Enhanced Bioactivity of the Anti-LOX-1 scFv Engineered by Multimerization Strategy

Abstract

Single-chain variable fragment (scFv) antibodies as therapeutic agents have the potential to reduce the production cost and immunogenicity relative to monoclonal antibodies, but their monovalency and lack of a fragment crystallizable region can lead to reduced function. Multimerization is one strategy for recovering the function; however, their application is limited by the production of multimeric proteins. In our previous study, an anti-lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) scFv showed potential use in diagnosis and therapy of atherosclerotic diseases, but is limited by its inherent low antigen-binding activity. In this study, to improve the efficacy of the anti-LOX-1 scFv, we constructed the anti-LOX-1 scFv multimers by modifying the linker length between the variable domains of the scFv or by fusing the scFv with self-merization domains and expressed these scFv multimers in Brevibacillus choshinensis hosts. After optimization, all of the scFv multimers obtained efficient secretion expression. Compared with the scFv monomer, the multimers that are successfully fractionated displayed increased neutralization activity and showed elevated antigen-binding avidity, especially the tetramer, which improved the antigen avidity by two orders of magnitude. Moreover, the scFv dimer and the tetramer both displayed better stability and longer half-life in serum, which can be attractive candidates for the next-generation anti-LOX-1 therapeutic antibody.

OxHDL controls LOX-1 expression and plasma membrane localization through a mechanism dependent on NOX/ROS/NF-κB pathway on endothelial cells

“…Because oxidation levels increase robustly during systemic inflammatory diseases, the participation of ROS in pathological activity has been well accepted [54,55]. It has been reported that ROS can regulate gene expression and act as signaling molecules to modulate protein function [56][57][58][59][60][61]. H 2 O 2 and other reactive intermediates are involved in the expression of a number of proteins, such as cyclooxygenase-2 and prostaglandins [62,63], the proto-oncogenes c-jun, cfos, and c-myc [64], TRP ion channels [65], the vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 [66,67], and also control the regulation of cell proliferation and survival, oncogene activation, immune response, apoptosis, and necrosis [67][68][69].…”

Scavenger Receptors as Biomarkers and Therapeutic Targets in Cardiovascular Disease

“…These scFvs exhibit specific binding affinity, enhanced bio-distribution, and low immunogenicity compared with humanized monoclonal antibodies, whilst being substantially cheaper to produce using bacterial expression systems. LOX-1-specific scFvs are in the early stages of development and ongoing studies are aiming to improve the binding affinity, thermostability, and serum half-life, and using multimerization techniques [ 172 , 173 ].…”