WO2013053008A2 - Molécules liant clec9a - Google Patents

Molécules liant clec9a Download PDF

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Publication number
WO2013053008A2
WO2013053008A2 PCT/AU2012/001233 AU2012001233W WO2013053008A2 WO 2013053008 A2 WO2013053008 A2 WO 2013053008A2 AU 2012001233 W AU2012001233 W AU 2012001233W WO 2013053008 A2 WO2013053008 A2 WO 2013053008A2
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Prior art keywords
actin
cells
fragment
polypeptide
compound
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PCT/AU2012/001233
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English (en)
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WO2013053008A3 (fr
Inventor
Peter Malcolm Colman
Ross Leon Coppel
Peter Edward Czabotar
Mireille Hanna Lahoud
Nicos Anthony Nicola
Antonia Natalie POLICHENI
Kenneth Douglas Shortman
Kirsteen McInnes TULLETT
Jian-Guo Zhang
Jacob Baum
Wilson Wong
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The Walter And Eliza Hall Institute Of Medical Research
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Publication of WO2013053008A2 publication Critical patent/WO2013053008A2/fr
Publication of WO2013053008A3 publication Critical patent/WO2013053008A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present invention relates to the identification of molecules which bind the dendritic cell marker known as Clec9A.
  • the present invention provides new compounds for targeting therapeutic agents such as antigens to dendritic cells.
  • methods of modulating a humoral and/or T-cell mediated immune response to the antigen methods of delivery of a cytotoxic agent to dendritic cells thereof involved in diseased states, methods of modulating the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, and methods of modulating antigen recognition, processing and/or presentation, as well as immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • DCs Dendritic cells
  • PAMPs pathogen-associated molecular patterns
  • DAMPs damaged cell-associated molecular patterns
  • the CD8 + DC (Shortman and Heath, 2010) are especially efficient at the uptake and processing of material from dead cells, and in "cross-presenting" such exogenous antigenic material on class I HC (Shortman and Heath, 2010).
  • Human CD141 + (BDCA-3 + ) DCs have been identified as the lineage and functional equivalents of the mouse CD8 + DCs (Jongbloed et al., 2010; Bachem et al., 2010; Crozat et al., 2010; Poulin et al., 2010), thus they would be expected to have a particular pattern of PAMP and DAMP receptors related to these functions.
  • Clec9A also called DNGR-1 (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008; Sancho et al., 2009; WO 2009/026660; WO 2009/137871; WO 2010/108215) which binds to dead cells.
  • Clec9A also regulates the cross-presentation of dead cell-associated antigens in a Syk-dependent manner (Sancho et al., 2009).
  • Clec9A has been found to be an especially effective target for delivery of antigens to DCs, so promoting immune responses (Caminschi et al., 2008; Sancho et al., 2008; Idoyaga et al., 2011; Lahoud et al., 2011). This has great promise as a strategy for enhancing the effectiveness of vaccines.
  • the present inventors have identified further ligands of Clec9A. These ligands can be used to target therapeutic agents or detectable labels to Clec9A expressing. cells such as dendritic cells.
  • the present invention provides a polypeptide complex conjugated to a therapeutic agent or a detectable label, wherein the polypeptide complex comprises
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A.
  • the first polypeptide can be selected from a large variety of proteins which bind actin. Examples include, but are not limited to, spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titin, myosin, tubulin, catenin, keratin, cytokeratin, nestin, larnin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin.
  • the first polypeptide comprises
  • the second polypeptide consists of actin, or a fragment thereof which binds the actin binding domain.
  • the actin is cytoskeletal actin or muscle actin.
  • the actin is filamentous actin or a filamentous fragment thereof.
  • the second polypeptide comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 26 to 40; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 26 to 40; and/or
  • the actin is cytoskeletal actin.
  • the second polypeptide comprises
  • the Clec9A comprises
  • the polypeptide complex further comprises Clec9A.
  • the complex when bound to Clec9A, further comprises RNF41 bound to the Clec9A.
  • the RNF41 comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 21 to 25; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 21 to 25; and/or
  • Suitable therapeutic agents include, but are not limited to, an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
  • the antigen can be any molecule that induces an immune response in an animal. Examples include, but are not limited to, a cancer antigen, a self-antigen, an allergen, and or an antigen from a pathogenic and/or infectious organism.
  • the antigen from a pathogenic and/or infectious organism can be from, but not limited to, Plasmodium falciparum or Plasmodium vivax.
  • the first and second polypeptides are separate polypeptide chains which form the complex at least by the actin binding domain binding the actin.
  • first and second polypeptides form part of a single polypeptide chain.
  • the first and second polypeptides are separated by a peptide linker.
  • the present invention provides a compound that binds a polypeptide complex which comprises:
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec A, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A.
  • the compound is not an antibody which binds Clec9A alone or a fragment of Clec9A which binds Clec9A alone such as a soluble fragment.
  • the compound is a polypeptide.
  • the compound is an antibody or antigen-binding fragment thereof.
  • antibodies or antigen-binding fragment thereof include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the actin is filamentous actin or a filamentous fragment thereof.
  • the compound of the above aspect is detectably labelled.
  • the present invention provides a composition comprising a polypeptide complex of the invention, or a compound of the invention, and a pharmaceutically acceptable carrier.
  • composition further comprises an adjuvant.
  • the present invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject at least one of
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • the immune response to an antigen is induced and/or enhanced.
  • the immune response is modulated by enhancing a helper T-cell response.
  • the immune response is modulated by the activation of CD4 + and/or CD8 + T-cells.
  • the immune response is modulated by enhancing B-cell antibody production.
  • antibodies produced include, but are not necessarily limited to, IgGl, IgG2b, IgG2c, IgG3, IgG4 ⁇ IgM, IgAl, IgA2, IgE and/or IgD antibody isotypes.
  • the immune response is modulated by generating a memory response.
  • the subject is administered with a compound comprising the antigen.
  • an immune response to a self-antigen or allergen is reduced.
  • the immune response is modulated by suppressing a T-cell response and/or a B-cell antibody response.
  • the present invention provides a method of modulating an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to at least one of
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • composition of the invention iv) a composition of the invention, and administering said cells to the subject.
  • the cells have been isolated from the subject.
  • a humoral and/or T-cell mediated response is modulated.
  • naive CD8 + T-cell activation, and/or naive CD4 + T- cell activation is modulated.
  • the humoral response comprises the production of IgGl, IgG2b, IgG2c, IgG3, IgG4, IgM, IgAl, IgA2, IgE, and/or IgD antibody isotypes.
  • the humoral response at least comprises the production of IgGl antibody isptype.
  • the dendritic cell is an animal dendritic cell or precursor of an animal dendritic cell. More preferably, the dendritic cell is a human dendritic cell. Even more preferably, the human dendritic cell is Necl-2+, HLA DR+ and/or BDCA-3+.
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • the infection is a Plasmodium sp., such as Plasmodium falciparum or Plasmodium vivax, infection.
  • the present invention provides a method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding.
  • the present invention provides a method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of i) a polypeptide complex comprising
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is increased.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased.
  • the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is increased.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased.
  • the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • diseases include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
  • actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A,
  • the present invention provides a method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
  • actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the compound is an antibody or antigen-binding fragment thereof.
  • examples include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the compound is detectably labelled.
  • the method is performed in vivo on a subject. In an alternate embodiment, the method is performed in vitro on a sample obtained from a subject.
  • diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury .
  • GVHD graft versus host disease
  • SIRS systemic inflammatory reaction syndrome
  • the present invention provides a method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
  • the cell in step i) is in vivo. In an alternate embodiment, the cell in step i) is in vitro.
  • the therapy is administered to a subject.
  • the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells.
  • the subject has cancer or an infection.
