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

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

Research output: Contribution to journalArticle

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.

Original languageEnglish
Article number56
JournalJournal of Biological Engineering
Volume13
Issue number1
DOIs
Publication statusPublished - 2019 Jun 21

Fingerprint

Immunotoxins
Immunoglobulin G
Proteins
Antibodies
Cysteine
Amino acids
Peptide Elongation Factor 2
Amino Acids
Neoplasms
Immunoglobulin Fragments
Molecules
Pathogens
Cytotoxicity
Half-Life
Pseudomonas aeruginosa toxA protein
human ERBB2 protein
Epidermal Growth Factor
Tumors
Elongation
Bacteria

Keywords

  • Immunoglobulin G
  • Immunotoxin
  • Pseudomonas Exotoxin A
  • Site-specific conjugation
  • Unnatural amino acid

ASJC Scopus subject areas

  • Environmental Engineering
  • Biomedical Engineering
  • Molecular Biology
  • Cell Biology

Cite this

Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A. / Lee, Byeong Sung; Lee, Yumi; Park, Jisoo; Jeong, Bo Seok; Jo, Migyeong; Jung, Sang Taek; Yoo, Tae Hyeon.

In: Journal of Biological Engineering, Vol. 13, No. 1, 56, 21.06.2019.

Research output: Contribution to journalArticle

Lee, Byeong Sung ; Lee, Yumi ; Park, Jisoo ; Jeong, Bo Seok ; Jo, Migyeong ; Jung, Sang Taek ; Yoo, Tae Hyeon. / Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A. In: Journal of Biological Engineering. 2019 ; Vol. 13, No. 1.
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AU - Jo, Migyeong

AU - Jung, Sang Taek

AU - Yoo, Tae Hyeon

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AB - 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.

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