EP0859845A1 - Neuartige verbindungen von zelloberflaechenproteinen - Google Patents

Neuartige verbindungen von zelloberflaechenproteinen

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Publication number
EP0859845A1
EP0859845A1 EP96935009A EP96935009A EP0859845A1 EP 0859845 A1 EP0859845 A1 EP 0859845A1 EP 96935009 A EP96935009 A EP 96935009A EP 96935009 A EP96935009 A EP 96935009A EP 0859845 A1 EP0859845 A1 EP 0859845A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
dna
cell surface
surface protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96935009A
Other languages
English (en)
French (fr)
Inventor
John Edward Smithkline Beecham Pharm. Hodgson
Martin K.R. SmithKline Beecham Pharm. BURNHAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Ltd
Original Assignee
SmithKline Beecham Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9521148.8A external-priority patent/GB9521148D0/en
Priority claimed from GBGB9604594.3A external-priority patent/GB9604594D0/en
Application filed by SmithKline Beecham Ltd filed Critical SmithKline Beecham Ltd
Publication of EP0859845A1 publication Critical patent/EP0859845A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides and recombinant host cells transformed with the polynucleotides.
  • the LPXTG motif has been identified as characteristic of surface proteins in Gram-positive bacteria (Navarre.W.W. and Schneewind.O. [ 1994] Molecular Microbiology 14( 1) 1 15- 121); Fischetti et al. [ 1990] Mol. Microbiol 4 1603-5. Schneewind et al. [ 1993] EMBO J. 4803-481 1).
  • fibronectin binding proteins EP0294349, EP0397633, W094/ 18327
  • fibrinogen binding protein WO94/06830
  • collagen binding protein WO92/07002
  • bone sialoprotein binding protein WO94/13310
  • the binding proteins or binding fragments thereof are used as antibacterial agents to block binding of the organism to host tissue, as vaccines to raise antibodies to the organism in the host animal or as antigens to raise therapeutic antibodies which can be used to block binding of the organism to host tissue
  • novel approaches have been described which purport to follow global gene expression during infection (Chuang, S et al [ 1993] Global Regulation of Gene Expression in Escherichia colt J Bacteriol 175, 2026-2036, Mahan, M J et al [ 1993] Selection of Bacte ⁇ al Virulence Genes That Are Specifically Induced in Host Tissues SCIENCE 259, 686-688
  • a suitable oligonucleotide useful for applying this method to genes expressed in Staphylococcus aureus is
  • the present invention relates to a novel cell surface protein from S aureus WCUH 29, characterised in that it comprises the amino acid sequence given in SEQ ID NO 1, or a fragment, analogue or derivative thereof.
  • the invention also relates to a polypeptide fragment of the cell surface protein, having the amino acid sequence given in SEQ ID NO 1 , or a derivative thereof
  • polynucleotides (DNA or RNA) which encode such polypeptides
  • the invention provides a polynucleotide having the DNA sequence given in SEQ ID NO 2.
  • the present invention also provides a novel protein from Staphylococcus aureus WCUH29 obtainable by expression of a gene characterised in that it comprises the DNA sequence given SEQ ID NO 2, or a fragment, analogue or derivative thereof.
  • the invention also relates to novel oligonucleotides, including SEQ ID NOs 3 and 4, derived from the sequences SEQ ID NO 2.
  • the present invention includes va ⁇ ants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of the polypeptide characterised by the deduced ammo acid sequence of SEQ ID NO 1
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • a polypeptide of the invention for therapeutic or prophylactic purposes, for example, as an antibacterial agent or a vaccine
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunisation
  • a method for identifying compounds which bind to and inhibit an activity of the polypeptide of SEQ ID NO: 1 comprising: contacting a cell expressing on the surface thereof a binding means for the polypeptide, said binding means being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said binding means, with a compound to be screened under conditions to permit binding to the binding means; and determining whether the compound binds to and activates or inhibits the binding by detecting the presence or absence of a signal generated from the interaction of the compound with the binding means.
  • an antagonist which inhibits the activity of the polypeptide of SEQ ID NO: l .
  • a method for the treatment of an individual having need to inhibit the polypeptide of SEQ ID NO: 1 comprising: administering to the individual a therapeutically effective amount of an antagonist against the polypeptide of the invention.
  • a process for diagnosing a disease related to expression of the polypeptide of the invention comprising:determining a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 1.
