WO2004026888A2 - Recepteur tlr9 derive de diverses especes mammaliennes - Google Patents

Recepteur tlr9 derive de diverses especes mammaliennes Download PDF

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Publication number
WO2004026888A2
WO2004026888A2 PCT/US2003/029577 US0329577W WO2004026888A2 WO 2004026888 A2 WO2004026888 A2 WO 2004026888A2 US 0329577 W US0329577 W US 0329577W WO 2004026888 A2 WO2004026888 A2 WO 2004026888A2
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tlr9
seq
species
ofthe
amino acid
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PCT/US2003/029577
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WO2004026888A3 (fr
Inventor
Grayson B. Lipford
Neeloffer Mookherjee
Lorne Babiuk
Robert Brownlie
Philip Griebel
George Mutwiri
Rolf Hecker
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Coley Pharmaceutical Gmbh
University Of Saskatchewan
Qiagen Gmbh
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Priority to AU2003278845A priority Critical patent/AU2003278845A1/en
Publication of WO2004026888A2 publication Critical patent/WO2004026888A2/fr
Publication of WO2004026888A3 publication Critical patent/WO2004026888A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • Synthetic oligodeoxynucleotides (ODN) and DNA containing immunostimulatory CpG motifs (CpG DNA) function as potent adjuvants and activators ofthe innate immune system.
  • ODN organic nucleotide
  • CpG DNA DNA containing immunostimulatory CpG motifs
  • Toll-like receptor 9 (TLR9) is known to be involved in innate immunity and to signal in response to CpG DNA. To date, the amino acid sequences only of human and murine TLR9 have been reported, and, interestingly, these two species are known to prefer different CpG motifs. The structural basis for this species-specific CpG motif preference has not yet been fully elucidated.
  • the instant invention provides, in part, novel amino acid and nucleotide sequences of rat, pig, cow, and horse TLR9. These novel TLR9 sequences are useful for elucidating certain key structural features of TLR9.
  • comparison of sequences of murine, human, and these novel TLR9 sequences permits identification of areas of highly conserved sequence, areas of group conservation, and areas of hypervariability.
  • comparisons permit an assessment of evolutionary relatedness among TLR9 molecules ofthe various species, as well as an assessment of inter-species homologies.
  • the invention provides isolated polypeptides having amino acid sequences for rat, pig (porcine), cow (bovine), horse (equine), and sheep (ovine) TLR9 polypeptides. These amino acid sequences correspond to SEQ ID NOs 1, 5, 9, 13, and 17, respectively.
  • each of these sequences is believed to include at least a majority of an extracellular domain, as well as a transmembrane region and at least part of a TLR/IL-1 receptor (TIR) domain.
  • TIR TLR/IL-1 receptor
  • the invention provides isolated polypeptides having amino acid sequences for essentially the whole extracellular domain, optionally including a signal peptide, of each of rat, porcine, bovine, equine, and ovine TLR9. These amino acid sequences correspond to SEQ ID NOs 2, 6, 10, 14, and 18, respectively.
  • Such extracellular domains are believed to include sequence specifically involved in binding to TLR9 ligand, such as CpG DNA.
  • such extracellular domains are believed to include sequence that confers species specificity for particular CpG motifs.
  • Isolated nucleic acid molecules encoding the polypeptides just described above are also provided according to further aspects ofthe invention.
  • Such nucleic acid molecules include, but are not limited to, nucleic acid molecules having sequences provided by SEQ ID NOs 3, 7, 11, 15, 19; and 4, 8, 12, 16, and 20, respectively.
  • Isolated nucleic acid molecules encoding the TLR9 polypeptides of SEQ ID NOs 1, 5, 9, 13, 17; and 2, 6, 10, 14, and 18 also include nucleic acid molecules that differ in sequence from SEQ ID NOs 3, 7, 11, 15, 19; and 4, 8, 12, 16, and 20, respectively, due to degeneracy ofthe genetic code.
  • nucleic acid molecules will hybridize, under stringent conditions, with suitably selected nucleic acid molecules having sequences selected from SEQ ID NOs 3, 4, 7, 8, 11, 12, 15, 16, 19, and 20.
  • the invention provides a vector which includes an isolated nucleic acid molecule ofthe invention, hi one embodiment the vector is an expression vector and the isolated nucleic acid molecule ofthe invention is operably linked to a regulatory sequence in the vector.
  • an expression vector according to this aspect ofthe invention causes the cell to express a polypeptide ofthe invention.
  • the invention according to another aspect provides a cell in which a vector ofthe invention is present.
  • the cell containing the vector expresses a polypeptide ofthe invention, h certain embodiments the cell also contains a reporter construct that transduces a TLR9-mediated signal in response to contact ofthe polypeptide of the invention or a TLR9 with a suitable TLR9 ligand.
  • the cell containing the vector, and optionally containing the reporter construct can be used in screening methods also provided by the invention.
  • the invention provides an antibody or antibody fragment that binds specifically to an isolated polypeptide ofthe invention.
  • the antibody or antibody fragment binds uniquely to one of rat, porcine, bovine, equine, or ovine TLR9 polypeptide. More specifically, the antibody or antibody fragment binds uniquely to one ofthe isolated polypeptides ofthe invention.
  • the antibody or antibody fragment that binds uniquely to one of rat, porcine, bovine, equine, or ovine TLR9 polypeptide also binds to either mouse or human TLR9.
  • the antibody or antibody fragment that binds uniquely to one of rat, porcine, bovine, equine, or ovine TLR9 polypeptide does not also bind to either mouse or human TLR9.
  • the antibody or antibody fragment binds selectively to a chimeric TLR9 polypeptide ofthe invention.
  • the antibody or antibody fragment ofthe invention is a monoclonal antibody or fragment of a monoclonal antibody.
  • the invention provides a method for identifying key amino acids in a TLR9 of a first species which confer specificity for CpG DNA optimized for TLR9 ofthe first species.
  • the method involves aligning protein sequences of TLR9 of a first species, TLR9 of a second species, and TLR9 of a third species, wherein the TLR9 ofthe third species preferentially generates a signal when contacted with a CpG DNA optimized for TLR9 ofthe first species rather than when contacted with a CpG DNA optimized for TLR9 ofthe second species; generating an initial set of candidate amino acids in the TLR9 ofthe first species by excluding each amino acid in the TLR9 ofthe first species which (a) is identical with the TLR9 ofthe second species or (b) differs from the TLR9 ofthe second species only by conservative amino acid substitution; generating a refined set of candidate amino acids by selecting each amino acid in the initial set of candidate amino acids in the TLR9 ofthe first species which (a) is identical with the TLR9 of
  • the invention provides a method for identifying key amino acids in human TLR9 which confer specificity for CpG DNA optimized for human TLR9.
  • the method according to this aspect ofthe invention involves aligning protein sequences of human TLR9, murine TLR9, and TLR9 of a third species, wherein the TLR9 ofthe third species preferentially generates a signal when contacted with a CpG DNA optimized for human TLR9 rather than when contacted with a CpG DNA optimized for murine TLR9; generating an initial set of candidate amino acids in human TLR9 by excluding each amino acid in human TLR9 which (a) is identical with murine TLR9 or (b) differs from murine TLR9 only by conservative amino acid substitution; generating a refined set of candidate amino acids by selecting each amino acid in the initial set of candidate amino acids in human TLR9 which (a) is identical with the TLR9 ofthe third species or (b) differs from the TLR9 ofthe third species only by conservative amino acid substitution; and identifying as key amino acids in
  • the method according to this aspect ofthe invention is performed iteratively with a plurality of TLR9s derived from different species other than human and mouse, wherem for each TLR9 the refined set of candidate amino acids is assigned a weight corresponding to a ratio equal to (responsiveness to human-preferred CpG DNA)/(responsiveness to murine-preferred CpG DNA).
