Poster N20 - Stabilization of TPP1 for the Treatment of Late Infantile Neuronal Ceroid Lipofuscinosis

Introduction: Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a hereditary lysosomal storage disease defined by the mutation of Cln2 gene. This in turn causes a deficiency in activity of tripeptidyl peptidase 1 (TPP1) lysosomal enzyme, leading to debilitating symptoms in patients. The onset of LINCL is marked by seizers along with visual impairment, progressing to dementia and loss of locomotor function, and finally resulting in the death of the patient – typically between 10 and 15 years of age. Currently, the only clinically approved treatment for slowing the loss of locomotor function due to LINCL is an enzyme replacement therapy. Recombinant TPP1 enzyme is administered bi-weekly via cerebral spinal fluid (CSF)-mediated intracerebroventricular injections. However, the frequency and associated costs of the treatment place significant burden on patient compliance and finance. For this reason, we aim to improve the potency of the treatment by increasing the enzyme’s half-life within the lysosome. With this, dosage and/or treatment frequency could be reduced to lessen patient burden. To achieve this, we propose to conjugate the recombinant TPP1 proenzyme with a polymer to generate polymer-protein hybrids (PPH) that can resist proteolytic degradation and improve stability of the enzyme in the harsh lysosomal environment.

Materials and

Methods: Copolymers of 2-hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) with varying degrees of polymerization (DP) were generated using photoinduced electron/energy transfer reversible addition-fragmentation chain transfer (PET-RAFT) polymerization and confirmed using gel permutation chromatography. To generate the PPHs, a solution comprised of polymers and the TPP1 proenzyme in a pH 8.7 buffer was reacted overnight at 4 OC. This allows the GMA to covalently bind to the exposed lysines of the recombinant TPP1 proenzyme. Following this, the PPHs were activated by pH 3.5 buffer solution and incubated at 37 OC for two hours to induce the release of the pro-domain thus exposing the active site of TPP1. Once the PPHs were in their active state, their activity was evaluated by placing them in a pH 4.5 buffer solution with a fluorogenic substrate (Ala-Ala-Phe-7-amido-4-methylcoumarin) and incubated for 1 hour at 37 OC. The fluorescent signal was then measured using a plate reader (excitation: 360 nm/emission: 460 nm) and an increase in the fluorescent signal was indicative of higher enzymatic activity.

Results, Conclusions, and Discussions: TPP1 is known to be stable under mildly acidic conditions. However, the generation of PPHs require more basic conditions to facilitate efficient conjugation of polymer to TPP1. As such, the first step was to confirm that the required pH of 8.7 would not cause premature denaturation. Towards this end, we compared the activity of the TPP1 enzyme at pH 5 and pH 8.7. After 24 hours, the activity at both conditions was found to be similar (Figure 1A), indicating that the conjugation conditions were suitable for TPP1 modification. Next, we sought to evaluate if the activity of TPP1 enzyme decreases over time after being activated. We found the activity of TPP1 was sustained over the four days post activation. Based on these results, we evaluated the impact of polymers on TPP1 activity. To achieve this, we synthesized HEMA-GMA copolymers with DPs of 50, 100, and 200. We then conjugated these polymers to the TPP1 proenzyme overnight at varying polymer-to-enzyme ratios of 1, 2, and 4 to form PPHs. After activation, PPHs with DP 50 polymer were successful in maintaining TPP1 activity (Figure 1C). This is most likely due to longer polymer chains conjugating to both the enzyme and pro-domain such that the enzyme is unable to release the pro-domain to expose the active site; this will be confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Interestingly, the smaller polymer chains were only able to achieve this when the ratio of polymer to enzyme was 4:1.

Method development was conducted to optimize conditions for synthesis of novel TPP1 PPHs. This work indicated that mildly basic conjugation conditions could be used without loss of enzyme activity, thus enabling the use of GMA copolymers for PPH preparation. When TPPs were reacted with shorter polymer chains (50 DP) in excess (4:1), enzymatic activity of the PPH was maintained versus the native enzyme. As a result, for future studies will focus on evaluation of PPHs formed from polymers with smaller DPs. Resistance to proteolytic degradation will then be assessed to determine the protective effect of the polymer conjugation.

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