  • a diagnostic process comprising: analyzing for the presence of the polypeptide of SEQ ID NO: 1 in a sample derived from a host.
  • an antibody against the polypeptide of SEQ ID NO: l is also provided.
  • an antagonist which inhibits the activity of the polypeptide of SEQ ID NO: l is also provided.
  • a method for the treatment of an individual having need to inhibit binding polypeptide of the invention comprising: administering to the individual a therapeutically effective amount of an antagonist against such polypeptide.
  • a process for diagnosing a disease related to expression of the polypeptide of the invention comprising:determining a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 1.
  • a diagnostic process comprising analyzing for the presence ot the polvpeptide of SEQ ID NO 1 in a sample denved from a host
  • inhibitors to such polypeptides useful as antibacterial agents
  • Another aspect of the invention is a pharmaceutical composition comprising the above polypeptide, polynucleotide or inhibitor of the invention and a pharmaceutically acceptable carner.
  • the invention provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the immediate physical interaction between a pathogen and mammalian host responsible for sequelae of infection
  • the invention further relates to the manufacture of a medicament for such uses
  • Figure 1 shows the polypeptide sequence of novel cell surface protein [SEQ ID NO: 1]
  • Figure 2 shows the polynucleotide sequence of novel cell surface protem [SEQ ID NO 2] deduced from the polynucleotide sequence of Figure 1 DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to a novel cell surface protein from S aureus
  • WCUH 29 characterised in that it comprises the amino acid sequence given in SEQ ID NO 1 , or a fragment, analogue or derivative thereof
  • Staphylococcus aureus WCUH 29 has been deposited at the National Collection of Industrial and Marine Bacteria Ltd (NCIMB), Aberdeen, Scotland under number NCIMB 40771 on 1 1 September 1995
  • the invention also relates to a polypeptide fragment ot the cell surface protein, having the ammo acid sequence given in SEQ ID NO 1 , or a de ⁇ vative thereof.
  • the amino acid sequence of SEQ ID NO 1 displays homology to bacte ⁇ al rodA (SWISSPROT ACCESSION RODA_ECOLI) (LPXTG motif)
  • polypept ⁇ de(s) will be used to refer to the cell surface protein, its fragments, analogues or derivatives as well as the polypeptide fragment or its derivatives.
  • the invention provides a polynucleotide encoding a cell surface protein from S. aureus WCUH 29 and characterised in that it comprises the DNA sequence given
  • the invention also relates to novel oligonucleotides, including SEQ ID NOs 3 and 4, derived from the sequences SEQ ID NO 2 which can act as PCR primers in the process herein described to determine whether or not the Staphylococcus aureus genes identified herein in whole or in part are transc ⁇ bed in infected tissue It is recognised that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • the polynucleotide having the DNA sequence given in SEQ ID NO 2 was obtained from the sequencing of a library of clones of chromosomal DNA of S.aureus WCUH 29 in E.coli. It has been demonstrated by the process herein described that it is transcribed in vivo in an established infection of S.aureus WCUH29 in a mouse model of infection.
  • a library of clones of chromosomal DNA of S.aureus WCUH 29 in E.coli or some other suitable host is probed with a radiolabelled oligonucleotide, preferably a 17mer or longer, derived from the partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using high stringency washes.
  • sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence Conveniently such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Maniatis, T Fritsch E F and Sambrook.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA
  • the DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence shown in SEQ ID NO 2 or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide
  • the present invention includes variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of the polypeptide characterised by the deduced amino acid sequence of SEQ ID NO 1
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occumng variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same polypeptide characterised by the deduced ammo acid sequence of SEQ ID NO 1 as well as variants of such polynucleotides which variants encode for a fragment, de ⁇ vative or analog of the polypeptide
  • nucleotide variants include deletion variants, substitution variants and addition or insertion va ⁇ ants
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence characterised by the DNA sequence of SEQ ID NO 2
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide
  • polynucleotide which encodes for the mature polypeptide, I e the native cell surface protein may include only the coding sequence for the mature polypeptide or the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence
  • additional coding sequence such as a leader or secretory sequence or a proprotein sequence
  • polynucleotide encoding a polypeptide' encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence
  • the present invention therefore includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell
  • a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide
  • the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein.