  • the invention also provides an isolated polypeptide having an amino acid sequence identical to SEQ ID NO:30 (extracellular domain (ECD) of murine TLR9) except for substitution of at least one key amino acid identified according to the method above.
  • the polypeptide according to this aspect ofthe invention is a chimeric TLR9 polypeptide.
  • the polypeptide according to this aspect ofthe invention binds to CpG DNA optimized for human TLR9 better than does the isolated polypeptide having an amino acid sequence identical to SEQ ID NO:30 (ECD of murine TLR9).
  • the polypeptide includes only one substituted amino acid.
  • the isolated polypeptide according to this aspect ofthe invention may further include sequence involved in TLR/IL-1R signal transduction, e.g., intracellular domain of TLR9 as provided in SEQ ID NOs 29 and 33.
  • a polypeptide according to this aspect of the invention is an isolated polypeptide having an amino acid sequence identical to SEQ ID NO:29 (full length murine TLR9) except for substitution of at least one key amino acid identified according to the method above.
  • the invention provides an isolated nucleic acid molecule including a nucleic acid sequence encoding a chimeric TLR9 polypeptide just described.
  • the isolated nucleic acid molecule has a nucleic acid sequence encoding a chimeric TLR9 polypeptide just described.
  • the invention provides a screening method to identify a TLR9 ligand.
  • the method involves contacting a polypeptide (including a chimeric TLR9 polypeptide) ofthe invention with a candidate TLR9 ligand; measuring a signal in response to the contacting; and identifying the candidate TLR9 ligand as a TLR9 ligand when the signal in response to the contacting is consistent with TLR9 signaling.
  • the candidate TLR9 ligand is an immunostimulatory nucleic acid.
  • the candidate TLR9 ligand is a CpG DNA.
  • the invention also provides, in yet a further aspect, a screening method to identify species-specific CpG-motif preference of an isolated polypeptide ofthe invention.
  • the method according to this aspect ofthe invention involves contacting an isolated polypeptide ofthe invention with a CpG DNA including a hexamer sequence selected from the group consisting of GACGTT, AACGTT, CACGTT, TACGTT, GGCGTT, GCCGTT, GTCGTT, GATGTT, GAAGTT, GAGGTT, GACATT, GACCTT, GACTTT, GACGCT, GACGAT, GACGGT, GACGTC, GACGTA, and GACGTG; measuring a signal in response to the contacting; and identifying a species- specific CpG-motif preference when the signal in response to the contacting is consistent with TLR9 signaling.
  • the CpG DNA is an oligodeoxynucleotide having a sequence selected from the group consisting of
  • TCCATGACGGTTTTGATGTT (SEQ IDNO:54), TCCATGACGTCTTTGATGTT (SEQ LDNO:55), TCCATGACGTATTTGATGTT (SEQ ID NO:56), and TCCATGACGTGTTTGATGTT (SEQ IDNO:57).
  • the signal includes expression of a reporter gene responsive to TLR/IL-IR signal transduction pathway.
  • the reporter gene is operatively linked to a promoter sensitive to NF- ⁇ B.
  • the signal in response to contacting is binding ofthe candidate TLR9 ligand or CpG DNA to the polypeptide ofthe invention.
  • the screening method is performed on a plurality of test compounds.
  • the response mediated by the TLR9 signal transduction pathway is measured quantitatively and the response mediated by the TLR9 signal transduction pathway associated with each ofthe plurality of test compounds is compared with a response arising as a result of an interaction between the functional TLR9 and a reference immunostimulatory compound.
  • Figure 1 depicts a Clustal W multiple sequence alignment of deduced amino acid sequences for cat (feline), dog (canine), cow (bovine), mouse (murine), sheep (ovine), pig (porcine), horse (equine), human, and rat TLR9 polypeptides.
  • the deduced amino acid sequences for feline, canine, bovine, murine, ovine, porcine, equine, human, and rat TLR9 polypeptides shown in the figure correspond to SEQ ID NOs 25, 21, 9, 29, 17, 5, 13, 33, and 1, respectively. Lines labeled "multiple" refer to the multiple sequence alignment of all six sequences shown.
  • FIG. 1 Lines labeled "mo/hu” refer to a paired sequence alignment of mouse and human TLR9 sequences alone.
  • Figure 2 is a cladogram depicting an evolutionary relatedness tree for rat, murine, porcine, bovine, equine, and human TLR9 polypeptides in Figure 1.
  • Figure 3 is a graph depicting species specificity of TLR9 signaling with selected oligonucleotides having strong specificity for human (2006), mouse (5890), or neither (1982).
  • the present invention provides novel amino acid and nucleotide sequences for TLR9 derived from rat, pig, cow, horse, and sheep. These sequences can be used to identify key features ofthe primary sequences of these and related TLR molecules, including previously known primary sequences of human and mouse (murine) TLR9. Such key features include binding site information and species specificity toward particular CpG motifs.
  • Native and novel chimeric TLR9 polypeptides designed with the aid of this information can be expressed in vitro or in vivo and used in screening assays to identify and to design novel TLR9 ligands. Additionally, the native and novel chimeric TLR9 polypeptides designed with the aid of this information can be expressed in vitro or in vivo and used in screening assays to compare various TLR9 ligands, including CpG DNA.
  • the invention provides isolated TLR9 polypeptides, and isolated nucleic acid molecules encoding them, from rat, pig, cow, horse, and sheep.
  • isolated as used herein with reference to a nucleic acid molecule or polypeptide means substantially free of or separated from components with which it is normally associated in nature, e.g., other nucleic acids, proteins, lipids, carbohydrates or in vivo systems to an extent practical and appropriate for its intended use.
  • the nucleic acids or polypeptides are sufficiently pure and are sufficiently free from other biological constituents of host cells so as to be useful in, for example, producing pharmaceutical preparations.
  • nucleic acid or polypeptide ofthe invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the nucleic acid or polypeptide may represent only a small percentage by weight of such a preparation.
  • the nucleic acid or polypeptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.
  • SEQ ID NO:l An amino acid sequence of rat TLR9 is provided as SEQ ID NO:l. Based on comparison with known amino acid sequences of human and murine TLR9, it appears that SEQ ID NO:l includes sequence for at least a majority ofthe extracellular domain, all ofthe transmembrane domain, and at least a portion ofthe intracellular domain of rat TLR9 (See Figure 1). Amino acids numbered 1-821 of SEQ ID NO:l are presumptively extracellular domain and correspond to SEQ ID NO:2.
  • SEQ ID NO:3 is a nucleotide sequence of rat TLR9 cDNA having an open reading frame corresponding to nucleotides 1-3096.
  • SEQ ID NO:4 is a nucleotide sequence of rat cDNA encoding amino acids 1-821 of SEQ ID NO:l.
  • An amino acid sequence of porcine TLR9 is provided as SEQ ID NO:5. Based on comparison with known amino acid sequences of human and murine TLR9, it appears that SEQ ID NO: 5 includes sequence for at least a majority ofthe extracellular domain, all ofthe transmembrane domain, and at least a portion ofthe intracellular domain of porcine TLR9 (See Figure 1). Amino acids numbered 1-819 of SEQ ID NO:5 are presumptively extracellular domain and correspond to SEQ ID NO:6.
  • SEQ ID NO:7 is a nucleotide sequence of porcine TLR9 cDNA having an open reading frame corresponding to nucleotides 77-3166.
