A V_L-linker-V_H orientation dependent single chain variable antibody fragment against rabies virus G protein with enhanced neutralizing potency in vivo.

Abstract

Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo

“…This study focused on the preparation of a novel, fully human bispecific antibody (BsAb), which not only inhibited tumor angiogenesis, but also activated immune responses. We first constructed BsDb co-targeting VEGF165 and PD-1 with a light-chain variable domains (VL)-to-heavy-chain variable domains (VH) orientation because some studies have reported that enhanced binding ability, affinity and in vivo biological activity were required for scFv or a diabody with a VL-to-VH orientation [ 24 , 25 , 26 ]. To express this protein, although the E. coli host system is considered the preferred choice for the production of recombinant proteins due to its affordability, short culture time, and high protein yield, the bioactivity of recombinant proteins expressed in E. coli might be lower compared to counterparts produced from Pichia pastoris or mammalian cells due to a lower ratio of correct folding [ 27 ].…”

Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid Latex Agglutination Test

“…Thus, to overcome the non-functional insoluble molecule, we made some changes in the scFvStx1(I) gene (then designated scFvStx1 ), in which the orientation was changed to VL-linker-VH. This arrangement allowed the CDH3 to be free at the C-terminal end, since this CDR is likely the most important contributor to antigen binding for most natural antibodies [25]. In addition, based on the fact that the linker could also interfere with molecule stability during expression or storage [26,27], the linker that connects the VL and VH domains was optimized for a common framework by phage display technology (data not shown).…”

Arrangement of VL and VH Domains in VRC01-Based Chimeric Antigen Receptor (CAR) Affects Functions and Exhaustion Status of CAR-T Cells

“…4,12,13 It has previously been shown that the arrangement of V L and V H domains could affect the characteristics of the scFv expressed in Escherichia coli, such as expression level, antigen binding, cross-reactivity, immunotoxin activity, and viral neutralization activity. [14][15][16][17][18][19][20] It has also been reported that the artificial GD2 and CD3-bispecific antibodies could redirect cytotoxic T cells to tumor cells and the activity depended on the arrangement of V L and V H domains. 21 In another study, the functions of the CAR-T cells targeted to C-type lectin-like molecule-1 for human acute myeloid leukemia cells have been compared by chang-ing the arrangement of V L and V H domains in the context of CAR with 4-1BB-derived CSSDs.…”