  • the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence)
  • leader sequence a methionine residue at the NH,-term ⁇ nus
  • this invention contemplates the use of both the methionine- containing and the methionmeless amino terminal variants of the protein of the invention.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence at either the 5' or 3' terminus of the gene which allows for purification of the polypeptide of the present invention
  • the marker sequence may be a hexa-histidine tag supplied by the pQE series of vectors (supphed commercially by Quiagen Inc ) to provide for purification of the polypeptide fused to the marker in the case of a bacterial host
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-desc ⁇ bed sequences if there is at least 50% and preferably at least 70% identity between the sequences
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove- described polynucleotides
  • stringent conditions means hybridization ill occur only if there is at least 95% and preferably at least 97% identity between the sequences
  • the polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the polypeptide characterised by the deduced amino acid sequence of SEQ ID NO 1
  • the invention also provides an isolated polynucleotide compnsing a member selected from the group consisting of a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide compnsing amino acids
  • fragment when referring to the polypeptide characterised by the deduced amino acid sequence of SEQ ID NO 1, means a polypeptide which retains essentially the same biological function or activity as such polypeptide
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide
  • the fragment, de ⁇ vative or analog of the polypeptide characterised by the deduced amino acid sequence of SEQ ID NO 1 may be (1) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (n) one in which one or more of the ammo acid residues includes a substituent group, or (in) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the poly
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the mate ⁇ al is removed from its original environment (e g , the natural environment if it is naturally occumng).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials m the natural system, is isolated
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • polypeptide of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide in a host and recovering the expressed product
  • polypeptides of the invention can be synthetically produced by conventional peptide synthesizers
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector
  • the vector may be, for example, in the form of a plasmid, a cosmid, a phage, etc
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate tor activating promoters selecting transformants or amplifying the genes
  • the culture conditions such as temperature pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan
  • Suitable expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e g , bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA
  • any other vector may be used as long as it is replicable and viable in the host
  • the appropriate DNA sequence may be
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis
  • promoter an appropriate expression control sequence(s)
  • LTR or SV40 promoter the E coli lac or trp
  • phage lambda PL promoter the phage lambda PL promoter
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator
  • the vector may also include appropriate sequences for amplifying expression
  • the expression vectors preferably contain one or more selectable marker genes to pro ide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic ceil culture, or such as tetracycline or ampicillin resistance m E coli
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction
  • the coding sequence may or may not contain a signal peptide or leader sequence
  • the polypeptides of the present invention can be expressed using, for example, the E coli tac promoter or the protein A gene (spa) promoter and signal sequence Leader sequences can be removed by the bacterial host in post-translational processing.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • CAT chloramphenicol transferase
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and t ⁇ .
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metalloth ⁇ one ⁇ n-I.
  • regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • Modification of the coding sequences may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate onentation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E coli and S cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired character ⁇ istics, e g , stabilization or simplified purification of expressed recombinant product
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence may be employed to transform an appropriate host to permit the host to express the protein
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse onentation
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pET-3 vectors (Stratagene), pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescnpt, ps ⁇ X174, pbluesc ⁇ pt SK, pbsks, pNH8A, pNHl ⁇ a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) Eukaryotic- pBlueBacIII (Invitrogen), pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia)
  • any other plasmid or vector may be used as long as they are replicable and viable in the host
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage 1 (E coli), pBR322 (E coli), pACYC177 (£ coli), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFR l (gram-negative bacteria), pME290 (non-£ coli gram-negative bacteria), pHV14 (£ coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces). YIp5 (Saccharomyces), a baculovirus insect cell system.
  • Polypeptides can be expressed in host cells under the control of appropriate promoters Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • the polypeptide of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed The polypeptide is then isolated from the host cells and purified If the expression system secretes the polypeptide into growth media, the polypeptide can be purified directly from the media. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction.
  • polypeptide is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product.
  • Polypeptide expressed in bacte ⁇ al hosts such as £. coli may require isolation from inclusion bodies and refolding.
  • the mature protein has a very hydroophobic region (normally at the C-terminus) which leads to an insoluble product of overexpression, it may be desirable to express a truncated protein in which the hydrophobic region has been deleted. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be glycosylated or may be non- glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • a “repiicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • double-stranded DNA molecule refers to the polymeric form of deoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in a double- stranded helix, both relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e g , restriction fragments), viruses, plasmids, and chromosomes
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i e , the strand having the sequence homologous to the mRNA)
  • a DNA 'coding sequence of or a "nucleotide sequence encoding a particular protein is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase m a cell and initiating transcription of a downstream (3' direction) coding sequence
  • the promoter sequence is bound at the 3' terminus by a translation start codon (e g , ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transc ⁇ ption at levels detectable above background
  • a transcription initiation site (conveniently defined by mapping with nuclease S l), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the - 10 and -35 consensus sequences
  • control sequences refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (1 e , the transcription and translation) of a coding sequence in a host cell
  • a control sequence "directs the expression ' of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence
  • a "host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence
  • a cell has been "transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
  • a polypeptide of the invention for therapeutic or prophylactic pu ⁇ oses for example, as an antibacterial agent or a vaccine.