  • SEQ ID NO:8 is a nucleotide sequence of porcine cDNA encoding amino acids 1- 819 ofSEQ ID NO:5.
  • SEQ ID NO:9 An amino acid sequence of bovine TLR9 is provided as SEQ ID NO:9. Based on comparison with known amino acid sequences of human and murine TLR9, it appears that SEQ ID NO:9 includes sequence for at least a majority ofthe extracellular domain, all ofthe transmembrane domain, and at least a portion ofthe intracellular domain of bovine TLR9 (See Figure 1). Amino acids numbered 1-818 of SEQ ID NO:9 are presumptively extracellular domain and correspond to SEQ ID NO: 10.
  • SEQ ID NO:l 1 is a nucleotide sequence of bovine TLR9 cDNA having an open reading frame corresponding to nucleotides 84-3170.
  • SEQ ID NO:12 is a nucleotide sequence of bovine cDNA encoding amino acids 1- 818 of SEQ ID NO:9.
  • An amino acid sequence of equine TLR9 is provided as SEQ ID NO: 13. Based on comparison with known amino acid sequences of human and murine TLR9, it appears that SEQ ID NO:13 includes sequence for at least a majority ofthe extracellular domain, all ofthe transmembrane domain, and at least a portion ofthe intracellular domain of equine TLR9 (See Figure 1). Amino acids numbered 1-820 of SEQ ID NO: 13 are presumptively extracellular domain and correspond to SEQ ID NO: 14.
  • SEQ LD NO: 15 is a nucleotide sequence of equine TLR9 cDNA having an open reading frame corresponding to nucleotides 115-3207.
  • SEQ ID NO: 16 is a nucleotide sequence of equine cDNA encoding amino acids 1- 820 ofSEQ ID NO:13.
  • SEQ ID NO:17 An amino acid sequence of ovine TLR9 is provided as SEQ ID NO:17. Based on comparison with known amino acid sequences of human and murine TLR9, it appears that SEQ ID NO:17 includes sequence for at least a majority ofthe extracellular domain, all ofthe transmembrane domain, and at least a portion ofthe intracellular domain of ovine TLR9 (See Figure 1). Amino acids numbered 1-818 of SEQ ED NO:17 are presumptively extracellular domain and correspond to SEQ DD NO: 18.
  • SEQ ID NO: 19 is a nucleotide sequence of ovine TLR9 cDNA having an open reading frame corresponding to nucleotides 92-3178.
  • SEQ DD NO:20 is a nucleotide sequence of ovine cDNA encoding amino acids 1-818 of SEQ DD NO:17.
  • MGPCHGALQP SLLVQAAM AVA AQGTLPPFLPCE QPHGLVNCN LFLKSVPHFSAAAPRDNVTSLS LSNRI HHLHDSDFAQ SNLQKLNLK NCPPAG SPMHFPCHMTIEPMTFLAVPTLE ⁇ LN SYNGITTVPALPSS VSLIL SRTNILQLDPTS TG HALRF YMDGNCYYKNPCGRA EVAPGAL GLGNLTH SLKYNNLTTVPRSLPPSLEY SYNHIVTLAPEDLA LTALRV DVGGNCRRCDHARMPCVECPHKFPQLHSDTFSH SR EGLVLKDSS YQLN PR FRGLGNLTV DLSENF YDCITKTKAFQGLAQLRRLNLSFNYHKKVSFAHLTLAPSFGSL S QE DMHGIF FRSLSQKTLQPLARLPMLQRLYLQMNFINQAQ GIFKDFPGLRYID SDNRISGAV ⁇ PVATTGE ⁇ DGGKK
  • SEQ ID NO:17 (Ovine TLR9) MGPYCAPHPLSLLVQAAALAAA AQGT PAFLPCELQPRG T ⁇ CN F KSVPRFSAGAPRANVTSLSLISNRIH H HDSDFVH SNLRVLNLfWNCPPAGLSPMHFPCRMTIEPNTF AVPTLEELNLSYNGITTVPALPSS VSLSLS RTSI VLGPTHFTGL__ALRFLYMDGNCYY_0_-PCQQAVEVAPGALLG1_GNLTHLSLKYNNLTEVPRRLPPS DT L SY_miITLAPEDLA_.LTALRV DVGGNCRRCDHARNPCRECP_aTFPKLHPDTFSH SRLEGLVL__DSS YKLEK DWFRGLGRLQVLD SENFLYDYITKTTIFRNLTQLRR NLSFNYHKKVSFAHLQLAPSFGGLVSLEKLDMHGIFF RSLTNTTLRPLTQLPK QSLSLQLNFINQAELS
  • nucleotide and amino acid sequences for canine and feline TLR9 are publicly availiable.
  • an amino acid sequence for canine TLR9 is available as GenBank accession number BAC65192 and its corresponding nucleotide sequence is available as GenBank accession number ABI 04899.
  • An amino acid sequence for feline TLR9 is available as GenBank accession number AAN15751 and its corresponding nucleotide sequence is available as GenBank accession number AY137581.
  • Complete nucleotide and amino acid sequences for canine and feline TLR9 were also determined independently from those available from public databases.
  • SEQ DD NO:21 An amino acid sequence of canine TLR9 is provided as SEQ DD NO:21. Based on comparison with known amino acid sequences of human and murine TLR9, it appears that SEQ ED NO:21 includes sequence for at least a majority ofthe extracellular domain, all ofthe transmembrane domain, and at least a portion ofthe intracellular domain of canine TLR9 (See Figure 1). Amino acids numbered 1-822 of SEQ ED NO:21 are presumptively extracellular domain and correspond to SEQ ID NO:22.
  • SEQ ID NO:23 is a nucleotide sequence of canine TLR9 cDNA having an open reading frame corresponding to nucleotides 91-3186.
  • SEQ ID NO:24 is a nucleotide sequence of canine cDNA encoding amino acids 1- 822 of SEQ ID NO:21.
  • SEQ ID NO:25 An amino acid sequence of feline TLR9 is provided as SEQ ID NO:25. Based on comparison with known amino acid sequences of human and murine TLR9, it appears that SEQ ED NO:25 includes sequence for at least a majority ofthe extracellular domain, all ofthe transmembrane domain, and at least a portion ofthe intracellular domain of feline TLR9 (See Figure 1). Amino acids numbered 1-820 of SEQ DD NO:25 are presumptively extracellular domain and correspond to SEQ DD NO:26.
  • SEQ DD NO:27 is a nucleotide sequence of feline TLR9 cDNA having an open reading frame corresponding to nucleotides 87-3179.
  • SEQ DD NO:28 is a nucleotide sequence of feline cDNA encoding amino acids 1-820 of SEQ DD NO:25.
  • SEQ DD NO:29 An amino acid sequence ofmurine TLR9 is available as GenBank accession no. AAK29625, provided as SEQ DD NO:29. Amino acids numbered 1- 821 of SEQ DD NO:29 presumptively include the entire extracellular domain and correspond to SEQ ED NO:30. SEQ ID NO:31 corresponds to GenBank accession number AF348140, which is a nucleotide sequence of murine TLR9 cDNA. SEQ DD NO:32 is a nucleotide sequence of murine cDNA encoding amino acids 1-821 of SEQ DD NO:29.
  • GenBank accession no. AAF78037 An amino acid sequence of human TLR9 is available as GenBank accession no. AAF78037, provided as SEQ DD NO:33. Amino acids numbered 1-820 of SEQ DD NO:33 presumptively include the entire extracellular domain and correspond to SEQ DD NO:34.
