Construction of a circularly connected VHH bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites

https://doi.org/10.1016/j.bbrc.2019.12.018Get rights and content

Highlights

  • Backbone cyclization of bispecific VHHs has been constructed.

  • The cyclized bispecific VHHs, termed Cyclobody, show bispecificity.

  • The new format will expand the construction strategy of antibody engineering.

Abstract

A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.

Introduction

A bispecific antibody (bsAb) is an artificially engineered antibody that possesses two different antigen-binding sites within one molecule [1]. The bispecificity enables the bridging of two different antigens to present spatially close orientations. This ability of bsAbs makes them attractive agents for clinical applications, diagnosis, and imaging [2]. For example, a bsAb that binds to a cancer antigen and an antigen on immune cells can recruit immune cells (e.g., T-cells and NK cells) to the tumor site and effectively attack cancer cells [3]. The key features of a bsAb are the two different antigen-binding portions in one molecule; thus, from the IgG-type to the tandem repeat of the antigen-binding single domains, various formats, which reach nearly 100 constructs, have been proposed to improve drug activity, stability, and solubility, among others [4].

Among the several antibody structures, the variable region of the camelid heavy-chain antibody, called VHH or nanobody, is the smallest single-domain antigen-binding domain [5]. Thus, the tandem repeat of two kinds of VHHs forms one of the smallest bsAb constructs [6]. We have previously reported on the construction of a tandem bispecific VHH using a protein trans-splicing reaction [7]. By using the split intein, which mediates the intermolecular protein ligation, two separate VHHs are covalently connected with traceless peptide-bond formation by the ligation reaction. Such a backbone ligation technique is a promising approach for generating a new type of engineered antibody, including the efficient production of an IgG-type bispecific antibody [8].

The split-intein technology can also be used to create a circular backbone protein by connecting both termini of one protein molecule. Two halves of the split intein are fused to the N and C termini of a protein of interest. The cyclization reaction, called the split-intein-mediated circular ligation of peptides and proteins (SICLOPPS) [9,10], then links the backbone. The circular proteins are generally protected from exopeptidase digestion and show higher stability [11,12]. The cyclization of scFv has also reported recently without compromising its stability [22].

We construct herein a novel circular topology of bispecific VHHs, termed a cyclobody, via the SICLOPPS reaction. Although numerous variations of bispecific antibodies have been proposed thus far, the circular topology of a bispecific antibody has never been investigated. The orientation of the cyclobody presents two antigen-binding sites to opposite sides by connecting both termini (Fig. 1). We successfully generated cyclobodies and found that they retain the antigen-binding activity for two different antigens. The successful construction of a new topology of a bispecific antibody will thus expand the construction strategy of next-generation antibodies.

Section snippets

Sample preparation

Genes for the cyclobody were constructed on the pSKBAD2 vector [13]. Npu C-intein and N-intein were fused to the N and C termini of the tandem VHH constructs (Fig. 1b). E×G and E×16 had linkers with sequences EPKIPQPQPKPQPQPQPQPQPKPQPKPEP (Pro linker) from llama IgG2 upper hinge [14] and GGGGS (G1 linker), respectively. The transformed BL21(DE3) cells were grown in an LB medium. The expression was then induced by 0.04 wt% arabinose. The cells were collected and sonicated after 4 h of induction.

Construction of circularly connected VHH bispecific constructs (cyclobody)

By inspecting the crystal structure of a variable region of a heavy-chain antibody (VHH), we found that the basic architecture of the VHH, which has a β-sandwich immunoglobulin fold, is suitable for circular connection with another VHH molecule because the two termini of the VHH domain are located at opposite sides of the β-strand direction (Fig. 1a). Two VHHs can be connected with an antiparallel orientation, and each VHH is like a rigid linker to position the termini close to each other. This

Acknowledgements

We thank Drs. Shinya Honda and Takamitsu Miyafusa for providing carboxy peptidase Y. We thank Dr. Chikako Yokoyama for helpful discussions. This study was supported by Takahashi Industrial and Economic Research Foundation, Kurata Grant from the Kurata Memorial Hitachi Science and Technology Foundation, and partially supported by Grant-in-Aid from JSPS for Scientific Research (KAKENHI Category B (19H03511)).

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