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While achieving rapid developments in recent years, bispecific antibodies are still difficult to design and manufacture, due to mispair of both heavy and light chains. Here we report a novel technology to make bispecific molecules. The knob-into-hole method was used to pair two distinct heavy chains as a heterodimer. IgG4 S228P CH1-CL interface was then partially replaced by T-cell receptor α/β constant domain to increase the efficiency of cognate heavy and light chain pairing. Following expression and purification, the bispecific antibody interface exchange was confirmed by Western blotting and LC–MS/MS. To ensure its validity, we combined a monovalent bispecific antibody against PD-1 (sequence from Pembrolizumab) and LAG3 (sequence from Relatlimab). The results showed that the molecule could be assembled correctly at a ratio of 95% in cells. In vitro functional assay demonstrated that the purified bispecific antibody exhibits an enhanced agonist activity compared to that of the parental antibodies. Low immunogenicity was predicted by an open-access software and ADA test. [ABSTRACT FROM AUTHOR]

The red arrow indicates a Tm1 value of 62.45°C." now reads: "C, The Tm1 of protein was quantitatively detected by Uncle biologics stability screening platform. S5C, the melting temperature (Tm1) is 62.45 °C, lower than the control IgG SB 4 sb S228P whose Tm1 is 69.00 °C, indicating that PD-1 × LAG3 TiMab is relatively stable." now reads: "The thermostability was also examined with Uncle biologics stability screening platform (Unchained Labs). [Extracted from the article]