Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1999-02-23
2002-10-08
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S157100, C424S164100, C424S167100, C424S178100, C424S179100, C424S184100, C424S235100, C424S234100, C424S236100, C424S239100, C424S247100, C530S300000, C530S350000, C530S825000, C435S069100, C435S070100, C435S071100, C435S071200, C435S069700, C435S252330, C536S023400, C536S023700
Reexamination Certificate
active
06461617
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to recombinant toxin fragments, to DNA encoding these fragments and to their uses such as in a vaccine and for in vitro and in vivo purposes.
BACKGROUND OF THE INVENTION
The clostridial neurotoxins are potent inhibitors of calcium-dependent neurotransmitter secretion in neuronal cells. They are currently considered to mediate this activity through a specific endoproteolytic cleavage of at least one of three vesicle or pre-synaptic membrane associated proteins VAMP, syntaxin or SNAP-25 which are central to the vesicle docking and membrane fusion events of neurotransmitter secretion. The neuronal cell targeting of tetanus and botulinum neurotoxins is considered to be a receptor mediated event following which the toxins become internalised and subsequently traffic to the appropriate intracellular compartment where they effect their endopeptidase activity.
The clostridiai neurotoxins share a common architecture of a catalytic L-chain (LC, ca 50 kDa) disulphide linked to a receptor binding and translocating H-chain (HC, ca 100 kDa). The HC polypeptide is considered to comprise all or part of two distinct functional domains. The carboxy-terminal half of the HC (ca 50 kDa), termed the H
C
domain, is involved in the high affinity, neurospecific binding of the neurotoxin to cell surface receptors on the target neuron, whilst the amino-terminal half, termed the H
N
domain (ca 50 kDa), is considered to mediate the translocation of at least some portion of the neurotoxin across cellular membranes such that the functional activity of the LC is expressed within the target cell. The H
N
domain also has the property, under conditions of iow pH, of forming ion-permeable channels in lipid membranes, this may in some manner relate to its translocation function.
For botulinum neurotoxin type A (BoNT/A) these domains are considered to reside within amino acid residues 872-1296 for the H
C
, amino acid residues 449-871 for the H
N
and residues 1-448 for the LC. Digestion with trypsin effectively degrades the H
C
domain of the BoNT/A to generate a non-toxic fragment designated LH
N
, which is no longer able to bind to and enter neurons (FIG.
1
). The LH
N
fragment so produced also has the property of enhanced solubility compared to both the parent holotoxin and the isolated LC.
It is therefore possible to provide functional definitions of the domains within the neurotoxin molecule, as follows:
(A) Clostridial Neurotoxin Light Chain:
a metalloprotease exhibiting high substrate specificity for vesicle and/or plasma-membrane associated proteins involved in the exocytotic process. In particular, it cleaves one or more of SNAP-25, VAMP (synaptobrevin/cellubrevin) and syntaxin.
(B) Clostridial Neurotoxin Heavy Chain H
N
Domain:
a portion of the heavy chain which enables translocation of that portion of the neurotoxin molecule such that a functional expression of light chain activity occurs within a target cell.
the domain responsible for translocation of the endopeptidase activity, following binding of neurotoxin to its specific cell surface receptor via the binding domain, into the target cell.
the domain responsible for formation of ion-permeable pores in lipid membranes under conditions of low pH.
the domain responsible for increasing the solubility of the entire polypeptide compared to the solubility of light chain alone.
(C) Clostridial Neurotoxin Heavy Chain H
C
Domain.
a portion of the heavy chain which is responsible for binding of the native holotoxin to cell surface receptor(s) involved in the intoxicating action of clostridial toxin prior to internalisation of the toxin into the cell.
The identity of the cellular recognition markers for these toxins is currently not understood and no specific receptor species have yet been identified although Kozaki et al. have reported that synaptotagmin may be the receptor for botulinum neurotoxin type B. It is probable that each of the neurotoxins has a different receptor.
It is desirable to have positive controls for toxin assays, to develop clostridial toxin vaccines and to develop therapeutic agents incorporating desirable properties of clostridial toxin.
However, due to its extreme toxicity, the handling of native toxin is hazardous.
The present invention seeks to overcome or at least ameliorate problems associated with production and handling of clostridial toxin.
SUMMARY OF THE INVENTION
Accordingly, the invention provides a polypeptide comprising first and second domains, wherein said first domain is adapted to cleave one or more vesicle or plasma-membrane associated proteins essential to neuronal exocytosis and wherein said second domain is adapted (i) to translocate the polypeptide into the cell or (ii) to increase the solubility of the polypeptide compared to the solubility of the first domain on its own or (iii) both to translocate the polypeptide into the cell and to increase the solubility of the polypeptide compared to the solubility of the first domain on its own, said polypeptide being free of clostridial neurotoxin and free of any clostridial neurotoxin precursor that can be converted into toxin by proteolytic action. Accordingly, the invention may thus provide a single polypeptide chain containing a domain equivalent to a clostridial toxin light chain and a domain providing the functional aspects of the H
N
of a clostridial toxin heavy chain, whilst lacking the functional aspects of a clostridial toxin H
C
domain.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
For the purposes of the invention, the functional property or properties of the H
N
of a clostridial toxin heavy chain that are required to be exhibited by the second domain of the polypeptide of the invention are either (i) translocation of the polypeptide into a cell, or (ii) increasing solubility of the polypeptide compared to solubility of the first domain on its own or (iii) both (i) and (ii). References hereafter to a H
N
domain or to the functions of a H
N
domain are references to this property or properties. The second domain is not required to exhibit other properties of the H
N
domain of a clostridial toxin heavy chain.
A polypeptide of the invention can thus be soluble but lack the translocation function of a native toxin-this is of use in providing an immunogen for vaccinating or assisting to vaccinate an individual against challenge by toxin. In a specific embodiment of the invention described in an example below a polypeptide designated LH
423
/A elicited neutralising antibodies against type A neurotoxin. A polypeptide of the invention can likewise thus be relatively insoluble but retain the translocation function of a native toxin—this is of use if solubility is imparted to a composition made up of that polypeptide and one or more other components by one or more of said other components.
The first domain of the polypeptide of the invention cleaves one or more vesicle or plasma-membrane associated proteins essential to the specific cellular process of exocytosis, and cleavage of these proteins results in inhibition of exocytosis, typically in a non-cytotoxic manner. The cell or cells affected are not restricted to a particular type or subgroup but can include both neuronal and non-neuronal cells. The activity of clostridial neurotoxins in inhibiting exocytosis has, indeed, been observed almost universally in eukaryotic cells expressing a relevant cell surface receptor, including such diverse cells as from Aplysia (sea slug), Drosophila (fruit fly) and mammalian nerve cells, and the activity of the first domain is to be understood as including a corresponding range of cells.
The polypeptide of the invention may be obtained by expression of a recombinant nucleic acid, preferably a DNA, and is a single polypeptide, that is to say not cleaved into separate light and heavy chain domains. The polypeptide is thus available in convenient and large quantities using recombinant techniques.
In a polypeptide according to the invention, said first domain preferably comprises a clostridial toxin light chain or a fragment
Foster Keith Alan
Quinn Conrad Padraig
Shone Clifford Charles
Baskar Padma
Microbiological Research Authority
Smith Lynette R. F.
Sterne Kessler Goldstein & Fox P.L.L.C.
LandOfFree
Recombinant toxin fragments does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant toxin fragments, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant toxin fragments will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2995504