Site-specific polyethylene glycolylated (pegylated) asparaginase, preparation method therefor and use thereof
Inventors
MA, Bruce Yong • Wang, Jun • Wang, He • Xu, Chunlin • Chen, Yifei • WANG, Yaofang
Assignees
GENSUN INSTITUTE OF BIOMEDICINE Co Ltd • Zonhon Biopharma Institute Inc
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Publication Number
US-10537620-B2
Publication Date
2020-01-21
Expiration Date
Abstract
The present application discloses a PEGylated asparaginase and use thereof. In this application, the polyethylene glycol (PEG) is coupled to the N-terminal amino of 1 or 2 subunits of L-asparaginase, and the molecular weight of the PEG is 30-40 KDa. The PEG is preferably branched and has an aldehyde group serving as an activating group. The PEGylated asparaginase is useful in the preparation of anti-tumor drugs.
Core Innovation
The invention relates to a site-specific diPEGylated L-asparaginase in which each N-terminal amino of 2 out of 4 subunits is coupled separately to one PEG molecule. The PEG molecule prior to coupling is branched and has a molecular weight of 30-40 KDa, producing a defined diPEGylated L-asparaginase structural formula with specified parameter ranges.
A key aspect is the defined general structural formula of the diPEGylated L-asparaginase, where R is H or a C1-C4 alkyl, n is an integer from 320 to 455, P is an integer from 1 to 4, AA is an N-terminal L-amino acid residue, m is an integer from 0 to 5, S is 2, and ASP is L-asparaginase. The PEG component includes aldehyde activating groups, and the framework is used to distinguish mono vs diPEGylated subunit occupancy and to confirm N-terminal PEGylation.
The document further states that the diPEGylated L-asparaginase provides improved characterization outcomes and functional performance relative to asparaginase and marketed controls. Reported benefits include high purification purity and uniform electrophoresis patterns, peptide mapping confirming N-terminal PEGylation, and circular dichroism and fluorescence indicating minimal secondary and tertiary structure change.
The document also reports enhanced pharmacokinetics, improved thermal stability, improved in-vitro enzyme activity retention, and improved tumor cell inhibition, and it describes use in preparing anti-tumor drugs for ALL and non-Hodgkin lymphoma including NK/T cell lymphoma.
Claims Coverage
The independent claim covers a site-specific diPEGylated L-asparaginase (or a pharmaceutically acceptable salt or complex) with a defined diPEGylation pattern across 4 subunits, implemented by coupling separate branched 30-40 KDa PEG molecules to N-terminal aminos of 2 subunits. The claim specifies a general structural formula with defined parameter ranges, yielding a composition defined by both site-specific PEGylation and PEG structural constraints.
Site-specific diPEGylation at N-terminal aminos on 2 of 4 subunits
A diPEGylated L-asparaginase (or a pharmaceutically acceptable salt or complex) wherein each of N-terminal aminos of 2 out of 4 subunits is coupled to one PEG molecule separately.
Branched 30-40 KDa PEG for separate coupling
The PEG molecule prior to coupling is branched and has a molecular weight of 30-40 KDa.
Defined diPEGylated L-asparaginase general structural formula parameters
The diPEGylated L-asparaginase has a general structural formula wherein R is H or a C1-C4 alkyl, n is an integer from 320 to 455, P is an integer from 1 to 4, AA is an N-terminal L-amino acid residue, m is an integer from 0 to 5, S is 2, and ASP is L-asparaginase.
Aldehyde-activated PEG coupling chemistry
The PEG used for coupling has an aldehyde group that acts as an activating group.
Overall claim coverage centers on a site-specific diPEGylated L-asparaginase defined by coupling branched 30-40 KDa PEG molecules separately to N-terminal aminos of 2 out of 4 subunits, together with a defined general structural formula and aldehyde-activating PEG coupling chemistry.
Stated Advantages
High purification purity (>98%).
Uniform SDS-PAGE/electrophoresis patterns distinguishing mono vs diPEGylated subunit occupancy.
Peptide mapping confirms N-terminal PEGylation.
Circular dichroism and fluorescence indicate minimal secondary and tertiary structure change.
Improved pharmacokinetics, including extended half-life and increased AUC versus asparaginase and Pegaspargase.
Enhanced thermal stability, retaining approximately 80% activity after 1 h at 55°C compared with near-loss for Pegaspargase.
Higher in-vitro activity retention (about 85%-90% versus about 53%-63% for marketed controls).
Improved tumor cell inhibition, with lower IC50 versus Pegaspargase/Oncaspar across THP-1, Raji, and L1210.
Documented Applications
Preparing anti-tumor drugs for acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma, including NK/T cell lymphoma.
Use in preparing anti-tumor drugs for other diseases as stated in the document.
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