EP1951914A2 - Methodes et agents diagnostiques et therapeutiques - Google Patents

Methodes et agents diagnostiques et therapeutiques

Info

Publication number
EP1951914A2
EP1951914A2 EP06837256A EP06837256A EP1951914A2 EP 1951914 A2 EP1951914 A2 EP 1951914A2 EP 06837256 A EP06837256 A EP 06837256A EP 06837256 A EP06837256 A EP 06837256A EP 1951914 A2 EP1951914 A2 EP 1951914A2
Authority
EP
European Patent Office
Prior art keywords
tlr7
hbv
hbsag
subject
signaling pathway
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Withdrawn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
EP06837256A
Other languages
German (de)
English (en)
Other versions
EP1951914A4 (fr
Inventor
George Ka-Kit Lau
Chee-Kin Hui
Hai Ying Zhang
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.)
Cheng Si Yuan (China-International) Hepatitis Research Foundation
Original Assignee
Cheng Si Yuan (China-International) Hepatitis Research Foundation
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 Cheng Si Yuan (China-International) Hepatitis Research Foundation filed Critical Cheng Si Yuan (China-International) Hepatitis Research Foundation
Publication of EP1951914A2 publication Critical patent/EP1951914A2/fr
Publication of EP1951914A4 publication Critical patent/EP1951914A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to the fields of therapy and diagnosis of Hepatitis B virus (HBV) infection in animal species including humans.
  • the present invention further provides compounds and compositions useful in the treatment of HBV infection in animal species such as humans including agents which facilitate clearance of HBV, and, in particular, chronic HBV infection.
  • Hepatitis B virus causes debilitating disease conditions and can lead to acute liver failure or chronic infection that can also lead to severe liver damage, hepatocellular carcinoma and liver failure.
  • HBV is a DNA virus which replicates via an RNA intermediate and utilizes reverse transcription in its replication strategy.
  • the HBV genome is of a complex nature having a partially double-stranded DNA structure with overlapping open reading frames encoding envelope, pre-core, core, polymerase and X genes.
  • the polymerase gene is the largest open reading frame and it encodes for the multi-functional polymerase (Pol) protein.
  • the polymerase gene overlaps all six other genes including the core gene that encodes for HBc and the precore gene that encodes for the hepatitis Be antigen
  • HBVAg the three envelope genes PreSl, PreS2 and S that encodes for the large, middle and small envelope proteins respectively (LHBs, MHBs and SHBs)
  • LHBs, MHBs and SHBs the small envelope protein
  • HBsAg the hepatitis B surface antigen
  • the X gene encoding for the multifunctional X protein.
  • HBeAg negative chronic hepatitis currently represents the predominant form of chronic hepatitis due to HBV in several parts of the world where non-genotype A infection is common such as in Africa, Asia, the Middle- East, the Mediteranean Basin and South America.
  • Chronic HBV infection is defined as the persistence of HBsAg for more than six months. HBV persistence may be due to the stable nature of covalently closed circular (cccDNA), infection of immunologically privileged sites and/or HBV-specific immune suppression., 1998).
  • cccDNA covalently closed circular
  • CD8+ T-cell functional HBV-specific CD4+ and CD8+ T-cell in persistent HBV infection when compared with individuals who successfully clear infection.
  • the HBV-specific CD8+ T-cell response is significantly diminished when evaluated by proliferative responses to whole HBV antigens or defined epitopes in HLA-A2 positive chronic carriers (Ferrari et al, J Immunol. 145:3442-9, 1990; Maini et al, J Exp Med 191:1269-80, 2000).
  • the host virus relationship is a dynamic process in which many viruses such as HBV attempt to maximize their invisibility while the host attempts to prevent and eradicate infection. Initially, a virus must bind and enter a target cell and migrate to the appropriate cellular compartment in order to replicate and infect other cells. Infected cells may be triggered by the virus to produce cyokines (e.g. TNF- ⁇ and IFN- ⁇ ) that inhibit one or more stages of the viral replication cycle, thereby limiting the extent of the infection.
  • cyokines e.g. TNF- ⁇ and IFN- ⁇
  • Host monocytes and macrophages play a key role in the early response to the virus as they secrete pro-inflammatory cytokines, such as IL-I, TNF- ⁇ , IL-6, IL- 12 and IL-18 that have indirect and direct effects on the infection. They can recruit further monocytes, natural killer (NK) cells and T-cells to perform functions and they can also help switch to the appropriate Th function to help eradicate the virus.
  • pro-inflammatory cytokines such as IL-I, TNF- ⁇ , IL-6, IL- 12 and IL-18 that have indirect and direct effects on the infection. They can recruit further monocytes, natural killer (NK) cells and T-cells to perform functions and they can also help switch to the appropriate Th function to help eradicate the virus.
  • NK natural killer
  • TLRs Toll Like Receptors
  • TLRs have been identified as a major class of pattern-recognition receptors.
  • the role of TLRs involving bacterial products, e.g. endotoxin and peptidoglycan has recently been clarified (Akashi et al, J Immunol. 164: 3471-3475, 2000; Takeuchi et al, Immunity, 11: 443-451, 1999; Tapping et al, J Immunol. 165: 5780-5787, 2000).
  • TLRs More than 13 TLRs have been identified and they play an important role in activation by a number of different bacteria. Recently, this has been extended to viruses with the demonstration that respiratory syncytial virus (RSV) stimulates TLR-4 in a murine model (Kurt- Jones et a!, Nat Immunol. 1: 398-401, 2000; Haeberle et al, J Infect Dis. 186: 1199-1206, 2002). In addition, Measles Virus (MV) has been shown to activate TLR-2 dependent signals (Bieback et al, J Virol.
  • RSV respiratory syncytial virus
  • TLR7 and TLR9 both located in the endosomal membrane.
  • TLR7 and TLR8 recognize viral single-stranded RNA as well as imidazoquinolines such as imiquimod and resiquimod and guanosine analogs.
  • TLR9 recognizes bacterial or viral DNA, including synthetic CpG oligodeoxynucleotides.
  • TLRs Stimulation of TLRs by their ligands initiates the activation of complex networks of intracellular signal transduction pathways to coordinate the ensuing inflammatory response.
  • Important components of these signaling networks are the adaptor protein MyD88 (and related proteins), several protein kinases (including IRAK-I, p38 MAP kinase and IKB kinase), TRAF6 and the transcription factor NF- ⁇ B.
  • Activation of NF- ⁇ B leads to the expression of a variety of pro-inflammatory mediators (e.g. TNF ⁇ , IL-I, IL-6 and MCP-I) (Akira, S. J Biol Chem 278, 38105-8; 2003; Barton, G. M. & Medzhitov, R.
  • TLR7 The activation of adaptive immune response by TLR7 is mediated by MyD88-dependent pathway.
  • TLR7, TLR8, and TLR9 induce interferon (IFN)-alpha in cells such as plasmacytoid dendritic cells (pDCs).
  • IFN interferon
  • pDCs plasmacytoid dendritic cells
  • This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7.
  • TNF tumor necrosis factor
  • TRF6 IFN regulatory factor
  • TLR3 and TLR4 are also capable of signaling via MyD88-independent pathways, involving the adaptor molecules TRIF (for TLR3 and 4) and TRAM (for TLR4) [Lien, E. & Golenbock, D. T. Nat Immunol 4, 1162-4, 2003].
  • T-cell receptors require co-stimulatory molecules, such as CD80 and CD86, to be expressed on the surface of the antigen-presenting cell in association with the peptide-MHC complex in order for activation to occur.
  • co-stimulatory molecules such as CD80 and CD86
  • the expression of these co-stimulatory molecules is controlled in part by TLRs (Pasare, C. & Medzhitov, R. Curr Opin Immunol 15, 677-82, 2003). They are also important in activating B-cells to produce rheumatoid factors (Leadbetter, E. A. et al. Nature 416, 603-7 2002).
  • the present invention identifies that up-regulation of Toll-like receptors and in particular TLR7 or another component of the TLR7-signaling pathway is associated with clearance of HBV from infected subjects and hence represent useful targets to treat chronic HBV infection. More particularly, the present invention identifies that up-regulation of TLR7 is required for HBsAg clearance and is associated with the generation of anti-HBsAg antibodies and resolution of HBV infection.
  • TLR7 is, therefore, a useful target for therapeutic agents to treat subjects such as humans infected with HBV.
  • the therapeutic agents up-regulate TLR7 and promote a decrease in HBsAg and the generation of anti-HBsAg antibodies which assist in the clearance and resolution of HBV infection.
  • TLR7 is also a useful diagnostic target to monitor therapeutic protocols and hence TLR7 is useful as an epidemiological management tool.
  • the present invention provides, therefore, therapeutic agents capable of up-regulating levels or activity of TLR7 or a component in the TLR7-signaling pathway.
  • agents may be used alone or in combination with other anti-HBV treatment protocols including nucleoside or nucleotide analogs, antiviral and/or immunomodulatory agents such as interferon (IFN) and vaccine compositions capable of stimulating a cellular and/or humoral response against HBV or its components.
  • IFN interferon
  • the present invention contemplates a method for monitoring infection by HBV during or subsequent to treatment in a subject, said method comprising determining changes in levels of TLR7 or a component in the TLR7 signaling pathway during or subsequent to treatment.
  • the present invention provides a method for monitoring acute, chronic or persistent infection by HBV during or subsequent to treatment in a subject, said method comprising determining changes in levels of TLR7 or a component in the TLR7-signaling pathway during or subsequent to treatment wherein elevated levels of TLR7 is indicative of improved potential for HBsAG clearance and resolution of HBV infection.
  • TLR7 may be measured in any cells such as but not limited to Kupffer cells, liver cells and/or peripheral monocytes.
  • the present invention contemplates, therefore, therapeutic and diagnostic agents and compositions including vaccine compositions comprising same useful in the treatment and/or diagnosis of infection by HBV or its clearance during or subsequent to treatment.
  • the present invention also provides a method of treating a subject infected with HBV, said method comprising administering to said subject an effective amount of an agent including a composition comprising an agent which up- regulates the level of TLR7.
  • an agent includes a single agent or two or more agents.