  • step ii) is performed on a sample obtained from a subject.
  • the therapy can be any type of procedure. Examples include, but are not limited to, drug therapy or radiotherapy.
  • the present invention provides a method of distinguishing between an early stage apoptotic cell and a late stage apoptotic cell, necrotic cell or dead cell, the method comprising
  • actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
  • the compound binding to the polypeptide indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the present invention provides a method of enriching dendritic cells, or a subset or precursors thereof, from a sample comprising
  • the cells obtained from step ii) are administered to a subject.
  • the cells are administered to treat and or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy.
  • the present invention provides a method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising
  • the present invention provides a method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
  • the polypeptide complex or compound is detectably labelled.
  • a detectably labelled secondary antibody that binds the polypeptide complex or compound.
  • the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, Clec9A, XCRI and BDCA3.
  • the dendritic cells are human dendritic cells that express one or more of the following markers, Necl-2, HLADR and BDC A3.
  • the dendritic cells are murine dendritic cells that express one or more of the following markers, CD24, Necl-2, CD 11c, Clec9A and CD8.
  • the precursor dendritic cells are intermediate or late precursor
  • dendritic cells which are capable of differentiating into dendritic cells in culture and/or on transfer into irradiated recipients.
  • actin or fragment thereof is bound to the actin binding domain, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A, and
  • the compound binding to the polypeptide complex indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying or is dead.
  • the compound is not an antibody which binds Clec9A, Clec9A per se or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the present inventors have also identified two tryptophan residues which are important for the biological function of Clec9A.
  • the present invention provides a compound which
  • ii) binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex which comprises:
  • actin or fragment thereof is bound to the actin binding domain, or
  • the compound is a polypeptide.
  • the compound is an antibody or antigen-binding fragment thereof.
  • antibodies or antigen-binding fragment thereof include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the compound does not affect the binding of RNF41 to Clec9A.
  • composition comprising a compound of the above aspect, and a pharmaceutically acceptable carrier.
  • present invention provides a method of reducing an immune response in a subject, the method comprising administering to the subject a compound of the above aspect and/or a composition of the above aspect.
  • the present invention provides a method of reducing an immune response to an antigen in a subject, the method comprising exposing dendritic cells or precursors thereof in vitro to a compound of the above aspect and or a composition of the above aspect, and administering said cells to the subject.
  • the present invention provides a method of treating and/or preventing a disease involving dendritic cells or precursors thereof, the method comprising administering to the subject a compound of the above aspect and/or a composition of the above aspect.
  • the disease is an autoimmune disease or inflammation.
  • the present invention provides a method of reducing the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
  • the present invention provides a method of reducing the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
  • the present invention provides a method of reducing an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering a compound of the above aspect and/or a composition of the above aspect.
  • a compound of the above aspect and/or a composition of the above aspect for use in at least one of 3 i) reducing an immune response in a subject,
  • the present invention provides an isolated and/or exogenous polynucleotide encoding
  • polypeptide complex of the invention wherein the first polypeptide and the second polypeptide form part of a single polypeptide chain
  • a vector comprising a polynucleotide of the invention.
  • the vector is an expression vector.
  • the present invention provides a host cell comprising a polynucleotide of the invention, and/or a vector of the invention.
  • the cell can be any cell type such as, but not limited to, a bacterial, yeast, animal, insect or plant cell.
  • the present invention provides an enriched population of dendritic cells and/or precursors thereof, obtained by a method of the invention.
  • the present invention provides an expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof of the invention.
  • composition comprising a polynucleotide of the invention, a vector of the invention, a host cell of the invention, and/or a cell population of the invention, and a pharmaceutically acceptable carrier.
  • the present invention provides a crystal of the C-type lectin- like domain of human Clec9A.
  • the present invention provides a set of atomic coordinates, or subset thereof, provided in Appendix I.
  • the present invention provides a computer-readable medium having recorded thereon data representing the atomic coordinates, or subset thereof, provided in Appendix I and/or a model produced using the atomic coordinates.
  • the present invention provides a computer-assisted method of identifying a compound that binds Clec9A, the method comprising
  • the method further comprises synthesising or obtaining an identified candidate compound and determining if the compound binds Clec9A.
  • the present invention provides a computer-assisted method for identifying a compound which binds a polypeptide complex which comprises
  • the method comprises the steps of:
  • the method further comprises synthesising or obtaining a selected candidate compound and detennining if the compound binds the polypeptide complex.
  • the subset at least comprises a structure defined by the atomic coordinates provided in Appendix II.
  • the present invention provides a method of identifying a compound which binds a polypeptide complex comprising
  • the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, the method comprising a) exposing the polypeptide complex to a binding partner which binds the polypeptide complex, and a candidate compound,
  • the present invention provides a method of identifying a compound which binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO: 1 , the method comprising
  • the present inventors have also identified that Clec9A can bind actin in the absence of a polypeptide which comprises an actin binding domain.
  • the present invention provides an isolated actin or a fragment thereof conjugated to a therapeutic agent or a detectable label, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain.
  • the actin or fragment thereof is filamentous actin (F- actin) or a filamentous fragment thereof.
  • the actin is cytoskeletal actin or muscle actin.
  • the actin is cytoskeletal actin.
  • the second polypeptide comprises
  • the Clec9A comprises
  • the antigen can be any molecule that induces an immune response in an animal. Examples include, but are not limited to, a cancer antigen, a self-antigen, an allergen, and/or an antigen from a pathogenic and/or infectious organism.
  • the conjugated actin or fragment thereof is encapsulated in, or exposed on the surface of, a liposome.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the immune response to an antigen is induced and/or enhanced.
  • the immune response is modulated by enhancing a helper T-cell response.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9 A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the cells have been isolated from the subject.
  • naive CD8 + T-cell activation, and/or naive CD4 + T- cell activation is modulated.
  • the method comprises administering a conjugated aetin or fragment thereof of the invention comprising the cytotoxic agent, drug and/or pharmacological agent.
  • diseases involving dendritic cells or precursors thereof include, but are not limited to, cancer, an infection, an autoimmune disease or an allergy.
  • the autoimmune disease is lupus erythematosus.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the present invention provides a method of modulating the antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the present invention provides a method of modulating an immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells, in a subject, the method comprising administering to the subject at least one of i) actin or a fragment thereof, wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain,
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is increased. In an alternate embodiment, the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, is decreased.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is increased.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased.
  • the immune response to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is increased.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased.
  • actin or a fragment thereof wherein the actin or fragment thereof is capable of binding Clec9A, and wherein the actin or fragment thereof is not bound to a polypeptide comprising an actin binding domain
  • the present invention provides a method of diagnosing, prognosing and/or monitoring the status of a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, the method comprising
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the compound is an antibody or antigen-binding fragment thereof.
  • examples include, but are not limited to, a monoclonal antibody, humanized antibody, single chain antibody, diabody, triabody, or tetrabody.
  • the compound is detectably labelled.
  • the method is performed in vivo on a subject. In an alternate embodiment, the method is performed in vitro on a sample obtained from a subject.
  • diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not limited to, graft versus host disease (GVHD), an autoimmune disease, an infection, a neurodegenerative disease, systemic inflammatory reaction syndrome (SIRS), cancer and injury.
  • GVHD graft versus host disease
  • SIRS systemic inflammatory reaction syndrome
  • the present invention provides a method of monitoring the effectiveness of a therapy for killing a cell, the method comprising
  • the cell in step i) is in vivo. In an alternate embodiment, the cell in step i) is in vitro.
  • the therapy is administered to a subject.
  • the subject is suffering from a disease associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells.
  • the subject has cancer or an infection.
  • step ii) is performed on a sample obtained from a subject.
