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Fusion proteins

Inactive Publication Date: 2011-04-21
HEALTH PROTECTION AGENCY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical conjugation is, however, often imprecise.
Chemical conjugation is also difficult to control.
This is problematic when attachment to only one of said components (preferably at a single site) is desired for therapeutic efficacy.
Thus, chemical conjugation results in a mixed population of modified toxin molecules, which is undesirable.

Method used

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Examples

Experimental program
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Effect test

example 1

Preparation of a LC / A and HN / A Backbone Clones

The following procedure creates the LC and HN fragments for use as the component backbone for multidomain fusion expression. This example is based on preparation of a serotype A based clone (SEQ ID1 and SEQ ID2), though the procedures and methods are equally applicable to the other serotypes [illustrated by the sequence listing for serotype B (SEQ ID3 and SEQ ID4) and serotype C (SEQ ID5 and SEQ ID6)].

Preparation of Cloning and Expression Vectors

pCR 4 (Invitrogen) is the chosen standard cloning vector, selected due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector, which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (BamHI-SalI-PstI-HindIII). A fragment of the expression vector has been removed to create a non-mobilisable p...

example 2

Preparation of a LC / A-nociceptin-HN / A Fusion Protein (Nociceptin is N-Terminal of the HN-Chain)

Preparation of Linker-nociceptin-Spacer Insert

The LC-HN linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and HN) is 23 amino acids long and has the sequence VRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood that proteolytic activation in nature leads to an HN domain that has an N-terminus of the sequence ALNDL. This sequence information is freely available from available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO). Into this linker a Factor Xa site, nociceptin and spacer are incorporated; and using one of a variety of reverse translation software tools [for example EditSeq best E. coli reve...

example 3

Preparation of a Nociceptin-LC / A-HN / A Fusion Protein (Nociceptin is N-Terminal of the LC-Chain)

The LC / A-HN / A backbone is constructed as described in Example 2 using the synthesised A serotype linker with the addition of a Factor Xa site for activation, arranged as BamHI-SalI-linker-protease site-linker-PstI-XbaI-stop codon-HindIII (SEQ ID8). The LC / A-HN / A backbone and the synthesised N-terminal presentation nociceptin insert (SEQ ID9) are cleaved with BamHI+HindIII restriction enzymes, gel purified and ligated together to create a nociceptin-spacer-LC-linker-HN. The ORF (SEQ ID15) is then cut out using restriction enzymes AvaI+XbaI for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID16.

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Abstract

A single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a dynorphin Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the dynorphin Targeting Moiety; and a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent. Nucleic acid sequences encoding the polypeptide fusion proteins, methods of preparing same and uses thereof are also described.

Description

STATEMENT REGARDING SEQUENCE LISTINGThe sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 35492_SEQ_FINAL.txt. The text file is 372 KB; was created on Aug. 23, 2010; and is being submitted via EFS-Web with the filing of the specification.FIELD OF THE INVENTIONThis invention relates to non-cytotoxic fusion proteins, and to the therapeutic application thereof as analgesic molecules.BACKGROUND OF THE INVENTIONToxins may be generally divided into two groups according to the type of effect that they have on a target cell. In more detail, the first group of toxins kill their natural target cells, and are therefore known as cytotoxic toxin molecules. This group of toxins is exemplified inter alia by plant toxins such as ricin, and abrin, and by bacterial toxins such as diphtheria toxin, and Pseudomonas exotoxin A. Cy...

Claims

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Application Information

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IPC IPC(8): A61K38/48C12N9/96C12N15/57C12N15/63C12P21/02A61P29/00
CPCC07K2319/06C07K2319/50A61K38/4893A61K38/482A61K38/4886C12N9/52C12Y304/24069A61K38/00A61K38/1709A61K47/48261A61K47/48338C07K14/665C07K2319/055C12N15/62
Inventor FOSTER, KEITHCHADDOCK, JOHNMARKS, PHILIPSTANCOMBE, PATRICKAOKI, KEI ROGERFRANCIS, JOSEPHSTEWARD, LANCE
Owner HEALTH PROTECTION AGENCY
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