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

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.

Intein mediated high throughput screening for bispecific antibodies

“…Split inteins are reported to be a powerful tool to avoid LC mispairing during bsAb production. [22,23] Split inteins are auto-processing domains found in nearly every organism. [24] The mechanism mediated by split inteins is called Protein Trans Splicing (PTS), and, since its discovery in 1988, it has been used for biotechnical applications, such as protein ligation, [25] purification or labeling.…”

Construction of Novel Bispecific Single-Domain Antibodies (BiSdAbs) with Potent Antiangiogenic Activities

“…1 However, long-term efficacy of mAbs was limited by resistance mechanisms, such as tumors evaded immune control. 2 Bispecific antibodies (BsAbs), such as bispecific T-cell engager (BiTE), 3 dual affinity re-targeting (DART), knob-inhole, and bispecific antibody by protein trans-splicing (BAPTS), 4 can redirect the cytotoxic potential of immune cells to eliminate tumor cells with one arm directly engaging T cells and the other recognizing cancer cells. As it has been shown previously, additional advantages of BsAbs are that drug resistance and severe adverse effects were much reduced.…”

Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

“…[13][14][15][16] Bispecific Antibody by Protein Trans-Splicing (BAPTS) has been reported to synthesize BsAbs with natural human immunoglobulin G (IgG) structure and no chain mispairing. 17,18 Nanobodies, such as camel-derived antibodies, that naturally lack the light chains and contain only the variable region of the heavy chain are the smallest antigenbinding fragments. 19 Compared with traditional monoclonal antibodies, nanobodies have the advantages of small size, high solubility, high stability, high affinity to their targets, low immunogenicity, and excellent tissue penetration.…”