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

Abstract

Background 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. 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. Results We 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. Conclusions We 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. Electronic supplementary material The online version of this article (10.1186/s13036-019-0188-x) contains supplementary material, which is available to authorized users.

Temperature responsive smart polymer for enabling affinity enrichment of current coronavirus (SARS-CoV-2) to improve its diagnostic sensitivity

“…To the best of our knowledge, this may be the first report that introduces the strained alkyne group into NIPAAm copolymer, but these findings were agreed with our previous report where we introduced carboxy isopropyl acrylamide into the NIPAAm copolymer and mentioned the relative LCST elevation [38] also, our findings were parallel with Maeda et al, who introduced HIPAAm into the NIPAAm copolymer [39] . SARS-CoV-2 antibody conjugation with azido groups was achieved by the addition of azido-(EG) 4 -NHS to the antibody lysine residues that were confirmed by the fluorescamine reduction method where increasing azido-(EG) 4 -NHS feeding during the reaction showed a higher azido conjugation to the antibody and fewer amine residues, these results were in agreement with Byeong results who labelled the anti-Her2 IgG to azido groups previously [40] . Our SDS-PAGE gel images showed different polymeric concentrations after conjugation with a constant concentration of SARS-CoV-2 antibody.…”

Natural and Designed Toxins for Precise Therapy: Modern Approaches in Experimental Oncology

“…The two protein molecules were then conjugated site-specifically using a bifunctional linker. The resulting construct demonstrated specific toxicity towards HER2-positive cancer cell in a picomolar range of concentrations [ 26 ]. In some cases, the coupling of antibody with a protein toxin can be provided by non-covalent binding of pre-modified modules, for example, with the use of streptavidin and biotin [ 27 ].…”

Immunotoxins: Targeted Toxin Delivery for Cancer Therapy

“…76,77 Both PE and DT enzymatically ADP-ribosylate elongation factor-2 (EF-2) in the cytosol. 76,77 They each catalyze the ADP-ribosylation of histidine-699 of EF-2, which is posttranslationally modified to a diphthamide residue 35. Despite their similar action, PE and DT have quite different amino acid sequences.…”

“…The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines. 76 Researchers utilized split inteins to generate immunotoxins against HER1/2, 113 while Wang et al reported reduction of nonspecific toxicity of immunotoxin by intein-mediated reconstitution on target cells. 5 Nanobody-based immunotoxins on several targets including CD7, CD38, and VEGFR2 were constructed and tumor-inhibitory effects were evaluated.…”