  • Another agent which is an anti-HBV agent such as a nucleoside or nucleotide analog or antiviral and/or immunomodulatory agents such as interferon (IFN), may also be in the composition or administered prior to, simultaneously with or subsequent to administration of the TLR7-up-regulating agent.
  • IFN interferon
  • Reference to a composition includes a vaccine or vaccine composition or medicament.
  • the present invention contemplates a method for monitoring a response to a therapeutic protocol directed against infection by HBV said method comprising determining the level or activity of TLR7 wherein the level of TLR7 or a component within the TLR7-signaling pathway is indicative of the clearing or resolving capacity of the therapeutic protocol.
  • Preferred subjects are mammals or avian species. Most preferred subjects are humans. Animal models are also contemplated by the present invention.
  • Figure 1 is a diagrammatic representation of the time line of treatment with a pegylated IFN and either oral placebo or lamivudine.
  • Figure 2 is a graphical representation showing TLR3, 5, 7 and 9 expression in PBMC.
  • Figure 3 is a graphical representation showing TLR7 expression following treatment with pegylated IFN.
  • Figure 4 is a graphical representation showing (A) Longitudinal TLR7 mRNA expression in the T-cells in HBsAg clearance, HBeAg seroconversion and nonresponder to pegylated interferon alfa-2a; (B) cycle threshold of GADPH; (C) HBcAg specific interferon- ⁇ producing CD4+ T cells in HBsAg clearance and HBeAg seroconversion; (D) HBsAg specific interferon- ⁇ producing CD4+ T cells in HBsAg clearance andwith HBeAg seroconversion; (E) HBV Core peptide 18-27 specific interferon- ⁇ producing CD8+ T cells in HBsAg clearance and HBeAg seroconversion; and (F) the characteristics of a HLA-A2 positive patient with HBsAg clearance.
  • Figure 5 is a graphical representation showing a dynamic time course suppression of TLR7 expression in HBsAg + patients compared to anti-HBc+/anti-HBs+ in co-culture model.
  • Figure 6 is a graphical representation showing the effects of a TLR7 agonist on (A) expression of TLR7; (i) TLR7 levels based on real time PCR; (ii) TLR levels based on Western Blot and (B) MTT assay.
  • Figure 7 is a graphical representation showing HBV RNA levels after co-culture with HBsAg or HBsAg + TLR relative to controls.
  • Figure 8 is a photographical representation showing specificity of HBA RNA detection.
  • Figure 9 is a graphical representation showing cccDNA in PBMCs after co-culture with HBV.
  • Figure 10 is a graphical representation showing (A) CD4 responsive as (B) CDS responsive with respect to the number of IFN- ⁇ -positive lymphocytes.
  • Figure 11 is a graphical representation showing HBV DNA in co-culture in the presence or absence of HBsAg and/or TLR7.
  • Figure 12 is a graphical representation showing pathways up-regulated at mRNA level on Day 7 of co-culture.
  • Figure 13 is a graphical representation showing (A) TLR7 mRNA expression on T-cells of patient with natural immunity to HBV (HBV immune) and HBsAg plus HBeAg positive patients with serum ALT more than 2 times the upper limit of normal (HBV Positive) after incubation wild-type HBV in the presence of autologous antigen presenting cells; (B) A real time PCR for TLR7 RNAi showing potent silencing of TLR7 with no effect on GADPH; (C) HBcAg specific interferon- ⁇ producing CD4+ T-cells in the in vitro model; and (D) HBV Core peptide 18-27 specific interferon- ⁇ producing CD8+ T cells in the in vitro model.
  • A TLR7 mRNA expression on T-cells of patient with natural immunity to HBV (HBV immune) and HBsAg plus HBeAg positive patients with serum ALT more than 2 times the upper limit of normal (HBV Positive) after incubation wild-type HBV in the
  • Figure 14 is a graphical representation showing (A) HBcAg specific interferon- ⁇ producing CD4+ T-cells in the HBV positive group after treatment with different doses of TLR7 agonist; and (B) HBV Core peptide 18-27 specific interferon- ⁇ producing CD8+ T cells in the HBV positive group after treatment with different doses of TLR7 agonist.
  • Figure 15 is a graphical representation showing (A) the enhancement in interferon- ⁇ release from CD4+ T-cells of HBV Positive patients by TLR7 agonist 2.0 ng/ml in in vitro culture was significantly reduced when both interferon- ⁇ and interferon- ⁇ Ab was added into the in vitro culture The addition of either interferon- ⁇ Ab or interferon- ⁇ Ab independently into the in vitro culture did not result in a significant reduction in the interferon- ⁇ release from CD4+ T-cells of HBV Positive patients by TLR7 agonist 2.0 ng/ml; and (B) TLR7 agonist can stimulated enhanced type I interferon (interferon- ⁇ / ⁇ ) secretion by monocytes matured dendritic cells.
  • Figure 16 is a representation showing oligonucleotide primers and probes used for Taqman analysis.
  • the present invention is predicated in part on the determination that up-regulation of a TLR and in particular TLR7, in at least liver cells, Kupffer cells and peripheral blood monocytes, dendritic cells or other antigen presenting cells and T-cells facilitates resolution of HBV infection and clearance of HBsAg.
  • the elevation of levels of TLR7 is proposed, therefore, to enhance both the innate immune response and the HBV specific immune response via inter alia generation of anti-HBsAg antibodies.
  • the presence, absence, levels or activity of TLR7 provides, therefore, the focus for the development of therapeutic and diagnostic protocols for
  • HBV infection or for monitoring the infection before, during or after therapeutic intervention.
  • the present invention is particularly relevant for subjects with chronic HBV infection.
  • the present invention provides, therefore, agents which up-regulate TLR7 or a component of the TLR7 signaling pathway and methods for the treatment of HBV infection.
  • Compositions such as vaccine compositions useful in generating a cellular or humoral response to HBV and/or its components is also contemplated by the present invention when used in combination with a TLR7-upregulating agent or agents.
  • the agents of the present invention may be in the form of a composition. When the composiition also comprises an antigen to induce an immune response then the composition may also be called a vaccine composition.
  • the TLR7-upregulating agents or compositions may be included used together with the vaccine composition or maintained separately.
  • compositions may, therefore, comprise a single agent or multiple TLR7-modulating agents or one or more TLR7-modulating agents and one or more anti-HBV agents such as a nucleoside or nucleotide analog or antiviral and/or immunomodulatory agents such as interferon (IFN) and/or an antigen which can stimulate an immune response against HBV and/or its components.
  • TLR7-modulating agents such as a nucleoside or nucleotide analog or antiviral and/or immunomodulatory agents such as interferon (IFN) and/or an antigen which can stimulate an immune response against HBV and/or its components.
  • anti-HBV agents such as a nucleoside or nucleotide analog or antiviral and/or immunomodulatory agents such as interferon (IFN) and/or an antigen which can stimulate an immune response against HBV and/or its components.
  • IFN interferon
  • compositions may be employed given simultaneously or sequentially wherein each composition comprises a single agent or single group of agents, which are selected from a TLR7-modulating agent, an antibody, a nucleotide or nucleoside analog or other anti-HBV agent or antiviral and/or immunomodulatory agents such as interferon (IFN) or an antigen useful in a vaccine composition.
  • TLR7-modulating agent an antibody
  • nucleotide or nucleoside analog or other anti-HBV agent or antiviral and/or immunomodulatory agents
  • IFN interferon
  • the present invention further contemplates a method for monitoring a response to a therapeutic protocol as well as a means for determining the efficacy of a therapeutic regimen.
  • the present invention provides a clinical or epidemiological management tool for HBV infection in animals, such as mammals and in particular humans.
  • the method of this aspect of the present invention is directed to determining levels of TLR7 or monitoring TLR7 levels or activity over time during or subsequent to treatment.
  • one aspect of the present invention contemplates a method for monitoring acute, chronic or persistent infection by HBV in a subject said method comprising determining the levels or activity of TLR7 or a component in the TLR7 signaling pathway wherein the level or activity of TLR7 is indicative of the capacity of the subject to induce clearance of HBsAg, to generate anti-HBsAg antibodies and to facilitate resolution of HBV infection.
  • Another aspect of the present invention is directed to a method for monitoring acute, chronic or persistent infection by HBV during or subsequent to treatment in a subject, said method comprising determining changes in levels of TLR7 or a component in the TLR7-signaling pathway during or subsequent to treatment wherein elevated levels of TLR7 is indicative of improved potential for HBsAG clearance and resolution of HBV infection.
  • a related embodiment of the present invention provides a method for monitoring a response to a therapeutic protocol directed against infection by HBV in a subject said method comprising determining the level or activity of a TLR7 or a component of the TLR7 signaling pathway wherein the level or activity of TLR7 or component of the TLR7 signaling pathway is indicative of the capacity of the subject to resolve HBV infection.
  • Still yet another aspect of the present invention is directed to method of treating a subject infected with HBV said method comprising administering to said subject an effective amount of an agent which up-regulates TLR7 or a component of the TLR7 signaling pathway.
  • the latter embodiment includes providing the subject with a TLR7-up-regulating agent together with other forms of anti-HB V therapy such as a nucleoside or nucleotide or antibody or other anti-HBV agent or antiviral and/or immunomodulatory agents such as interferon (IFN) or an antigen useful in a vaccine composition.
  • a TLR7-up-regulating agent such as a nucleoside or nucleotide or antibody or other anti-HBV agent or antiviral and/or immunomodulatory agents such as interferon (IFN) or an antigen useful in a vaccine composition.
  • IFN interferon
  • the combination therapy may be in the form of a single composition or multiple compositions given simultaneously or sequentially.
  • compound used interchangeably herein to refer to a chemical compound that induces a desired pharmacological and/or physiological effect.
