Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl. In contrast, Li+ (1-3 mM) promotes the cyclic AMP-induced increase of cysteine proteinase-2 mRNA levels, and induces the expression of this prestalk-associated gene in the absence of cyclic AMP stimuli. At concentrations exceeding 4-5 mM, LiCl inhibits cysteine proteinase-2 gene expression. LiCl reduces basal Ins(1,4,5)P3 levels and decreases the cyclic AMP-induced accumulation of Ins(1,4,5)P3; both effects occur half-maximally at 2-3 mM-LiCl. These results indicate that the induction of the cysteine proteinase-2 gene by Li+ is not due to elevated levels of Ins(1,4,5)P3. It is, however, possible that inhibition of prespore gene expression by Li+ is caused by Li+-induced reduction of basal and/or stimulated Ins(1,4,5)P3 levels

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8