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Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof

a technology of phenylalanine ammonialyase and composition, which is applied in the field of prokaryotic phenylalanine ammonialyase (pal) variants, can solve the problems of limited use of pal and lack of concerted efforts to improve these parameters, and achieves enhanced properties, potent catalytic activity, and effective treatment

Inactive Publication Date: 2013-02-14
BIOMARIN PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method for making and using prokaryotic PAL enzymes for therapeutic purposes. The enzymes can be derived from various prokaryotic organisms such as Streptomyces maritimus, Nostoc punctiforme, Anabaena variabilis, and others. The patent also describes variants of the enzymes that have increased activity or reduced immunogenicity. The variants can be chemically modified or linked to other molecules to provide additional benefits such as increased half-life or decreased immunogenicity. The patent also describes pharmaceutical compositions containing prokaryotic PAL enzymes and their use for treating various disorders.

Problems solved by technology

Although PAL potentially has various therapeutic applications, the use of PAL can be limited by reduced specific activity and proteolytic instability.
Further, a delicate balance is required between substrate affinity and enzyme activity to achieve and maintain control of plasma phenylalanine levels within a normal somewhat narrow range in disorders characterized by hyperphenylalaninemia.
As yet, a concerted effort toward improving these parameters has not been made due to a paucity of structural and biochemical knowledge regarding this protein.

Method used

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  • Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof
  • Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof
  • Compositions of prokaryotic phenylalanine ammonia-lyase variants and methods of using compositions thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of Nostoc punctiforme and Anabaena variabilis PAL DNA Manipulations

[0221]N. punctiforme genomic DNA was purchased from ATCC (29133D) and the PAL gene (ZP—00105927) was PCR-amplified from primers 5′-CACTGTCATATGAATATAACATCTCTACAACAGAACAT-3′ (SEQ ID NO:12) and 5′-GACAGTGGCGGCCGCTCACGTTGACTTTAAGCTCGAAAAAATATG-3′ (SEQ ID NO:13). The resulting PCR product was digested with NdeI and NotI and the 1.7 kb fragment was ligated into pET-28a(+) and pET-30a(+) (Novagen) for N-His tagged and untagged, respectively.

[0222]A. variabilis cells were purchased from ATCC (29413). Genomic DNA was extracted (Qiagen) and the PAL gene (YP—324488) was amplified by SOE-PCR to remove an NheI site. Primer 1 (5′-CACTGTGCTAGCATGAAGACACTATCTCAAGCACAAAG-3′) (SEQ ID NO:14) and primer 2 (5′-GGAAATTTCCTCCATGATAGCTGGCTTGGTTATCAACATCAATTAGTGG-3′) (SEQ ID NO:15) were used to amplify nucleotides 1-1190 and primer 3 (5′-CCACTAATTGATGTTGATAACCAAGCCAGCTATCATGGAGGAAATTTCC-3′) (SEQ ID NO:16) and primer 4 (5′-CACTGTGCGG...

example 2

Purification of NpPAL and AvPAL

[0227]The cultures were centrifuged in a bench-top centrifuge at 5,000 g for 20 minutes and the supernatant discarded. The cell pellets were typically frozen at −70° C. prior to further processing. Upon thawing, the cell pellets were suspended to approximately 80 optical density units (600 nm) in TBS (25 mM Tris, 150 mM NaCl, pH 7.8). The cells were lysed by two passes through an APV pressure homogenizer at 12-14,000 psi. The crude lysate was then heat-treated at 55° C. for 2 hours. The lysate is centrifuged at 10,000 g for 30 minutes and the supernatant retained and filtered with a 0.2 μm vacuum filter (Corning).

[0228]The PAL was purified from the clarified lysate by passage sequentially over a butyl 650M column (Tosoh BioSciences) and a MacroPrep High Q column (BioRad). The eluted product showed a high level of purity by both SDS PAGE and reverse phase HPLC.

example 3

Generation of Pegylated PAL Variants

[0229]A method for pegylation of PAL from Rhodosporidium toruloides (RtPAL) is described below. Similar methods are used for pegylation of prokaryotic PAL (e.g., NpPAL or AvPAL) are described in EXAMPLE 6.

Protein Pegylation

[0230]Pegylation uses modifications of literature methods (Hershfield, et al., (1991), ibid.; U.S. Pat. No. 6,057,292; Lu, et al., Biochemistry 40(44):13288-13301 (2001); Nektar Therapeutics, 2003 catalog). Activated PEGs include both the linear PEG succinimidyl succinates (mPEG-SPA, MW 5 kDa or MW 20 kDa) and the branched PEG hydrosuccinimides (mPEG2-NHS ester, MW 10 kDa or MW 40 kDa), which are both capped on one end with a methoxy group and available from Nektar Therapeutics; experimental determination of optimal pegylated proteins is normally required (Veronese, et al., J. Bioactive Compatible Polymers 12:196-207 (1997)). Optimal pegylation conditions are determined using different ratios of PAL:PEG (taking into account the ...

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Abstract

Provided herein are phenylalanine ammonia-lyase (PAL) variants produced by prokaryotes, wherein such prokaryotic PAL variant has a greater phenylalanine-converting activity and / or a reduced immunogenicity as compared to a wild-type PAL. Further provided are compositions of prokaryotic PAL and biologically active fragments, mutants, variants or analogs thereof, as well as methods for the production, purification, formulation, and use of such compositions for industrial and therapeutic purposes, e.g., treating hyperphenylalaninemia, including phenylketonuria, and other disorders, including cancer.

Description

FIELD[0001]Provided herein are compositions related to prokaryotic phenylalanine ammonia-lyase (PAL) variants, including optimization of such compositions to enhance prokaryotic PAL catalytic activity and / or stability, while reducing immunogenicity and / or proteolytic sensitivity of prokaryotic PAL. Also provided herein are uses of such optimal compositions of prokaryotic PAL variants for therapeutic purposes, e.g., treating hyperphenylalaninemia (HPA), including phenylketonuria (PKU), and other disorders, including cancer.BACKGROUND[0002]Phenylalanine ammonia-lyase (PAL) is a non-mammalian enzyme widely distributed in plants (Koukol, et al., J. Biol. Chem. 236:2692-2698 (1961); Hanson, et al., The Enzymes 7:75-166 (1972); Poppe, et al., Curr. Org. Chem. 7:1297-1315 (2003)), some fungi (Rao, et al., Can. J. Biochem. 4512:1863-1872 (1967); Abell, et al., Methods Enzymol. 142:242-253 (1987)) and bacteria (Bezanson, et al., Can. J. Microbiol. 16:147-151 (1970); Xiang, et al., J. Biol. C...

Claims

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Application Information

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IPC IPC(8): A61K38/51A61P3/00A61P7/00C12N9/88
CPCA61K38/51A61K47/48215A61K9/0019C12Y403/01024C12N9/88C12N9/96A61K47/60A61P3/00A61P35/00A61P35/02A61P7/00C07K1/14
Inventor OKHAMAFE, AUGUSTUS O.BELL, SEAN M.ZECHERLE, G. NICKANTONSEN, KRISZHANG, YANHONGTRAN, KIEU LYFITZPATRICK, PAUL A.KAKKIS, EMIL D.VELLARD, MICHEL CLAUDEWENDT, DANIEL J.MUTHALIF, MUBARACK
Owner BIOMARIN PHARMA INC
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