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Butyrylcholinesterase variants that alter the activity of chemotherapeutic agents

A kind of butyrylcholinesterase, variant technology, applied in the field of production and therapeutic use, can solve problems such as restricted use

Inactive Publication Date: 2006-01-11
APPLIED MOLECULAR EVOLUTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both enzymes from non-human species and intercellular enzymes are immunogenic, which severely limits their usefulness

Method used

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  • Butyrylcholinesterase variants that alter the activity of chemotherapeutic agents
  • Butyrylcholinesterase variants that alter the activity of chemotherapeutic agents
  • Butyrylcholinesterase variants that alter the activity of chemotherapeutic agents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0118] Example 1 Butyrylcholinesterase variant library

[0119] This example describes the synthesis and characterization of a library of butyrylcholinesterase variants expressed in mammalian cells. The initial library of butyrylcholinesterase variants was generated by mutating residues determined to be important for the catalytic activity of butyrylcholinesterase. On the Silicone Graphics Octane computer, the FlexiDock program (Tripos Inc., St. Louis, MO) in the Sybyl 6.4 software was used to dock the substrate to the active site of butyrylcholinesterase to determine the internal pair of butyrylcholinesterase. Potentially important residues for catalytic activity. Using PCR-based mutagenesis or codon-based mutagenesis as described herein, residues important for catalytic activity are mutated and packaged into a library.

[0120] For example, Pfu polymerase (Stratagene, La Jolla, CA) is used to generate a butyrylcholinesterase variant library by PCR-site-directed mutagenesis of hu...

Embodiment II

[0130] Example II Carboxyesterase activity of butyrylcholinesterase variants

[0131] This example demonstrates the carboxylesterase activity of some butyrylcholinesterase variants. The standard assay for carboxylesterase activity is the o-nitrophenyl acetate (o-NPA) hydrolysis assay (see Beaufay et al., J.Cell.Biol. 61: 188-200 (1974)). A modified method of this assay is implemented as described below, which measures the o-NPA activity of butyrylcholinesterase variants standardized by capture with an anti-butyrylcholinesterase antibody.

[0132] The protocol for determining the carboxylesterase activity of the captured butyrylcholinesterase by o-nitrophenyl acetate:

[0133] 1) Coat a 96-well Immulon2 plate with 10mg / ml rabbit anti-human butyrylcholinesterase (Dako#A0032) in PBS (100ml / well) at 4°C overnight.

[0134] 2) Remove the coating solution and block the plate with 3% BSA in PBS (250 ml / well) at room temperature for 2 hours.

[0135] 3) Add 200 ml of butyrylcholinesterase...

Embodiment V

[0154] Example V Antibody-butyrylcholinesterase fusion polypeptide for CTP-11 targeting activation

[0155] This example illustrates the construction and characterization of an anti-EGF receptor-BChE fusion polypeptide, which can be used for antibody-directed enzyme prodrug therapy (ADEPT) using CPT-11.

[0156] The N-terminus of BChE (truncated L530 monomer) was fused to the C-terminus of the CH1 domain of the anti-epidermal growth factor receptor (EGFR) antibody ch225 to construct a model antibody-BChE fusion polypeptide. The two domains are connected by a GGGS linker or natural antibody hinge region.

[0157] A model antibody-enzyme fusion polypeptide was produced, which exhibited antigen binding and catalytic enzyme functions. The nucleotide and amino acid sequences of the mouse anti-EGF variable light chain are shown in Figure 9. Figure 10 shows the nucleotide and amino acid sequences of mouse anti-EGF variable heavy chain and constant heavy chain 1 hinge region and L530.

[...

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Abstract

The invention provides a butyrylchinesterase variant, a method of converting a camptothecin derivative to a topoisomerase inhibitor by contacting the camptothecin derivative with a butyrylcholinesterase variant and a method of treating cancer by administering to an individual an effective amount a butyrylcholinesterase variant exhibiting increased capability to convert a camptothecin derivative to a topoisomerase inhibitor compared to butyrylcholinesterase.

Description

Technical field [0001] The present invention relates to butyrylcholinesterase variants, and more particularly, to its production and therapeutic use. Background of the invention [0002] Cancer is one of the leading causes of death in the United States. Every year, more than half a million Americans die of cancer, and more than one million people are newly diagnosed with the disease. In cancer, neoplastic cells escape their normal growth regulation mechanisms and proliferate in an uncontrolled manner, leading to the formation of malignant tumors. If the treatment of the primary tumor is incomplete or not initiated before the actual deterioration of the disease, the tumor cells can be transferred to another site. Therefore, early diagnosis and effective treatment of malignant tumors are necessary for survival. [0003] Current methods of treating cancer include surgery, radiation therapy, and chemotherapy. A major problem with each of these treatments is their lack of specificity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/43C12N9/00A61K38/00C12N9/18
CPCC12N9/18A61K38/00A61P35/00A61P35/04
Inventor J·D·沃特金斯J·D·潘库克
Owner APPLIED MOLECULAR EVOLUTION