  • the compound binding to the actin or a fragment thereof indicates that the cell is a late stage apoptotic cell, necrotic cell or dead cell.
  • the compound is not an antibody which binds Clec9A, or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the cells obtained from step ii) are administered to a subject.
  • the cells are administered to treat and/or prevent a disease selected from cancer, an infection, an autoimmune disease or an allergy.
  • the present invention provides a method of detecting dendritic cells, or a subset or precursors thereof, in a sample comprising
  • the present invention provides a method of detecting dendritic cells, or a subset or precursor thereof, in a subject comprising
  • the dendritic cells express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, Clec9A, XCRI and BDCA3.
  • the dendritic cells are human dendritic cells that express one or more of the following markers, Necl-2, HLADR and BDCA3.
  • the dendritic cells are murine dendritic cells that express one or more of the following markers, CD24, Necl-2, CDl lc, Clec9A and CD8.
  • the precursor dendritic cells are intermediate or late precursor dendritic cells which are capable of differentiating into dendritic cells in culture and/or on transfer into irradiated recipients.
  • the present invention provides a method of detecting a cell with a disrupted cell membrane, a cell infected with a pathogen, a dying cell or a dead cell, the method comprising
  • the compound binding to the actin or fragment thereof indicates that the cell has a disrupted cell membrane, is infected with a pathogen, is dying, or is dead.
  • the compound is not an antibody which binds Clec9A, Clec9A per se or a fragment of Clec9A which binds Clec9A such as a soluble fragment.
  • the present invention provides an enriched population of dendritic cells and/or precursors thereof, obtained by a method of the invention.
  • the present invention provides an expanded dendritic cell population, and/or precursors thereof, obtained by culturing an enriched population of dendritic cells and/or precursors thereof of the invention.
  • composition comprising a cell population of the invention, and a pharmaceutically acceptable carrier.
  • the present invention provides a method of identifying a compound which binds actin or a fragment thereof, the method comprising
  • the present invention provides a kit comprising one or more of a conjugated actin or fragment thereof of the invention, a cell population of the invention, and a composition of the invention.
  • FIG. 1 A schematic representation of the endogenous and recombinant soluble mClec9A proteins.
  • the endogenous protein includes the Clec9A extracellular domains, the transmembrane (TM) and the cytoplasmic (Cyto) domains.
  • mClec9A-ecto which consists of the full Clec9A ectodomain, a FLAG tag and a biotinylation consensus sequence (predicted mol wt of 27 kDa);
  • mClec9A-CTLD which consists of the Clec9A-CTLD, FLAG tag and biotinylation consensus sequence (predicted mol wt of 19.7 kDa);
  • mClec9A-stalk which consists of the Clec9A-stalk region, FLAG tag and biotinylation consensus sequence (predicted mol wt of 12 kDa).
  • (C) Viable or freeze-thawed mouse fibroblasts (3T3 cell line) were incubated with biotinylated mClec9A or hCLEC9A ectodomains (solid line), or with biotinylated control (Cire-ecto, dashed line). Binding in (C)-(F) was detected using SA-PE and flow cytometry.
  • Control untreated MEFs (viable) and ABT-737 treated MEFs (2.5 ⁇ ABT- 737 for 16 h; late apoptotic) were incubated in PBS alone or in the presence of DNasel, RNaseA, protease K or trypsin. Cells were washed extensively to remove nucleases and proteases, then incubated with biotinylated mClec9A-ecto (solid line), or biotinylated Cire-ecto as a control (dashed line). 80% of the untreated MEFs were viable based on normal FSC and PI exclusion, whereas 97% of the ABT-737 treated MEFs were dead based on reduced FSC and high PI staining.
  • Clec9A-ecto binding to the ⁇ N-4 + muscle actin was significantly greater than binding to ⁇ N-4 alone (*** p ⁇ 0.0001) or to muscle actin alone (*** p ⁇ 0.0005).
  • Clec9A-ecto binding to ⁇ N + muscle actin was significantly greater than binding to ⁇ N alone or to muscle actin alone (*** pO.OOOl);
  • Clec9A-ecto binding to ⁇ N-4 + platelet actin and to ⁇ N + platelet actin was significantly greater than binding to ⁇ N or ⁇ N-4 alone, respectively, or to platelet actin alone (*** p ⁇ 0.0001).
  • C Clec9A binding to dead cells is inhibited by pre-incubation of Clec9A with GST-tagged erythrocytic spectrin ⁇ N plus actin. Pre-associated spectrin ⁇ N plus platelet actin, was incubated with mClec9A-ecto (0.5 h, 21°C), before incubation with freeze-thawed 293F cells.
  • Clec9A-ecto binding to the spectrin ⁇ N-4 + actin was significantly greater than binding to ⁇ N-4 alone (*** pO.0001) or to actin alone (*** p ⁇ 0.0005).
  • Clec9A-ecto binding to the spectrin ⁇ N + actin was significantly greater than binding to ⁇ N or to actin alone (*** p ⁇ 0.0001);
  • Clec9A- ecto binding to a-actinin- 1 N + actin was significantly greater than binding to a- actinin- 1 N or to actin alone (* * * p ⁇ 0.0001 ).
  • human 293F cells were freeze-thawed to induce membrane disruption then incubated with the original hCLEC9A-CTLD, or hCLEC9A-CTLD (S225D) at 5 ⁇ g/ml (solid lines). Binding was detected using FITC-conjugated anti- FLAG mAb, and flow cytometry. Staining with secondary reagents alone is demonstrated (dashed line). This confirmed that the hCLEC9A-CTLD (S225D) maintained binding activity to dead cells.
  • FIG. 1 Schematic representation of RNF41 protein.
  • A, B Mouse Clec9A-ecto binding to GST-RNF41.
  • GST-tagged RNF41-C- terminal domain (CTD), RNF41-N -terminal domains (RBCC) and GST control were coated onto ELISA plates at 10 ⁇ g/ml.
  • FLAG-tagged mClec9A-ecto and mClecl2A- ecto were incubated on the ELISA plates at concentrations from ⁇ to 0.04 g/ml.
  • Clec binding to RNF41 was detected using anti-FLAG-HRP. Cumulative data of 3 experiments is shown, demonstrating the means of absorbance values.
  • C, D Human CLEC9A-ecto binding to GST-RNF41.
  • GST-tagged RNF41-C- terminal domain (CTD), RNF41-N-terminal domains (RBCC) and GST control were coated onto ELISA plates at ⁇ g/ml.
  • FLAG-tagged wildtype CLEC9A (hCLEC9A- ecto) and mutant CLEC9A (W131A, W227A, represented as hCLEC9A-ecto (W->A)) were incubated on the ELISA plates at concentrations from 10 ⁇ g/ml to 0.12 ⁇ / ⁇ 1. CLEC9A binding to RNF41 was detected using anti-FLAG-HRP. Data is shown for 1 representative experiment.
  • mClec9A-ecto 5 ⁇ g ml
  • control mClecl2A-ecto
  • platelet actin 25 ⁇ g/ml
  • GST-tagged ABD 25ug/ml
  • eythrocytic spectrin spectrin ⁇ N
  • non erthyroctic spectrin spectrin ⁇ N
  • a-actinin-1 a-actinin-1 N
  • Wildtype hCLEC9A-ecto, mutant hCLEC9A-ecto (W131A, W227A) and control mClecl2A-ecto ⁇ g/ml) were pre-incubated with platelet actin (25 ⁇ g ml) complexed with GST-tagged ABD (25 ⁇ g ml) of eythrocytic spectrin (spectrin ⁇ N), non erthyroctic spectrin (spectrin ⁇ N) and a-actinin-1 (a-actinin-1 N).
