LAUSR

Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two VHH domains within bispecific antibodies recognizing human TNF and the surface molecules of murine myeloid cells (F4/80 or CD11b) on TNF retention and inhibition. TNF‐neutralizing properties in vitro and in vivo of MYSTI‐2 and MYSTI‐3 antibodies were compared with new variants with interchanged VHH domains and different linker sequences. The most effective structure of MYSTI‐2 and MYSTI‐3 proteins required the Ser/Gly‐containing ‘superflexible’ linker. The orientation of the modules was crucial for the activity of the proteins, but not for MYSTI‐3 with the Pro/Gln‐containing ‘semi‐rigid’ linker. Our results may contribute toward the development of more effective drug prototypes.