Manufacture of active highly phosphorylated human lysosomal sulfatase enzymes and uses thereof

Inventors

Pungor, Erno • Hague, Charles • Chen, Zhi • Dvorak-Ewell, Melita • Vellard, Michel Claude • Koppaka, Vish

Assignees

Biomarin Pharmaceutical Inc

Abstract

This invention provides compositions of active highly phosphorylated lysosomal sulfatase enzymes, their pharmaceutical compositions, methods of producing and purifying such lysosomal sulfatase enzymes and compositions and their use in the diagnosis, prophylaxis, or treatment of diseases and conditions, including particularly lysosomal storage diseases that are caused by, or associated with, a deficiency in the lysosomal sulfatase enzyme.

Core Innovation

The invention provides a platform for producing active, highly phosphorylated human lysosomal sulfatases, including recombinant human arylsulfatase A (ARSA) and recombinant human GALNS (N-acetylgalactosamine-6-sulfatase). The produced sulfatase is characterized by at least 0.5 bis-phosphorylated oligomannose chains per protein chain and by a catalytic cysteine characterized by FGly conversion. The platform is focused on human lysosomal sulfatases relevant to lysosomal storage diseases, including metachromatic leukodystrophy (MLD) and Morquio A (MPS IVa).

In the disclosed platform, CHO-K1-derived END3 complementation group cells are engineered to co-express SUMF1 together with the target sulfatase. The disclosure references CHO-K1-derived END3 complementation group cells as producing contexts, including G71/G71S and related clones, and describes recombinant human lysosomal sulfatase enzymes produced from G71S cells with co-expressed SUMF1. The target output is a sulfatase enzyme product with desired enzymatic and biochemical product attributes that support activity and phosphorylation-related features.

The disclosure also defines scalable production and purification concepts for the produced enzyme, including multi-column chromatography trains, purification and polishing steps, ultrafiltration/diafiltration, and chromatographic steps. The document includes example performance metrics such as production yields, purification recoveries and purities, enzyme specific activity, serum stability half-life, receptor binding and uptake measures, binding to an M6P receptor, uptake-related binding to synoviocytes, and FACE and CE measurements of bis-phosphorylated species and percent FGly activation.

The document further describes in vitro and in vivo evaluation focused on uptake, substrate degradation, biodistribution, and delivery to lysosome-rich tissues. It includes use of fluorescently labeled GALNS for biodistribution and confocal microscopy with lysosome marker (LAMP-1), and describes in vitro cell-based substrate degradation and tissue-associated distribution outcomes.

Claims Coverage

The partial content provides one independent claim. This claim includes tightly specified inventive features: a specific treatment indication, an enzyme identity, a quantitative structural/biochemical constraint on phosphorylation, and a defined production cell context using a CHO-derived END3 complementation group cell.

Across the single provided independent claim, the claimed invention combines treatment of MLD by administration of an effective amount of lysosomal sulfatase, specification that the enzyme is recombinant human ARSA with at least 0.5 bis-phosphorylated oligomannose chains per protein chain, and production by a CHO-derived END3 complementation group cell.

Stated Advantages

Documented Applications