Trans-splicing Reaction Research Articles

Development of a β-lactamase activity assay for detecting ligand–protein interactions using an engineered split intein and β-lactamase

Abstract Several synthetic compounds bind to proteins of interest and inhibit protein–protein interactions. To develop a detection method for the interactions between the synthetic compounds and the target proteins, we used an engineered split intein derived from Nostoc punctiforme PCC73102 (Npu) DnaE and TEM-1 β-lactamase as reporter proteins. We constructed synthetic ligands bearing a 6-residue C-terminal peptide from Npu DnaE and Cys-Trp as the C-extein, and target proteins bearing the N-terminal region of the engineered Npu DnaE and residues 24–284 of β-lactamase. Specific ligand–protein interactions such as phosphopeptide–Src homology domain 2 (SH2) of c-Src and imatinib–quinone reductase 2 (NQO2) increased the protein trans-splicing (PTS) reaction rates and yields. The PTS product showed the enhanced β-lactamase activity compared with the starting materials. The PTS-based β-lactamase activity assay was used for the quantitative analysis of the ligand–protein interactions. The signal sequence and 9-residue N-terminal sequence of Escherichia coli (E. coli) lipoprotein (Lpp) and residues 46–159 of outer membrane protein A (OmpA) (LppOmpA) were conjugated with the target proteins bearing split intein and β-lactamase to display them on live E. coli cell surfaces. PTS on live E. coli surfaces provided enhanced resistance to carbenicillin.

In vivo Validation of Hsp90 Trans-splicing in Giardia lamblia: Highlighting the Role of Cis-elements

Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.

Construction of IgG–Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart’s chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG–Fab2–type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG–Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG–Fab2 type bsAb will expand the bsAb production method.

Use of the redox-dependent intein system for enhancing production of the cyclic green fluorescent protein

To expand the reported redox-dependent intein system application, in this work, we used the split intein variant with highly trans-splicing efficiency and minimal extein dependence to cyclize the green fluorescent protein variant reporter in vitro. The CPG residues were introduced adjacent to the intein's catalytic cysteine for reversible formation of a disulfide bond to retard the trans-splicing reaction under the oxidative environment. The cyclized reporter protein in Escherichia coli cells was easily prepared by organic extraction and identified by the exopeptidase digestion. The amounts of extracted cyclized protein reporter in BL21 (DE3) cells were higher than those in hyperoxic SHuffle T7 coexpression system for facilitating the disulfide bond formation. The double His6-tagged precursor was purified for in vitro cyclization of the protein for 3 h. Compared with the purified linear counterpart, the cyclic reporter showed about twofold increase in fluorescence intensity, exhibited thermal and hydrolytic stability, and displayed better folding efficiency in BL21 (DE3) cells at the elevated temperature. Taken together, the developed redox-dependent intein system will be used for producing other cyclic disulfide-free proteins. The cyclic reporter is a potential candidate applied in certain thermophilic aerobes.

Construction and evaluation of GPC3-targeted immunotoxins as a novel therapeutic modality for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) accounts for ∼90% of all liver cancer cases, which was the third most common cause of cancer death worldwide in 2020. Glypican-3 (GPC3) is highly and specifically expressed in HCC, which makes it a promising therapeutic target. We discovered novel antibody sequences against GPC3 from a phage display library and ranked the candidates by their binding affinity and epitope bins. Candidates with single- to double-digit nanomolar affinity were selected and expressed in Fab format and linked to a deimmunized bacterial exotoxin moiety via an intein trans-splicing reaction. The resulting immunotoxins were evaluated for their in vitro binding specificity and affinity, cell surface binding on the HepG2 or Huh7, rate of internalization, and potency of cytotoxicity. The immunotoxin called GT5 exhibited strong antigen binding and cell surface binding, as well as high internalization efficiency. The molecule GT5 was further evaluated for cytotoxicity in HepG2 and Huh7 cell-based assay and assessed for its pharmacokinetics and antitumor activity in a murine tumor xenograft model. GT5 significantly inhibited tumor growth and showed stronger potency than the chemotherapeutic drug sorafenib. In conclusion, GT5, a novel GPC3 targeting immunotoxin, was shown to have a high affinity towards GPC3 and effectively inhibit hepatocellular tumor growth in vitro and in vivo, thus providing the basis for further development of GT5 immunotoxin as a novel therapeutic modality for the treatment of liver cancer.