  • CLEC-ABD-Actin complexes were incubated onto ELISA plates and binding to RNF41 was detected using anti-FLAG- HRP. Cumulative data of 2 experiments is shown, demonstrating the means of absorbance values.
  • Ectodomains of mClec9A (5 ⁇ ) were incubated with various concentrations of actin in the form of preformed muscle actin filaments, then centrifuged.
  • SEQ ID NO: 9 Open reading frame encoding human Clec9A.
  • SEQ ID NO: 10 Open reading frame encoding murine Clec9A.
  • SEQ ID NO: 12 Open reading frame encoding rhesus monkey Clec9A.
  • SEQ ID NO: 16 Open reading frame encoding rat Clec9A.
  • SEQ ID NO: 17 Soluble mouse Clec9A including stalk.
  • SEQ ID NO:20 Soluble human Clec9A without stalk.
  • SEQ ID NO:21 Human RNF41 RING(Really Interesting New Gene) finger protein 41 (isoform 1).
  • SEQ ID NO:26 Human muscle alpha actin.
  • SEQ ID NO:27 Human cytoplasmic beta actin.
  • SEQ ID NO:28 Human cytoplasmic gamma actin.
  • SEQ ID NO:31 Mouse cytoplasmic gamma actin.
  • SEQ ID NO:34 Chimpanzee cytoplasmic gamma actin.
  • SEQ ID NO:36 Rat cytoplasmic beta actin.
  • SEQ ID NO:37 Rat cytoplasmic gamma actin.
  • SEQ ID NO:38 Horse muscle alpha actin.
  • SEQ ID NO:39 Horse cytoplasmic beta actin.
  • SEQ ID NO:41 Human erythrocytic spectrin, alpha 1 (elliptocytosis 2 or SPTA1).
  • SEQ ID NO:42 Human non-erythrocytic spectrin, alpha 1 (alpha-fodrin or SPTANl)
  • SEQ ID NO:43 Human non-erythrocytic spectrin, alpha 1 (alpha-fodrin or SPTANl) 30 (isoform 2)
  • SEQ ID NO:46 Human non-erythrocytic beta spectrin 1, or SPTBN1 (isoform 1)
  • SEQ ID NO:47 Human non-erythrocytic beta spectrin 1
  • SPTBN1 isoform 2
  • SEQ ID NO-.48 Human non-erythrocytic beta spectrin 2
  • SEQ ID NO:49 Mouse spectrin alpha 1 , or SPNA 1.
  • SEQ ID NO:50 Mouse spectrin alpha 2, or SPNA2.
  • SEQ ID NO:51 Mouse spectrin beta 1 , or SPNB 1.
  • SEQ ID NO:54 Mouse spectrin beta 3, or SPNB3.
  • SEQ ID NO:56 Mouse spectrin beta 5, or SPNB5.
  • SEQ ID NO:57 Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 1).
  • SEQ ID NO:58 Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 2).
  • SEQ ID NO:59 Chimpanzee erythrocytic alpha 1 spectrin, (elliptocytosis 2 or SPTA1) (isoform 3).
  • SEQ ID NO:60 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 1).
  • SEQ ID NO:61 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 2).
  • SEQ ID NO:62 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 3).
  • SEQ ID NO:63 Chimpanzee erythrocytic beta spectrin, or SPTB (isoform 4).
  • SEQ ID NO:64 Chimpanzee non-erythrocytic beta spectrin 1, or SPTBNl (isoform 1).
  • SEQ ID NO:65 Chimpanzee non-erythrocytic beta spectrin 2, or SPTBN2 (isoform 1).
  • SEQ ID NO:66 Horse erythrocytic alpha spectrin 1 (elliptocytosis 2 or SPTA1).
  • SEQ ID NO:67 Horse erythrocytic beta spectrin, or SPTB.
  • SEQ ID NO:68 Horse non-erythrocytic beta spectrin 1 , or SPTBN 1.
  • SEQ ID NO:73 Human filamin isoform 1.
  • SEQ ID NO:74 Human dystrophin isoform 1.
  • SEQ ID NO:75 Human actinin.
  • SEQ ID NO:76 Human alpha actinin 2.
  • SEQ ID NO:78 Human calponin-1 actin binding domain.
  • SEQ ID NO:79 Human calponin-2 actin binding domain.
  • SEQ ID NO:80 Human calponin-3 actin binding domain.
  • SEQ ID NO:81 Human plectin isoform 1 actin binding domain.
  • SEQ ID NO:83 Human dystrophin isoform 1 actin binding domain.
  • SEQ ID NO:86 Human alpha actinin 1 (isoform a) actin binding domain.
  • SEQ ID NO:88 Human spectrin beta chain brain 1 isoform 2 actin binding domain.
  • SEQ ID NO:89 Human spectrin beta chain erythrocyte isoform b actin binding domain.
  • the term about refers to +/- 20%, more preferably +/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value.
  • cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells includes situations where the cell can have, where possible, one or more of these features.
  • the cell could have a disrupted membrane, be infected with a pathogen and be dying.
  • eyely stage apoptotic cell or “early stage apoptotic cells” includes cells that are AnnexinV + and ⁇ .
  • the term "dead cell” or “dead cells” refers to cell(s) that has passed a point of no return in the death prbcess and which changes cannot be reversed. The cell(s) may have died through apoptosis or necrosis.
  • pathogen includes any organism which can infect a cell. Examples include, but are not limited to, viruses, protozoa and bacteria.
  • the term "uptake and/or clearance" of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof refers to the removal of cellular material, such a proteins or fragments thereof, of the cells.
  • dendritic cells are responsible, at least in part, for the uptake and/or clearance of the cells.
  • the dendritic cells are Clec9A + .
  • the term “surrounding cells” refers to cells in close proximity to one or more of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells.
  • treating include administering a therapeutically effective amount of a molecule of the invention (such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein) useful for the invention sufficient to reduce or eliminate at least one symptom of the specified condition.
  • a molecule of the invention such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein
  • preventing include administering a therapeutically effective amount of a molecule of the invention (such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein) useful for the invention sufficient to stop or hinder the development of at least one symptom of the specified condition.
  • a molecule of the invention such as a polypeptide complex, actin or fragment thereof, conjugate or compound as defined herein
  • diagnosis refers to the detection of a disease.
  • the term “prognosing” or variations thereof refers to an assessment of the future outcome of a disease.
  • monitoring the status refers to determining the stage of a disease.
  • the status can be determined before, during and/or after a subject has been administered with a treatment for the disease.
  • Clec9A refers to a polypeptide which comprises; i) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 8;
  • the polypeptide is at least expressed on a subset of dendritic cells.
  • the dendritic cells also express one or more of the following markers, CD8, CD24, Necl-2, CD1 lc, HLADR, XCR1, Clec9A and BDCA3.
  • markers CD8, CD24, Necl-2, CD1 lc, HLADR, XCR1, Clec9A and BDCA3.
  • soluble fragments include those with the CTLD domain (for instances fragments comprising sequences as provided in SEQ ID NOs 17 to 20).
  • Clec9A has also been referred to in the art as 5B6 and HEEE9341.
  • tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l refer to the relative position of the amino acid compared to surrounding amino acids.
  • a polypeptide of the invention may have deletional or substitutional mutations which alters the relative positioning, of the amino acid when aligned against, for example, SEQ ID NO:l.
  • tryptophan residues 131 and 227 of full length human Clec9A correspond to residues 65 and 161 respectively of the fragment provided as SEQ ID NO: 18.
  • the position of the tryptophan residues may vary between orthologs of Clec9A.
  • the phrase "tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l" encompasses tryptophan residues 155 and 250 respectively of SEQ ID NO:2.
  • the polypeptide comprises the defined amino acid at the nominated residue number.