  • SEQ ED NO:35 corresponds to GenBank accession number AF245704, which is a nucleotide sequence of human TLR9 cDNA.
  • SEQ ID NO:36 is a nucleotide sequence of human cDNA encoding amino acids 1-820 of SEQ ED NO:33.
  • chimeric TLR9 polypeptides and nucleic acid molecules encoding them are provided by the invention.
  • the chimeric polypeptides include at least one amino acid subsititution based on a comparison of conserved and non-conserved amino acids among at least two of rat, murine, porcine, bovine, equine, ovine, canine, feline, and human TLR9.
  • TLR9 polypeptide sequences can be used to identify and select individual amino acid positions and even individual amino acids to substitute in designing a chimeric TLR9.
  • substitution or substitutions can be effected using methods known to those of ordinary skill in molecular biology.
  • Nucleic acids encoding the native or chimeric polypeptides ofthe invention can be inserted into an expression vector and used to express TLR9 polypeptide.
  • a conservative amino acid substitution shall refer to a substitution of a first amino acid for a second amino acid, wherein side chains ofthe first amino acid and the second amino acid share similar features in terms of hydrophobicity, size, aromaticity, or tendency to alter conformation.
  • conservative amino acid substitutions generally may be made between members within each ofthe following groups: hydrophobic (A, I, L, M, V), neutral (C, S, T), acidic (D, E), basic (H, K, N, Q, R), and aromatic (F, W, Y).
  • a non- conservative amino acid substitution refers to any other amino acid substitution.
  • An expression vector for TLR9 will include at least a nucleotide sequence coding for a TLR9, or a fragment thereof coding for a functional TLR9 polypeptide, operably linked to a gene expression sequence which can direct the expression ofthe TLR9 nucleic acid within a eukaryotic or prokaryotic cell.
  • a "gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation ofthe nucleic acid to which it is operably linked.
  • the "gene expression sequence” is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation ofthe TLR9 nucleic acid to which it is operably linked.
  • the gene expression sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter.
  • Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, ⁇ -actin promoter, and other constitutive promoters.
  • Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the simian virus (e.g., SN40), papillomavirus, adenovirus, human immunodeficiency virus (HIN), Rous sarcoma virus (RSN), cytomegalovirus (CMV), the long terminal repeats (LTR) of Moloney murine leukemia virus and other retro viruses, and the thymidine kinase (TK) promoter of herpes simplex virus.
  • simian virus e.g., SN40
  • papillomavirus e.g., papillomavirus
  • adenovirus e.g., human immunodeficiency virus (HIN), Rous sarcoma virus (RSN), cytomegalovirus (CMV), the long terminal repeats (LTR) of Moloney murine leukemia virus and other retro viruses
  • LTR long terminal repeats
  • Inducible promoters are expressed in the presence of an inducing agent.
  • the metallothionein (MT) promoter is induced to promote transcription and translation in the presence of certain metal ions.
  • Other inducible promoters are known to those of ordinary skill in the art.
  • the gene expression sequence shall include, as necessary, 5' non- transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control ofthe operably joined nucleic acid coding sequence for a TLR9 polypeptide.
  • the gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.
  • a nucleic acid coding sequence and a gene expression sequence are said to be "operably linked” when they are covalently linked in such a way as to place the transcription and/or translation ofthe nucleic acid coding sequence under the influence or control ofthe gene expression sequence.
  • the TLR9 nucleic acid coding sequence and the gene expression sequence are said to be "operably linked” when they are covalently linked in such a way as to place the transcription and/or translation ofthe TLR9 nucleic acid coding sequence under the influence or control ofthe gene expression sequence.
  • TLR9 sequence be translated into a functional protein
  • two DNA sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription ofthe TLR9 sequence and if the nature ofthe linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability ofthe promoter region to direct the transcription ofthe TLR9 sequence, or (3) interfere with the ability ofthe corresponding RNA transcript to be translated into a protein.
  • a gene expression sequence would be operably linked to a TLR9 nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that TLR9 nucleic acid sequence such that the resulting transcript might be translated into the desired TLR9 protein or polypeptide.
  • TLR9 ligand refers to a molecule that specifically binds a TLR9 polypeptide.
  • the TLR9 ligand specifically binds a TLR9 polypeptide corresponding to at least a ligand-binding portion ofthe extracellular domain of TLR9.
  • TLR9 signaling refers to TLR/IL-IR signal transduction mediated through the TLR9, as described in further detail elsewhere herein.
  • CpG nucleic acids have been reported to be TLR9 ligands, but TLR9 ligands may include other entities as well, including, for example, small molecules.
  • a species-preferred CpG DNA refers to a particular CpG DNA that is optimized for signal induction by a TLR9 of a particular species.
  • a CpG DNA that is optimized for signal induction by a TLR9 of a particular species refers to a CpG DNA having a sequence that preferentially binds to and/or induces signaling by TLR9 of that species.
  • a human-preferred CpG DNA shall refer to a CpG DNA that optimally stimulates human TLR9 to signal through its TIR domain.
  • a murine-preferred CpG DNA shall refer to a CpG DNA that optimally stimulates murine TLR9 to signal through its TER. domain.
  • Examples of human-preferred and murine-preferred CpG DNA are ODN 2006 (SEQ DD NO:58) and 1668 (SEQ DD NO:60), respectively.
  • TLR9s The binding and species specificity of TLR9s are believed to be influenced by key amino acids present in the extracellular domain of TLR9.
  • Key amino acids in a TLR9 as used herein refer to those amino acids which contribute significantly to ligand binding and ligand specificity of a particular TLR9 polypeptide.
  • CpG nucleic acid or a “CpG immunostimulatory nucleic acid” as used herein is a nucleic acid containing at least one unmethylated CpG dinucleotide (cytosine-guanine dinucleotide sequence, i.e., "CpG DNA” or DNA containing a 5' cytosine followed by 3' guanine and linked by a phosphate bond) which activates a component ofthe immune system.
  • the entire CpG nucleic acid can be unmethylated or portions may be unmethylated but at least the C ofthe 5' CG 3' must be unmethylated.
  • a CpG nucleic acid is represented by at least the formula: 5'-N ⁇ X ⁇ CGX 2 N 2 -3' wherein Xi and X 2 are nucleotides, N is any nucleotide, and Ni and N are nucleic acid sequences composed of from aboixt 0-25 N's each, hi some embodiments Xi is adenine, guanine, or thymine and/or X 2 is cytosine, adenine, or thymine. In other embodiments Xi is cytosine and/or X 2 is guanine.
  • Nucleic acids having modified backbones such as phosphorothioate backbones, also fall within the class of immunostimulatory nucleic acids.
  • U.S. Pat. Nos. 5,723,335 and 5,663,153 issued to Hutcherson, et al. and related PCT publication WO95/26204 describe immune stimulation using phosphorothioate oligonucleotide analogues. These patents describe the ability ofthe phosphorothioate backbone to stimulate an immxme response in a non-sequence specific manner.
  • An immunostimulatory nucleic acid molecule including for example a CpG DNA, may be double-stranded or single-stranded. Generally, double-stranded molecules may be more stable in vivo, while single-stranded molecules may have increased activity.
  • nucleic acid and “oligonucleotide” refer to multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)) or a modified base.
  • a substituted pyrimidine e.g., cytosine (C), thymine (T) or uracil (U)
  • a substituted purine e.g., adenine (A) or guanine (G)
  • nucleic acid and oligonucleotide refer to oligoribonucleotides as well as oligodeoxyribonucleotides.
  • the terms shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer.