  • the terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • compound When the terms “compound”, “active agent”, “pharmacologically active agent”, “medicament”, “active” and “drug” are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
  • the term “compound” is not to be construed as a chemical compound only but extends to peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and chemical analogs thereof. Reference to a “peptide”, “polypeptide” or “protein” includes molecules with a polysaccharide or lipopolysaccharide component.
  • agonist or “potentiator” is an example of a compound, active agent, pharmacologically active agent, medicament, active and drug which up-regulates the level of TLR7 or a component in the TLR7 signaling pathway.
  • compositions comprising combinations of compounds or agents such as an agonist of TLR7 or a component in the TLR7 signaling pathway and a nucleoside or nucleotide analog or anti-HBsAg antibody, or antiviral cytokines (eg., IFN-oc., IFN- ⁇ , IL-2, TNF-oc) or other immunomodulatory or anti-HBV agents.
  • compounds or agents such as an agonist of TLR7 or a component in the TLR7 signaling pathway and a nucleoside or nucleotide analog or anti-HBsAg antibody, or antiviral cytokines (eg., IFN-oc., IFN- ⁇ , IL-2, TNF-oc) or other immunomodulatory or anti-HBV agents.
  • the present invention contemplates, therefore, compounds useful in up-regulating levels of TLR7 or potentiating general or specific TLR7 signaling. It is proposed that up-regulating TLR7 signaling facilitates clearance of HBsAg and anti-HBsAg antibody production . This is proposed to lead to resolution of HBV infection.
  • the compounds have an effect on acute, chronic infection by HBV.
  • the cells carrying TLR7 to be modulated include inter alia liver cells, Kupffer cells and peripheral/monocytes. A liver cell includes a hepatocyte.
  • references to a "compound”, “active agent”, “pharmacologically active agent”, “medicament”, “active” and “drug” includes combinations of two or more actives such as an agonist or potentiator of TLR7 or TLR7 signaling.
  • a “combination” also includes multi-part such as a two-part pharmaceutical composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.
  • a multi-part pharmaceutical pack may have a modulator of TLR7 and one or more anti-viral agents.
  • modulating or its derivatives, such as “modulate” or “modulation”, are used to describe up- or down-regulation.
  • the aim is to up-regulate the TLR7 levels or activity to assist in HBsAg clearance.
  • TLR7 includes a component of TLR7 signaling pathway.
  • an effective TLR7-modulating amount of an agent is a sufficient amount of the agent to directly or indirectly up-regulate the function or level of TLR7 or to potentiate TLR7 signaling. This may be accomplished, for example, by the agents acting as an agonist (i.e. a potentiator) of the TLR7 or its signaling components such as agents which are, or mimic, components of the TLR7 signaling pathway, and which induce the TLR7 signaling pathway via other cellular receptors or by the agents antagonizing inhibitors of TLR7 signaling components.
  • an agonist i.e. a potentiator
  • an appropriate "effective amount” is a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
  • the exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount”. However, an appropriate "effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
  • pharmaceutically acceptable carrier excipient or diluent
  • a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
  • a pharmaceutical composition may also be described depending on the formulation as a vaccine composition.
  • a "pharmacologically acceptable" salt, ester, emide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.
  • treating and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms of infection or disease, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms of infection and/or their underlying cause and improvement or remediation of damage.
  • Collateral damage for example, following viral infection may be liver damage such as cirrhosis or hepatocellular carcinoma of the liver.
  • the treatment results in clearance of HBsAg via anti-HBsAg antibodies and resolution of HBV infection.
  • Treating" a patient may involve prevention of an adverse physiological event in an individual as well as treatment of a clinically symptomatic individual by resolving HBV infection or downstream condition such as liver damage or cancer.
  • the subject method of "treating" a patient with HBV infection treating chronic or persistent HBV infection and treating individuals presently on another therapeutic protocol.
  • Hepatitis B virus includes all variants including variants resistant to particular therapeutic agents such as nucleoside or nucleotide analogs or immunological agents. Particularly important variants of HBV comprise mutations in HBsAg. Different strains of HBV are also contemplated by this term.
  • patient or "subject” or “individual” as used herein refers to an animal, preferably a mammal and more preferably a human who can benefit from the pharmaceutical formulations and methods of the present invention. There is no limitation on the type of animal that could benefit from the presently described pharmaceutical formulations and methods. A patient regardless of whether a human or non-human animal may be referred to as an individual, subject, animal, host or recipient.
  • the compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry.
  • the compounds of the present invention which modulate the activity or levels of TLR& or a component in the TLR7 signaling pathway may be large or small molecules, nucleic acid molecules (including antisense or sense molecules), peptides, polypeptides or proteins or hybrid molecules such as RNAi- or siRNA-complexes (including RISC complexes and Dicer complexes), ribozymes or DNAzymes.
  • the compounds may need to be modified so as to facilitate entry into a cell. Examples of agents include chemical agents which interact with or agonize TLR7 or genetic molecules which modulate TLR7 expression.
  • the preferred animals are humans.
  • laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model. Livestock animals include sheep, cows, pigs, goats, horses and donkeys. Non-mammalian animals such as avian species (such as ducks), zebrafish, amphibians (including cane toads) and Drosophila species such as Drosophila melanogaster are also contemplated.
  • the present invention provides, therefore, agents which agonize TLR7 levels or activity or a component in the TLR7 signaling pathway.
  • the present invention contemplates methods of screening for such agents comprising, for example, contacting a candidate drug with TLR7 or a part thereof.
  • the TLR7 molecule is referred to herein as a "target” or "target molecule”.
  • the screening procedure includes assaying (i) for the presence of a complex between the drug and the target, or (ii) an alteration in the expression levels of nucleic acid molecules encoding the target.
  • One form of assay involves competitive binding assays.
  • the target is typically labeled. Free target is separated from any putative complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to target molecule.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a target and is described in detail in Geysen (International Patent Publication No. WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with a target and washed. Bound target molecule is then detected by methods well known in the art. This method may be adapted for screening for non-peptide, chemical entities. This aspect, therefore, extends to combinatorial approaches to screening for target modulators of TLR7.
  • Purified target can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the target may also be used to immobilize the target on the solid phase.
  • Antibodies specific for HBsAg may also be useful as inhibitors of HBsAg.
  • the present invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the target compete with a test compound for binding to the target or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the target. Such antibodies may also be useful as diagnostic agents.
  • Antibodies to HBsAg or TLR7 or a component in the TLR7 signaling pathway may be polyclonal or monoclonal although monoclonal antibodies are preferred. Antibodies may be prepared by any of a number of means.
  • antibodies are generally but not necessarily derived from non-human animals such as primates, livestock animals (e.g. sheep, cows, pigs, goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs, rabbits) and companion animals (e.g. dogs, cats).
  • livestock animals e.g. sheep, cows, pigs, goats, horses
  • laboratory test animals e.g. mice, rats, guinea pigs, rabbits
  • companion animals e.g. dogs, cats.
  • antibody based assays are conducted in vitro on cell or tissue biopsies.
  • an antibody is suitably deimmunized or, in the case of human use, humanized, then the antibody can be labeled with, for example, a nuclear tag, administered to a subject and the site of nuclear label accumulation determined by radiological techniques.
  • the HBsAg or TLR7 or a component in the TLR7 signaling pathway antibody is regarded, therefore, as a pathogenic marker targeting agent. Accordingly, the present invention extends to deimmunized forms of the antibodies for use in pathogenic target imaging in human and non-human subjects. This is described further below.
  • the molecule is required to be extracted from a biological sample whether this is from an animal including human tissue or from cell culture if produced by recombinant means.
  • monocytes and hepatocytes are a convenient source.
  • the HBsAg or TLR7 or a component in the TLR7 signaling pathway can be separated from the biological sample by any suitable means.
  • the separation may take advantage of any one or more of the surface charge properties, size, density, biological activity and its affinity for another entity (e.g.
  • separation of HBsAg or TLR7 or a component in the TLR7 signaling pathway from the biological sample may be achieved by any one or more of ultra-centrifugation, ion- exchange chromatography (e.g. anion exchange chromatography, cation exchange chromatography), electrophoresis (e.g. polyacrylamide gel electrophoresis, isoelectric focussing), size separation (e.g., gel filtration, ultra-filtration) and affinity-mediated separation (e.g.
  • ion- exchange chromatography e.g. anion exchange chromatography, cation exchange chromatography
  • electrophoresis e.g. polyacrylamide gel electrophoresis, isoelectric focussing
  • size separation e.g., gel filtration, ultra-filtration
  • affinity-mediated separation e.g.
  • immunoaffmity separation including, but not limited to, magnetic bead separation such as Dynabead (trademark) separation, immunochromatography, immuno- precipitation).