  • the "sample” can be any biological material suspected of having cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • the “sample” can be any biological material suspected of having Clec9A + dendritic cells Examples include, but are not limited to, blood, for example, whole peripheral blood, cord blood, foetus blood, bone marrow, plasma, serum, urine, cultured cells, saliva or urethral swab, lymphoid tissues, for example tonsils, peyers patches, appendix, thymus, spleen and lymph nodes, and any biopsy samples taken for routine screening, diagnostic or surgical reason such as tumour biopsy or bioposy of inflamed organs/ tissues.
  • the sample may be tested directly or may require some form of treatment prior to testing.
  • a biopsy sample may require homogenization to produce a cell suspension prior to testing.
  • a reagent such as a buffer
  • the mobilizing reagent may be mixed with the sample prior to placing the sample in contact with a compound as defined herein.
  • conjugate As used herein, the terms “conjugate”, “conjugated” or variations thereof are used broadly to refer to any form to covalent or non-covalent association between a compound useful for the invention and a therapeutic agent or a detectable label, or to placing a compound useful for the invention and a therapeutic agent or detectable label in close proximity to each other such as in a liposome.
  • the term "immune response” refers to an alteration in the reactivity of the immune system of a subject in response to an antigen and may involve antibody production, induction of cell-mediated immunity, complement activation and/or development of immunological tolerance.
  • the term "subject” preferably relates to an animal. More preferably, the subject is a mammal such as a human, dog, cat, horse, cow, or sheep. Most preferably, the subject is a human.
  • the present inventors have identified protein complexes which bind Clec9A.
  • the protein complexes comprise
  • actin or fragment thereof is bound to the actin binding domain.
  • present inventors have also identified that when bound to the protein complex, Clec9A is still capable of binding RNF41.
  • Clec9A binds actin without the need for an actin binding protein.
  • ABS actin binding domain
  • proteins include, but are not limited to, spectrin, actinin, calponin, plectin, filamin, dystrophin, utrophin, fimbrin, ankyrin, tropomodulin, troponin, tropomyosin, cofilin, gelsolin profilin, titiii, myosin, tubulin, catenin, keratin, cytokeratin, nestin, lamin, kinesin or dynein, or an actin binding fragment thereof, or a variant thereof which binds actin.
  • Actin binding domains relevant to the invention comprise a calponin homology domain (CHD).
  • CHD calponin homology domain
  • the ABD is typically about 100 residues.
  • Three major groups of ABD proteins have been recognized on the basis of sequence analysis, namely i) proteins containing a single N-terminal ABD including calponin, Vav, IQGAP and Cdc24, ii) proteins with an F-actin binding domain composed of two ABDs in tandem including spectrins, dystrophin, filamins and plakins, and iii) proteins of the fimbrin plastin family which also contain two ABDs in tandem (Gimona et al., 2002; Korenbaum and Rivero, 2002; Banuelos et al., 1998; Stradal et al., 1998).
  • a first polypeptide as defined herein can comprise one, two or possibily more calponin homology domains.
  • an actin binding domain relevant to the invention comprises a) an amino acid sequence as provided in any one of SEQ ID NOs 78 to 89; b) an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 78 to 89.
  • the first polypeptide comprises
  • the first polypeptide comprises
  • variant thereof which binds actin generally refers to naturally occurring proteins, or fragments thereof comprising an actin binding domain, which bind actin and which have been altered at one or more amino acids but still maintain the ability to bind actin.
  • variants are at least 75%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identical to a naturally occurring actin binding protein, preferably a mammalian actin binding protein such as those described herein.
  • the first polypeptide is itself a protein complex of two or more subunits (for instance spectrin may be present as a complex), in other embodiments the first polypeptide is a single polypeptide chain.
  • spectrin refers to membrane-associated cytoskeletal proteins involved in the crosslinking of filamentous actin which act as molecular scaffold proteins to link the actin cytoskeleton to the plasma membrane, and function in the determination of cell shape, arrangement of transmembrane proteins, and organization of organelles (Broderick and Winder, 2005).
  • Spectrins are traditionally divided into erythrocytic and non-erythrocytic forms, the former being exclusive to red blood cells and being responsible for the elasticity of BCs.
  • Spectrins are ubiquitous in cells and different isoforms may be expressed in different tissues in different organisms.
  • Spectrins are highly modular proteins, containing many repeating alpha-helical 106-amino acid units (or 'spectrin repeats').
  • Alpha forms generally contain 20 spectrin repeats and, in contrast to the beta forms, generally lack an actin binding domain (ABD).
  • Most alpha forms contain an SH3 (Src homology 3 domains) for binding polyproline-containing proteins.
  • Non- erythrocytic alpha isoforms generally contain an EF-hand motif for binding calcium.
  • Examples of erythrocytic alpha forms of spectrin are given as SEQ ID NOs: 41, 57-59 and 66.
  • Examples of non-erythrocytic alpha forms of spectrin are given as SEQ ID NOs: 42-43.
  • Mutations in the human SPTA1 gene (encoding erythrocytic spectrin alpha 1) are the cause of elliptocytosis type 2 (EL2), an autosomal dominant hematological disorder characterised by hemolytic anemia and elliptical or oval RBC shape.
  • SPTA1 mutations also cause hereditary pyropoikilocytosis (HPP) and spherocytosis type III (SPH3), both being hemolytic disorders.
  • HPP hereditary pyropoikilocytosis
  • SPH3 spherocytosis type III
  • SPTANl non- erythrocytic alpha 1 gene
  • Sjogrens syndrome Sjogrens syndrome
  • autoimmune diseaeses rheumatoid arthritis
  • multiple sclerosis multiple sclerosis
  • neurodegenerative diseases and xerostomia Non-erythrocytic forms of alpha 1 spectrin (encoded by the SPTANl gene) are also known as alpha-fodrin.
  • Beta forms generally contain 17 spectrin repeats and an actin binding domain (ABD).
  • ABDs generally contain two CH (calponin homology) domains, which enable beta forms of spectrin to interact with F-actin.
  • Non-erythrocytic forms of beta spectrin contain a PH (pleckstrin homology) domain for interaction with membrane phospholipids.
  • Beta forms of spectrin generally lack EF-hand motifs. Examples of erythrocytic beta forms of spectrin are given as SEQ ID NOs: 44-45, 60-63 and 67. , Examples of non-erythrocytic beta forms of spectrin are given as SEQ ID NOs: 46-48, 64-65 and 68.
  • erythrocytic spectrin beta Mutations in the human SPTB gene (encoding erythrocytic spectrin beta) are the cause of RBC disorders including elliptocytosis type 3 (EL3), spherocytosis type I (SPH1), muscular dystrophy, various anemic disease and pyropoikilocytosis. Mutations in the non-erythrocytic beta 1 gene (SPTBN1) cause neurofibromatosis type 2 and leukemia. Non-erythrocytic forms of beta 1 spectrin (encoded by the SPTBN1 gene) are also known as beta-fodrin.
  • Spectrin functions as a tetramer of alpha and beta dimers linked in a head-to- head arrangement.
  • Alpha and beta spectrin interact to form a dimer and two heterodimers form the functional tetramer.
  • Tetramers bind via their tail ends to a junctional complex consisting of filamentous actin and band 4.1 protein.
  • Spectrin also binds to integral membrane proteins via ankyrin and band 3 protein (especially in RBCs) and also via protein 4.1 and glycophorin C. Interactions also occur with phospholipids via the PH domains of beta spectrin.
  • the first polypeptide is not spectrin.
  • the polypeptide does not comprise
  • Actin is a well known globular protein about 42 kDa in size found in close to all eukaryotic cells (Rohn and Baum et al., 2010). Actin is one of the most highly- conserved proteins, differing by as little as 20% in primary amino acid sequence in species as diverse as algae and humans. Actin is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. Actin participates in many important cellular processes including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape.
