EP1039927A1 - Ugc (upstream-gen von cdsa) - Google Patents

Ugc (upstream-gen von cdsa)

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Publication number
EP1039927A1
EP1039927A1 EP98961915A EP98961915A EP1039927A1 EP 1039927 A1 EP1039927 A1 EP 1039927A1 EP 98961915 A EP98961915 A EP 98961915A EP 98961915 A EP98961915 A EP 98961915A EP 1039927 A1 EP1039927 A1 EP 1039927A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
polynucleotide
ugc
compnsmg
seq
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Withdrawn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
EP98961915A
Other languages
English (en)
French (fr)
Inventor
Jianzhong Huang
Damien Mcdevitt
Christopher M. Traini
Xinhe Jiang
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 Corp
Original Assignee
SmithKline Beecham Corp
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
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of EP1039927A1 publication Critical patent/EP1039927A1/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Withdrawn legal-status Critical Current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
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses
  • the invention relates to novel polynucleotides and polypeptides of the ugc family, hereinafter referred to as "ugc"
  • polynucleotides that encode ugc polypeptides particularly polynucleotides that encode the polypeptide herein designated ugc
  • the polynucleotide compnses a region encoding ugc polypeptides compnsing a sequence set out in Table 1 [SEQ ID NO 1] which includes a full length gene, or a variant thereof
  • a further aspect of the invention there are provided isolated nucleic acid molecules encoding ugc, particularly Pseudomonas aeruginosa ugc. including mRNAs, cDNAs, genomic DNAs Further embodiments of the invention include biologicalh diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same
  • a polynucleotide of the mvention for therapeutic or prophylactic purposes, in particular genetic immunization
  • the particularly preferred embodiments of the invention are naturally occurring allelic vanants of ugc and polypeptides encoded thereby
  • novel polypeptides of Pseudomonas aeruginosa referred to herein as ugc as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same
  • inhibitors to such polypeptides useful as antibacterial agents, including, for example, antibodies
  • a ugc polypeptide or polynucleotide for assessing ugc expression, treating disease, assaymg genetic vanation, and administering a ugc polypeptide or polynucleotide to an organism to raise an lmmunological response against a bactena, especially a Pseudomonas aeruginosa bactena
  • polynucleotides that hybndize to ugc polynucleotide sequences, particularly under stringent conditions
  • antibodies against ugc polypeptides there are provided antibodies against ugc polypeptides
  • methods for identifying compounds which bmd to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the mvention compnsmg contacting a polypeptide or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide or polynucleotide to assess the bmding to or other mteraction with the compound, such bmdmg or mteraction bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polyn
  • ugc agonists and antagonists preferably bactenostatic or bactenocidal agonists and antagonists
  • the mvention relates to novel ugc polypeptides and polynucleotides, which are essential to
  • the mvention relates to polypeptides and polynucleotides of a novel ugc of Pseudomonas aeruginosa
  • the mvention relates especially to ugc having the nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO
  • polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of ugc, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO l]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% similarity (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% similanty (still more preferably at least 95% identity) to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids
  • the mvention also includes polypeptides of the formula
  • X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the ammo terminus, X is hydrogen, and at the carboxyl terminus.