  • polynucleosides i.e., a polynucleotide minus the phosphate
  • nucleic acid and oligonucleotide also encompass nucleic acids or oligonucleotides with a covalently modified base and/or sugar.
  • nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 2' position and other than a phosphate group at the 5' position.
  • modified nucleic acids may include a 2'-O-alkylated ribose group.
  • modified nucleic acids may include sugars such as arabinose instead of ribose.
  • the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide-nucleic acids (which have amino acid backbone with nucleic acid bases).
  • the nucleic acids are homogeneous in backbone composition.
  • the substituted purines and pyrimidines ofthe immunostimulatory nucleic acids include standard purines and pyrimidines such as cytosine as well as base analogs such as C- 5 propyne substituted bases.
  • Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymine, 5- methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties.
  • the immunostimulatory nucleic acid is a linked polymer of bases or nucleotides.
  • linked or “linkage” means two entities are bound to one another by any physicochemical means. Any linkage known to those of ordinary skill in the art, covalent or non-covalent, is embraced. Such linkages are well known to those of ordinary skill in the art. Natural linkages, which are those ordinarily found in nature connecting the individual units of a nucleic acid, are most common. The individual units of a nucleic acid maybe linked, however, by synthetic or modified linkages.
  • nucleic acid molecules useful according to the invention can be obtained from natural nucleic acid sources (e.g., genomic nuclear or mitochondrial DNA or cDNA), or are synthetic (e.g., produced by oligonucleotide synthesis).
  • nucleic acids isolated from existing nucleic acid sources are referred to herein as native, natural, or isolated nucleic acids.
  • the nucleic acids useful according to the invention may be isolated from any source, including eukaryotic sources, prokaryotic sources, nuclear DNA, mitochondrial DNA, etc.
  • nucleic acid encompasses both synthetic and isolated nucleic acids.
  • the immunostimulatory nucleic acids can be prodxxced on a large scale in plasmids, (see Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) and separated into smaller pieces or administered whole. After being administered to a subject the plasmid can be degraded into oligonucleotides.
  • One skilled in the art can purify viral, bacterial, eukaryotic, etc. nucleic acids using standard techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
  • the immunostimulatory nucleic acids can be synthesized de novo using any of a number of procedures well known in the art.
  • the ⁇ -cyanoethyl phosphoramidite method eaucage SL and Caruthers MH, Tetrahedron Let 22:1859 (1981)
  • nucleoside H-phosphonate method Gagg et al., Tetrahedron Let 27:4051-4054 (1986); Froehler et al., Nucl Acid Res 14:5399-5407 (1986); Garegg et al., Tetrahedron Let 27:4055-4058 (1986); Gaffhey et al, Tetrahedron Let 29:2619-2622 (1988)
  • These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market.
  • the immimostimulatory nucleic acid may be any size of at least 6 nucleotides but in some embodiments are in the range of between 6 and 100 or in some embodiments between 8 and 35 nucleotides in size.
  • Immunostimulatory nucleic acids can be produced on a large scale in plasmids. These may be administered in plasmid form or alternatively they can be degraded into oligonucleotides before administration.
  • a “stabilized immunostimulatory nucleic acid” shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g., via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Nucleic acids that are tens to hundreds of kbs long are relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase their effect. For example, if the 3' end of an oligonucleotide has self-complementarity to an upstream region, so that it can fold back and form a sort of stem loop structure, then the oligonucleotide becomes stabilized and therefore exhibits more activity.
  • Some stabilized immunostimulatory nucleic acids have a modified backbone. It has been demonstrated that modification ofthe oligonucleotide backbone provides enhanced activity ofthe immunostimulatory nucleic acids when administered in vivo. Nucleic acids, including at least two phosphorothioate linkages at the 5' end ofthe oligonucleotide and multiple phosphorothioate linkages at the 3' end, preferably 5, may provide maximal activity and protect the oligonucleotide from degradation by intracellular exo- and endo-nucleases.
  • modified oligonucleotides include phosphodiester modified oligonucleotide, combinations of phosphodiester and phosphorothioate oligonucleotide, methylphosphonate, methylphosphorothioate, phosphorodithioate, and combinations thereof. Each of these combinations and their particular effects on immune cells is discussed in more detail in U.S. Pat. Nos. 6,194,388 and 6,207,646, the entire contents of which are incorporated herein by reference. It is believed that these modified oligonucleotides may show more stimulatory activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, and/or altered intracellular localization. Both phosphorothioate and phosphodiester nucleic acids are active in immune cells.
  • Other stabilized immunostimulatory nucleic acids include: nonionic DNA analogs, such as alkyl- and aryl-phosphates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated.
  • Oligonucleotides which contain diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.
  • Phosphorothioate nucleic acid molecules may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries.
  • Aryl- and alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described. Uhlmann E and Peyman A (1990) Chem Rev 90:544; Goodchild J (1990) Bioconjugate Chem 1:165.
  • Other sources of immunostimulatory nucleic acids useful according to the invention include standard viral and bacterial vectors, many of which are commercially available.
  • a "vector" is any nucleic acid material which is ordinarily used to deliver and facilitate the transfer of nucleic acids to cells.
  • the vector as used herein may be an empty vector or a vector carrying a gene which can be expressed.
  • the vector In the case when the vector is carrying a gene the vector generally transports the gene to the target cells with reduced degradation relative to the extent of degradation that would result in the absence ofthe vector.
  • the vector optionally includes gene expression sequences to enhance expression ofthe gene in target cells such as immune cells, but it is not required that the gene be expressed in the cell.
  • Nucleic acid-binding fragments of TLRs are believed to include the extracytoplasmic (extracellular) domain or subportions thereof, such as those which include at least an MBD motif, a CXXC motif, or both an MBD motif and a CXXC motif.
  • Both mouse and human TLR9 have an N-terminal extension of approximately 180 amino acids compared to other TLRs.
  • An insertion also occurs at amino acids 253-268, which is not found in TLRs 1-6 but is present in human TLR7 and human TLR8.
  • This insert has two CXXC motifs which participate in forming a CXXC domain.
  • the CXXC domain resembles a zinc finger motif and is found in DNA-binding proteins and in certain specific CpG binding proteins, e.g., methyl-CpG binding protein-1 (MBD-1). Fujita N et al. (2000) Mol Cell Biol 20:5107-18.
  • Both human and mouse TLR9 CXXC domains occur at aa 253- 268:
  • MBD motif An additional motif believed to be involved in CpG binding is the MBD motif, also found in MBD-1, listed below as SEQ ED NO:53. Fujita, N et al.(2000) Mol Cell Biol 20:5107-18; Ohki I et al. (1999) EMBO J 18:6653-61. Amino acids 524-554 of hTLR9 and aa 525-555 of mTLR9 correspond to the MBD motif of MBD-1 as shown:
  • MBD motif MBD-1 R-XXXXXXX-R-X-D-X-Y-XXXXXXXXX-R-S-XXXXX-Y SEQ ID NO:65 hTLR9 Q-XXXXXXX-K-X-D-X-Y-XXXXXXXXX-R- -XXXXX-Y SEQ DD NO:66 mTLR9 Q-xxxxxxx- ⁇ -x-D-x- ⁇ -xxxxxxx-Q- -xxxxxx- ⁇ SEQ D NO:67
  • a screening method for identifying an immunostimulatory compound.
  • the method according to this aspect ofthe invention involves contacting a functional TLR9 with a test compound; detecting presence or absence of a response mediated by a TLR9 signal transduction pathway in the presence ofthe test compound arising as a result of an interaction between the functional TLR9 and the test compound; and dete ⁇ nining the test compound is an immunostimulatory compound when the presence of a response mediated by the TLR9 signal transduction pathway is detected.
