An off-the-Shelf Approach for the Production of Fc Fusion Proteins by Protein Trans-Splicing towards Generating a Lectibody In Vitro.

Anniina Jaakkonen,Gerrit Volkmann,Hideo Iwaï

doi:10.3390/ijms21114011

Abstract

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a “lectibody”. Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

Highlights

Immunoglobulin G (IgG) is a multidomain protein that consists of four polypeptide chains
We hypothesized that carbohydrate-binding proteins like lectins could be able to replace the role of the variable domain of VHH as a binding domain in IgG format when fused at the N-terminus of Fc domain (Figures 3 and 6d)
We demonstrated an off-the-shelf approach to producing Fc fusion proteins at their N-terminus by in vitro protein ligation using protein trans-splicing (PTS)

Summary

Introduction

Immunoglobulin G (IgG) is a multidomain protein that consists of four polypeptide chains. Two identical heavy chains and two identical light chains form a Y-shaped molecule connected by disulfide bonds (Figure 1a). While the heavy chain is composed of four structural domains called immunoglobulin domains (VH, CH1, CH2, and CH3), the light chain has two immunoglobulin domains (VL and CL) (Figure 1a) [1]. Variable domains VH and VL constitute an antigen-binding region, bearing two binding sites per IgG. The enormous diversity of the variable domains plays an essential role in recognizing specific antigens, such as pathogens and foreign molecules in immunity [1]. Antibodies from camelids (like camel and llama) lack light chains, and are termed as heavy-chain antibodies (Figure 1b) [2]. Antigen-binding regions of the heavy-chain antibodies contain only one immunoglobulin fold termed VHH. Jt.hMeolf.uScni.c2t0i2o0n, 2a1l, xroFOleRoPfEEthReREvVaIEriWable domains VH and VL in conventional immunoglob2uolfi1n7s [2]

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