  • Choice of the separation technique(s) employed may depend on the biological activity or physical properties of the HBsAg or TLR7 or a component in the TLR7 signaling pathway sought or from which tissues it is obtained.
  • the separation of HBsAg or TLR7 or a component in the TLR7 signaling pathway from the biological fluid preserves conformational epitopes present on the kinase and, thus, suitably avoids techniques that cause denaturation of the molecule.
  • an animal is immunized with a pre-core protein/HBeAg or a TLR or a component in the TLR7 signaling pathway or a sample comprising HBsAg or TLR7 or a component in the TLR7 signaling pathway by standard methods to produce antibody-producing cells, particularly antibody-producing somatic cells (e.g. B lymphocytes). These cells can then be removed from the immunized animal for immortalization.
  • a pre-core protein/HBeAg or a TLR or a component in the TLR7 signaling pathway or a sample comprising HBsAg or TLR7 or a component in the TLR7 signaling pathway by standard methods to produce antibody-producing cells, particularly antibody-producing somatic cells (e.g. B lymphocytes). These cells can then be removed from the immunized animal for immortalization.
  • antibody-producing somatic cells e.g. B lymphocytes
  • carrier any substance of typically high molecular weight to which a non- or poorly immunogenic substance (e.g. a hapten) is naturally or artificially linked to enhance its immunogenicity.
  • Immortalization of antibody-producing cells may be carried out using methods which are well-known in the art.
  • the immortalization may be achieved by the transformation method using Epstein-Barr virus (EBV) (Kozbor et al, Methods in Enzymology. 121: 140, 1986).
  • EBV Epstein-Barr virus
  • antibody-producing cells are immortalized using the cell fusion method (described in Coligan et al, supra, 1991-1997), which is widely employed for the production of monoclonal antibodies.
  • somatic antibody-producing cells with the potential to produce antibodies, particularly B cells are fused with a myeloma cell line.
  • somatic cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals, preferably rodent animals such as mice and rats. Mice spleen cells are particularly useful. It would be possible, however, to use rat, rabbit, sheep or goat cells, or cells from other animal species instead.
  • myeloma cell lines have been developed from lymphocytic tumors for use in hybridoma-producing fusion procedures (Kohler and Milstein, supra 1976; Shulman et al, Nature. 276:269-270, 1978; VoIk et al, J. Virol. 42 (l):220-227, 1982). These cell lines have been developed for at least three reasons. The first is to facilitate the selection of fused hybridomas from unfused and similarly indefinitely self-propagating myeloma cells. Usually, this is accomplished by using myelomas with enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of hybridomas.
  • lymphocytic tumor cells To eliminate the production of tumor cell antibodies by the hybridomas, myeloma cell lines incapable of producing endogenous light or heavy immunoglobulin chains are used. A third reason for selection of these cell lines is for their suitability and efficiency for fusion.
  • myeloma cell lines may be used for the production of fused cell hybrids, including, e.g. P3X63-Ag8, P3X63-AG8.653, P3/NSl-Ag4-l (NS-I), Sp2/0-Agl4 and S194/5.XXO.Bu.l.
  • the P3X63-Ag8 and NS-I cell lines have been described by Kohler and Milstein (supra, 1976).
  • Shulman et al, ⁇ supra, 1978 developed the Sp2/0-Agl4 myeloma line.
  • the S194/5.XXO.Bu.l line was reported by Trowbridge (J. Exp. Med. 148(1): 313-323, 1978).
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually involve mixing somatic cells with myeloma cells in a 10:1 proportion (although the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of an agent or agents (chemical, viral or electrical) that promotes the fusion of cell membranes. Fusion methods have been described (Kohler and Milstein, supra, 1975,; Kohler and Milstein, supra, 1976; Gefter et al, Somatic Cell Genet. 3: 231-236, 1977; VoIk et al, supra, 1982). The fusion-promoting agents used by those investigators were Sendai virus and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • fusion procedures produce viable hybrids at very low frequency (e.g. when spleens are used as a source of somatic cells, only one hybrid is obtained for roughly every IxIO 5 spleen cells), it is preferable to have a means of selecting the fused cell hybrids from the remaining unfused cells, particularly the unfused myeloma cells.
  • a means of detecting the desired antibody-producing hybridomas among other resulting fused cell hybrids is also necessary.
  • the selection of fused cell hybrids is accomplished by culturing the cells in media that support the growth of hybridomas but prevent the growth of the unfused myeloma cells, which normally would go on dividing indefinitely.
  • the somatic cells used in the fusion do not maintain long-term viability in in vitro culture and hence do not pose a problem.
  • myeloma cells lacking hypoxanthine phosphoribosyl transferase HPRT-negative
  • HPRT-negative hypoxanthine phosphoribosyl transferase
  • HAT hypoxanthine/aminopterin/thymidine
  • myeloma cells with different genetic deficiencies (drug sensitivities, etc.) that can be selected against in media supporting the growth of genotypically competent hybrids is also possible.
  • each cell line may be propagated in either of two standard ways.
  • a suspension of the hybridoma cells can be injected into a histocompatible animal. The injected animal will then develop tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration.
  • the individual cell lines may be propagated in vitro in laboratory culture vessels.
  • the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation, and subsequently purified.
  • the cell lines are tested for their specificity to detect HBsAg or TLR7 or a component in the TLR7 signaling pathway of interest by any suitable immunodetection means.
  • cell lines can be aliquoted into a number of wells and incubated and the supernatant from each well is analyzed by enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique, or the like.
  • ELISA enzyme-linked immunosorbent assay
  • the cell line(s) producing a monoclonal antibody capable of recognizing the target HBsAg or TLR7 or a component in the TLR7 signaling pathway but which does not recognize non-target epitopes are identified and then directly cultured in vitro or injected into a histocompatible animal to form tumors and to produce, collect and purify the required antibodies.
  • These antibodies are HBsAg- or TLR7-specific or specific to a component of the TLR7 signaling pathway. This means that the antibodies are capable of distinguishing a HBsAg or TLR7 or a component in the TLR7 signaling pathway from other molecules. Additional broad spectrum antibodies may be used provided that they do not cross-react with molecules in a normal cell.
  • the monoclonal antibody is destined for use as a therapeutic agent such as to activate TLR7 or a component in the TLR7 signaling pathway or inhibit HBsAg, then, it will need to be deimmunized with respect to the host into which it will be introduced (e.g. a human).
  • the deimmunization process may take any of a number of forms including the preparation of chimeric antibodies which have the same or similar specificity as the monoclonal antibodies prepared according to the present invention.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
  • CDRs complementary determining regions
  • the deimmunizing process is specific for humans. More particularly, the CDRs can be grafted onto a human antibody variable region with or without human constant regions.
  • the non-human antibody providing the CDRs is typically referred to as the "donor” and the human antibody providing the framework is typically referred to as the "acceptor”.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e. at least about 85-90%, preferably about 95% or more identical.
  • a humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a donor antibody is said to be “humanized”, by the process of "humanization”, because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs.
  • Reference herein to "humanized” includes reference to an antibody deimmunized to a particular host, in this case, a human host.
  • deimmunized antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • Exemplary conservative substitutions may be made according to Table 2.
  • the present invention contemplates a deimmunized antibody molecule having specificity for an epitope recognized by a monoclonal antibody to TLR7 or a component in the TLR7 signaling pathway or in HBsAg wherein at least one of the CDRs of the variable domain of said deimmunized antibody is derived from the said monoclonal antibody to the TLR7 or a component in the TLR7 signaling pathway or HBsAg and the remaining immunoglobulin-derived parts of the deimmunized antibody molecule are derived from an immunoglobulin or an analog thereof from the host for which the antibody is to be deimmunized.
  • This aspect of the present invention involves manipulation of the framework region of a non- human antibody.
  • the present invention extends to mutants and derivatives of the subject antibodies but which still retain specificity for TLR7 or a component in the TLR7 signaling pathway or HBsAg.
  • mutant or “derivatives” includes one or more amino acid substitutions, additions and/or deletions.
  • CDR includes CDR structural loops which covers to the three light chain and the three heavy chain regions in the variable portion of an antibody framework region which bridge ⁇ strands on the binding portion of the molecule. These loops have characteristic canonical structures (Chothia et al, J. MoI. Biol. 196: 901, 1987; Chothia et al, J. MoI. Biol. 227: 799, 1992).
  • framework region region of an immunoglobulin light or heavy chain variable region, which is interrupted by three hypervariable regions, also called CDRs.
  • CDRs hypervariable regions
  • a "human framework region” is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs.
  • the CDRs are primarily responsible for binding to an epitope of TLR7 or a component in the TLR7 signaling pathway or HBsAg.
  • the term “heavy chain variable region” means a polypeptide which is from about 110 to 125 amino acid residues in length, the amino acid sequence of which corresponds to that of a heavy chain of a monoclonal antibody of the invention, starting from the amino-terminal (N-terminal) amino acid residue of the heavy chain.
