RE:RE:RE:RE:RE:RE:RE:RE:RE:Scarpa LSD StudyThis discussion of I2S glycosylation is very interesting. For producing recombinant enzymes, it is not just about have a cell transcribe mRNA from the DNA and translate the mRNA into the corresponding protein. It is also important to have the proper post-translational modifications, including the proper glycosylation of certain amino acid residues. Anyone know what cell line Bioasis is using for MTfp-I2S and for other recombinant proteins? Shire is using a human cell line to make Eleprase (I didn't dig deeper to find out which one). I presume Bioasis is using a mammalian cell line, possibly human cell line. But I honestly never thought about this before.
Here's a description of Shire's Eleprase mechanism of action:
https://www.medicines.org.uk/emc/medicine/19764/SPC/Elaprase+2+mg+ml+concentrate+for+solution+for+infusion/
"Mechanism of action
Hunter syndrome is an X-linked disease caused by insufficient levels of the lysosomal enzyme iduronate-2-sulfatase. Iduronate-2-sulfatase functions to catabolize the glycosaminoglycans (GAG) dermatan sulfate and heparan sulfate by cleavage of oligosaccharide-linked sulfate moieties. Due to the missing or defective iduronate-2-sulfatase enzyme in patients with Hunter syndrome, glycosaminoglycans progressively accumulate in the cells, leading to cellular engorgement, organomegaly, tissue destruction, and organ system dysfunction.
Idursulfase is a purified form of the lysosomal enzyme iduronate-2-sulfatase, produced in a human cell line providing a human glycosylation profile, which is analogous to the naturally occurring enzyme. Idursulfase is secreted as a 525 amino acid glycoprotein and contains 8 N-linked glycosylation sites that are occupied by complex, hybrid, and high-mannose type oligosaccharide chains. Idursulfase has a molecular weight of approximately 76 kD.
Treatment of Hunter syndrome patients with intravenous Elaprase provides exogenous enzyme for uptake into cellular lysosomes. Mannose-6-phosphate (M6P) residues on the oligosaccharide chains allow specific binding of the enzyme to the M6P receptors on the cell surface, leading to cellular internalization of the enzyme, targeting to intracellular lysosomes and subsequent catabolism of accumulated GAG."
It looks like Armagen, at least for their Rhesus studies, were using a non-human, mammalian chinese hamster ovary (CHO) cell line (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176522/). In this paper, they indicate they did a dose range between 0 and 30 mg/kg in Rhesus. CHO cell ine should still have the proper glycosylation machinery found in human cell lines.
For Armagen's Phase I Hunter syndrome clinical trial (https://clinicaltrials.gov/ct2/show/NCT02262338) in collaboration with Shire, they are using Insulin Receptor Monoclonal Antibody-Human Iduronate 2-Sulfatase (IDS) Fusion Protein, AGT-182, at 1 to 3 mg/kg weekly infusions for 8 weeks (with a caveat that additional doses may be added). On Armagen's website, they refer to AGT-182 as the molecule they used in the Rhesus experiments, in which they used the CHO cell line. So it doesn't appear that Armagen is using any new cell line or I2S protein variant in this Phase I trial as a result of the Shire collaboration. In this case, their Rhesus studies only showed 1% of injected dose getting into the brain. I could be wrong here though.
So I don't think that Eleprase recombinant I2S is glycosylated in any proprietary way by Shire. I think it is simply glycosylated because they are using a mammalian cell line. If this is true, then Bioasis will obtain similar glycosylation profile of their MTfp-I2S if they are using a human or similar mammalian cell line.
In terms of dosing, the 6 mg/kg of MTfp-I2S used by Scarpa is middle of the range used in the Armagen Rhesus validation study. But keeping in mind that Armagen only reported 1% of injected dose to cross the BBB, then Bioasis MTfp-I2S would be expected to have a huge advantage because they have reported ~3-6% of injected doses of their various conjugates crossing the BBB. I don't remember off hand if they ever reported the number for MTfp-I2S though. Assuming 3-6%, then this gives great reasoning for Scarpa/Bioasis to follow up with lower dosing to determine if they can still obtain the same level of benefit at lower doses of MTfp-I2S. Also, the fact that the Armagen/Shire AGT-182 human Phase I study is using 1 and 3 mg/kg (and possibly other doses), is a testament that no one knows the optimized efficacious dose in humans (or animal models) for Hunter syndrome yet.
Best,
BDAZ