  • actin isoforms
  • alpha actins
  • beta actins
  • gamma actins
  • the alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus (examples provided as SEQ ID NOs 26, 29, 32, 35 and 38).
  • the beta and gamma actins co-exist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility (examples provided as SEQ ID NOs 27, 28, 30, 31 , 33, 34, 36, 37, 39 and 40).
  • G-actin is the monomeric form of the globular protein which assembles into actin filaments.
  • F-actin is the polymeric form of the globular, protein assembled into actin filaments.
  • the actin is filamentous actin or a filamentous fragment thereof.
  • the actin is a polymer comprising at least two actin monomer units or fragments thereof.
  • Determining whether a compound will bind acti can be determined using standard techniques such as using a simple actin binding assay (Corrado et al., 1994). Briefly, the candidate protein is expressed as fusion proteins with a tag, F-actin is absorbed on to microtiter plates, the candidate protein is incubated on the F-actin coated microtiter plates, the plates are washed, anti-tag rabbit antibody is added, anti- rabbit antibody conjugated to a detectable marker is added, and the plates screened for the detectable marker. As the skilled person would readily appreciate, a similar method can be used to identify fragments of actin which bind Clec9A.
  • RNF-41 protein is also known as RING (Really Interesting New Gene) finger protein, neuregulin receptor degradation protein- 1 (NRDP1), or fetal liver RING protein (FLRF), refers to a protein which acts as an E3-ubiquitin ligase and regulates the degradation of target proteins.
  • Target proteins for RNF-41 include members of the EGF (epidermal growth factor) receptor family, for example ErbB3 (or Her3).
  • Other targets of RNF-41 include ErbB4, ubiquitin-specific protease 8 (Usp8), Birc6 and reticulon 4 (Rtn4, also known as NogoA). Mutations in RNF-41 have been linked to tumour diseases. Overexpression of RNF-41 has been shown to decrease ErbB3 and inhibition of breast cancer growth. Decreased levels of RNF-41 are inversely correlated with ErbB3 levels in primary human breast cancer tissue.
  • Clec9A is expressed by a subset of dendritic cells, can be targetted to modulate an immune response, and binds a ligand on cells with a disrupted cell membrane, cells infected with a pathogen, dying cells and dead cells (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008; Sancho et al., 2009; WO 2009/026660; WO 2009/137871; WO 2010/108215).
  • the present inventors have now identified further molecules which bind Clec9A which can be used to, inter alia, target therapeutic molecules to dendritic cells.
  • the invention relates to a polypeptide complex comprising a first polypeptide comprising an actin binding domain and a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, wherein the actin or fragment thereof is bound to the actin binding domain, and which has been modified to deliver a therapeutic agent.
  • the invention in another embodiment, relates to a compound that binds a polypeptide complex which comprises a first polypeptide comprising an actin binding domain and a second polypeptide comprising actin, or a fragment thereof which binds the actin binding domain, wherein the actin or fragment thereof is bound to the actin binding domain, and wherein the polypeptide complex is capable of binding Clec9A, and wherein the compound does not detectably bind the first or second polypeptide in isolation, and wherein the compound is not Clec9A.
  • the present invention provides a compound which
  • ii) binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex which comprises:
  • actin or fragment thereof is bound to the actin binding domain, or
  • iii) competes with the one or both tryptophan residues of CIec9A for binding to the polypeptide complex.
  • the binding may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions.
  • the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of hydrophilic/lipophilic interactions.
  • the compound is a purified and/or recombinant polypeptide (which can be a polypeptide complex).
  • the polypeptide complex, conjugate or compound may bind specifically a target molecule.
  • the phrase "specifically binds”, means that under particular conditions, the polypeptide complex, conjugate or compound binds the target and does not bind to a significant amount to other, for example, proteins or carbohydrates.
  • the polypeptide complex, conjugate or compound specifically binds the target and not other molecules in a sample obtained from a subject comprising, for example, cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof.
  • a polypeptide complex, conjugate or compound is considered to "specifically binds" if there is a greater than 2-fold difference, and preferably a 5, 25, 50 or 100 fold greater difference between the binding of the polypeptide complex, conjugate or compound when compared to another protein.
  • a compound of, and/or useful for, the invention is an antibody or antigen-binding fragment thereof.
  • antibodies and “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds.
  • L light
  • H heavy
  • the structure of immunoglobulins has been well characterized, see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
  • each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as C H ).
  • the heavy chain constant region typically is comprised of three domains, CH I , Cm, and CH3.
  • Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL).
  • the light chain constant region typically is comprised of one domain, CL-
  • the VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and V L is typically composed of three CDRs and four FRs, arranged from amirio-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk, 1987).
  • the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (phrases such as variable domain residue numbering as in Kabat or according to Kabat herein refer to this numbering system for heavy chain variable domains or light chain variable domains).
  • the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • humanized antibody refers to herein an antibody derived from a non-human antibody, typically murine, that retains or substantially retains the antigen-binding properties of the parent antibody but which is less immunogenic in humans.
  • complementarity determining region refers to amino acid sequences which together define the binding affinity and specificity of a variable fragment (Fv) region of a immunoglobulin binding site.
  • framework region refers to amino acid sequences interposed between CDRs. These portions of the antibody serve to hold the CDRs in appropriate orientation (allows for CDRs to bind antigen).
  • a variable region either light or heavy, comprises a framework and typically three CDRs.
  • constant region refers to the portion of the antibody molecule which confers effector functions.
  • the constant regions of the subject humanized antibodies are derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, antibodies with desired effector function can be produced.
  • Preferred heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3) and gamma 4 (IgG4), more preferably gamma 4 (IgG4).
  • the light chain constant region can be of the kappa or lambda type, preferably of the kappa type.
  • Antibodies may exist as intact immunoglobulins, or as modifications in a variety of forms including, for example, but not limited to, domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light and heavy chain variable regions, or Fd fragments containing the heavy chain variable region and the CHI domain.
  • domain antibodies including either the VH or VL domain, a dimer of the heavy chain variable region (VHH, as described for a camelid), a dimer of the light chain variable region (VLL), Fv fragments containing only the light and heavy chain variable regions, or Fd fragments containing the heavy chain variable region and the CHI domain.
  • a scFv consisting of the variable regions of the heavy and light chains linked together to form a single-chain antibody (Bird et al., 1988; Huston et al., 1988) and oligomers of scFvs such as diabodies and triabodies are also encompassed by the term "antibody”. Also encompassed are fragments of antibodies such as Fab, (Fab') 2 and FabFc 2 fragments which contain the variable regions and parts of the constant regions. CDR-grafted antibody fragments and oligomers of antibody fragments are also encompassed.
  • the heavy and light chain components of an Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region.
  • the antibody may be of animal (for example mouse, rabbit or rat) or human origin or may be chimeric (Morrison et al., 1984) or humanized (Jones et al., 1986; UK 8707252).
  • the term "antibody” includes these various forms. Using the guidelines provided herein and those methods well known to those skilled in the art which are described in the references cited above and in such publications as Harlow & Lane ⁇ supra) the antibodies for use in the methods of the present invention can be readily made.
  • an "antigen-binding fragment” refers to a portion of an antibody as defined herein that is capable of binding the same antigen as the full length molecule.
  • Antibodies or antigen-binding fragments of, and/or useful for, the invention which are not from a natural source, such as a humanized antibody, preferably retain a significant proportion of the binding properties of the parent antibody.
  • such antibodies or fragments of, and/or useful for, the invention retain the ability to specifically bind the antigen recognized by the parent antibody used to produce the antibody or fragment such as a humanized antibody.
  • the antibody or fragment exhibits the same or substantially the same antigen-binding affinity and avidity as the parent antibody.