287 Development of a non-invasive, non-viral RNA therapy approach for dystrophic epidermolysis bullosa

In this study we aim to correct mutations within COL7A1 that cause malfunction, reduction or complete absence of type VII collagen in the skin’s basement membrane zone (BMZ), leading to dystrophic epidermolysis bullosa (DEB), a severe and rare skin blistering disease associated with a high risk of skin cancer as well as increased mortality. Therefore, we use a 3’-RTMS6m repair molecule to develop a safe, non-viral, non-invasive and efficient in vivo RNA therapy for DEB. This RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable to correct all mutations occurring between exon 65 and exon 108 of COL7A1 via a trans-splicing reaction between the repair molecule and the mutated mRNA.

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex.

Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

Kinetics study of the natural split Npu DnaE intein in the generation of bispecific IgG antibodies.

Rapid and efficient bispecific antibody (BsAb) production for industrial applications is still facing many challenges. We reported a technology platform for generating bispecific IgG antibodies, "Bispecific Antibody by Protein Trans-splicing (BAPTS)." While the "BAPTS" method has shown potential in high-throughput screening of BsAbs, further understanding and optimizing the methodology is desirable. A large number of BsAbs were selected to illustrate the conversion efficiency and kinetics parameters. The temperature of reaction makes no significant influence in conversion efficiency, which can reach more than 70% within 2h, and CD3 × HER2 BsAb can reach 90%. By fitting trans-splicing reaction to single-component exponential decay curves, the apparent first-order rate constants at a series of temperatures were determined. The rate constant ranges from 0.02 to 0.11min-1 at 37°C, which is a high rate reported for the protein trans-splicing reaction (PTS). The reaction process is activated rapidly with activation energy of 8.9kcal·mol-1 (CD3 × HER2) and 5.2kcal·mol-1 (CD3 × EGFR). The BsAbs generated by "BAPTS" technology not only had the similar post-translation modifications to the parental antibodies, but also demonstrated excellent in vitro and in vivo bioactivity. The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach. KEY POINTS: • The trans-splicing reaction of Npu DnaE intein in "BAPTS" platform is a rapid process with low reaction activation and high rate. • The BsAb generated by "BAPTS" remained effective in tumor cell killing. • The kinetics parameters and activation energy of the reaction illustrate feasible for high-throughput screening and industrial applications using the "BAPTS" approach.

The role of nuclear organization in trans-splicing based expression of heat shock protein 90 in Giardia lamblia.

Hsp90 gene of G. lamblia has a split nature comprising two ORFs separated by 777 kb on chromosome 5. The ORFs of the split gene on chromosome 5 undergo transcription to generate independent pre-mRNAs that join by a unique trans-splicing reaction that remains partially understood. The canonical cis-acting nucleotide elements such as 5'SS-GU, 3'SS-AG, polypyrimidine tract and branch point adenine are present in the independent pre-mRNAs and therefore trans-splicing of Hsp90 must be assisted by spliceosomes in vivo. Using an approach of RNA-protein pull down, we show that an RNA helicase selectively interacts with HspN pre-mRNA. Our experiments involving high resolution chromosome conformation capture technology as well as DNA FISH show that the trans-spliced genes of Giardia are in three-dimensional spatial proximity in the nucleus. Altogether our study provides a glimpse into the in vivo mechanisms involving protein factors as well as chromatin structure to facilitate the unique inter-molecular post-transcriptional stitching of split genes in G. lamblia.

An RNA Triangle with Six Ribozyme Units Can Promote a Trans-Splicing Reaction through Trimerization of Unit Ribozyme Dimers

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with a linker element. We introduced a kink-turn motif as a bent linker element of the ribozyme dimer to design a closed trimer with a triangular shape. The oligomeric states of the resulting ribozyme dimers (kUrds) were analyzed biochemically and observed directly by atomic force microscopy (AFM). Formation of kUrd oligomers also triggered trans-splicing reactions, which could be monitored with a reporter system to yield a fluorescent RNA aptamer as the trans-splicing product.

Intein-Mediated Protein trans-Splicing of the Recombinant Streptavidin on Magnetosomes

When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be detrimental. In the study, the streptavidin monomer fragment (S1-116) was fused with the intein N-terminal (termed precursor S1-116-IN), and S1-116-IN was expressed in E. coli (BL21). Meanwhile, the SA117-160 fragment was fused with the C-terminal intein, and then this chimeric polypeptide was expressed on magnetosomes by fusion with magnetosome membrance protein MamF. In the in vitro protein splicing system, the purified engineered magnetosomes (BMP-SA117-160-IC) and the S1-116-IN precursor were mixed. Intein-mediated trans-splicing reaction was induced to produce the functional magnetic beads BMP-SA. Our results indicate that intein-mediated protein trans-splicing may lead to efficient synthesis of the recombinant streptavidin on the magnetosomes, showing its promising potential to produce other functional magnetic nanoparticles.

