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A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells.

ABSTRACT: Use of monoclonal antibodies is emerging as a highly promising and fast-developing scenario for innovative treatment of viral, autoimmune and tumour diseases. The search for diagnostic and therapeutic antibodies currently depends on in vitro screening approaches, such as phage and yeast display technologies. Antibody production still represents a critical step for preclinical and clinical evaluations. Accordingly, improving production of monoclonal antibodies represents an opportunity, to facilitate downstream target validations. SINEUP RNAs are long non-coding transcripts, possessing the ability to enhance translation of selected mRNAs. We applied SINEUP technology to semi-stable production of monoclonal antibodies in HEK293E cells, which allows for episomal propagation of the expression vectors encoding the heavy and light chains of IgGs. Co-expression of SINEUP RNA with mRNAs encoding heavy and light chains of IgG4s was able to increase the production of different anti-CLDN1 antibodies up to three-fold. Improved production of monoclonal antibodies was achieved both in transiently transfected HEK293E cells and in cellular clones with stable expression of the SINEUP. Compared to antibody preparations obtained under standard conditions, the anti-CLDN1 IgG4s produced in the presence of the SINEUP transcript showed unaltered post-translational modifications, and retained the ability to recognize their target. We thus propose SINEUP technology as a valuable tool to enhance semi-stable antibody production in human cell lines.

SUBMITTER: Sasso E

PROVIDER: S-EPMC6150626 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells.

Sasso Emanuele E Latino Debora D Froechlich Guendalina G Succoio Mariangela M Passariello Margherita M De Lorenzo Claudia C Nicosia Alfredo A Zambrano Nicola N

mAbs 20180510 5

Use of monoclonal antibodies is emerging as a highly promising and fast-developing scenario for innovative treatment of viral, autoimmune and tumour diseases. The search for diagnostic and therapeutic antibodies currently depends on in vitro screening approaches, such as phage and yeast display technologies. Antibody production still represents a critical step for preclinical and clinical evaluations. Accordingly, improving production of monoclonal antibodies represents an opportunity, to facili ...[more]

PMID: 29658818

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Project description:Semi-immunity against the malaria parasite is defined by a protection against clinical episodes of malaria and is partially mediated by a repertoire of inhibitory antibodies directed against the blood stage of Plasmodium falciparum, in particular against surface proteins of merozoites, the invasive form of the parasite. Such antibodies may be used for preventive or therapeutic treatment of P. falciparum malaria. Here, the isolation and characterization of novel human monoclonal antibodies (humAbs) for such applications is described.B lymphocytes had been selected by flow cytometry for specificity against merozoite surface proteins, including the merozoite surface protein 10 (MSP10). After Epstein-Barr virus (EBV) transformation and identification of promising resulting lymphoblastoid cell lines (LCLs), human immunoglobulin heavy and light chain variable regions (Vh or Vl regions) were secured, cloned into plant expression vectors and transiently produced in Nicotiana benthamiana in the context of human full-size IgG1:?. The specificity and the affinity of the generated antibodies were assessed by ELISA, dotblot and surface plasmon resonance (SPR) spectroscopy. The growth inhibitory activity was evaluated based on growth inhibition assays (GIAs) using the parasite strain 3D7A.Supernatants from two LCLs, 5E8 and 5F6, showed reactivity against the second (5E8) or first (5F6) epidermal growth factor (EGF)-like domain of MSP10. The isolated V regions were recombinantly expressed in their natural pairing as well as in combination with each other. The resulting recombinant humAbs showed affinities of 9.27 × 10(-7) M [humAb10.1 (H5F6:?5E8)], 5.46 × 10(-9) M [humAb10.2 (H5F6:?5F6)] and 4.34 × 10(-9) M [humAb10.3 (H5E8:?5E8)]. In GIAs, these antibodies exhibited EC50 values of 4.1 mg/ml [95% confidence interval (CI) 2.6-6.6 mg/ml], 6.9 mg/ml (CI 5.5-8.6 mg/ml) and 9.5 mg/ml (CI 5.5-16.4 mg/ml), respectively.This report describes a platform for the isolation of human antibodies from semi-immune blood donors by EBV transformation and their subsequent characterization after transient expression in plants. To our knowledge, the presented antibodies are the first humAbs directed against P. falciparum MSP10 to be described. They recognize the EGF-like folds of MSP10 and bind these with high affinity. Moreover, these antibodies inhibit P. falciparum 3D7A growth in vitro.

Dataset Information

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells.

Publications

A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells.

Similar Datasets

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