Figure 1.

Schematic representation of bispecific anti-cytokine antibodies exemplified by myeloid-specific TNF inhibitors (MYSTI). (A,B) Generation of FITC-labeled bispecific antibody composed of anti-hTNF V_HH and anti-F4/80 V_HH (MYSTI, A) and control antibody composed of the same anti-hTNF V_HH and irrelevant V_HH [Systemic TNF Inhibitor, STI, (B)]. Briefly, antibodies were expressed and purified as previously described (19) and were subsequently labeled with FITC. Calculated F/P ratio was approximately four FITC molecules per protein molecule. (C–F) Schematic representation of MYSTI (C–E) and STI (F) binding to macrophages analyzed by flow cytometry and confocal microscopy. FITC-labeled MYSTI binds specifically to F4/80 on the surface of macrophages and can bind and retain exogenously added hTNF or hTNF produced by activated cells as detected by anti-hTNF phycoerythrin (PE)-labeled antibody (Miltenyi Biotec). This resulted in surface staining of macrophages both with FITC and PE (C). MYSTI can be quickly internalized by macrophages resulting in intracellular FITC staining only (D), or when hTNF was added exogenously—double staining for both FITC and PE (E). STI did not bind to macrophages, as suggested by the absence of FITC or PE staining (F). Red dotted line indicates the position of tmTNF cleavage by TACE (ADAM17). Adapted from (19).