Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-11-03
2002-11-12
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S005000, C435S006120, C435S173300, C435S235100, C536S023100, C536S023400, C536S023720
Reexamination Certificate
active
06479280
ABSTRACT:
The present invention relates to a recombinant bacteriophage, pseudovirion or phagemid that is capable of entering bacteria by specific binding to an artificial receptor. Said receptor does not comprise at its active binding site elements such as proteins or peptides that are derived from the natural receptor used in the specific initial bacteriophage-bacterium interaction.
BACKGROUND OF THE INVENTION
Bacteriophages, like bacteria, are very common in all natural environments. Bacteriophages (phages) are intracellular parasites. Bacteria and their phages have a common evolutionary history and phages may have adapted to their host species by multiple mechanisms. The phage genome may consist of double-stranded DNA, single-stranded DNA, double-stranded RNA or single-stranded RNA. Bacteriophages exist in several morphologies and can be spherical, cubic, filamentous, pleomorphic or tailed. Based on their life cycle, bacteriophages can be divided into three groups: the virulent phages capable of only lytic propagation (called lytic phages), the so-called temperate phages capable of either lytic propagation or lysogenic phase and the non-lysing phages where the mature phage is continuously extruded. The virulent life cycle of wild type phages consists of infection of the host cell, i.e. attachment to a specific receptor in the bacterial cell wall, followed by entering of the phage genome in the cell, replication of the phage genome, production of the phage structural components, phage assembly and release of the progeny phages after lysis of the host cell. In the lysogenic life cycle, the phage genome exists as a prophage resulting in coexistence of phage and host cell without lysis. Usually, this is achieved by integration of the phage genome into the bacterial chromosome. The life cycle of the non-lysing phages, like e.g. Bacteriophage M13, is similar to that of the lytic phages, but the infection is not followed by lysis. Bacteriophages have been extensively used in biotechnology. Phage genes or complete phages may be used to obtain lysis and/or killing of bacteria.
U.S. Pat. No. 4,637,980 describes the use of an
E. coli
strain containing defective temperature sensitive lambda lysogens as a method for cell disruption. Smith and coworkers (Smith et al., 1987
, J. Gen Microbiol
. 133; 1111-1126) describe the use of bacteriophages to treat diarrhea in calves, caused by seven different bovine enteropathogenic strains of
E. coli
WO95/27043 describes a method to treat infectious diseases caused by several bacterial genera, such as Mycobacterium Staphylococcus, Vibrio, Enterobacter, Enterococcus, Eschericia, Haemophilus, Neisseria, Pseudomonas, Shigella, Serratia, Salmonella and Stretococcus, comprising the administration of bacteriophages with delayed inactivation by the animal host defence system. WO98/51318 describes a diagnostic kit and a pharmaceutical composition, comprising bacteriophages to diagnose and to treat bacterial diseases caused by bacteria, such as Listeria, Klebsielia, Pneumococcus, Moraella, Legionelle, Edwardsiella, Yersinia, Proteus, Heliobacter, Salmonella, Chlamrydia, Aeromonas and Renibacterium.