  • the affinity of the antibody or fragment will not be less than 10% of the parent antibody affinity, more preferably not less than about 30%, and most preferably the affinity will not be less than 50% of the parent antibody.
  • Methods for assaying antigen-binding affinity are well known in the art and include half-maximal binding assays, competition assays, and Scatchard analysis.
  • immunoassay formats may be used to select antibodies or fragments that are specifically immunoreactive with the ligand.
  • surface labelling and flow cytometric analysis or solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See Harlow & Lane (supra) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • the antibodies may be Fv regions comprising a variable light (VL) and a variable heavy (VH) chain.
  • VL variable light
  • VH variable heavy chain
  • the light and heavy chains may be joined directly or through a linker.
  • a linker refers to a molecule that is covalently linked to the light and heavy chain and provides enough spacing and flexibility between the two chains such that they are able to achieve a conformation in which they are capable of specifically binding the epitope to which they are directed.
  • Protein linkers are particularly preferred as they may be expressed as an intrinsic component of the Ig portion of the fusion polypeptide.
  • recombinantly produced single chain scFv antibody preferably a humanized scFv
  • scFv antibody preferably a humanized scFv
  • Monoclonal antibodies of, and/or useful for, the invention can be readily produced by one skilled in the art.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against target epitopes can be screened for various properties; i.e. for isotype and epitope affinity.
  • Animal-derived monoclonal antibodies can be used for both direct in vivo and extracorporeal immunotherapy. However, it has been observed that when, for example, mouse-derived monoclonal antibodies are used in humans as therapeutic agents, the patient produces human anti-mouse antibodies. Thus, animal-derived monoclonal antibodies are not preferred for therapy, especially for long term use. With established genetic engineering techniques it is possible, however, to create chimeric or humanized antibodies that have animal-derived and human-derived portions.
  • the animal can be, for example, a mouse or other rodent such as a rat.
  • variable region of the chimeric antibody is, for example, mouse-derived while the constant region is human-derived
  • the chimeric antibody will generally be less immunogenic than a "pure" mouse-derived monoclonal antibody. These chimeric antibodies would likely be more suited for therapeutic use, should it turn out that "pure" mouse-derived antibodies are unsuitable.
  • the light and heavy chains can be expressed separately, using, for example, immunoglobulin light chain and immunoglobulin heavy chains in separate plasmids. These can then be purified and assembled in vitro into complete antibodies; methodologies for accomplishing such assembly have been described (see, for example, Sun et al., 1986).
  • a DNA construct may comprise DNA encoding functionally rearranged genes for the variable region of a light or heavy chain of an antibody linked to DNA encoding a human constant region. Lymphoid cells such as myelomas or hybridomas transfected with the DNA constructs for light and heavy chain can express and assemble the antibody chains.
  • the antibody is humanized, that is, an antibody produced by molecular modeling techniques wherein the human content of the antibody is maximised while causing little or no loss of binding affinity attributable to the variable region of, for example, a parental rat, rabbit or murine antibody.
  • variable domain framework residues have little or no direct contribution.
  • the primary function of the framework regions is to hold the CDRs in their proper spatial orientation to recognize antigen.
  • substitution of animal, for example, rodent CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework is highly homologous to the animal variable domain from which they originated.
  • a human variable domain should preferably be chosen therefore that is highly homologous to the animal variable domain(s).
  • a suitable human antibody variable domain sequence can be selected as follow.
  • Step 1 Using a computer program, search all available protein (and DNA) databases for those human antibody variable domain sequences that are most homologous to the animal-derived antibody variable domains.
  • the output of a suitable program is a list of sequences most homologous to the animal-derived antibody, the percent homology to each sequence, and an alignment of each sequence to the animal- derived sequence. This is done independently for both the heavy and light chain variable domain sequences. The above analyses are more easily accomplished if only human immunoglobulin sequences are included.
  • Step 2. List the human antibody variable domain sequences and compare for J homology. Primarily the comparison is performed on length of CDRs, except CDR3 of the heavy chain which is quite variable.
  • Human heavy chains and Kappa and Lambda light chains are divided into subgroups; Heavy chain 3 subgroups, Kappa chain 4 subgroups, Lambda chain 6 subgroups.
  • the CDR sizes within each subgroup are similar but vary between subgroups. It is usually possible to match an animal-derived antibody CDR to one of the human subgroups as a first approximation of homology. Antibodies bearing CDRs of similar length are then compared for amino acid sequence homology, especially within the CDRs, but also in the surrounding framework regions.
  • the human variable domain which is most homologous is chosen as the framework for humanisation.
  • An antibody may be humanized by grafting the desired CDRs onto a human framework according to standard procedures such as those decribed in EP 0239400.
  • a DNA sequence encoding the desired reshaped antibody can therefore be made beginning with the human DNA whose CDRs it is wished to reshape.
  • the animal- derived variable domain amino acid sequence containing the desired CDRs is compared to that of the chosen human antibody variable domain sequence.
  • the residues in the human variable domain are marked that need to be changed to the corresponding residue in the animal to make the human variable region incorporate the animal-derived CDRs. There may also be residues that need substituting in, adding to or deleting from the human sequence.
  • Oligonucleotides are synthesized that can be used to mutagenize the human variable domain framework to contain the desired residues. Those oligonucleotides can be of any convenient size. One is normally only limited in length by the capabilities of the particular synthesizer one has available. The method of oligonucleotide-directed in vitro mutagenesis is well known.
  • Synthetic gene sequences such as those encoding humanized antibodies or fragments thereof, can be commercially ordered through any of a number of service companies, including DNA 2.0 (Menlo Park, Calif.), Geneart (Regensburg, Germany), CODA Genomics (Irvine, Calif.), and GenScript, Corporation (Piscataway, N.J.).
  • humanisation may be achieved using the recombinant polymerase chain reaction (PCR) methodology of WO 92/07075.
  • PCR polymerase chain reaction
  • a CDR may be spliced between the framework regions of a human antibody.
  • the technique of WO 92/07075 can be performed using a template comprising two human framework regions, AB and CD, and between them, the CDR which is to be replaced by a donor CDR.
  • Primers A and B are used to amplify the framework region AB, and primers C and D used to amplify the framework region CD.
  • the primers B and C each also contain, at their 5' ends, an additional sequence corresponding to all or at least part of the donor CDR sequence.
  • Primers B and C overlap by a length sufficient to permit annealing of their 5' ends to each other under conditions which allow a PCR to be performed.
  • the amplified regions AB and CD may undergo gene splicing by overlap extension to produce the humanized product in a single reaction.
  • the mutagenised DNAs can be linked to an appropriate DNA encoding a light or heavy chain constant region, cloned into an expression vector, and transfected into host cells, preferably mammalian cells. These steps can be carried out in routine fashion.
  • a reshaped antibody may therefore be prepared by a process comprising:
  • the DNA sequence in step (a) encodes both the variable domain and each constant domain of the human antibody chain.
  • the humanized antibody can be prepared using any suitable recombinant expression system.
  • the cell line which is transformed to produce the altered antibody may be a Chinese Hamster Ovary (CHO) cell line or an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line.
  • the cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus.
  • the immortalised cell line is a myeloma cell line or a derivative thereof.
  • the CHO cells used for expression of the antibodies may be dihydrofolate reductase (DHFR) deficient and so dependent on thymidine and hypoxanthine for growth.
  • DHFR dihydrofolate reductase
  • the parental DHFR * CHO cell line is transfected with the DNA encoding the antibody and DHFR gene which enables selection of CHO cell transformants of DHFR positive phenotype. Selection is carried out by culturing the colonies on media devoid of thymidine and hypoxanthine, the absence of which prevents untransformed cells from growing and transformed cells from resalvaging the folate pathway and thus bypassing the selection system.
