Autolytic degradation of ocriplasmin: a complex mechanism unraveled by mutational analysis

Abstract

Ocriplasmin, a truncated form of plasmin, is commercialized in the USA and in Europe under the trade name Jetrea(®), and indicated for the treatment of symptomatic vitreomacular adhesion and vitreomacular traction including when associated with macular hole ≤400 µm, respectively. We have shown in a previous study that ocriplasmin undergoes autolytic degradation when injected in eye vitreous, which leads to its rapid inactivation. In order to investigate this process further, we have introduced in ocriplasmin a variety of amino acid substitutions within or in the immediate vicinity of the three major autolytic cleavage sites. We demonstrate here that autolytic inactivation of ocriplasmin is a sequential process where initial cleavage occurs primarily between residues 156 and 157. Reduction or even blocking of autolysis can be achieved by mutating a limited number of key residues. In this study, we also report the identification of a series of ocriplasmin variants with improved resistance to autolysis and unimpaired catalytic activity. Such variants represent useful tools for the exploration of therapeutic approaches aiming at non-surgical resolution of vitreomacular adhesion.

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia Pastoris

“…The autolytic and mixed variants also exhibited reduced K cat and V max values. This results were in agreement with Noppen et al [17]. Also, reduced K cat and V max values was observed in variant with A59T mutation.…”

“…Previous studies revealed that initial cleavage occurs at position 156-157 because it is faster and after this cleavage, sensitivity of the other two sites to autolytic cleavage greatly enhances [27]. Cleavage at 156-157 site was caused inactivation, and mutant with cleavage at 177-178 site was detected in wild-types [17].…”

“…Ocriplasmin is non-surgical alternative for the treatment of symptomatic vitreomacular traction. Its proteolytic activity at physiologic pH and natural vulnerability to autolytic inactivation can in uence its e cacy in vivo and lead to serious damages [17].…”

“…ocriplasmin comprises 2 polypeptides of 230 and 19 residues, which are connected to each other by 2 disul de bonds. Four intra-chain disul de bonds stabilize the domain of 230-residue [17]. Despite obviating the risks associated with the surgery, pharmacologic vitreolysis with ocriplasmin cannot be considered free of any risks [10].…”

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

“…Vigilant monitoring and pars plana vitrectomy constitute standard procedures for managing symptomatic vitreomacular adhesion [ 1 , 3 ]. Given the risk of damage to the retina caused by the surgery [ 7 ], proteolytic enzymes such as plasmin have been examined for the enzymatic release of retinal traction [ 8 ].…”