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

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

Quantifying trophic interactions and niche sizes of juvenile fishes in an invaded riverine cyprinid fish community

Quantifying feeding interactions between non-indigenous and indigenous fishes in invaded fish communities is important for determining how introduced species integrate into native food webs. Here, the trophic interactions of invasive 0+ European barbel Barbus barbus (L.) and the three other principal 0+ fishes in the community, Squalius cephalus (L.), Leuciscus leuciscus (L.) and Phoxinus phoxinus (L.), were investigated in the River Teme, a River Severn tributary in Western England. Barbus barbus has been present in the River Teme for approximately 40 years. Analyses of stomach contents from samples collected from three sites between June and September 2015 revealed that, overall, fishes displayed a generalist feeding strategy, with most prey having low frequency of selection. Relationships of diet composition versus body length and gape height were species-specific, with increasing dietary specialisms apparent as the 0+ fishes increased in length and gape height. The trophic niche size of invasive B. barbus was always significantly smaller than S. cephalus and L. leuciscus, and was significantly smaller than P. phoxinus at two sites. This was primarily due to differences in the functional morphology of the fishes; 0+ B. barbus were generally restricted to foraging on the benthos, whereas the other fishes were able to forage on prey present throughout the water column. Nevertheless, the invasive B. barbus were exploiting very similar prey items to populations in their native range, suggesting these invaders were strongly pre-adapted to the River Teme and this arguably facilitated their establishment and invasion