Treatment of intracellular infection

Details Treatment of intracellular infection Treatment of intracellular infection

2001-11-20

2003-12-09

Housel, James (Department: 1648)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Virus or bacteriophage

C424S093400, C424S093210, C424S093700, C424S205100, C424S168100, C424S248100, C435S320100, C435S002000, C435S005000, C435S007220, C435S069100, C435S091310, C435S091320

Reexamination Certificate

active

06660264

ABSTRACT:

The present application is a 371 of PCT/GB00/01350 filed on Apr. 10, 2000, and published in English on Oct. 19, 2000, the disclosure of which is incorporated herein by reference in its entirety.
The present invention relates to an agent for causing lysis of a microorganism residing within a cell, to a method for preparing said agent, to compositions comprising said agent, and to the use of said agent. In particular, the agent of the present invention is suitable for the treatment of an intracellular infection by a microorganism.
Many microorganisms are capable of forming intracellular infections. These include: infections caused by species of Salmonella, Yersinia, Shigella, Campylobacter and Chlamydia. Live Salmonella and Yersinia can survive within the cells of mucosa of the gastrointestinal tract and fibroblasts, provide antigenic material continuously into the blood circulation and stimulate chronic inflammation and lead to arthritis; infections caused by the survival of
Legionella pneumophila
within alveolar macrophages and epithelial cells; infections caused by the survival of
Listeria monocytogenes
within cell cytosol; infections caused by an intracellular protozoan
Toxoplasma gondii
; and infections caused by the intracellular survival of Bordetella species (macrophages),
Staphylococcus aureus
(epithelial cells) and Group B streptococci (macrophages). Some of these infections are exclusively intracellular, others contain both intracellular and extracellular components. However, it is the intracellular survival cycle of bacterial infection which is suspected as a main supportive factor for disease progression.
Generally, these microorganisms do not circulate freely in the body, for example, in the bloodstream. Accordingly, intracellular microorganisms are often not amenable to drug treatment regimes. Where drugs are available, this problem has been exacerbated by the development of multiple drug resistant. microorganisms. For similar reasons, vaccine therapies are not effective against such intracellular microorganisms. Also, increased systemic concentration of antibiotics to improve bioavailability within cells may result in severe side effects.
As an example of an intracellular disease-causing microorganism, reference is made to
Mycobacteria tuberculosis
. This bacterium is responsible for causing the disease tuberculosis which is responsible for more than three million deaths a year world-wide.
M. tuberculosis
infects macrophage cells within the body. Soon after macrophage infection, most
M. tuberculosis
bacteria enter, persist and replicate within cellular phagosome vesicles, where the bacteria are sequestered from host defenses and extracellular factors.
A number of drug therapy regimes have been proposed for combating
M. tuberculosis
infections, with the best results to date having been achieved with the drug isoniazid. As an alternative, bacteriophage therapy has been suggested in the early 1980's based on results of the treatment of experimental tuberculosis with rabbits infected with
M. bovis
BSG and
M. microti
, and guinea pigs infected with the human pathogen
M. tuberculosis
strain H37Rv. However, the highest therapeutic effect obtained with bacteriophage was not higher than that achieved with isoniazid.
Phage, in particular bacteriophage, have been known for many years and have been employed as delivery vehicles in conventional treatment regiments for alleviating conditions associated with defective or aberrant cells.
For example, WO 98/05344 teaches the use of bacteriophage for delivering an exogenous gene, such as a therapeutic polynucleotide, to a mammalian cell. Targeting of the bacteriophage to a pre-selected cell is achieved by use of a targeting moiety linked to the bacteriophage, said targeting moiety effecting binding and initiating internalisation of the bacteriophage into the pre-selected cell. Once delivered to the pre-selected mammalian target cell, the exogenous genetic material can be transcribed and translated, thereby increasing the concentration of the therapeutic molecule encoded by the therapeutic polynucleotide in the target cell.
Aberrant cell treatment regiments such as those disclosed in WO 98/05344 are conventionally known as gene therapy methods. Such regiments, however, do not address the problem and/or persistence of intracellular infections by microorganisms.
WO 97/29185 teaches the preparation of recombinant phages, and the use thereof in the treatment or prophylaxis of bacterial infections. According to WO 97/29185, an anti-bacterial antibody is presented from an exposed surface of a bacteriophage, thereby rendering the bacteriophage capable of binding to and inhibiting growth of the targeted bacterial cell. WO 97/29185 does not, however, teach how to combat intracellular infections by microorganisms.
Additional background art relating to modified bacteriophage is provided in:
WO 99/10485, which teaches a bacteriophage system for identifying ligands susceptible to cell internalisation. Such ligands may provide suitable targets for bacteriophage gene delivery vehicles; and
WO 94/24959, which teaches a method of detecting compounds by utilising a chemically modified lambdoid bacteriophage. In more detail, a bacteriophage is modified to form a phage-target molecule complex, said complex being non-infective. Upon challenge with a molecule of interest, the target molecule is cleaved and the bacteriophage becomes infective. Thus, the presence of a molecule of interest may be detected by the presence of infective bacteriophage.
Neither of WO 99/10485 or WO 94/24959 addresses problems associated with microbial infections, least of all the problem of combating intracellular microbial infections.
There is therefore a need for a system for combating intracellular infections by microorganisms. In particular, there is a need for a system for combating intracellular infections by mycobacteria.
The above problem is alleviated by the present invention which, according to a first aspect, provides an agent for causing lysis of a microorganism residing within a target cell, comprising a targeting moiety capable of binding to a target cell and a phage associated with the targeting moiety, wherein following binding of the targeting moiety of the cell the phage enters the target cell and effects lysis of the microorganism residing within the target cell.
The term “targeting moiety” means any structure which is capable of binding to the cell of interest. Examples include an antibody or fragment thereof, a receptor capable of binding to a ligand on the cell of interest, and a ligand capable of binding to a receptor on the cell of interest. Preferably, the targeting moiety is a ligand for a cell-surface receptor. Good results have been achieved in a specific embodiment of the invention using a transferrin molecule as targeting moiety. The targeting moiety need not demonstrate 100% specificity for the cell of interest, though naturally a degree of specificity is desirable for a highly efficient system. The targeting moiety may be capable of binding and internalisation, in which case the phage and targeting moiety may be delivered as a complex (ie. associated) into the target cell. Identification of potential targeting moieties susceptible to internalisation may be achieved by, for example, conventional methods such as those disclosed in WO 99/10485, or on a trial-and-error basis. Alternatively, the targeting moiety may be capable of binding but not internalisation, in which case the phage alone may be delivered into the target cell.
The term “binding” includes any interaction between the targeting moiety and the cell of interest which permits the phage to be delivered into the cell. This delivery process is one in which the whole phage enters the cell of interest. The targeting moiety may become separated from the phage during this delivery process. Without being bound by any theory, it is believed that binding involves the formation of a complex between the agent and a receptor present on the target cell. It is believed that for

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