  • Y is hydrogen or a metal
  • Ri and R3 are any ammo acid residue
  • m is an integer between 1 and 1000 or zero
  • n is an integer between 1 and 1000 or zero
  • R 2 is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 In the formula above R 2 is onented so that its ammo terminal residue is at the left, bound to R ⁇ and its carboxy terminal residue is at the nght, bound to R3
  • Any stretch of ammo acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer
  • a fragment is a variant polypeptide having an ammo acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned polypeptides
  • fragments may be "free-standing,” or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region, a smgle larger polypeptide
  • Preferred fragments m clude, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that mcludes the ammo terminus, or a continuous senes of residues that mcludes the carboxyl terminus
  • Degradation forms of the polypeptides of the mvention m a host cell, particularly a Pseudomonas aeruginosa are also preferred
  • fragments characterized by structural or functional attributes such as fragments that comp
  • biologically active fragments which are those fragments that mediate activities of ugc, including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • fragments compnsmg receptors or domains of enzymes that confer a function essential for viabihty of Pseudomonas aeruginosa or the abihty to initiate, or maintain cause disease in an individual, particularly a human
  • Vanants that are fragments of the polypeptides of the mvention may be employed for producmg the corresponding full-lengtii polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention
  • the term "X” or "Xaa” may also be used m desc ⁇ bmg certain polypeptides of the invention "X” and "Xaa” mean that any of the twenty naturally occu ⁇ ng ammo acids may appear at such a designated position m the polypeptide sequence
  • Polynucleotides Another aspect of the mvention relates to isolated polynucleotides, including the full length gene, that encode the ugc polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
  • a polynucleotide of the mvention encoding ugc polypeptide may be obtained usmg standard cloning and screening methods, such as those for clonmg and sequencmg chromosomal DNA fragments from bactena usmg Pseudomonas aeruginosa strain 4 cells as starting matenal, followed by obtaining a full length clone
  • a polynucleotide sequence of the mvention such as a sequence given m Table 1 [SEQ ID NO 1]
  • typically a library of clones of chromosomal DNA of Pseudomonas aeruginosa strain 4 m E cob or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, denved from a
  • the mvention provides a polynucleotide sequence identical over its entire length to a coding sequence in Table 1 [SEQ ID NO 1] Also provided b the mvention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment m reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence
  • the polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5' and 3 " sequences, such as the transcnbed, non-translated sequences, termination signals, nbosome bmdmg sites, sequences that stabilize mRNA, introns, pohadenylation signals, and additional coding sequence which encode additional ammo acids
  • a marker sequence that facilitates purification of the fused polypeptide can be encoded In certam embodiments of the mvention, the
  • Polynucleotides of the mvention also mclude, but are not limited to, polynucleotides compnsing a structural gene and its naturally associated sequences that control gene expression
  • a preferred embodiment of the mvention is a polynucleotide of compnsmg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 754 set forth in SEQ ED NO 1 of Table 1, both of which encode the ugc polypeptide
  • the mvention also mcludes polynucleotides of the formula
  • R j and R3 are independently any nucleic acid residue
  • m is an mteger between 1 and 3000 or zero
  • n is an mteger between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 In the polynucleotide formula above R is oriented so that its 5' end residue is at the left, bound to Ri and its 3' end residue is at the ⁇ ght, bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropol
  • polynucleotides of the inventions are denved from Pseudomonas aeruginosa, however, they may preferably be obtained from organisms of the same taxonomic genus They may also be obtained, for example, from organisims of the same taxonomic family or order
  • polynucleotide encodmg a polypeptide encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Pseudomonas aeruginosa ugc having an amino acid sequence set out in Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a smgle continuous region or discontmuous regions encodmg the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences
  • the invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2].
  • Variants that are fragments of the polynucleotides of the invention may be used to synthesize full- length polynucleotides of the invention.
  • Further particularly preferred embodiments are polynucleotides encoding ugc variants, that have the amino acid sequence of ugc polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination.
  • silent substitutions, additions and deletions that do not alter the properties and activities of ugc.
  • polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding ugc polypeptide having an amino acid sequence set out in Table 1 [SEQ ED NO:2], and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding ugc polypeptide and polynucleotides complementary thereto.
  • polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
  • Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA ofTable l [SEQ ID NO:l].
  • the invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions and “stringent hybridization conditions” mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50%) formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0. lx SSC at about 65°C.
  • Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein.
  • the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: l under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or a fragment thereof; and isolating said DNA sequence.
  • Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
  • polynucleotides of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding ugc and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the ugc gene.
  • Such probes generally will comprise at least 15 bases.
  • such probes will have at least 30 bases and may have at least 50 bases.
  • Particularly preferred probes will have at least 30 bases and will have 50 bases or less .
  • the coding region of the ugc gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO: 1] to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays.
  • Polynucleotides of the invention that are oligonucleotides derived from the sequences of Table 1 [SEQ ID NOS: 1 or 2] may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • the invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things
  • the additional ammo acids may be processed away from the mature protem by cellular enzymes
  • a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • N may also be used m desc ⁇ bmg certam polynucleotides of the mvention "N” means that any of the four DNA or RNA bases may appear at such a designated position m the DNA or RNA sequence, except it is preferred that N is not a base that when taken in combmation with adjacent nucleotide positions, when read in the correct readmg frame, would have the effect of generatmg a premature termination codon in such readmg frame
  • a polynucleotide of the mvention may encode a mature protein, a mature protem plus a leader sequence (which may be referred to as a preprotem), a precursor of a mature protem having one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein. which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide Vectors, host cells, expression
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques
  • Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the mvention
  • Introduction of a polynucleotide into the host cell can be effected by methods descnbed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Sprmg Harbor, NY (1989), such as, calcium
  • appropnate hosts include bactenal cells, such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtihs cells, fiingal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells, and plant cells
  • vectors include, among others, chromosomal, episomal and virus-denved vectors, e g , vectors denved from bactenal plasmids, from bactenophage, from transposons from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression system constructs may contam control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography.
