TY - JOUR
T1 - Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A
AU - Lee, Byeong Sung
AU - Lee, Yumi
AU - Park, Jisoo
AU - Jeong, Bo Seok
AU - Jo, Migyeong
AU - Jung, Sang Taek
AU - Yoo, Tae Hyeon
N1 - Funding Information:
This research was supported by the National Research Foundation of Korea funded by the Ministry of Science and ICT (2014M3C1A3051470 and 2015M3D3A1A01064878).
Publisher Copyright:
© 2019 The Author(s).
PY - 2019/6/21
Y1 - 2019/6/21
N2 - 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.
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.
KW - Immunoglobulin G
KW - Immunotoxin
KW - Pseudomonas Exotoxin A
KW - Site-specific conjugation
KW - Unnatural amino acid
UR - http://www.scopus.com/inward/record.url?scp=85068509723&partnerID=8YFLogxK
U2 - 10.1186/s13036-019-0188-x
DO - 10.1186/s13036-019-0188-x
M3 - Article
AN - SCOPUS:85068509723
SN - 1754-1611
VL - 13
JO - Journal of Biological Engineering
JF - Journal of Biological Engineering
IS - 1
M1 - 56
ER -