Research Article: Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells

Date Published: June 30, 2017

Publisher: Tabriz University of Medical Sciences

Author(s): Mohammad Tasyriq Che Omar.

http://doi.org/10.15171/apb.2017.036

Abstract

Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, the production of functional recombinant antibodies in target cell line is necessary to be evaluated before used in therapeutic application such intrabodies against HIV-1.

Partial Text

The development of smaller antibody fragments, which maintain the antigen affinity of the parental antibody, is preferable for better antibody production. Furthermore, the advance in smaller fragments production is the primary source for the in vitro antibody generation systems.1-3 The Fv fragment that comprises of variable (V) regions is the smallest antigen-binding fragment that maintains the parental antibodies antigenicity properties. To connect the V regions, a soluble and flexible amino acid peptide linker is applied to a scFv (single chain fragment variable) fragment.4-6 Nowadays, two standard formats of recombinant antibodies that are produced in eukaryotes expression system are scFv and Fab that show relevant for the clinical evaluation purposes.7 Moreover, a fusion of the Fc domain towards antibody fragments result in regaining effector functions, stability, and avidity.8,9

Many studies highlighting the use of antibody from commercial hybridoma cell (clone 183-H12-5C) in detecting the HIV-1 capsid protein p24,32,33 however until now it has yet to be used for therapeutic purpose of HIV-1/AIDS. With the fact that monoclonal antibodies (mAbs) are highly specific and stable, it becomes an attractive candidate for in vivo treatment. Since 1992 once FDA approved the first therapeutic mAb muromonab-CD3, 61 therapeutic mAbs approved by FDA until mid-2016.34 Although the number of approved mAbs is encouraging, most of them are limited to target surface antigens that make an intracellular protein such HIV-1 p24 is challenging. The initial part of developing antibody-based biotherapeutic is to test the antigenicity of anti-p24 mAb inside the target cells. However, antibodies do not readily permeate cell membranes, and thus the use of smaller fragment like scFv which easily expressing in the cytoplasm of mammalian is an option. Previously, the monovalent scFv antibody fragment against HIV-1 CA p24 had successfully isolated by phage display technology (data not shown). Expression of the scFv fusion in bacterial cells had shown high potential for production of functional scFv which then being used as detection reagent (data not shown). Although there was one recombinant antibody scFv against HIV-1 CA p24 produced using bacterial-based system had been reported recently, however no antiviral assays were performed using the format in order to validate its function in HIV replication and infectivity.35 Here the expression of scFv in the human mammalian-based system was reported and discussed for establishment of antibody-based biotherapeutic against HIV-1.

In this work, the mammalian plasmid vectors expressing anti-p24 scFv were successfully engineered. ScFv and scFv-Fc were specifically expressed in a human mammalian-based expression system. The purified scFv and scFv-Fc showed good binding activity and specificity towards recombinant p24 and HIV-1 derived p24 (gag). Intracellular expression in relevant cells also demonstrated the effectiveness of inhibiting HIV-1 replication and infectivity. The antibody could be used as candidates for the development of antibody-based biotherapeutics against HIV-1/AIDS.

Cluster of Oncology and Radiological Sciences, AMDI, USM provided the facilities that were used for the experiments. Mohammad Tasyriq Che Omar was sponsored by the ASTS (Academic Staff Training Scheme USM) (KS4049) and Ministry of Higher Education (MOHE) (KPT860818025037) scholarships during the study.

Not applicable.

The author declare that there is no conflict of interests regarding the publication of this article.

 

Source:

http://doi.org/10.15171/apb.2017.036

 

Leave a Reply

Your email address will not be published.