A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60–114, PDP_60–114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP_60–114 had no impact on activity. The specific activity of the GDF11/PDP_60–114 complex (EC₅₀ = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP_60–114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP_60–114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP_60–114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC₅₀ = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC₅₀ = 2 nM) by protease treatment. Complex formation with PDP_60–114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-β family that form latent pro/mature domain complexes

Additional file 3: of CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study

CDP7657 immune complex ( IC ) and aglycosyl hu5c8 IC do not induce platelet aggregation in rhesus monkey platelets both in the absence of presence of sub-aggregatory amounts of the platelet agonist (ADP). In vitro aggregation assay of rhesus monkey washed platelets. A CDP7657 IC did not aggregate rhesus monkey washed platelets in the absence (blue and red), or presence (black and green) of ADP. B Similarly, aglycosyl hu5c8 IC did not aggregate rhesus monkey washed platelets in the absence (blue and red), or presence (black and green) of ADP. (PDF 171 kb

Additional file 1: of CDP7657, an anti-CD40L antibody lacking an Fc domain, inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study

Inhibition of secondary immune response in Cynomolgus monkeys. CDP7657 at 5 or 20 mg/kg (60 mg/kg was not evaluated in this study) was compared with hu5c8 at 20 mg/kg. Animals were administered a single dose of antibody or i.v saline. and challenged with tetanus toxoid (TT) on day 1; they then received a second dose of antibody and TT on day 30. Data are expressed as the mean anti-TT IgG titer ± standard deviation; approximately 50 % inhibition was observed at 20 mg/kg CDP7657, although this was not statistically significant. ***P <0.001 (one-way analysis of variance) compared with control; NS not significant. (PDF 66 kb