In the PTS reaction, the N-terminal and the C-terminal a part of a split intein (intein-N and intein-C) are fused to the target proteins and ligated with each other to form a peptide bond, and the intein moiety is released without any structural trace at the ligation site. a bsAb construction method using intein-mediated protein trans-splicing to produce IgGCFab2Ctype 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 usually circumvented by individual 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 producing bsAb, IgGCFab2 (Her2/CD3), exhibited target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgGCFab2 type bsAb will expand the bsAb production method. Subject terms: Proteins, Biochemistry, Medical research, Molecular medicine Introduction A bispecific antibody (bsAb) is BMP2 an designed antibody having two different antigen-binding portions within one molecule, while general monoclonal antibodies (mAbs) target only one target antigen1C4. The dual binding ability of bsAbs has multiple applications, which cannot be achieved by general mAbs, including recruiting killer immune cells to malignancy cells2 and activation of receptor molecules by co-cauterization5. Such ability makes bsAbs an emerging class of new antibody therapeutics. One difficulty for immunoglobulin G (IgG) bsAb development is usually a chain-pairing problem that four different polypeptide chains, consisting of two heavy chains and two light chains, should form correct BH3I-1 pairings with each other, where only one combination out of 10 combinations is the correct pairing, although it has great potential. Several antibody engineering techniques have been developed to overcome this chain-pairing problem, such as knobs-into-holes mutation for heavy chain pairing, which introduces convexCconcave mutations around the interface of the Fc dimer6 and CrossMab for heavy chain-light chain pairing, achieved by exchanging the order of domains in the Fab region7. A split intein-mediated protein ligation can be used for generating bsAb molecules among such antibody engineering methods. The reaction, termed protein trans-splicing (PTS), is usually a widely used protein engineering technique to connect separately expressed two target proteins8C10. In the PTS reaction, the N-terminal and the C-terminal a part of a split intein (intein-N and intein-C) are fused to the target proteins and ligated with each BH3I-1 other to form a peptide bond, and the intein moiety is usually released without any structural trace at the ligation site. Connecting two single-domain nanobodies is the simplest usage of the ligation technique for the bsAb construction. We previously reported the construction of tandem VHHs in a bacterial cell11. Various combinations of tandem VHH bsAb can be created using this method. We further utilized the ligation technique to construct circularly connected VHH bsAb by ligating the N- and C-terminus12. The intein-mediated ligation between one Fab arm and the BH3I-1 rest of the IgG molecule was also reported for building IgG-type bispecific antibodies13C15. This study utilized the PTS reaction to construct the IgGCFab2 bsAb (Fig.?1). The IgGCFab2 format was initially developed to construct multivalent mono-specific antibodies16. The heavy chain-light chain-pairing problem, caused by the similarity of two different light chains, humpers its construction by the general recombinant expression method although IgGCFab2 is an interesting format for bsAb. Thus, the use of a common light chain17 or exchanging one light chain with one of the VH-CH1 portions, FIT-Ig, was previously reported to overcome the mispairing issue18,19. Obtaining the common light chain is usually a cumbersome process and the FIT-Ig production potentially results in undesired Fab formation although these techniques are interesting. In this study, we statement the PTS-based method for the IgGCFab2 bsAb production by ligating the separately prepared IgG portion and the Fab portion. The heavy chain/light chain-pairing problem was avoided because the IgG part and Fab parts were separately expressed. Here, we demonstrate the construction of IgGCFab2 bsAb, which binds to the Her2 and CD3 antigens. Open in a separate window Physique 1 Reaction plan of IgGCFab2 construction via PTS reaction. Results and conversation Design of IgG and Fab parts for IgGCFab2 bsAb We designed intein-C fused to the N-terminus of the heavy chain of an IgG and.
