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Modified L-Asparaginase

a technology of lasparaginase and l-asparaginase, which is applied in the field of lasparaginases, can solve the problems of inability to meet the needs of patients, etc., and achieves the effect of reducing the activity of pegylated biopharmaceuticals compared to unmodified biopharmaceuticals

Inactive Publication Date: 2019-06-06
JAZZ PHARMA IRELAND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a modified protein that combines two types of proteins, L-asparaginase and (poly)peptides. These (poly)peptides are made up of proline and alanine amino acid residues. This can be done by either chemical conjugation or expressing the modified protein as a fusion protein. The invention also provides nucleic acids, vectors, and host cells that can be used to produce this modified protein. The modified protein can be used in medicine, specifically for the treatment of cancer. The L-asparaginase used in this invention can be derived from Erwinia or has at least 85% identity to the amino acid sequence of SEQ ID NO:1.

Problems solved by technology

These properties decrease both in vitro and in vivo stability and can impair drug safety.
However, in many cases, PEGylated biopharmaceuticals show significantly reduced activity compared to the unmodified biopharmaceutical.

Method used

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Examples

Experimental program
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Effect test

example 1

[0185]Optimization of Coupling Ratio for the Preparation of Pyroglutamoyl-P / A(20)-aminohexanoyl-Crisantaspase

[0186]4.38 mg Pga-P / A#1(20)-Ahx peptide (Part A of FIG. 1; TFA salt, purity 98%; PSL Peptide Specialty Laboratories, Heidelberg, Germany) (SEQ ID NO: 16, amino acid sequence shown in SEQ ID NO: 5) was dissolved in 66.3 μl DMSO. The chemical activation of the P / A peptide via its terminal carboxylate group was started by addition of 23.7 μL of a solution of 500 mM TBTU (CAS#125700-67-6; Iris Biotech, Marktredwitz, Germany) in DMSO and 2.7 μL DIPEA to the peptide solution and vortexing (cf. Part C of FIG. 1). In this setup, the concentration of the peptide was 25.8 mM and the molar ratio between DIPEA, TBTU and Pga-P / A#1(20)-Ahx was 5:5:1. After 10 min incubation at 25° C. the mixture was diluted in Eppendorf tubes according to Table 1.

[0187]A solution of Dickeya chrysanthemi L-Asparaginase (Crisantaspase, SEQ ID NO: 1, recombinant, produced in E. coli (lot RE-LAP-P57D) with a c...

example 2

[0190]Preparation of Pyroglutamoyl-P / A(40)-aminohexanoyl-Crisantaspase

[0191]28 mg of the Pyroglutamoyl-P / A#1(40)-Ahx peptide (SEQ ID NO. 17, amino acid sequence shown in SEQ ID NO: 15), Part B of FIG. 1, TFA salt, purity 98%; Almac Group, Craigavon, UK) was dissolved in 1324 μL of anhydrous DMSO (99.9%; Sigma-Aldrich, Taufkirchen, Germany). To achieve chemical activation of the P / A peptide via its terminal carboxylate group, 162 μL of a solution of 500 mM TBTU (CAS# 125700-67-6; Iris Biotech, Marktredwitz, Germany) in DMSO and, after mixing, 14 μL DIPEA (99.5%, biotech. Grade, Sigma-Aldrich) were added. The whole mixture was vortexed briefly and incubated for 20 min at 25° C. (cf. Part C of FIG. 1). In this setup, the peptide concentration was 5.41 mM and the molar ratio between DIPEA, TBTU and Pga-P / A#1(40)-Ahx was 10:10:1.

[0192]3.5 mL of an ice-cold Crisantaspase solution (SEQ ID NO: 1)(2 mg / mL in PBS) was mixed with the activated peptide solution (1.5 mL), resulting in a mass rat...

example 3

[0194]Preparation of Pyroglutamoyl-P / A(20)-aminohexanoyl-Crisantaspase

[0195]21 mg of the Pyroglutamoyl-P / A#1(20)-Ahx peptide (SEQ ID NO: 5, Part A of FIG. 1; TFA salt, purity 98%; PSL Peptide Specialty Laboratories, Heidelberg, Germany) was dissolved in 1376 μL of anhydrous DMSO (99.9%; Sigma-Aldrich, Taufkirchen, Germany). To achieve chemical activation of the P / A peptide via its terminal carboxylate group, 114 μL of a solution of 500 mM TBTU (CAS# 125700-67-6; purchased from Iris Biotech, Marktredwitz, Germany) in DMSO and, after mixing, 10 μL DIPEA (99.5%, biotech. Grade, Sigma-Aldrich) were added. The whole mixture was vortexed briefly and incubated for 20 min at 25° C. (Part C of FIG. 1). In this setup, the peptide concentration was 7.58 mM and the molar ratio between DIPEA, TBTU and Pga-P / A#1(20)-Ahx was 5:5:1.

[0196]3.5 mL of an ice-cold Crisantaspase solution (SEQ ID NO: 1)(2 mg / mL in PBS) was mixed with the activated peptide solution (1.5 mL), resulting in a mass ratio betwe...

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Abstract

The disclosure provides a modified protein that is a combination of (i) an L-asparaginase and (ii) one or more (poly)peptide(s), wherein the (poly)peptide consists solely of proline and alanine amino acid residues, and methods of preparation and use thereof.

Description

[0001]The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: sequencelisting; date recorded: Jun.28, 2017; file size: 34 KB).BACKGROUND[0002]Proteins with L-asparagine aminohydrolase activity, commonly known as L-asparaginases, have successfully been used for the treatment of Acute Lymphoblastic Leukemia (ALL) in children for many years. ALL is the most common childhood malignant cancers (Avramis (2005) Clin. Pharmacokinet. 44, 367-393).[0003]L-asparaginase has also been used to treat Hodgkin's disease, acute myelocytic Leukemia, acute myelomonocytic Leukemia, chronic lymphocytic Leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Kotzia (2007) J. Biotechnol. 127, 657-669). The anti-tumor activity of L-asparaginase is believed to be due to the inability or reduced ability of certain malignant cells to synthesize L-asparagine (Id). These maligna...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/82A61K38/48A61K38/02
CPCC12N9/82A61K38/48A61K38/02C07K2319/00C07K14/00C12Y305/01001
Inventor FRIEDRICH, LARSO'DONNELL, ANNE
Owner JAZZ PHARMA IRELAND LTD
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