  • An immunostimulatory compound is a natural or synthetic compound that is capable of inducing an immune response when contacted with an immune cell.
  • a TLR9 ligand that is an immunostimulatory compound is a natural or synthetic compound that is capable of inducing an immune response when contacted with an immtxne cell that expresses TLR9.
  • a TLR9 ligand that is an immunostimulatory compound is also a natural or synthetic compound that is capable of inducing a TLR IL-IR signal transduction pathway when contacted with a TLR9.
  • Immunostimulatory compounds include but are not limited to immunostimulatory nucleic acids.
  • the immunostimulatory compound can be, for example, a nucleic acid molecule, polynucleotide or oligonucleotide, a polypeptide or oligopeptide, a lipid or lipopolysaccharide, a small molecule.
  • a basis for certain ofthe screening assays is the presence of a functional TLR9 in a cell.
  • the functional TLR9 in some instances is naturally expressed by a cell.
  • expression ofthe functional TLR9 can involve introduction or reconstitution of a species-specific TLR9 into a cell or cell line that otherwise lacks the TLR9 or lacks responsiveness to immunostimulatory nucleic acid, resulting in a cell or cell line capable of activating the TLR/IL-IR signaling pathway in response to contact with an immunostimulatory nucleic acid.
  • expression ofthe functional TLR9 can involve introduction of a chimeric or modified TLR9 into a cell or cell line that otherwise lacks the TLR9 or lacks responsiveness to immxmostimulatory nucleic acid, resulting in a cell or cell line capable of activating the TLR/IL-IR signaling pathway in response to contact with an immunostimulatory nucleic acid.
  • cell lines lacking TLR9 or immunostimulatory nucleic acid responsiveness include, but are not limited to, 293 fibroblasts (ATCC CRL-1573), MonoMac-6, THP-1, U937, CHO, and any TLR9 knock-out.
  • the introduction ofthe species-specific, chimeric or modified TLR9 into the cell or cell line is preferably accomplished by transient or stable transfection ofthe cell or cell line with a TLR9-encoding nucleic acid sequence operatively linked to a gene expression sequence (as described above). Methods for transient and for stable transfection of a cell are well known in the art.
  • the screening assays can have any of a number of possible readout systems based upon either TLR/IL-IR signaling pathway or other assays useful for assessing response to immunostimulatory nucleic acids. It has been reported that immune cell activation by CpG immunostimulatory sequences is dependent in some way on endosomal processing.
  • the readout for the screening assay is based on the use of native genes or, alternatively, cotransfected or otherwise co-introduced reporter gene constructs which are responsive to the TLR/IL-IR signal transduction pathway involving MyD88, TRAP, p38, and/or ERK.
  • TLR/IL-IR signal transduction pathway involving MyD88, TRAP, p38, and/or ERK.
  • EMBO J 18:6913-6982 activate kinases including KB kinase complex and c-Jun N-terminal kinases.
  • reporter genes and reporter gene constructs particularly useful for the assays can include a reporter gene operatively linked to a promoter sensitive to NF- ⁇ B.
  • the reporter gene operatively linked to the TLR-sensitive promoter can include, without limitation, an enzyme (e.g., luciferase, alkaline phosphatase, ⁇ -galactosidase, chloramphenicol acetyltransferase (CAT), etc.), a bioluminescence marker (e.g., green- fluorescent protein (GFP, U.S. Pat. No.
  • an enzyme e.g., luciferase, alkaline phosphatase, ⁇ -galactosidase, chloramphenicol acetyltransferase (CAT), etc.
  • CAT chloramphenicol acetyltransferase
  • bioluminescence marker e.g., green- fluorescent protein (GFP, U.S. Pat. No.
  • the reporter is selected from IL-8, TNF- ⁇ , NF- ⁇ B-luciferase (NF- ⁇ B- luc; hacker H et al. (1999) EMBO J ' 18:6973-6982), IL-12 p40-luc (Murphy TL et al. (1995) Mol Cell Biol 15:5258-5267), and TNF-luc (Hacker H et al. (1999) EMBO J 18:6973-6982).
  • NF- ⁇ B-luc reporter constructs
  • substrate can be supplied as part ofthe assay, and detection can involve measurement of chemiluminescence, fluorescence, color development, incorporation of radioactive label, drug resistance, or other marker of enzyme activity.
  • detection can be accomplished using FACS analysis or functional assays.
  • Secreted molecules can be assayed using enzyme-linked immunosorbent assay (ELISA) or bioassays. Many such readout systems are well known in the art and are commercially available.
  • comparison can be made to a reference immunostimulatory nucleic acid.
  • the reference immunostimulatory nucleic acid may be any suitably selected immunostimulatory nucleic acid, including a CpG nucleic acid.
  • the screening method is performed using a plurality of test nucleic acids.
  • comparison of test and reference responses is based on comparison of quantitative measurements of responses in each instance.
  • the invention provides a screening method for identifying species specificity of an immunostimulatory nucleic acid.
  • the method involves contacting a TLR9 of a first species with a test immunostimulatory nucleic acid; contacting a TLR9 of a second species with the test immunostimulatory nucleic acid; measuring a response mediated by a TLR signal transduction pathway associated with the contacting the TLR9 ofthe first species with the test immunostimulatory nucleic acid; measuring a response mediated by the TLR signal transduction pathway associated with the contacting the TLR9 ofthe second species with the test immunostimulatory nucleic acid; and comparing the two responses.
  • the TLR9 may be expressed by a cell or it may be part of a cell-free system.
  • the TLR9 may be part of a complex, with either another TLR or with another protein, e.g., MyD88, IRAK, TRAF, I ⁇ B, NF- ⁇ B, or functional homologues and derivatives thereof.
  • another protein e.g., MyD88, IRAK, TRAF, I ⁇ B, NF- ⁇ B, or functional homologues and derivatives thereof.
  • a given ODN can be tested against a panel of human fibroblast 293 fibroblast cells transfected with TLR9 from various species and optionally cotransfected with a reporter construct sensitive to TLR/IL-IR activation pathways.
  • the invention provides a method for screening species selectivity with respect to a given nucleic acid sequence.
  • Test compounds can include but are not limited to peptide nucleic acids (PNAs), antibodies, polypeptides, carbohydrates, lipids, hormones, and small molecules. Test compounds can further include variants of a reference immunostimulatory nucleic acid incorporating any one or combination ofthe substitutions described above. Test compoxmds can be generated as members of a combinatorial library of compounds.
  • PNAs peptide nucleic acids
  • Test compoxmds can be generated as members of a combinatorial library of compounds.
  • the screening methods can be performed on a large scale and with high throughput by incorporating, e.g., an array-based assay system and at least one automated or semi-automated step.
  • the assays can be set up using multiple- well plates in which cells are dispensed in individual wells and reagents are added in a systematic manner using a multiwell delivery device suited to the geometry ofthe multi well plate.
  • Manual and robotic multiwell delivery devices suitable for use in a high throughput screening assay are well known by those skilled in the art.
  • Each well or array element can be mapped in a one-to-one manner to a particular test condition, such as the test compound.
  • Readouts can also be performed in this multiwell array, preferably using a multiwell plate reader device or the like. Examples of such devices are well known in the art and are available through commercial sources. Sample and reagent handling can be automated to further enhance the throughput capacity ofthe screening assay, such that dozens, hundreds, thousands, or even millions of parallel assays can be performed in a day or in a week. Fully robotic systems are known in the art for applications such as generation and analysis of combinatorial libraries of synthetic compounds. See, for example, U.S. Pat. Nos. 5,443,791 and 5,708,158.