  • a polynucleotide of the invention for therapeutic or prophylactic pu ⁇ oses in particular genetic immunisation.
  • Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds.
  • the encoded protein upon expression can be used as a target for the screening of antibacterial drugs.
  • the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
  • inhibitors to such polypeptides useful as antibacterial agents
  • antibodies against such polypeptides are provided.
  • Another aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising the above polypeptide, polynucleotide or inhibitor of the invention and a pharmaceutically acceptable carrier
  • the invention provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the immediate physical interaction between a pathogen and mammalian host responsible for sequelae of infection
  • the molecules of the invention may be used
  • the invention further relates to the manufacture of a medicament for such uses
  • the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bactena to damaged tissue
  • tissue damage include wounds in skin or connective tissue caused e g by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina
  • the polypeptides or cells expressing them can be used as an immunogen to produce antibodies thereto These antibodies can be. for example, polyclonal or monoclonal antibodies
  • the term antibodies also includes chimeric, single chain. and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Various procedures known in the art may be used for the production of such antibodies and fragments
  • Antibodies generated against the polypeptides of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman The antibody so obtained will then bind the polypeptides itself In this manner, e en a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide
  • Polypeptide derivatives include antigenically or immunologically equivalent derivatives which form a particular aspect of this invention
  • antigenically equivalent derivative encompasses a polypeptide or its equivalent which will be specifically recognised by certain antibodies which, when raised to the protein or polypeptide according to the present invention, interfere with the immediate physical interaction between pathogen and mammalian host
  • immunologically equivalent derivative' encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host
  • polypeptides in which one or more of the amino acid residues are modified may be used.
  • Such peptides may, for example, be prepared by substitution, addition, or rearrangement of amino acids or by chemical modification thereof All such substitutions and modifications are generally well known to those skilled in the art of peptide chemistry
  • N-terminal fragment of the protein relative to the LPXTG motif, I e not in the cytoplasm, is most relevant for the preparation of antibodies to the regions of proteins (see - Binding and activation of plasminogen at the surface of Staphylococus aureus Kuusela. P and Saksela. O [ 1990] Eur J Biochem 193 759- 65)
  • the polypeptide such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken
  • the fusion protein may provide stability to the polypeptide
  • the antigen may be associated, for example by conjugation , with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH)
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256 495-497), the t ⁇ oma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hyb ⁇ doma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96).
  • hybridomas are screened to select a cell line with high binding affinity and favorable cross reaction with other staphylococcal species using one or more of the original polypeptide and/or the fusion protein The selected cell line is cultured to obtain the desired Mab
  • Hybridoma cell lines secreting the monoclonal antibody are another aspect of this invention
  • phage display technology could be utilised to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- enes of lymphocytes from humans screened for possessing anti-Fbp or from naive libraries (McCafferty, J. et al , ( 1990), Nature 348. 552-554; Marks, J. et al. ⁇ 1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et ai., ( 1991 ) Nature 352, 624-628).
  • the antibody should be screened again for high affinity to the polypeptide and/or fusion protein.
  • a fragment of the final antibody may be prepared.
  • the antibody may be either intact antibody of M r approx 150,000 or a de ⁇ vative of tt. for example a Fab fragment or a Fv fragment as described in Skerra, A and Pluckthun, A ( 1988) Science 240 1038- 1040. If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies.
  • the antibody of the invention may be prepared by conventional means for example by established monoclonal antibody technology (Kohler, G. and Milstein, C. (1975) , Nature, 256, 495-497) or using recombinant means e.g.
  • the antibody is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system such as described above for the expression of polypeptides of the invention.
  • the choice of vector for the expression system will be determined in part by the host, which may be a prokaryotic cell, such as £. coli (preferably strain B) or Streptomyces sp. or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell.
  • the host may also be a transgenic animal or a transgenic plant [for example as described in Hiatt.A et -...,( 1989) Nature 34, 76-78].
  • Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived from, for example, baculoviruses and vaccinia.
  • the Fab fragment may also be prepared from its parent monoclonal antibody by enzyme treatment, for example using papain to cleave the Fab portion from the Fc portion.
  • the antibody or derivative thereof is modified to make it less immunogenic in the patient.
  • the antibody may most preferably be 'humanised' ; where the complimentarity determining reg ⁇ on(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al ( 1986), Nature 321, 522-525 or Tempest et al.,( 1991 ) Biotechnology 9, 266-273.
  • the modification need not be restricted to one of 'humanisation' ; other primate sequences (for example Newman, R. et al .1992, Biotechnology, 10, 1455- 1460) may also be used.
  • the humanised monoclonal antibody, or its fragment having binding activity form a particular aspect of this invention.
  • This invention provides a method of screening drugs to identify those which interfere with the interaction of the cell surface protein or active fragment to mammalian cells, the method comprising incubating a mammalian cell or membrane preparation with labeled polypeptide in the presence of the drug and measuring the ability of the drug to block this interaction.
  • a polynucleotide of the invention in genetic immunisation will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff er al.. Hum Mol Genet 1992, 1 :363, Mantho ⁇ e et al., Hum. Gene Ther.
  • Suitable promoters for muscle transfection include CMV, RSV, SRa, actin, MCK. alpha globin, adenovirus and dihydrofolate reductase.
  • the active agent may be administered to a patient as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic
  • compositions may be formulated for topical application for example in the form of ointments, creams lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams
  • topical formulations may also contain compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions
  • Such carriers may constitute from about 1 % to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
  • In-dwelling dev ices include surgical implants, prosthetic devices and catheters, l e , devices that are introduced to the body of a patient and remain in position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters continuous ambulatory peritoneal dialysis (CAPD) catheters, etc
  • composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in ⁇ dwelling device Treatment may be continued after surgery during the in-body time of the device
  • composition could also be used to broaden penoperative cover for any surgical technique to prevent staphylococcal wound infections
  • Many orthopaedic surgeons consider that patients with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteraemia Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.
  • composition of the invention may be used to bathe an indwelling device immediately before insertion.
  • the active agent will preferably be present at a concentration of l ⁇ g/ml to lOmg/mi for bathing of wounds or indwelling devices
  • a vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response
  • a suitable unit dose for vaccination is 0 5-5ug/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks.
  • the antibodies described above may also be used as diagnostic reagents to detect the presence of bacteria containing the cell surface protein.
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordina ⁇ lv skilled artisan
  • Digestion of DNA refers to catalytic cleavage of the DNA with a rest ⁇ ction enzyme that acts only at certain sequences in the DNA
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan For analytical pu ⁇ oses.
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D et al , Nucleic Acids Res., 8 4057 (1980)
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another o gonucleotide without adding a phosphate with an ATP in the presence of a kinase A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al , Id., p 146) Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA gase ("ligase") per 0 5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated
  • ligase T4 DNA gase
  • the polynucleotide having the DNA sequence given in SEQ ID NO 2 was obtained from a library of clones of chromosomal DNA of S.aureus WCUH 29 in E.coli. In some cases the sequencing data from two or more clones containing overlapping S.aureus WCUH 29 DNA was used to construct the contiguous DNA sequence in SEQ ID No 2. Libraries may be prepared by routine methods, for example: Methods ] and 2
  • Total cellular DNA is isolated from Staphylococcus aureus strain WCUH29 (NCIMB 40771) according to standard procedures and size-fractionated by either of two methods.
  • Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures. DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli infected with the packaged library. The library is amplified by standard procedures. Method 2.
  • Total cellular DNA is partially hydrolsed with a combination of four restriction enzymes (Rsal, Pall, Alul and Bsh 12351) and size-fractionated according to standard procedures.
  • EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library.
  • the library is amplified by standard procedures.
  • Necrotic fatty tissue from a four day groin infection of Staphylococcus aureus WCUH29 in the mouse is efficiently disrupted and processed in the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bacterial RNA
  • the optimal conditions for disruption and processing to give stable preparations and high yields of bacterial RNA are followed by the use of hybridisation to a radiolabelled o gonucleotide specific to Staphylococcus aureus 16S RNA on Northern blots
  • the RNAase free, DNAase free, DNA and protein free preparations of RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) using unique primer pairs designed from the sequence of each gene of Staphylococcus aureus WCUH29
  • mice should be monitored regularly during the first 24 hours after infection, then daily until termination of study Animals with signs of systemic infection, I e lethargy, ruffled appearance, isolation from group, should be monitored closely and if signs progress to mo ⁇ bundancy, the animal should be culled immediately
  • the abscess/muscle sheet and other infected tissue may require cutting in sections, prior to flash-freezing in liquid nitrogen, thereby allowing easier storage in plastic collecting vials.