  • the term “light chain variable region” means a polypeptide which is from about 95 to 130 amino acid residues in length, the amino acid sequence of which corresponds to that of a light chain of a monoclonal antibody of the invention, starting from the N-terminal amino acid residue of the light chain.
  • Full-length immunoglobulin "light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH 2 -terminus (about 110 amino acids) and a K or ⁇ constant region gene at the COOH-terminus.
  • Full-length immunoglobulin "heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g. ⁇ (encoding about 330 amino acids).
  • immunoglobulin or "antibody” is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized immunoglobulin genes include the K. ⁇ , ⁇ . ⁇ (IgGi, IgG 2 , IgG 3 , IgG 4 ), ⁇ . ⁇ and ⁇ constant region genes, as well as the myriad immunoglobulin variable region genes.
  • One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain.
  • immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, Fab' and (Fab') 2 .
  • the present invention also contemplates the use and generation of fragments of monoclonal antibodies produced by the method of the present invention including, for example, Fv, Fab, Fab' and F(ab') 2 fragments.
  • fragments may be prepared by standard methods as for example described by Coligan et al. (supra, 1991-1997).
  • the present invention also contemplates synthetic or recombinant antigen-binding molecules with the same or similar specificity as the monoclonal antibodies of the invention.
  • Antigen- binding molecules of this type may comprise a synthetic stabilized Fv fragment.
  • Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N terminus or C terminus of a V # domain with the C terminus or N-terminus, respectively, of a Vi domain.
  • sFv single chain Fv fragments
  • scFv single chain Fv fragments
  • ScFv lack all constant parts of whole antibodies and are not able to activate complement.
  • Suitable peptide linkers for joining the Y H and Vz, domains are those which allow the V # and V/, domains to fold into a single polypeptide chain having an antigen binding site with a three dimensional structure similar to that of the antigen binding site of a whole antibody from which the Fv fragment is derived.
  • Linkers having the desired properties may be obtained by the method disclosed in U.S. Patent No 4,946,778. However, in some cases a linker is absent.
  • ScFvs may be prepared, for example, in accordance with methods outlined in Krebber et al. J. Immunol Methods. 201(l):35-55, 1997. Alternatively, they may be prepared by methods described in U.S. Patent No 5,091,513, European Patent No 239,400 or the articles by Winter and Milstein (Nature. 349:293, 1991) and Pl ⁇ ckthun et al. (In Antibody engineering: A practical approach, 203- 252, 1996).
  • the synthetic stabilized Fv fragment comprises a disulphide stabilized Fv (dsFv) in which cysteine residues are introduced into the V # and NL domains such that in the fully folded Fv molecule the two residues will form a disulphide bond there between.
  • dsFv disulphide stabilized Fv
  • Suitable methods of producing dsFv are described, for example, in (Glockshuber et al, Biochem. 29:1363-1367, 1990; Reiter et al, J. Biol. Chem. 269 ⁇ S327-18331, 1994; Reiter et al, Biochem. 33:5451-5459, 1994; Reiter et al, Cancer Res. 54:2714-2718, 1994; Webber et al, MoI. Immunol. 32:249-258, 1995).
  • dAbs single variable region domains
  • the synthetic or recombinant antigen-binding molecule may comprise a "minibody".
  • minibodies are small versions of whole antibodies, which encode in a single chain the essential elements of a whole antibody.
  • the minibody is comprised of the Vy and Vi domains of a native antibody fused to the hinge region and CH3 domain of the immunoglobulin molecule as, for example, disclosed in U.S. Patent No 5,837,821.
  • the synthetic or recombinant antigen binding molecule may comprise non-immunoglobulin derived, protein frameworks.
  • non-immunoglobulin derived, protein frameworks For example, reference may be made to (Ku & Schutz, Proc. Natl. Acad. ScI USA. 92:6552-6556, 1995) which discloses a four-helix bundle protein cytochrome b562 having two loops randomized to create CDRs, which have been selected for antigen binding.
  • the synthetic or recombinant antigen-binding molecule may be multivalent (i.e. having more than one antigen binding site). Such multivalent molecules may be specific for one or more antigens.
  • Multivalent molecules of this type may be prepared by dimerization of two antibody fragments through a cysteinyl-containing peptide as, for example disclosed by (Adams et al, Cancer Res. 53:4026-4034, 1993; Cumber et al, J. Immunol. 149:l20- ⁇ 26, 1992).
  • dimerization may be facilitated by fusion of the antibody fragments to amphiphilic helices that naturally dimerize (Pl ⁇ nckthun, Biochem 37:1579-1584, 1992) or by use of domains (such as leucine zippers jun and fos) that preferentially heterodimerize (Kostelny et al, J. Immunol. 148: 1547- 1553, 1992).
  • Multivalent antibodies are useful, for example, in detecting different forms of TLRs such as TLR-2 and TLR-4.
  • Analogs of proteinaceous molecules contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS);
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D- isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table 3.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and Cp atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • another aspect of the present invention contemplates any compound which binds or otherwise interacts with TLR7 or a component of the TLR7 signaling pathway resulting in enhanced clearage of HBsAg and resolution of HBV infection.
  • peptide mimetic is intended to refer to a substance which has some chemical similarity to the target but which antagonizes or agonizes or mimics the target.
  • the target in this case may be a ligand of TLR7 or a component in the TLR7 signaling pathway or HBsAg.
  • a peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson et al, "Peptide Turn Mimetics” in Biotechnology and Pharmacy, Pezzuto et al, Eds., Chapman and Hall, New York, 1993).
  • peptide mimetics The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions such as those of antibody and antigen, enzyme and substrate or scaffolding proteins.
  • a peptide mimetic is designed to permit molecular interactions similar to the natural molecule.
  • Peptide or non-peptide mimetics may be useful, for example, to competitively inhibit or otherwise bind to TLR7 or a component in the TLR7 signaling pathway or HBsAg.
  • the compounds of the present invention may be selected to interact with a target alone or single or multiple compounds may be used to affect multiple targets.
  • multiple targets may include an HBsAg and HBV.
  • one useful therapeutic combination would be an agonist of TLR7 or a component in the TLR7 signaling pathway and a nucleoside or nucleotide analog and/or antiviral cytokine
  • the target or fragment employed in screening assays may either be free in solution, affixed to a solid support, or borne on a cell surface.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing TLR7 or a component in the TLR7 signaling pathway or a fragment thereof, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • a substance identified as a modulator of target function or gene activity may be a peptide or non-peptide in nature.
  • Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
  • the designing of mimetics to a pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are unsuitable active agents for oral compositions as they tend to he quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
  • a range of sources e.g. spectroscopic techniques, x-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modeling process.
  • the three-dimensional structure of TLR7 or a component in the TLR7 signaling pathway and a ligand thereof are modeled. This can be especially useful where the TLR7 or a component in the TLR7 signaling pathway and/or its ligand change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modeling can be used to generate inhibitors which interact with the linear sequence or a three-dimensional configuration.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson ⁇ Bio/Technology. P: 19-21 , 1991).
  • one first determines the three-dimensional structure of TLR7, TLR7 ligand or HBsAg by x-ray crystallography, by computer modeling or most typically, by a combination of approaches.
  • the present invention extends to a genetic approach to up-regulating expression of a gene encoding a TLR7 or a component in the TLR7 signaling pathway.
  • General techniques can be used to up-regulate expression of TLR7 or a component in the TLR7 signaling pathway by increasing gene copy numbers or antagonizing inhibitors of gene expression.
  • nucleic acids include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g.
  • phosphorothioates phosphorodithioates, etc.
  • pendent moieties e.g. polypeptides
  • intercalators e.g. acridine, psoralen, etc.
  • chelators e.g. acridine, psoralen, etc.
  • alkylators e.g. ⁇ -anomeric nucleic acids, etc.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • Antisense polynucleotide sequences are useful in silencing transcripts of the HBsAg gene. Expression of such an antisense construct within a cell interferes with HBsAg gene transcription and/or translation. Furthermore, co-suppression and mechanisms to induce RNAi such as using short interfering RNA (siRNA) or ONA-derived RNAi (ddRNAi) may also be employed. Hence, antisense or sense molecules may be directly administered. In this latter embodiment, the antisense or sense molecules may also be formulated in a composition and then administered by any number of means to target cells or administered via an expression construct.
  • siRNA short interfering RNA
  • ddRNAi ONA-derived RNAi
  • morpholinos are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development. 7:187-195, 1997).
  • morpholinos which are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages
  • Such compounds are injected into embryos and the effect of interference with mRNA is observed.
  • the present invention employs compounds such as oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules such as those encoding a HBsAg, i.e. the oligonucleotides induce pre-transcriptional or post- transcriptional gene silencing.
  • oligonucleotides which specifically hybridize to, or have complementing with a nucleic acid molecule encoding the HBsAg.
  • the oligonucleotides may be provided directly to a cell or generated within the cell.
  • target nucleic acid and “nucleic acid molecule encoding the HBsAg gene transcript” have been used for convenience to encompass DNA encoding the HBsAg, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