  • Lymphoid tissues primarily spleen or blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • RNAlaterTM (Ambion ® , Austin, TX, USA), stabilized at 4°C overnight and stored at -70°C. Blood samples were centrifuged at 500 x g for 25 min at room temperature and the buffy coat, containing enriched PBMC, was then removed and stored at -70°C. The mouse specimen was used as a comparative positive control.
  • First-strand cDNA synthesis Total RNA from the spleen and PBMC samples was isolated using a monophasic solution of phenol and guanidine isothiocyanate: TRIzolTM reagent (GIBCO BRL ® , Burlington, ON, Canada) according to the manufacturer's instructions. First-strand cDNA was synthesized from the total RNA using SUPERSCRIPTTM II reverse transcriptase (GEBCO BRL ® , Burlington, ON, Canada).
  • RNA was added to 50 pmoles of oligo(dT) primer [poly T( 18 )]; the mixture was heated to 70°C for 10 min and subsequently chilled on ice. The following was added to the cooled reaction mixture: 1 ⁇ l of mixed dNTP stock containing 10 mM each dATP, dCTP, dGTP and dTTP (Amersham Pharmacia Biotech Inc., Baie de Urfe, Quebec) at vomral pH, IX first strand buffer (50 mM Tris-HCl pH 8.3/ 75 mM KCl/ 3 mM MgCl 2 ) and 2 ⁇ l of 0.1 M DTT.
  • mixed dNTP stock containing 10 mM each dATP, dCTP, dGTP and dTTP (Amersham Pharmacia Biotech Inc., Baie de Urfe, Quebec) at embarkral pH
  • IX first strand buffer 50 mM Tris-HCl pH 8.3/ 75 mM KCl/
  • TLR9 gene was PCR amplified from each of the above- mentioned species using primers designed from known mouse and human TLR9 sequence in Genbank: Accession AF314224 and AF259262, respectively. The primers were designed using the primer design software, Clone Manager 5 (Scientific and Educational Software, Durham, NC, USA).
  • TLR9 gene-specific primers used were: forward primer 5'-ACCTTGCCTGCCTTCCTACCCTGTGA-3' (SEQ DD NO:37) and reverse primer 5'-GTCCGTGTGGGCCAGCACAAA-3' (SEQ DD NO:38).
  • the 2.7 Kbp fragment was PCR amplified using Advantage ® 2 DNA polymerase mix (BD Biosciences Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions.
  • PCR reaction volumes of 25 ⁇ l contained 15 pmoles of each primer, 0.2 mM of dNTP mix and 1 ⁇ l of reverse transcription reaction.
  • PCR amplification was conducted by initial denaturation at 94°C for 1 min followed by 30 cycles of 94°C denaturation (15 sec), 65°C annealing (45 sec) and 72°C extensions (2 min), with a final extension at 72°C for 5 min.
  • PCR amplified fragment was treated with 500 units of T4 DNA polymerase (Amersham Pharmacia Biotech Inc., Baie de Urfe, Quebec) for 15 min at room temperature prior to cleaning the reaction with QIAquick PCR purification kit
  • TLR9 were extended and completed using standard 5' and 3' RACE PCR and primers designed using the sequences obtained from the 2.7 Kbp fragments.
  • Nucleotide sequences of rat, porcine, bovine, equine, canine, and feline TLR9 cDNA obtained by the methods above are provided as SEQ DD NOs 3, 7, 11, 15, 19, 23, and 27, respectively.
  • Deduced amino acid sequences are provided as SEQ ID NOs 1, 5, 9, 13, 17, 21, and 25, respectively.
  • Deduced amino acid sequences of full-length murine and human TLR9 are provided as SEQ ID NOs 29 and 33, respectively.
  • Example 2 Comparison of Aligned Sequences for TLR9 from Narious Mammalian Species. Multiple sequence alignment of deduced amino acid sequences for feline, canine, bovine, mouse, ovine, porcine, horse, human, and rat TLR9 polypeptides was performed using Clustal W 1.82 (see, for example, www.cmbi.kun.nl/bioinf/tools/clustalw.shtml). In addition, paired sequence alignment of deduced amino acid sequences for murine and human TLR9 polypeptides was performed using Clustal W 1.82. The results ofthe multiple sequence alignment are presented in Figure 1. As will be appreciated from Figure 1, certain amino acids are highly conserved across all species examined.
  • the extracellular domains of feline, canine, bovine, mouse, ovine, porcine, horse, human, and rat TLR9 correspond to amino acids numbered 1-820, 1-822, 1-818, 1- 821, 1-818, 1-819, 1-820, 1-820, and 1-821, respectively, as shown in Figure 1.
  • Figure 2 presents an evolutionary relatedness tree for six TLR9 polypeptides examined.
  • the cladogram in Figure 2 was prepared using Clustal W (see above).
  • murine and human TLR9 are nearly the most divergent TLR9s in this group.
  • human and horse TLR9 appear relatively closely related.
  • Mouse TLR9 cDNA (SEQ ID NO:31) and human TLR9 cDNA (SEQ ID NO:35) in pT-Adv vector were individually cloned into the expression vector pcDNA3.
  • l(-) from Invitrogen using the EcoRI site.
  • hTLR9 and mTLR9 (mTLR9) signaling were transfected into 293 fibroblast cells using the calcium phosphate method. Since NF- ⁇ B activation is central to the IL-l/TLR signal transduction pathway
  • human fibroblast 293 cells were transiently transfected with mTLR9 and the NF- ⁇ B-luc construct or with mTLR9 alone.
  • CpG-ODN (1668, 2 ⁇ M; TCCATGACGTTCCTGATGCT, SEQ ID NO:60)
  • GpC-ODN (1668-GC, 2 ⁇ M; TCCATGAGCTTCCTGATGCT, SEQ DD NO:61)
  • LPS 100 ng/ml
  • NF- ⁇ B activation by luciferase readout (8h) or IL-8 production by ELISA (48h) were monitored.
  • Results showed that expression of TLR9 (human or mouse) in 293 cells results in a gain of function for CpG-DNA stimulation.
  • TLR9 human TLR9
  • murine TLR9 or either TLR9 with the NF- ⁇ B-luc reporter plasmid
  • 293 cells were transfected in 10 cm plates (2x10 cells/plate) with 16 ⁇ g of DNA and selected with 0.7 mg/ml G418 (PAA Laboratories GmbH, C ⁇ lbe, Germany).
  • Clones were tested for TLR9 expression by RT-PCR.
  • the clones were also screened for IL-8 production or NF- ⁇ B-luciferase activity after stimulation with ODN. Four different types of clones were generated.
  • 293-hTLR9-luc expressing human TLR9 and 6-fold NF- ⁇ B-luciferase reporter
  • 293-mTLR9-luc expressing murine TLR9 and 6-fold NF- ⁇ B-luciferase reporter
  • Example 4 Similar ODN Sequence Specificity of TLR9 of Human and Equine TLR9. 3xl0 6 293T cells were electroporated with 5 ⁇ g NF- ⁇ B-luc plasmid and 5 ⁇ g of either horse TLR9-pcDNA3.1 plasmid or humanTLR9-pcDNA3.1 plasmid at 200V, 975 ⁇ F. After the electroporation the cells were plated in 96-well cell culture plates at 2.5x10 4 cells per well and grown overnight at 37°C. The cells were stimulated with the indicated concentration of ODN for 16h, after which the supernatant was removed and the cells lysed in lysis buffer and frozen for at least 2 hours at -80°C. Luciferase activity was measured by adding Luciferase Assay substrate from Promega. Values are given as fold specific induction over non- stimulated control. Results are shown in Figure 3.