Targeted suicide gene therapy for liver cancer based on ribozyme-mediated RNA replacement through post-transcriptional regulation

Hepatocellular carcinoma (HCC) has high fatality rate and limited therapeutic options. Here, we propose a new anti-HCC approach with high cancer-selectivity and efficient anticancer effects, based on adenovirus-mediated Tetrahymena group I trans-splicing ribozymes specifically inducing targeted suicide gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To confer potent anti-HCC effects and minimize hepatotoxicity, we constructed post-transcriptionally enhanced ribozyme constructs coupled with splicing donor and acceptor site and woodchuck hepatitis virus post-transcriptional regulatory element under the control of microRNA-122a (miR-122a). Adenovirus encoding post-transcriptionally enhanced ribozyme improved trans-splicing reaction and decreased human TERT (hTERT) RNA level, efficiently and selectively retarding hTERT-positive liver cancers. Adenovirus encoding miR-122a-regulated ribozyme caused selective liver cancer cytotoxicity, the efficiency of which depended on ribozyme expression level relative to miR-122a level. Systemic administration of adenovirus encoding the post-transcriptionally enhanced and miR-regulated ribozyme caused efficient anti-cancer effects at a single dose of low titers and least hepatotoxicity in intrahepatic multifocal HCC mouse xenografts. Minimal liver toxicity, tissue distribution, and clearance pattern of the recombinant adenovirus were observed in normal animals administered either systemically or via the hepatic artery. Post-transcriptionally regulated RNA replacement strategy mediated by a cancer-specific ribozyme provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

Giardia lamblia Hsp90 pre-mRNAs undergo self-splicing to generate mature RNA in an in vitro trans-splicing reaction.

We have previously shown that the Heat Shock Protein 90 (Hsp90) gene in G. lamblia is expressed from two ORFs localized 777 kb apart. The pre-mRNAs transcribed from these ORFs are stitched by a trans-splicing mechanism. Here, we provide mechanistic details of this process by reconstituting the reaction using in vitro synthesized pre-mRNA substrates. Using RT-PCR, northern blot and nanostring technology, we demonstrate that the in vitro synthesized pre-mRNAs have the capability to self-splice in the absence of nuclear proteins. Inhibition of the trans-splicing reaction using a ssDNA oligo corresponding to a 26-nucleotide complementary sequence confirmed their role in juxtapositioning the pre-mRNA substrates during the self-splicing reaction. Our study provides the first example of a self catalysed, trans-splicing reaction in eukaryotes.

Reduction of non-specific toxicity of immunotoxin by intein mediated reconstitution on target cells

Recombinant immunotoxins are chimeric proteins composed of a targeting peptide that binds to a specific tumor antigen and a toxin protein killing target cells. Recombinant immunotoxin exhibits potent cancer inhibiting effects both in vivo and in vitro. However, the non-specific toxicity causes severe syndromes limiting their clinical application. To reduce toxicity caused by recombinant immunotoxins in general, we divided an immunotoxin into two nontoxic segments that may restore toxic bioactivity on tumor cell surface based on the intein mediated trans-splicing reaction. Both split and reconstituted immunotoxins were tested for their biological activities. We found that the reconstituted immunotoxin retained antigen specificity and affinity toward cancer cells overexpressing HER2/neu. After being internalized into HER2/neu positive cells, the reconstituted immunotoxin showed comparable cytotoxicity as the original immunotoxin, while the split immunotoxin fragments showed no toxic activity to cells with or without HER2/neu expression. This approach can potentially be used under clinical settings to reduce non-specific toxicity by administering patients with inactive immunotoxin fragments. Cytotoxic effect only occurs at tumor sites where the inactive fragments bind, trans-splice and become active toxin.

Naturally split intein Npu DnaE mediated rapid generation of bispecific IgG antibodies

High product purity, preserving natural IgG architecture, and excellent production efficiency are highly desirable in bispecific antibody manufacturing. We have reported a platform called Bispecific Antibody by Protein Trans-Splicing (BAPTS) to synthesize BsAbs with natural human IgG structure and no chain mispairing. In the method, two antibody fragments carrying different target-specificities are separately expressed in mammalian cells and subsequently fused to form BsAbs by utilizing the trans-splicing property of the split intein Npu DnaE. The hinge region of antibody, a region with less functional impact, is selected for conjugating the two fragments. The method involves the following steps: (i) constructing five plasmids coding antibody components; (ii) separately expressing and purifying two antibody fragments A and B. Fragment A contains one Fab, “Knobs-into-Holes” mutations in the CH3 domain and NPU DnaEC. Fragment B contains another Fab and NPU DnaEN; (iii) mixing of fragments A and B under permissive reducing conditions in vitro to enable trans-splicing reaction; (iv) removing the reductant to allow re-oxidation of disulfide bonds; (v) isolating BsAb product from unreacted precursors by affinity chromatography. The method allows correct assembly of two heavy and two light chains to form bispecific IgG antibodies in natural structure with no synthetic linkers. No chain mispairing was observed in the product by UPLC-MASS. In addition, the observed kinetics and low reaction activation energy confirmed that the trans-splicing is thermodynamically favored reaction. The BAPTS technology is feasible for industrial applications.

Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules.

Live-cell-based biosensors have emerged as a useful tool for biotechnology and chemical biology. Genetically encoded sensor cells often use bimolecular fluorescence complementation or fluorescence resonance energy transfer to build a reporter unit that suffers from nonspecific signal activation at high concentrations. Here, we designed genetically encoded sensor cells that can report the presence of biologically active molecules via fluorescence-translocation based on split intein-mediated conditional protein trans-splicing (PTS) and conditional protein trans-cleavage (PTC) reactions. In this work, the target molecules or the external stimuli activated intein-mediated reactions, which resulted in activation of the fluorophore-conjugated signal peptide. This approach fully valued the bond-making and bond-breaking features of intein-mediated reactions in sensor construction and thus eliminated the interference of false-positive signals resulting from the mere binding of fragmented reporters. We could also avoid the necessity of designing split reporters to refold into active structures upon reconstitution. These live-cell-based sensors were able to detect biologically active signaling molecules, such as Ca2+ and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca2+ stimuli and the glucocorticoid receptor agonist, dexamethasone. These live-cell-based sensing systems hold large potential for applications such as drug screening and toxicology studies, which require functional information about targets.

Group I Intron-Based Therapeutics Through Trans-Splicing Reaction.

In 1982, the Cech group discovered that an intron structure in an rRNA precursor of Tetrahymena thermophila is sufficient to complete splicing without assistance from proteins. This was the first moment that scientists recognized RNAs can have catalytic activities derived from their own unique three-dimensional structures and thus play more various roles in biological processes than thought before. Several additional catalytic RNAs, called ribozymes, were subsequently identified in nature followed by intense studies to reveal their mechanisms of action and to engineer them for use in fields such as molecular cell biology, therapeutics, imaging, etc. Naturally occurring RNA-targeting ribozymes can be broadly classified into two categories by their abilities: Self-cleavage and self-splicing. Since ribozymes use base-pairing to recognize cleavage sites, identification of the catalytic center of naturally occurring ribozymes enables to engineer from "self" to "trans" acting ones which has accelerated to design and use ribozyme as valuable tools in gene therapy fields. Especially, group I intron-based trans-splicing ribozyme has unique property to use as a gene therapeutic agent. It can destroy and simultaneously repair (and/or reprogram) target RNAs to yield the desired therapeutic RNAs, maintaining endogenous spatial and temporal gene regulation of target RNAs. There have been progressive improvements in trans-splicing ribozymes and successful applications of these elements in gene therapy and molecular imaging approaches for various pathogenic conditions. In this chapter, current status of trans-splicing ribozyme therapeutics, focusing on Tetrahymena group I intron-based ribozymes, and their future prospects will be discussed.

The Mitochondrion-Targeted PENTATRICOPEPTIDE REPEAT78 Protein Is Required for nad5 Mature mRNA Stability and Seed Development in Maize

Pentatricopepetide repeat (PPR) proteins are a large family of RNA-binding proteins involved in RNA metabolism in plant organelles. Although many PPR proteins have been functionally studied, few of them are identified with a function in mitochondrial RNA stability. By using a reverse genetic approach, we characterized the role of the mitochondrion-targeted PPR78 protein in nad5 mature mRNA stability and maize (Zea mays) seed development. Loss of PPR78 function leads to a dramatic reduction in the steady-state level of mitochondrial nad5 mature mRNA, blocks the assembly of complex I in the electron transport chain, and causes an arrest in embryogenesis and endosperm development. Characterization of a second strong allele confirms the function of PPR78 in nad5 mRNA accumulation and maize seed development. The generation of mature nad5 requires the assembly of three distinct precursor RNAs via trans-splicing reactions, and the accumulation of nad5T1 precursor is reduced in the ppr78 mutants. However, it is the instability of mature nad5 rather than nad5T1 causing loss of the full-length nad5 transcript, and degradation of nad5 losing both translation start and stop codons is enriched in the mutant. Our data imply the assembly of mature nad5 mRNA precedes the protection of PPR78.

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

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.