  • the hybridization of a compound of the subject invention with its target nucleic acid is generally referred to as "antisense” or may be part of a complex with dicer such as a RISC.
  • hybridization means the pairing of complementary strands of oligomeric compounds.
  • the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • nucleobases complementary nucleoside or nucleotide bases
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances.
  • An antisense or RNAi compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non- specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
  • oligomeric compound refers to a polymer or oligomer comprising a plurality of monomeric units.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non- naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
  • the open reading frame (ORF) or "coding region” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is a region which may be effectively targeted. Within the context of the present invention, one region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
  • nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • linear compounds are generally preferred.
  • linear compounds may have internal nucleobase complementarity and may, therefore, fold in a manner as to produce a fully or partially double-stranded compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • oligonucleotides containing modified backbones or non-natural internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotri- esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 '-5' linkages, 2 '-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2
  • Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • the antisense or RNAi oligonucleotides may be administered by any convenient means including by inhalation, local or systemic means.
  • the present invention extends, therefore, to a pharmaceutical composition, medicament, drug or other composition including a patch or slow release formulation comprising a modulator of TLR7 activity or gene expression or the activity or gene expression of a component of the TLR7 signaling pathway.
  • Another aspect of the present invention contemplates a method comprising administration of such a composition to a subject such as for treatment or prophylaxis of an infection or other disease condition.
  • the present invention contemplates a method of making a pharmaceutical composition comprising admixing an agent of the instant invention with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients. Where multiple compositions are provided, then such compositions may be given simultaneously or sequentially. Sequential administration includes administration within nanoseconds, seconds, minutes, hours or days. Preferably, sequential administration is within seconds or minutes.
  • another aspect of the present invention contemplates a method for the treatment or prophylaxis of an infection or other disease condition in a subject, said method comprising administering to said subject an effective amount of a compound as described herein or a composition comprising same.
  • the subject is a mammal such as a human or an animal model system such as a mouse, rat, rabbit, guinea pig, hamster, zebrafish or amphibian or avian species such as a duck.
  • This method also includes providing a wild-type or mutant target gene function to a cell. This is particularly useful when generating an animal model. Alternatively, it may be part of a gene therapy approach. A target gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. If a gene portion is introduced and expressed in a cell carrying a mutant target allele, the gene portion should encode a part of the target protein.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art and any suitable vector may be used. Methods for introducing DNA into cells such as electroporation calcium phosphate co- precipitation and viral transduction are known in the art. This aspect of the present invention extends to constructs which encode ddRNAi.
  • Gene transfer systems known in the art may be useful in the practice of genetic manipulation. These include viral and non-viral transfer methods.
  • viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including papovaviruses (e.g. SV40, Madzak et al, J. Gen. Virol. 73:1533-1536, 1992), adenovirus (Berkner, Curr. Top. Microbiol. Immunol. 158:39-66, 1992; Berkner et al, BioTechniques 6; 616-629, 1988; Gorziglia and Kapikian, J Virol. 66:4407-4412, 1992; Quantin et al, Proc. Natl. Acad Sci.
  • papovaviruses e.g. SV40, Madzak et al, J. Gen. Virol. 73:1533-1536, 1992
  • adenovirus e.g. SV40, Madzak et al, J
  • herpesviruses including HSV and EBV (Margolskee, Curr. Top., Microbiol. Immunol. 158:67-95, 1992; Johnson et al, J. Virol. 66:2952-2965, 1992; Fink et al, Hum. Gene Ther. 3:11-19, 1992; Breakefield and Geller, MoI. Neurobiol. 7:339-371, 1987; Freese et al, Biochem. Pharmacol. ⁇ 0:2189-2199, 1990; Fink et al, Ann. Rev. Neurosci.
  • Non-viral gene transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor- mediated DNA transfer.
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viralvectors to particular cells.
  • the retroviral vector producer cell line can be injected into particular tissue. Injection of producer cells would then provide a continuous source of vector particles.
  • plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization and degradation of the endosome before the coupled DNA is damaged.
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is non-specific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration.
  • the compounds, agents, medicaments, nucleic acid molecules and other target antagonists or agonists of the present invention can be formulated in pharmaceutical compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18 th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. topical, intravenous, oral, intrathecal, epineural or parenteral.
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698.
  • WO 92/19195 WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.
  • the vector could be targeted to the target cells.
  • the cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent.
  • the present invention further contemplates diagnostic protocols such as to determine the presence or absence of infection or other disease condition, whether an infection has become chronic, the susceptibility of a subject to infection and/or the efficacy of a therapeutic protocol.
  • Immunological based TLR7 or a component in the TLR7 signaling pathway or HBsAg detection protocols may take a variety of forms.
  • a plurality of antibodies may be immobilized in an array each with different specificities to particular TLR7 or a component in the TLR7 signaling pathway or HBsAg or monocytes or hepatocytes comprising TLR7 or a component in the TLR7 signaling pathway or HBsAg.
  • Cells from a biopsy are then brought into contact with the antibody array and a diagnosis may be made as to the level and type of TLR7 or a component in the TLR7 signaling pathway or HBsAg elevated or down-regulated on or in the cell.
  • Another antigen-binding molecule suitably a second antibody specific to the antigen, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may be either qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including minor variations as will be readily apparent.
  • a first antibody having specificity for the antigen or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient and under suitable conditions to allow binding of any antigen present to the antibody.
  • the antigen-antibody complex is washed and dried and incubated With a second antibody specific for a portion of the antigen.
  • the second antibody has generally a reporter molecule associated therewith that is used to indicate the binding of the second antibody to the antigen.
  • the amount of labeled antibody that binds, as determined by the associated reporter molecule is proportional to the amount of antigen bound to the immobilized first antibody.
  • reporter molecule associated with the antigen-binding molecule may include the following:
  • the reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a paramagnetic ion, a lanthanide ion such as Europium (Eu 34 ), a radioisotope including other nuclear tags and a direct visual label.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by International Patent Publication No. WO 93/06121.
  • a solution containing the appropriate substrate is then added to the complex of antibody- antigen-antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
  • fluorescent compounds such as fluorescein, rhodamine and the lanthanide, europium (EU) may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labeled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent-labeled antibody is allowed to bind to the first antibody- antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest.
  • Immunofluorometric assays are well established in the art and are particularly useful for the present method.
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.
  • Monoclonal antibodies to TLR7 or a component in the TLR7 signaling pathway or HBsAg may also be used in ELISA-mediated detection of the TLR. This may be undertaken in any number of ways such as immobilizing anti-TLR7 or antibodies to a component of the TLR7 signaling pathway or anti-HBsAg antibodies to a solid support and contacting these with liver cells.
  • Labeled anti-TLR7 or anti-component of TLR7 signaling pathway or anti-HBsAg antibodies are then used to detect immobilized TLR7 or HBsAg.
  • antibodies to other liver cell surface markers are used.
  • This assay may be varied in any number of ways and all variations are encompassed by the present invention. This approach enables rapid detection and quantitation of TLR7 or a component in the TLR7 signaling pathway or HBsAg levels.
  • the method for detection comprises detecting the level of expression in a cell of a polynucleotide encoding TLR7 or a component in the TLR7 signaling pathway or HBsAg.
  • Expression of such a polynucleotide may be determined using any suitable technique.
  • a labeled polynucleotide encoding TLR7 or a component in the TLR7 signaling pathway or HBsAg may be utilized as a probe in a Northern blot of an RNA extract obtained from the cell.
  • a nucleic acid extract from the animal is utilized in concert with oligonucleotide primers corresponding to sense and antisense sequences of a polynucleotide encoding the kinase, or flanking sequences thereof, in a nucleic acid amplification reaction such as RT PCR.
  • a variety of automated solid-phase detection techniques are also appropriate.
  • VLSIPS very large scale immobilized primer arrays (trademark)) are used for the detection of nucleic acids as, for example, described by Fodor et al. ⁇ Science. 251:161-111, 1991) and Kazal et al ⁇ Nature Medicine. 2:153-159, 1996).