  • ODN 2006 (TCGTCGTTTTGTCGTTTTGTCGTT; SEQ DD NO:58) has a strong specificity for human TLR9.
  • ODN 1982 (TCCAGGACTTCTCTCAGGTT; SEQ ED NO:70) was the negative control ODN.
  • ODN 5890 (TCCATGACGTTTTTGATGTT; SEQ ID NO:39) has a strong specificity for mouse TLR9.
  • This experiment demonstrates the similarity of horse TLR9 to human TLR9 in binding specificity, a result predicted by the evolutionary relatedness of horse TLR9 to human TLR9.
  • Mouse TLR9 is more distant from horse TLR9 and human TLR9 in sequence homology, and ODN 5890 was not detected by either human or horse TLR9.
  • Example 5 Non-human, Non-murine Native Mammalian TLR9 Useful in Screening for Human-Preferred CpG DNA.
  • Native rat, porcine, bovine, equine, and ovine TLR9 polypeptides are screened for binding or TLR9 signaling activity when contacted with human-preferred CpG DNA (ODN 2006).
  • Rat, porcine, bovine, equine, or ovine TLR9 polypeptides which exhibit significant TLR9 binding or TLR9 signaling activity in this assay are then used as the basis for screening for additional human-preferred CpG DNA.
  • An expression vector containing a nucleic acid sequence encoding a selected native rat, porcine, bovine, equine, or ovine TLR9 polypeptide, and optionally a reporter construct, is introduced into cells which do not express TLR9.
  • the cells expressing the selected native rat, porcine, bovine, equine, or ovine TLR9 polypeptide are contacted with candidate human-preferred CpG DNA.
  • candidate human-preferred CpG DNA exhibiting significant TLR9 binding or TLR9 signaling activity are selected as human- preferred CpG DNA.
  • Chimeric TLR9 polypeptides are screened for binding or TLR9 signaling activity when contacted with human-preferred CpG DNA (ODN 2006). Chimeric TLR9 polypeptides which exhibit significant TLR9 binding or TLR9 signaling activity in this assay are then used as the basis for screening for additional human-preferred CpG DNA.
  • An expression vector containing a nucleic acid sequence encoding a selected chimeric TLR9 polypeptide, and optionally a reporter construct, is introduced into cells which do not express TLR9. The cells expressing the selected chimeric TLR9 polypeptide are contacted with candidate human- preferred CpG DNA.
  • Candidate human-preferred CpG DNA exhibiting significant TLR9 binding or TLR9 signaling activity are selected as human-preferred CpG DNA.
  • Example 7 Chimeric TLR9 Responsive to Both Human-Preferred and Murine-Pref erred CpG DNA.
  • Chimeric TLR9 polypeptides are screened for binding or TLR9 signaling activity when contacted with human-preferred CpG DNA (ODN 2006) and also screened for binding or TLR9 signaling activity when contacted with murine-preferred CpG DNA (ODN 1668).
  • Chimeric TLR9 polypeptides which exhibit significant TLR9 binding or TLR9 signaling activity in each of these assays are then used as the basis for screening for additional human- preferred CpG DNA and for screening for additional murine-preferred CpG DNA.
  • An expression vector containing a nucleic acid sequence encoding a selected chimeric TLR9 polypeptide, and optionally a reporter construct, is introduced into cells which do not express TLR9.
  • the cells expressing the selected chimeric TLR9 polypeptide are contacted with candidate human-preferred CpG DNA or candidate murine-preferred CpG DNA.
  • candidate human-preferred CpG DNA or candidate murine-preferred CpG DNA are selected as human-preferred CpG DNA.
  • Candidate murine-preferred CpG DNA exhibiting significant TLR9 binding or TLR9 signaling activity are selected as murine- preferred CpG DNA.

Abstract

La présente invention a trait à de nouvelles séquences d'acides aminés et de nucléotides pour le récepteur TLR9 de rat, de porc (porcin), de vache (bovin), de cheval (équin), et de mouton (ovin). L'invention à également trait à des séquences d'acides aminés et de nucléotides pour le récepteur TLR9 pour chien (canin), chat (félin), souris (murin), et humain. La comparaison desdites séquences, particulièrement en combinaison avec une évaluation fonctionnelle pour des préférences de motifs CpG spécifiques aux espèces permet l'identification de régions spécifiques et de résidus d'acides aminés d'intérêt dans l'interaction du ligand TLR9. L'invention a également trait à des nouvelles molécules chimériques du récepteur de TLR9, des cellules exprimant ces molécules, et des procédés pour leur utilisation dans des analyses de criblage pour des ligands TLR9.
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US8202688B2 (en) 1997-03-10 2012-06-19 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
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US7820379B2 (en) 2000-09-15 2010-10-26 Coley Pharmaceutical Gmbh Process for high throughput screening of CpG-based immuno-agonist/antagonist
US8834900B2 (en) 2001-08-17 2014-09-16 University Of Iowa Research Foundation Combination motif immune stimulatory oligonucleotides with improved activity
US8153141B2 (en) 2002-04-04 2012-04-10 Coley Pharmaceutical Gmbh Immunostimulatory G, U-containing oligoribonucleotides
US9428536B2 (en) 2002-04-04 2016-08-30 Zoetis Belgium Sa Immunostimulatory G, U-containing oligoribonucleotides
US8658607B2 (en) 2002-04-04 2014-02-25 Zoetis Belgium Immunostimulatory G, U-containing oligoribonucleotides
US7807803B2 (en) 2002-07-03 2010-10-05 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US8114419B2 (en) 2002-07-03 2012-02-14 Coley Pharmaceutical Group, Inc. Nucleic acid compositions for stimulating immune responses
US8283328B2 (en) 2002-08-19 2012-10-09 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US8304396B2 (en) 2002-08-19 2012-11-06 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids
US7998492B2 (en) 2002-10-29 2011-08-16 Coley Pharmaceutical Group, Inc. Methods and products related to treatment and prevention of hepatitis C virus infection
US7956043B2 (en) 2002-12-11 2011-06-07 Coley Pharmaceutical Group, Inc. 5′ CpG nucleic acids and methods of use
US8163506B2 (en) * 2003-06-17 2012-04-24 Meiji Co., Ltd. Use of toll-like receptor-expressing cells
US8188254B2 (en) 2003-10-30 2012-05-29 Coley Pharmaceutical Gmbh C-class oligonucleotide analogs with enhanced immunostimulatory potency
US7795235B2 (en) 2004-10-20 2010-09-14 Coley Pharmaceutical Gmbh Semi-soft c-class immunostimulatory oligonucleotides
US7662949B2 (en) 2005-11-25 2010-02-16 Coley Pharmaceutical Gmbh Immunostimulatory oligoribonucleotides
US8580268B2 (en) 2006-09-27 2013-11-12 Coley Pharmaceutical Gmbh CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity
US10260071B2 (en) 2006-09-27 2019-04-16 Coley Pharmaceutical Gmbh CpG oligonucleotide analogs containing hydrophobic T analogs with enhanced immunostimulatory activity
WO2014001422A3 (fr) * 2012-06-28 2014-02-27 Intervet International B.V. Récepteurs de type toll
CN104718221A (zh) * 2012-06-28 2015-06-17 英特维特国际股份有限公司 Toll-样受体及免疫刺激性寡核苷酸
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US10112985B2 (en) 2012-06-28 2018-10-30 Intervet Inc. Toll-like receptors

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