Another application of bacteriophages is the in vitro selection of proteins displayed on the tip of filamentous phages on immobilised target (=biopanning), which is a powerful technique for the isolation of interacting protein-ligand pairs from large libraries, such as antibody libraries (for a recent review: Rodi and Makowski, 1999
, Curr. Opin. Biotechn
., 10: 87-93). However, for optimal in vitro biopanning, a purified target protein is needed. Moreover, high quality of the library is a prerequisite for success. Enrichment for selfligated vector, phages carrying incomplete sequences, incorrect reading frames, deletions and amber stop codons are very often observed (Beekwilder et al, 1999
, Gene
, 228, 23-31 and de Bruin et al, 1999
, Nature Biotechnology
, 17: 397-399). In the search to avoid the problems encountered with panning using imperfect libraries, several alternative techniques, both bacteriophage based and non bacteriophage based, have been developed. Non bacteriophage based techniques are, amongst others ribosome display (Dall'Acqua and Carter, 1998
, Curr. Opin. Struct. Biol
., 8: 443-450) and the yeast two-hybrid system (Drees, 1999
, Curr. Opin. Chem. Biol
., 3: 64-70). Bacteriophage based techniques comprise display on phage lambda, SIP (Spada and Pluckthun, 1997
, Biol. Chem
., 378: 445-456; EP0614989) and CLAP (Malmborg et al, 1997
, J. Mol. Biol
., 273: 544-551; WO9710330). SIP and CLAP are in vivo selection techniques and have the advantage that the F
+
E.coli
host cells can only be infected by bacteriophages or pseudovirions when a matched pair is formed. Both systems are based on the fact that pilin on the F-pili of
E.coli
cells serve as the natural receptor for binding of the D2-domain of pill from the phage (Deng et al., 1999
, Virology
, 253:271-277). This results in retraction of the pilus, so that an interaction between the D1 domain of pill with the TOL protein complex located in the
E.coli
cell membrane leads to the infection (Deng et al, 1999
, Virology
, 253: 271-277). SIP has the disadvantage that it only works for high affinities of the binding pairs and that each target needs to be cloned, expressed and purified as a fusion with the D2 domain of pill. Therefore, with SIP, normally only one target can be screened at the time. For CLAP only small peptides (15-18 amino acids) can be expressed on the F-pilus, hence, this technique can only be used for small linear epitopes. An additional disadvantage is the need for modified M13 to avoid natural infection of host cells. Therefore, removal of the D2 domain of pill is essential. This results in a truncated form of M13 and concomitant difficulties to obtain good titres.
It is known that bacteriophages use specific receptors on the host cell wall as a way to recognise the host cell and to start the infection process. In all the applications cited above, the propagation of phages, pseudovirions or phagemids is dependent on the use of the natural phage receptor, or part of it, on the host cell wall. For M13, mainly used in these systems, the natural receptor is pilin (Malmborg et al., 1997
, J. Mol. Biol
. 273: 544-551). Other examples of natural receptors are lamB for bacteriophage lambda (Werts et al, 1994
, J. Bacteriol
. 176: 941-947), the outer membrane protein OmpA for bacteriophages K3, Ox2 and M1 (Montag et al, 1987
, J. Mol. Biol
., 196: 165-174), the outer membrane proteins OmpF and Ttr for bacteriophage T2 (Montag et al, 1987
, J. Mol. Biol
., 196, 165-174), the outer membrane protein OmpC for the T4 phage family (T4, Tula, Tulb) (Montag et al.,1990
, J. Mol. Biol
., 216: 327-334). The T4 bacteriophage family is using a C-terminal region of protein 37 as natural ligand (Montag et al., 1990
, J. Mol. Biol
., 216: 327-334), bacteriophages T2, K3, Ox2 and M1 are using protein 38 as natural ligand (Montag et al, 1987
, J. Mol. Biol
., 196, 165-174) whereas phage lambda is using the C-terminal portion of the lambda tail fibre protein as natural ligand (Wang et al., 1998
, Res. Microbiol
, 149: 611-624). Bacteriophage—receptor independent phage binding to mammalian cells expressing the growth factor receptor ErbB2 followed by receptor mediated endocytosis was also described: Marks and collaborators (Poul and Marks, 1999
, J. Mol Biol
., 288: 203-211 and Becceril and Marks, 1999
, Biochem. Biophys. Res. Commun
., 255: 386-393) successfully isolated phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor mediated endocytosis by selection of a phage antibody library on breast tumour cells and recovery of infectious phage from within the cell. However, the phage could not propagate in the mammalian cell, and the detection of the cells carrying bacteriophage could only be realised in an indirect way, by expression green fluorescent protein as a reporter gene.
SUMMARY OF THE INVENTION
One aspect of the invention is a ge
Muyldermans Serge
Silence Karen
Steyaert Jan
Torreele Els
Fish & RIchardson, P.C., P.A.
Vlaams Interuniversitair Institutuut voor Biotechnologie VZW
Winkler Ulrike
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