights

Immunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics
Trastuzumab, a human epidermal growth factor receptor 2 (Her2)-targeting immunoglobulin G (IgG), was engineered with an unpaired Cys, and an unnatural amino acid having the azido group was incorporated into PE24 to install the bioorthogonal reactive moiety at the toxin protein
The two proteins were conjugated via a linker having bifunctional groups of maleimide and dibenzocyclooctyne (DBCO), which are reactive with the thiol and azido groups, respectively (Scheme 1)

Summary

Introduction

Immunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. One of the intensively studied immunotoxins is a fusion protein composed of an antibody fragment, such as variable fragment (Fv), single-chain Fv (scFv), or antigen binding fragment (Fab), and an engineered Pseudomonas exotoxin A (PE) [3, 9]. To improve the efficacy and the toxicity of PE, portions of the toxin that were not necessary for the translocation and the modification of eEF-2 were deleted, resulting in PE24, which showed much lower off-target toxicity than the original [14], and its immunogenicity was reduced by removing T- and B-cell epitopes [15, 16]

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