Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2000-06-23
2002-12-24
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093600, C435S235100, C435S320100
Reexamination Certificate
active
06497874
ABSTRACT:
TECHNICAL FIELD
This invention relates to bacteriophages useful for the treatment of bacterial infections, especially mucosal bacterial infections such as
Helicobacter pylori
infections.
BACKGROUND ART
Bacteriophages and Antibiotic Resistance
Resistance to antibiotics is a global problem of increasing medical and economical importance. There is thus a great need for alternative methods to eradicate bacteria which will circumvent the problem of such resistance.
A bacteriophage, or phage, is a virus which specifically infects bacteria. Phages bind to their host bacterium and transfer genes encoding various phage proteins. They utilize the protein-synthesizing machinery, amino acids etc., and the energy provided by the host bacterium for their replication (Maloy et al. (eds.): Microbial genetics. Jones and Bartlett Publishers, 1994).
Most phages lyse or by other mechanisms destroy specific strains of bacteria. The present invention stems from the realisation that genetic modification of phages, in particular filamentous bacteriophages, offers a means for designing new bacterium-specific phages capable of eradicating certain bacteria, e.g.
Helicobacter pylori
, and having the potential to overcome problems related to antibiotic resistance.
Filamentous Phages
E. coli
cells bearing hair-like F-pili are hosts for filamentous phages such as M13, fd and f1. These Ff (F pili, filamentous) phages are nearly identical in sequence and behaviour (Rashed & Oberer (1986) Microbiological reviews 50, 401-427; Kornberg & Baker, in: DNA Replication, p. 557-570, W.H. Freeman and Co., New York 1992). Ff phages alone among the bacterial viruses do not produce a lytic infection, but rather induce a state in which the infected host cells produce and secrete phage particles without undergoing lysis.
The single-stranded genome of phage M13 encodes 10 different proteins. The DNA is enclosed in a protein coat comprised of approximately 2700 copies of the gene 8 protein (g8p). A viable M13 phage also expresses five copies of the 43 kDa gene 3 protein (g3p) on its tip, which protein is responsible for adsorption to
E. coli
pili. The gene 3 protein is anchored to the virus coat via the C-terminal part of the polypeptide chain, whereas the N-terminal globular domain is exposed and mediates the attachment of the phage to the tip of a host F pilus. By electron microscopy, the adsorption complex appears as a “knob-on-stem” structure at one end of the phage. During infection, the leader sequences of g3p and g8p direct the transport of these proteins into the inner membrane of the bacterial cell.
The Ff phages have gained popularity as cloning vectors because they have no physical constraints limiting the length of DNA that can be packaged and because they allow the easy purification of single-stranded DNA. A phagemid is a vector which carries both the M13 (single-stranded) and plasmid (double-stranded) origins of replication. Phagemids can be grown as plasmids or packaged as recombinant M13 phage with the aid of a helper phage such as M13K07 (Veira & Messing (1987) Methods in Enzymol. 153, 3-11).
Recombinant Antibody Production
Antibody molecules contain discrete fragments which can be isolated by protease digestion or produced by recombinant techniques. One such fragment is the Fv (fragment variable) which is composed only of the V
L
and V
H
regions of the antibody. In U.S. Pat. No. 4,946,778 a recombinant version of the Fv fragment, designated single-chain Fv (ScFv), is described, where the two variable regions are artificially joined with a neutral linker and expressed as a single polypeptide chain.
A technology for recombinant antibody production has been developed by McCafferty and coworkers (McCafferty (1990) Nature 348, 552-554; Winter & Milstein (1991) Nature 349, 293). This approach relies on a phage-display system in which V
H
(variable heavy) and V
L
(variable light) genes are cloned into a phage vector whereafter fragments of antibodies are expressed as fusion proteins displayed on the phage surface. With this approach, antibodies of defined specificity and affinity can be selected from a population. It has been suggested that antibodies isolated and manufactured in prokaryotic systems should be called “coliclonal” antibodies (Chiswell & McCafferty (1992) Trends in Biotechnology 10, 80-84).
The commercially available phagemid pCANTAB5 is designed such that antibody variable region genes can be cloned between the leader sequence and the main body of the M13 gene 3. The g3p leader sequence directs transport of the resulting fusion protein to the inner membrane and/or periplasm of
E. coli
where the main g3p domain attaches the fusion protein to the tip of the assembling phage. The expression of the antibody-g3p gene is controlled by an inducible lac promoter on the phagemid.
Helicobacter pylori Infection
It is widely accepted that the bacterium
Helicobacter pylori
is the main cause of gastric and duodenal ulcer, responsible for 84% and 95%, respectively, of reported cases (Kuipers, E. L. et al. (1995) Aliment. Pharmacol. Ther. 9 (suppl.2), 59-69).
H. pylori
colonises the wall of the stomach, protected from the acid environment by a layer of mucus which lines the stomach wall, and by a metabolic process which enables the organism to secrete ammonia to neutralise acid.
Conventional antibiotic treatment appears to have little effect on
H. pylori.
This is probably due to: (i) poor access of the antimicrobial agent to the organism which is not directly exposed to the blood circulation; and (ii) rapid passage of many oral antibiotics through the stomach, or degradation of such antibiotics in the acid conditions of the stomach.
PURPOSE OF THE INVENTION
The purpose of the present invention is to provide new forms of treatment for eradication of bacteria, especially eradication of bacteria responsible for mucosal bacterial infections such as
Helicobacter pylori
. In particular, it provides filamentous bacteriophages genetically modified to have binding specificity towards another bacterial host for use in therapy.
Methods of treatment of mucosal bacterial infections based on recombinant phages are believed to be superior to conventional antibiotic treatment for several reasons, e.g. the following:
it will be possible to eradicate bacteria resistant to conventional antibiotics;
the high specificity of the recombinant phage towards specific bacterial species;
propagation of the phage is self-limiting;
in the case of
Helicobacter pylori
infections, the motility of
Helicobacter pylori
could help to distribute the phage to all parts of the gastric mucosa.
DISCLOSURE OF THE INVENTION
In the present description and examples, the terms “standard protocols” and “standard procedures” are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
In a first aspect, this invention provides a modified bacteriophage for use in the treatment or prophylaxis of a bacterial infection, which bacteriophage presents at its surface a recombinant protein comprising
(i) a first component derived from a bacteriophage surface protein; and
(ii) a second component comprising variable region sequences of an antibody to provide a bacterial antigen binding site, said second component rendering said bacteriophage capable of binding to and thereby inhibiting growth of bacterial cells involved in the etiology of said infection.
The said modified bacteriophage can e.g. be a modified filamentous phage, such as a modified M13 phage.
The said bacterial infection can e.g. be a mucosal bacterial infection such as
Helicobacter pylori
infection. However, the present invention is not restricted to phages capable of incapacitating
Helicobacter pylori
cells, but rather comprises phages with altered properties which can be used for incapacitating a wide range of bacteria. It will be understood that a phage according to the invention which is
Chen Shin-Lin
Priebe Scott D.
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