A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system

Abstract

BackgroundAdvances in antibody engineering provide strategies to construct recombinant antibody-like molecules with modified pharmacokinetic properties. Multermerization is one strategy that has been used to produce antibody-like molecules with two or more antigen binding sites. Multimerization enhances the functional affinity (avidity) and can be used to optimize size and pharmacokinetic properties. Most multimerization strategies involve genetically fusing or non-covalently linking antibody fragments using oligomerization domains. Recent studies have defined guidelines for producing antibody-like molecules with optimal tumor targeting properties, which require intermediates size (70–120 kDa) and bi- or tri-valency.ResultsWe described a highly modular antibody-engineering platform for rapidly constructing synthetic, trivalent single chain variable fragments (Tri-scFv) using the SpyCatcher/SpyTag protein ligase system. We used this platform to construct an anti-human epidermal growth factor receptor 3 (HER3) Tri-scFv. We generated the anti-HER3 Tri-scFv by genetically fusing a SpyCatcher to the C-terminus of an anti-HER3 scFv and ligating it to a synthetic Tri-SpyTag peptide. The anti-HER3 Tri-scFv bound recombinant HER3 with an apparent KD of 2.67 nM, which is approximately 12 times lower than the KD of monomeric anti-HER3 scFv (31.2 nM). Anti-HER3 Tri-scFv also bound endogenous cell surface expressed HER3 stronger than the monomer anti-HER3 scFv.ConclusionWe used the SpyTag/SpyCatcher protein ligase system to ligate anti-HER3 scFv fused to a SpyCatcher at its C-termini to a Tri-SpyTag to construct Tr-scFv. This system allowed the construction of a Tri-scFv with all the scFv antigen-binding sites pointed outwards. The anti-HER3 Tri-scFv bound recombinant and endogenously expressed HER3 with higher functional affinity (avidity) than the monomeric anti-HER3 scFv. The Tri-scFv had the size, valency, and functional affinity that are desired for therapeutic and imaging applications. Use of the SpyTag/SpyCatcher protein ligase system allows Tri-scFvs to be rapidly constructed in a simple, modular manner, which can be easily applied to scFvs or other antibody fragments targeting other antigens.

Biomaterials for Sequestration of Growth Factors and Modulation of Cell Behavior

“…Recently, novel molecules based on the function of antibodies (particularly single-chain variable fragments) have begun to be engineered in order to retain the original avidity while improving pharmacokinetic properties. [140][141][142] These developments may open another avenue of research, but so far the majority of the work on the subject has been dedicated to two other types of molecules: peptides and aptamers.…”

Solubility, Stability, and Avidity of Recombinant Antibody Fragments Expressed in Microorganisms

“…One example in clinic is blinatumomab ( BLINCYTO , ® 2014 ), a bispecific scFv for CD19 and CD3 ( Table 1 ), which functions as a T lymphocyte engager to cancerous B lymphocytes for the treatment of acute lymphoblastc leukemia ( Holliger et al, 1993 ; Mack et al, 1995 ; Suresh et al, 2014 ; Wu et al, 2015 ). In addition, constructing anti-HER3 trivalent scFv using SpyCatcher ligase system enhanced affinity by 12-fold as compared to a monomeric anti-HER3 counterpart ( Alam et al, 2018 ). Another general approach of antibody fragments is the utilization of targeting ligands on nanoparticles in nanomedicine ( Richards et al, 2017 ).…”

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

“…Specifically, the SpyTag/SpyCatcher fragments can either be fused C- or N-terminally or to internal positions within the protein, unlike, for example, split inteins, which are restricted to C- or N-terminal fusions. Within the years, a plethora of applications has been published, ranging from the generation of bispecific antibodies to ROBO1 [ 68 ], a generic trivalent scFv platform [ 69 ], and the generation of site-specific conjugated ADCs with high efficiency [ 70 ] for therapeutic approaches. Akiba et al described an elegant technology for generating biparatopic antibodies through two-step targeting using a pair antibody-SpyTag or SpyCatcher-fusions targeting different epitopes of the same target that spontaneously react to form a covalent bond between fragments to generate a biparatopic antibody in situ [ 71 ].…”