  • tissue samples (each approx 0.5-0 7g) in 2ml screw-cap tubes are removed from -80°C.storage into a dry ice ethanol bath
  • the samples are disrupted individually whilst the remaining samples are kept cold in the dry ice ethanol bath.
  • TRIzol Reagent Gibco BRL, Life Technologies
  • the lid is replaced taking care not to get any beads into the screw thread so as to ensure a good seal and eliminate aerosol generation.
  • the sample is then homogenised in a Mini-BeadBeater Type BX-4 (Biospec Products) Necrotic fatty tissue is treated for 100 seconds at 5000 ⁇ m in order to achieve bacterial lysis In vivo grown bacteria require longer treatment than in yttro grown 5 aureus WCUH29 which are disrupted bv a 30 -econd bead-beat
  • RNA extraction is then continued according to the method given by the manufacturers of TRIzol Reagent l e -
  • the aqueous phase approx 0 6 ml, is transferred to a stenle eppendorf tube and 0 5 ml of isopropanol is added
  • the supematant is removed and discarded then the RNA pellet is washed with 1 ml 75% ethanol
  • a brief vortex is used to mix the sample before centrifuging at 7,500 x g, 4°C for 5 minutes
  • the ethanol is removed and the RNA pellet dried under vacuum for no more than 5 minutes Samples are then resuspended by repeated pipetting in 100 microlitres of DEPC treated water,
  • RNA preparations are stored at -80 °C for up to one month
  • the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 °C
  • RNA isolation Quality of the RNA isolated is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA yields
  • 2 2M formaldehyde gels are run and vacuum blotted to Hybond-N
  • DNAase was inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the manufacturers protocol DNAase treated RNA was resuspended in 73 microlitres of DEPC treated water with the addition of Rnasin as described in Method 1
  • RNA samples derived from infected tissue 10 microhtre samples of DNAase treated RNA are reverse transcribed usmg.a SuperSc ⁇ pt Preamplification System for First Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) according to the manufacturers instructions. 1 nanogram of random hexamers is used to prime each reaction. Controls without the addition of SuperScnptll reverse transcriptase are also run. Both +/-RT samples are treated with RNaseH before proceeding to the PCR reaction
  • PCR reactions are set up on ice in 0 2ml tubes by adding the following components- 45 microlitres PCR SUPERMIX (Gibco BRL, Life Technologies)
  • each primer at lOmM initial concentration 2 microlitres cDNA PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 as follows
  • RT/PCR controls may include +/- reverse transcriptase reactions, 16s rRNA primers or DNA specific pnmer pairs designed to produce PCR products from non- transcribed S aureus WCUH29 genomic sequences
  • Primer pairs which fail to give the predicted sized product in either DNA PCR or RT PCR are PCR failures and as such are uninformative Of those which give the correct size product with DNA PCR two classes are distinguished in RT PCR.
  • SEQ ID NO 2 Genes which are transcribed in vivo reproducibly give the correct size product in RT PCR and show a stronger signal in the +RT samples than the signal (if at all present) in -RT controls
  • the following nucleotide sequence (SEQ ID NO 2) was identified in the above test as transcribed in vivo Deduced amino acid sequence is given as SEQ ID NO 1
  • a pair of PCR primers useful to identify the gene are for example, 5'- ctatacatat agtagtgg-3'[SEQ ID NO 3] and 5'-ttacttttgg atggtata-3' [SEQ ID NO 4]
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 610-270-4478 I B ) TELEFAX 610-270-5090 (C) TELEX
  • Lys Ala lie Gly Ser Gly Gin Leu Leu Gly Lys Gly Tyr Asn Xaa Gly
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • AAATTGGAGA TCAATTTACC AAAATCTTTA TCGTTGGTTT CGTCACTTTA CTTGTGTTCC 660
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE ORIGINAL SOURCE:
  • MOLECULE TYPE Genomic DNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:

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AU5740194A (en) * 1992-12-07 1994-07-04 Magnus Hook A bone sialoprotein binding protein as well as its preparation
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