Abstract
Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPBWT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPBΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297linked GnGn-type glycan contains α1,3-fucose and β1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.
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Abbreviations
- ER:
-
endoplasmic reticulum
- Fab:
-
fragment antigen-binding
- Fc:
-
fragment crystallizable
- FT:
-
α1,3-fucosyltransferase
- GlcNAc:
-
N-acetylglucosamine
- HC:
-
antibody heavy chain
- HER2 and HER3:
-
human epidermal growth factor receptors 2 and 3
- IgG:
-
immunoglobulin G
- LC:
-
antibody light chain
- MALDI-TOF/TOF:
-
matrix-assisted laser desorption/ionization-timeof-flight/time-of-flight tandem mass spectrometry
- MAPP:
-
monoclonal antibody produced in plants
- PNGase F:
-
peptide-N-glycosidase F
- PPB:
-
pertuzumab plant biosimilar
- PPB-ΔXTFT:
-
pertuzumab plant biosimilar obtained from N. benthamiana ΔXTFT
- TFA:
-
trifluoroacetic acid
- TMA:
-
therapeutic monoclonal antibody
- TPB:
-
trastuzumab plant biosimilar
- TPB-ΔXTFT:
-
trastuzumab plant biosimilar obtained from N. benthamiana ΔXTFT
- XT:
-
β1,2-xylosyltransferase
- ΔXTFT:
-
transgenic N. benthamiana plants with knockout of α1,3-fucosyltransferase (FT) and β1,2-xylosyltransferase (XT) genes
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Original Russian Text © T. V. Komarova, E. V. Sheshukova, E. N. Kosobokova, M. V. Serebryakova, V. S. Kosorukov, V. N. Tashlitsky, Y. L. Dorokhov, 2017, published in Biokhimiya, 2017, Vol. 82, No. 4, pp. 687-699.
These authors contributed equally to this work.
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Komarova, T.V., Sheshukova, E.V., Kosobokova, E.N. et al. Trastuzumab and pertuzumab plant biosimilars: Modification of Asn297-linked glycan of the mAbs produced in a plant with fucosyltransferase and xylosyltransferase gene knockouts. Biochemistry Moscow 82, 510–520 (2017). https://doi.org/10.1134/S0006297917040137
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DOI: https://doi.org/10.1134/S0006297917040137