  • VLSIPS very large scale immobilized primer arrays
  • RNA is isolated from a cellular sample suspected of containing pre-core protein/HBeAg or TLR RNA.
  • RNA can be isolated by methods known in the art, e.g. using TRIZOL (trademark) reagent (GIBCO-BRL/Life Technologies, Gaithersburg, Md.).
  • Oligo-dT, or random-sequence oligonucleotides, as well as sequence-specific oligonucleotides can be employed as a primer in a reverse transcriptase reaction to prepare first-strand cDNAs from the isolated RNA.
  • Resultant first-strand cDNAs are then amplified with sequence-specific oligonucleotides in PCR reactions to yield an amplified product.
  • amplification of specific nucleic acid sequences by PCR relies upon oligonucleotides or "primers" having conserved nucleotide sequences wherein the conserved sequences are deduced from alignments of related gene or protein sequences, e.g. a sequence comparison of mammalian TLR genes.
  • one primer is prepared which is predicted to anneal to the antisense strand and another primer prepared which is predicted to anneal to the sense strand of a cDNA molecule which encodes a HBsAg or TLR such as TLR7.
  • Isolation, purification and characterization of the amplified TLR7 or a component in the TLR7 signaling pathway or HBsAg DNA may be accomplished by excising or eluting the fragment from the gel (for example, see references Lawn et al, Nucleic Acids Res. 2:6103, 1981; Goeddel et al, Nucleic Acids Res. 8:4057-1980), cloning the amplified product into a cloning site of a suitable vector, such as the pCRII vector (Invitrogen), sequencing the cloned insert and comparing the DNA sequence to the known sequence of TLR7 or a component in the TLR7 signaling pathway or HBsAg. The relative amounts of TLR7 or a component in the TLR7 signaling pathway or HBsAg mRNA and cDNA can then be determined.
  • the present invention further provides gene arrays and/or gene chips to screen for the up- or down-regulation of mRNA transcripts.
  • This aspect of the present invention is particularly useful in identifying conditions which result in the down- of HBsAg transcripts or up- regulation of gene transcripts of TLR7 or a component in the TLR7 signaling pathway.
  • C T The threshold cycle numbers (C T ) were determined with Sequence Detector Software (version 1.6; Applied Biosystems, Foster City, CA, USA) and transformed using the ⁇ C T or ⁇ C T methods as described by the manufacturer using GADPH as calibrator gene.
  • HBV-specific CD4 and CD8+ T-cells were enumerated by IFN- ⁇ producing CD4+ ELISPOT assays and tetramer staining for CD8+ T-cells.
  • Elispot assays The number of HBV-specific, interferon- ⁇ (IFN- ⁇ ) producing CD4 positive T cells will be determined, as described previously [Lau et. al. Gastroenterology 2002; 122:614-624]. Briefly, 96-well millilitre plates (Milipore, Bedford, MA, USA) are coated overnight with the primary antibody to human IFN- ⁇ (Mabtech, Nacka, Sweden) at 10 ⁇ g/ml at 4 0 C.
  • IFN- ⁇ interferon- ⁇
  • the plates are washed and incubated for further 2 hours with 100 ⁇ l of streptavidin-alkaline phosphatase (Mabtech, Nacka, Sweden).
  • the enzyme reaction is developed with freshly prepared nitro- blue tetrazolium chloride/bromo-chloro-inolyl-phosphate toluidine salt (NBT/BCIP, Roche Diagnostics Ltd., Lewes, UK).
  • NBT/BCIP nitro- blue tetrazolium chloride/bromo-chloro-inolyl-phosphate toluidine salt
  • the reaction is stopped with distilled water and the spots are counted using ELISPOT reader (ImmunoSpot (Registered) 3B Analyzers from Cellular Technology Ltd, Ohio, USA).
  • the number of specific spot-forming cells (SFC) per IxIO 6 PBMC is determined as the mean number of spots in the presence of an antigen minus the mean number of spots in the wells with medium only.
  • Serum HBV DNA was measured by Roche Amplicor HBV Monitor (Trade Mark) (Cobas) assay.
  • HBV replicative intermediate of the covalently closed circular (ccc) DNA was measured using real time PCR Assay as described by He et al, (Biochem Biophys Res Commun. 2002; 295:1102-7) HBV genotype determined by direct sequencing and comparison to known genotypes.
  • the reaction mixture was created with the Taqman one-step Master Mix kit (cat no: 4309169, ABI). Briefly, 5 ⁇ l of templates RNA was mixed with 1 ⁇ l of RNA free water, 10 ⁇ l of MASTER MIX, 1 ⁇ l of HBV forward and reverse primers and 1 ⁇ l of probe, 1 ⁇ l of GAPDH primers and 1 ⁇ l of probe MIX in a single reaction tube giving a total volume of 20ul.
  • Real time PCR was performed on MJ Chromo qPCR machine (New Jersey, NJ, USA) at 48 0 C for 30 mins, 95 0 C for 10 mins, 95 0 C for 15 sec and finally at 60 0 C for 1 min for a total of 40 cycles. Relative quantity of the HBV RNA is calculated by the formula of ⁇ Ct 2 .
  • TLR- 1 to 10 were compared by real-time PCR, in PBMCs collected from patients with chronic HBV infection (HBeAg +/-) and patients with resolved HBV infection HBsAg -ve (results for TLR-3, TLR-5, TLR-7 and TLR-9 are shown in Figure 2).
  • the TLR- 7 was significantly increased in patients who had resolved HBV (ie., HBsAg -ve, anti-HBs positive).
  • Patients with a natural immunity to HBV (Anti-HBs positive/ HBsAg negative) and HBeAg negative CHB had a significantly higher mRNA expression of TLR7 when compared with patients who were HBeAg positive or HBeAg negative (p 0.02) [Table 5].
  • TLR7 expression by real time PCR was also studied in serial PBMCs collected from patients treated with pegylated interferon (IFN)- ⁇ 2a at baseline, every 12 weeks during the 48-weeks treatment period and 24 weeks after the end-of-therapy (see Figure 3).
  • the baseline data is presented in Table 6).
  • TLR-7 expression was detected on week 12 and was persistently increased from week 12 to week 72 in patients who cleared HBsAg.
  • the HBV-specific CD4 and CD8+ T-cells were enumerated by IFN- ⁇ producing CD4+ ELISPOT assays and tetramer staining for CD8+ T-cells.
  • the CD4 and CD8 levels were correlated to TLR-7 was associated with an increase in HBV specific CD4+ T cells in patients with HBsAg clearance and also an increase in HBV specific CD8+ T cells in patients with HBsAg clearance.
  • HBV specific CD8+ response was also analyzed in parallel in 13 HLA-A2 patients (32.1%); 3 with HBsAg clearance, 4 with HBeAg seroconversion and 6 nonresponder; with ELISpot to Core 18-27 peptide and Surface 177-185 peptide. Enhancement in interferon- ⁇ production to Core 18-27 peptide was detected in all 3 groups beginning at week 24 of treatment. A peak in interferon- ⁇ production to Core 18-27 peptide occurred at week 24 of treatment. Patients with HBsAg clearance had a significantly higher frequency HBV specific interferon- ⁇ producing CD8+ T cells by ELISpot to Core 18-27 peptide at week 36 (mean ⁇ SEM 189.50 ⁇ 39.24 vs.
  • viral markers such as serum HBV DNA as measured by Roche Amplicor HBV Monitor (Trade Mark) (Cobas) assay and HBV genotype determined by direct sequencing, and ALT were also determined.
  • the levels of TLR-7 were measured by real-time PCR in a time course experiment (Figure 5). There was a significant increase in TLR-7 in patients who were HBsAg -ve versus HBsAg +ve.
  • TLR-7 agonist was added to the ex vivo co-cultured cells and the TLR-7 was measured by real-time PCR and western blot (Figure 6). In the presence of the TLR-7 agonist 2 ng/ ⁇ l there was an increase in TLR-7 RNA and protein expression in HBsAg +ve cells by 50% (Figure 7).
  • HBV RNA positive controls (marked Control +), was extracted RNA from liver tissue of HBsAg+/HBeAg+/ALT> 3 times the upper limit of normal (range 13-51 LVL) patient with HBV DNA>1, 000,000 copies/ml by the Amplicor assay while negative controls (Control -) contain the reaction mixture but no nucleic acids.
  • HBV cccDNa levels in PMBCs from patients that were HBsAg +ve increased over the 7 days.
  • HBV cccDNA levels in PBMCs from patients that were HBsAg +ve treated with the TLR-7 agonist were lower similar to the levels found in PBMCs from patients that were HBs Ag -ve.
  • the positive control (Control +) is DNA extracted from the liver tissue of an HBsAg+/HBeAg+/ALT> 3 times the upper limit of normal (range 13-51 LVL) patient with HBV DNA>l,000,000 copies/ml by the Amplicor assay.
  • the negative control (Control -) is the reaction mixture without nucleic acid.
  • HBV DNA levels from patients that were HBsAg +ve increased (Figure 11).
  • HBV DNA levels in PBMCs from patients that were HBsAg +ve treated with the TLR-7 agonist were lower similar to the levels found in PBMCs from patients that were HBsAg -ve.
  • TLR-7 up-regulation of TLR-7 plays an essential role in serological clearance of HBsAg in patients with chronic HBV infection.
  • the activation of adaptive immune response by TLR-7 is mediated by MyD88-dependent pathway.
  • TLR7 To examine the functional specificity of TLR7, isolated T-cells of HBV immune and HBV positive groups were incubated with WtHBV in the presence of autologous APCs for 168 hours. It was found that incubation with wtHBV resulted in suppression of TLR7 mRNA expression on the T-cells within 12 hours in both groups. However, the TLR7 mRNA expression in HBV immune group started to recover by 24 hours of incubation while in the HBV positive group, TLR7 mRNA expression remain persistently suppressed until 168 hours ( Figure 13A).
  • Interferon- ⁇ production was tested from CD4+ and CD8+ T-cells after incubation with wtHBV by ELISpot.
  • interferon- ⁇ production from CD4+ T cells by ELISpot started to increase at 24 hours and peaked at 168 hours. This increase in interferon- ⁇ production from CD4+ T cells corresponded to the increase in TLR7 mRNA expression in the T-cells.
  • the interferon- ⁇ production from CD4+ T cells was higher in the HBV immune group at 168 hours of incubation ( Figure 13B).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Communicable Diseases (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention a trait de manière générale aux domaines de la thérapie et du diagnostic d'une infection par le virus de l'hépatite B (VHB) chez une espèce animale, notamment chez les êtres humains. La présente invention concerne également des composés et des compositions, utiles dans le traitement d'une infection par le VHB chez une espèce animale telle que les êtres humains, comprenant des agents qui facilitent la clairance du VHB, et en particulier d'une infection chronique par le VHB.
EP06837256A 2005-11-09 2006-11-09 Méthodes et agents diagnostiques et thérapeutiques Withdrawn EP1951914A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US73548005P 2005-11-09 2005-11-09
PCT/US2006/043665 WO2007056527A2 (fr) 2005-11-09 2006-11-09 Methodes et agents diagnostiques et therapeutiques