  • phosphocellulose chromatography hydrophobic mteraction chromatography, affinity chromatography, hvdroxylapatite chromatography, and lectin chromatography
  • high performance hquid chromatography is employed for purification
  • Well known techmques for refoldmg protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification Diagnostic Assays
  • This mvention is also related to the use of the ugc polynucleotides of the mvention for use as diagnostic reagents Detection of ugc in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease Eukaryotes (herein also " ⁇ nd ⁇ v ⁇ dual(s)"), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the ugc gene may be detected at the nucleic acid level by a variety
  • Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin
  • Genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification technique pnor to analysis RNA, cDNA and genomic DNA may also be used in the same ways Usmg amplification, characterization of the species and strain of prokaryote present m an individual, may be made by an analysis of the genotype of the prokaryote gene
  • Deletions and insertions can be detected by a change m size of the amplified product m companson to the genotype of a reference sequence
  • Point mutations can be identified by hybndizing amplified DNA to labeled ugc polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures DNA sequence differences may also be detected by alterations m the electrophor
  • RT-PCR can be used to detect mutations It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose, PCR or RT-PCR As an example.
  • PCR primers complementary to a nucleic acid encodmg ugc can be used to identify and analyze mutations
  • the mvention also mcludes primers of the formula
  • R 1 X-(R 1 ) m -(R 2 )-(R 3 ) n -Y
  • X is hydrogen
  • Y is hydrogen or a metal
  • R ⁇ and R3 is any nucleic acid residue
  • m is an mteger between 1 and 20 or zero
  • n is an integer between 1 and 20 or zero
  • R 2 is a primer sequence of the mvention, particularly a primer sequence selected from Table 2
  • R 2 is onented so that its 5' end residue is at the left, bound to Ri and its 3' end residue is at the nght, bound to R3
  • Any stretch of nucleic acid residues denoted by either R group, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucleotide of Table 1
  • the mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These primers may be used for, among other things, amplifying ugc DNA isolated from a sample denved from an individual
  • the primers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techmques for elucidation of the DNA sequence In this way, mutations m the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections by Pseudomonas aeruginosa.
  • comp ⁇ smg determining from a sample denved from an individual a mcreased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1]
  • Increased or decreased expression of ugc polynucleotide can be measured usmg any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hyb ⁇ dization methods
  • a diagnostic assay in accordance with the mvention for detecting over- expression of ugc protem compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a ugc protein, m a sample denved from a host are well-known to those of skill m the art
  • Assay techmques that can be used to determine levels of a ugc protein, m a sample denved from a host are well-known to those of skill m the art
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Antibodies
  • Antibodies as used herem mcludes monoclonal and polyclonal antibodies, chimenc, smgle chain, simianized antibodies and humanized antibodies, as well as Fab fragments, mcludmg the products of an Fab immunolglobuhn expression library
  • Antibodies generated against the polypeptides of the mvention can be obtained by administering the polypeptides or epitope-bea ⁇ ng fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols.
  • any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
  • phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-ugc 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 al, (1991) Nature 352, 624-628). If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies.
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
  • antibodies against ugc- polypeptide may be employed to treat infections, particularly bacterial infections.
  • Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of this invention.
  • the term "antigenically equivalent derivative” as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host.
  • the term “immunologically equivalent derivative” as used herein 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.
  • 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 lmmunogemc carrier protem for example bovme serum albumin (BSA) or keyhole limpet haemocyanin (KLH)
  • BSA bovme serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comp ⁇ smg multiple copies of the protem 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
  • the antibody or vanant thereof is modified to make it less lmmunogemc m the individual
  • the antibody may most preferably be "humanized", where the complimentanty determining reg ⁇ on(s) of the hyb ⁇ doma-de ⁇ ved antibody has been transplanted mto a human monoclonal antibody , for example as descnbed m Jones, P et al (1986), Nature 321, 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273
  • a polynucleotide of the mvention m genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA mto muscles (Wolff et al , Hum Mol Genet 1992, 1 363, Manthorpe et al .
