Efficient generation of bispecific IgG antibodies by split intein mediated protein trans-splicing system

Lei Han,Hua Jiang,Jianwei Zhu,Kai Ding,Junsheng Chen,Yueqing Xie,Weihan Yin,Baohong Zhang,John Gilly,Huili Lu,Huifang Zong

doi:10.1038/s41598-017-08641-3

Abstract

Many methods have been developed to produce bispecific antibodies (BsAbs) for industrial application. However, huge challenges still remain in synthesizing whole length BsAbs, including their assembly, stability, immunogenicity, and pharmacodynamics. Here we present for first time a generic technology platform of generating bispecific IgG antibodies, “Bispecific Antibody by Protein Trans-splicing (BAPTS)”. Different from published methods, we assembled two parental antibody fragments in the hinge region by the protein trans-splicing reaction of a split intein to generate BsAbs without heavy/heavy and light/heavy chain mispairing. Utilizing this simple and efficient approach, there have been several BsAbs (CD3×HER2, CD3×EGFR, EGFR×HER2) synthesized to demonstrate its broad applicability. Correctly paired mAb arms were assembled to form BsAbs that were purified through protein A affinity chromatography to demonstrate industrial applicability at large scale. Further, the products were characterized through physical-biochemistry properties and biological activities to confirm expected quality of the products from “BAPTS”. More importantly, correct pairing was confirmed by mass spectrum. Proof-of-concept studies with CD3×HER2 BsAb (T-cell recruitment) demonstrated superior bioactivity compared with trastuzumab. The results of undetectable mispairing and high biological activity have indicated that this method has the potential to be utilized to manufacture BsAbs with high efficiency at industrial scale.

Highlights

IntroductionTherapeutic monoclonal antibodies (mAbs) are important therapeutic proteins[1]. Bispecific antibodies (BsAbs) have demonstrated enhanced biological functions in many cases[2,3,4,5]
Therapeutic monoclonal antibodies are important therapeutic proteins[1]
Efforts to create bispecific antibodies with an Fc region resulted in dual variable domain IgGs (DVD-Ig)[5] and IgG-scFv12, which are tetravalent unnatural formats, different in size and geometry from conventional IgG antibodies and may create potential immunogenicity[13]

Summary

Introduction

Therapeutic monoclonal antibodies (mAbs) are important therapeutic proteins[1]. Bispecific antibodies (BsAbs) have demonstrated enhanced biological functions in many cases[2,3,4,5]. Correct pairing of the light chains is achieved by exchanging the CH1 domain of one heavy chain with the CL domain of the corresponding light chain This approach has been used to create therapeutic BsAbs for anti-virus applications[4, 20, 21]. In “CrossMab” technology, unnatural domain junctions were generated and natural antibody architecture was replaced Another approach is to express mAbs separately[22,23,24], combine the two mAbs under mild refolding conditions to form a hybrid BsAb molecule. A similar strategy was presented by Spiess et al, who co-cultured two E. coli strains expressing corresponding half of each mAb that was refolded to synthesize BsAb. Lacking post-translational modification may result in differences in biological functions, stability and in vivo half-life[25, 26]. It is only applicable to kappa but not lambda variable domains[26, 27]

Methods

Results

Conclusion