Publications (2)

Publication Number Publication Date
EP1951914A2 true EP1951914A2 (fr) 2008-08-06
EP1951914A4 EP1951914A4 (fr) 2009-11-04

Family

ID=38023993

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06837256A Withdrawn EP1951914A4 (fr) 2005-11-09 2006-11-09 Méthodes et agents diagnostiques et thérapeutiques

Country Status (3)

Country Link
EP (1) EP1951914A4 (fr)
AU (1) AU2006311462A1 (fr)
WO (1) WO2007056527A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101979557B (zh) * 2010-10-28 2015-07-01 百奥迈科生物技术有限公司 一种siRNA分子及其在抗病毒药物中的应用
KR102139708B1 (ko) 2015-03-16 2020-07-31 에프. 호프만-라 로슈 아게 Tlr7 작용제 및 hbv 캡시드 조립 억제제를 사용하는 병용 치료
CN108802383B (zh) * 2018-06-21 2021-05-11 首都医科大学附属北京地坛医院 乙肝小三阳低复制期孕产期再激活监控设备

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003043572A2 (fr) * 2001-11-16 2003-05-30 3M Innovative Properties Company Procedes et compositions lies a des composes irm et a des voies de recepteurs de type toll (tlr)
WO2005020912A2 (fr) * 2003-08-25 2005-03-10 3M Innovative Properties Company Administration de composes modificateurs de la reponse immunitaire
WO2005025583A2 (fr) * 2003-09-05 2005-03-24 Anadys Pharmaceuticals, Inc. Administration de ligands tlr7 et de leurs prodrogues pour traiter l'infection au virus de l'hepatite c

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003043572A2 (fr) * 2001-11-16 2003-05-30 3M Innovative Properties Company Procedes et compositions lies a des composes irm et a des voies de recepteurs de type toll (tlr)
WO2005020912A2 (fr) * 2003-08-25 2005-03-10 3M Innovative Properties Company Administration de composes modificateurs de la reponse immunitaire
WO2005025583A2 (fr) * 2003-09-05 2005-03-24 Anadys Pharmaceuticals, Inc. Administration de ligands tlr7 et de leurs prodrogues pour traiter l'infection au virus de l'hepatite c

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DAVIES S L ET AL: "Isatoribine - Anti-hepatitis C virus drug TLR7 receptor agonist" DRUGS OF THE FUTURE, vol. 30, no. 9, September 2005 (2005-09), pages 886-891, XP002547239 ISSN: 0377-8282 *
HEMMI H ET AL: "SMALL ANTI-VIRAL COMPOUNDS ACTIVATE IMMUNE CELLLS VIA THE TLR7 MYD88-DEPENDENT SIGNALING PATHWAY" NATURE IMMUNOLOGY, NATURE PUBLISHING GROUP, GB, vol. 3, no. 2, 1 February 2002 (2002-02-01), pages 196-200, XP009000917 ISSN: 1529-2908 *
HORSMANS YVES ET AL: "Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection" HEPATOLOGY, vol. 42, no. 3, September 2005 (2005-09), pages 724-731, XP002547240 ISSN: 0270-9139 *
HUI CHEE-KIN ET AL: "Upregulation of toll-like receptor 7 is essential for serological clearance of hepatitis B surface antigen in chronic hepatitis B virus (HBV) infection" HEPATOLOGY, vol. 42, no. 4, Suppl. 1, October 2005 (2005-10), pages 707A-708A, XP002547237 56TH ANNUAL MEETING OF THE AMERICAN-ASSOCIATION-FOR-THE-STUDY-OF-LIVE R-DISEASES; SAN FRANCISCO, CA, USA; NOVEMBER 11 -15, 2005 ISSN: 0270-9139 *
ISOGAWA MASANORI ET AL: "Toll-like receptor signaling inhibits hepatitis B virus replication in vivo." JOURNAL OF VIROLOGY JUN 2005, vol. 79, no. 11, June 2005 (2005-06), pages 7269-7272, XP002547238 ISSN: 0022-538X *
LEE ET AL: "Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: Activation of Toll-like receptor 7" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 100, no. 11, 27 May 2003 (2003-05-27), pages 6646-6651, XP002307483 ISSN: 0027-8424 *
See also references of WO2007056527A2 *

Also Published As

Publication number Publication date
EP1951914A4 (fr) 2009-11-04
AU2006311462A1 (en) 2007-05-18
WO2007056527A3 (fr) 2009-05-14
WO2007056527A2 (fr) 2007-05-18

Similar Documents

Publication Publication Date Title
US8273527B2 (en) Viral variants
AU2009212828A1 (en) Therapeutic, Prophylactic and Diagnostic Agents
US20100003262A1 (en) Therapeutic, prophylactic and diagnostic agents for hepatitis b
US20070043512A1 (en) Therapeutic and prophylactic compositions and uses therefor
EP1951914A2 (fr) Methodes et agents diagnostiques et therapeutiques
AU2005243607B2 (en) Therapeutic, prophylactic and diagnostic agents for hepatitis B
WO1997004091A1 (fr) Nouveaux ligands de recepteurs et sequences genetiques codant ces ligands
US20050008643A1 (en) Diagnostic methods and agents
WO2004099412A1 (fr) Molecules d'acides nucleiques exprimees de facon differentielle chez des animaux presentant des troubles du comportement
US8895698B2 (en) Binding partners of antibodies specific for dendritic cell antigens
US20090054307A1 (en) Prophylactic and therapeutic agents and uses therefor
WO2006029462A1 (fr) Molecule d'acide nucleique exprimee de façon differentielle dans un systeme de modele comportemental de souris et utilisations de ladite molecule
AU2002318961A1 (en) Diagnostic methods and agents
AU2005313857A1 (en) Binding partners of antibodies specific for dendritic cell antigens

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20080605

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR MK RS

RIN1 Information on inventor provided before grant (corrected)

Inventor name: ZHANG, HAI YING,THE UNIVERSITY OF HONG KONG

Inventor name: HUI, CHEE-KIN,THE UNIVERSITY OF HONG KONG

Inventor name: LAU, GEORGE KA-KIT,THE UNIVERSITY OF HONG KONG

R17D Deferred search report published (corrected)

Effective date: 20090514

A4 Supplementary search report drawn up and despatched

Effective date: 20091005

RIC1 Information provided on ipc code assigned before grant

Ipc: C12Q 1/68 20060101ALI20090925BHEP

Ipc: C12Q 1/70 20060101AFI20070720BHEP

17Q First examination report despatched

Effective date: 20100120

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100602