  • Polypeptides of the mvention may also be used to assess the bmdmg of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • the mvention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of ugc polypeptides or polynucleotides, particularly those compounds that are bactenostatic and/or bactenocidal
  • the method of screening may mvolve high-throughput techmques
  • a synthetic reaction mix for example, to screen for agomsts or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg ugc polypeptide and a labeled substrate or hgand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a ugc agonist or antagonist
  • the abihty of the candidate molecule to agonize or antagonize the ugc polypeptide is reflected m decreased bmdmg of the labeled ligand or decreased production of product
  • ugc polypeptides that bmd well and mcrease the rate of product production from substrate are agomsts Detection of the rate or level of production of product from substrate may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colonmetnc labeled substrate converted mto product, a reporter gene that is responsive to changes in ugc polynucleotide or polypeptide activity, and bmding assays known m the art
  • an assay for ugc antagonists is a competitive assay that combines ugc and a potential antagonist with ugc-binding molecules, recombinant ugc bmding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay
  • Ugc can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of ugc molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • Potential antagonists mclude small orgamc molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide or polypeptide of the mvention and thereby inhibit or extinguish its activity
  • Potential antagonists also may be small orgamc molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducmg ugc-mduced activities, thereby preventing the action of ugc by excluding ugc from bmdmg
  • Potential antagonists m include a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventing bmding to cellular bmd g molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small orgamc molecules, peptides or peptide-like molecules
  • Other potential antagonists m clude antisense molecules (see Okano, J Neurochem 56 560 (1991), OLIGODEOXYNUCLEOTLDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a desc ⁇ ption of these molecules)
  • Prefened potential antagonists m include compounds related to and vanants of ugc
  • each of the DNA sequences provided herem may be used m the discovery and development of antibacterial compounds
  • the encoded protem upon expression, can be used as a target for the screening of antibacterial drugs
  • the DNA sequences encodmg the ammo terminal regions of the encoded protem or Shme-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
  • the mvention also provides the use of the polypeptide, polynucleotide or inhibitor of the mvention to interfere with the initial physical mteraction between a pathogen and mammalian host responsible for sequelae of infection
  • the molecules of the mvention may be used m the prevention of adhesion of bactena, m particular gram positive bactena, to mammalian extracellular matrix protems on m-dwelhng devices or to extracellular matnx protems in wounds, to block ugc protein-mediated mamma
  • the antagomsts and agomsts of the mvention may be employed, for instance, to inhibit and treat diseases
  • Hehcobacter pylori (herein H pylori) bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm)
  • the international Agency for Research on Cancer recently recognized a cause-and- effect relationship between H pylori and gastric adenocarcmoma, classifying the bactenum as a Group I (definite) carcinogen
  • Prefe ⁇ ed antimicrobial compounds of the mvention (agomsts and antagomsts of ugc) found usmg screens provided by the mvention, particularly broad- spectrum antibiotics, should be useful m the treatment of H pylori mfection Such treatment should decrease the advent of H
  • Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of ugc, or a fragment or a variant thereof, for expressing ugc, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not.
  • an immunological response such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not.
  • One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comprise DNA. RNA, a modified nucleic acid, or a DNA/RNA hybrid
  • a further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a ugc or protein coded therefrom, wherein the composition comprises a recombinant ugc or protein coded therefrom comprising DNA which codes for and expresses an antigen of said ugc or protein coded therefrom.
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.
  • a ugc polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties.
  • fused recombinant protein preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof.
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.
  • the co-protein may be attached to either the amino or carboxy terminus of the first protein.
