The structure of myeloid cell-specific TNF inhibitors affects their biological properties.

Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two VH H domains within bi-specific 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 VH H domains and different linker sequences. The most effective structure of MYSTI-2 and MYSTI-3 proteins required the Ser/Gly-containing "supeflexible" 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 towards the development of more effective drug prototypes.