  • compositions particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
  • this mvention provides methods usmg the desc ⁇ bed polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bactenal cell surface protems m DNA constructs used m such genetic immunization experiments m animal models of mfection with Pseudomonas aeruginosa will be particularly useful for identifymg protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resistmg or clearing mfection for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Pseudomonas aeruginosa mfection, m mammals, particularly humans
  • the polypeptide may be used as an antigen for vaccmation of a host to produce specific antibodies which protect against mvasion of bactena, for example by blocking adherence of bactena to damaged tissue
  • tissue damage mclude wounds m skin or connective tissue caused, e g , by mechanical, chemical or thermal damage or by implantation of mdwellmg devices, or wounds m the mucous membranes, such as the mouth, mammary glands, urethra or vagma
  • the mvention also mcludes a vaccine formulation which compnses an immunogenic recombinant protem of the mvention together with a suitable earner Smce the protein may be broken down in the stomach, it is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, mtravenous, or intradermal
  • Formulations suitable for parenteral administration mclude aqueous and non-aqueous sterile injection solutions which may contam anti-oxidants, buffers, bactenostats and solutes which render the formulation msotonic with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may mclude suspendmg agents or thickening agents
  • the formulations may be presented m umt-dose or multi-dose contamers, for example, sealed ampules and vials and may be stored m a freeze-dned condition requiring only the addition of the sten
  • compositions for purposes of compositions, kits and administration
  • the mvention also relates to compositions compnsing the polynucleotide or the polypeptides discussed above or their agomsts or antagomsts
  • the polypeptides of the mvention may be employed m combination with a non-sterile or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject
  • Such compositions compnse Such compositions compnse.
  • earners may mclude, but are not limited to, salme, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof
  • the formulation should suit the mode of administration
  • the mvention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • Polypeptides and other compounds of the mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • compositions may be administered m any effective, convenient manner mcludmg, for instance, administration by topical, oral, anal, vaginal, mtravenous, intrapentoneaL intramuscular, subcutaneous, lntranasal or tradermal routes among others
  • the active agent may be administered to an individual as an lnjectable composition, for example as a stenle aqueous dispersion, preferably isotomc
  • the composition may be formulated for topical application for example m the form of omtments, creams, lotions, eye omtments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contam appropnate conventional additives, mcludmg, for example, preservatives, solvents to assist drug penetration, and emollients in omtments and creams
  • Such topical formulations may also contam compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions
  • Such earners 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
  • the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg
  • the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of
  • composition of the mvention may be used to bathe an mdwellmg device immediately before msertion
  • the active agent will preferably be present at a concentration of l ⁇ g/ml to lOmg/ml for bathing of wounds or mdwellmg devices
  • a vaccme composition is convemently m lnjectable form
  • Conventional adjuvants may be employed to enhance the immune response
  • a suitable umt dose for vaccmation is 0 5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an mterval of 1-3 weeks With the mdicated dose range, no adverse toxicological effects will be observed with the compounds of the mvention which would preclude their administration to suitable individuals
  • “Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparmg the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity” and “simila ⁇ ty” can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press.
  • Isolated means altered “by the hand of man” from its natural state, i e , if it occurs in nature, it has been changed or removed from its ongrnal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is “isolated”, as the term is employed herem
  • a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said organism, which organism may be living or nonliving
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)"
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • 'Nariant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non- naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techmques, by direct synthesis, and by other recombmant methods known to skilled artisans EXAMPLES
  • the polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1] was obtamed from a library of clones of chromosomal DNA of Pseudomonas aeruginosa m E cob
  • the sequencmg data from two or more clones contammg overlappmg Pseudomonas aeruginosa DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libranes may be prepared by routme methods, for example Methods 1 and 2 below
  • Total cellular DNA is isolated from Pseudomonas aeruginosa strain 4 accordmg to standard procedures and size-fractionated by either of two methods
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate accordmg to standard procedures
  • DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E cob infected with the packaged library
  • the library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combmation of restnction enzymes approp ⁇ ate to generate a senes of fragments for clonmg mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E cob infected with the packaged library
  • the library is amplified by standard procedures
  • Example 2 ugc Characterization
  • Ts mutants m an attempt to identify essential P aeruginosa gene products
  • a genomic library contammg 5 to 6 kb DNA fragments of P aeruginosa was constructed to complement these Ts mutants
  • Nucleotide sequence analysis of plasmids complementmg the Ts mutants revealed many known essential genes as well as genes with unknown functions
  • One of the novel essential genes, encodmg a gene product with an unknown biochemical function is called ugc
  • Nucleotide sequence analysis of the Ts ugc allele revealed a C- T transition mutation at nucleotide position 404 m Table 1 [SEQ ID NO 1], which caused an ammo acid substitution resulting m the change of alanine at position 135 m Table 1 [SEQ ID NO 2] to valme m the Ugc ORF Flow cytometry studies of the Ts ugc allele revealed a C- T transition mutation at nucleotide

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