  • These transformants usually express low levels of the DNA of interest by virtue of co-integration of transfected DNA of interest and DNA encoding DHFR.
  • the expression levels of the DNA encoding the antibody may be increased by amplification using methotrexate (MTX).
  • MTX methotrexate
  • This drug is a direct inhibitor of the enzyme DHFR and allows isolation of resistant colonies which amplify their DHFR gene copy number sufficiently to survive under these conditions. Since the DNA sequences encoding DHFR and the antibody are closely linked in the original transformants, there is usually concomitant amplification, and therefore increased expression of the desired antibody.
  • GS glutamine synthetase
  • Msx methionine sulphoximine
  • the cell line used to produce the humanized antibody is preferably a mammalian cell line
  • any other suitable cell line such as a bacterial cell line or a yeast cell line
  • E. co/i ' -derived bacterial strains could be used.
  • the antibody obtained is checked for functionality. If functionality is lost, it is necessary to return to step (2) and alter the framework of the antibody.
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be recovered and purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (See, generally, Scopes, R., Protein Purification, Springer- Verlag, N.Y. (1982)).
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • a humanized antibody may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (See, generally, Lefkovits and Pernis (editors), Immunological Methods, Vols. I and II, Academic Press, ( 1979 and 1981 )). Studies carried out by Greenwood et al. (1993) have demonstrated that recognition of the Fc region of an antibody by human effector cells can be optimised by engineering the constant region of the immunoglobulin molecule.
  • variable region genes of the antibody with the desired specificity, to human constant region genes encoding immunoglobulin isotypes that have demonstrated effective antigen dependent cellular cytotoxicity (ADCC) in human subjects, for example the IgGl and IgG3 isotypes (Greenwood and Clark, Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man. Mike Clark (editor), Academic Titles, Section II, p.85-l l3, (1993)).
  • the resulting chimeric or humanized antibodies should be particularly effective in modulating humoral immunity and/or T-cell mediated immunity.
  • Antibodies with fully human variable regions can also be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Various subsequent manipulations can be performed to obtain either antibodies per se or analogs thereof (see, for example, US 6,075,181).
  • Genes encoding antibodies, both light and heavy chain genes or portions thereof, e.g., single chain Fv regions, may be cloned from a hybridoma cell line. They may all be cloned using the same general strategy such as RACE using a commercially available kit, for example as produced by Clontech. Typically, for example, poly(A) + mRNA extracted from the hybridoma cells is reverse transcribed using random hexamers as primers. For Fv regions, the VH and VL domains are amplified separately by two polymerase chain reactions (PCR).
  • PCR polymerase chain reactions
  • Heavy chain sequences may be amplified using 5' end primers which are designed according to the amino-terminal protein sequences of the heavy chains respectively and 3' end primers according to consensus immunoglobulin constant region sequences (Kabat et al., Sequences of Proteins of Immunological Interest. 5th edition. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • Light chain Fv regions are amplified using 5' end primers designed according to the amino- terminal protein sequences of light chains and in combination with the primer C-kappa.
  • One of skill in the art would recognize that many suitable primers may be employed to obtain Fv regions.
  • the PCR products are subcloned into a suitable cloning vector. Clones containing the correct size insert by DNA restriction are identified. The nucleotide sequence of the heavy or light chain coding regions may then be determined from double stranded plasmid DNA using sequencing primers adjacent to the cloning site. Commercially available kits (e.g., the SequenaseTM kit, United States Biochemical Corp., Cleveland, Ohio, USA) may be used to facilitate sequencing the DNA. DNA encoding the Fv regions may be prepared by any suitable method, including, for example, amplification techniques such as PCR and LCR.
  • amplification techniques such as PCR and LCR.
  • Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. While it is possible to chemically synthesize an entire single chain Fv region, it is preferable to synthesize a number of shorter sequences (about 100 to 150 bases) that are later ligated together.
  • sub-sequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence.
  • the sequences may be ligated together, either directly or through a DNA sequence encoding a peptide linker, using techniques well known to those of skill in the art.
  • heavy and light chain regions are connected by a flexible peptide linker (e.g., (Gh/ 4 Ser)3) which starts at the carboxyl end of the heavy chain Fv domain and ends at the amino terminus of the light chain Fv domain.
  • the entire sequence encodes the Fv domain in the form of a single-chain antigen-binding protein.
  • Polypeptide complexes, conjugates and compounds defined herein can be used to deliver a therapeutic agent.
  • therapeutic agents include, but are not limited to, an antigen, a cytotoxic agent, a drug and/or pharmacological agent.
  • the therapeutic agent may be a polypeptide fused to the polypeptide complex, actin or a fragment thereof, or compound as defined herein.
  • Fusion polypeptides comprising the polypeptide complex or compound may be prepared by methods known to one of skill in the art. For example, a gene encoding a polypeptide chain is fused to a gene encoding a therapeutic agent.
  • the gene is linked to a segment encoding a peptide connector.
  • the peptide connector may be present simply to provide space between the compound and the therapeutic agent or to facilitate mobility between these regions to enable them to each attain their optimum conformation.
  • the DNA sequence comprising the connector may also provide sequences (such as primer sites or restriction sites) to facilitate cloning or may preserve the reading frame between the sequence encoding the binding moiety and the sequence encoding the therapeutic agent.
  • sequences such as primer sites or restriction sites
  • the design of such connector peptides is well known to those of skill in the art.
  • Polypeptide complexes, actins or compounds useful for the invention may be fused to, or otherwise bound to the therapeutic agent by any method known and available to those in the art.
  • the two components may be chemically bonded together by any of a variety of well-known chemical procedures.
  • the linkage may be by way of heterobifunctional cross-linkers, e.g., SPDP, carbodiimide, glutaraldehyde, or the like.
  • Production of various immunotoxins, as well as chemical conjugation methods are well-known within the art (see, for example, “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, p. 168-190 (1982); Waldmann, 1991 ; Vitetta et al., 1987; Pastan et al., 1986; and Thorpe et al., 1987).
  • drugs and/or pharmacological agents include, but are not limited to, agents that promote DC activation (e.g. TLR ligands), agents that suppress DC activation or function (e.g. specific inhibitors or promotors of DC signalling molecules such as kinases and phosphatases), and agents that modulate DC death (e.g. promotors or suppressors of apoptosis).
  • agents that promote DC activation e.g. TLR ligands
  • agents that suppress DC activation or function e.g. specific inhibitors or promotors of DC signalling molecules such as kinases and phosphatases
  • agents that modulate DC death e.g. promotors or suppressors of apoptosis.
  • Such drugs and/or pharmacological agents are well known to those skilled in the art.
  • polypeptide toxins that are suitable for use as cytotoxic agents in the methods of the invention.
  • polypeptides include, but are not limited to, polypeptides such as native or modified Pseudomonas exotoxin (PE), diphtheria toxin (DT), ricin, abrin, gelonin, momordin II, bacterial RIPs such as shiga and shiga-Iike toxin a-chains, luffm, atrichosanthin, momordin I, Mirabilis anti- viral protein, pokeweed antiviral protein, byodin 2 (U.S. 5,597,569), gaporin, as well as genetically engineered variants thereof.
  • PE native or modified Pseudomonas exotoxin
  • DT diphtheria toxin
  • ricin abrin
  • gelonin gelonin
  • momordin II bacterial RIPs
  • shiga and shiga-Iike toxin a-chains such as shiga
  • Native PE and DT are highly toxic compounds that typically bring about death through liver toxicity.
  • Pseudomonas exotoxin and DT are modified into a form that removes the native targeting component of the toxin, e.g., domain la of Pseudomonas exotoxin and the B chain of DT.
  • the invention is not limited to a particular cytotoxic agent.
  • cytotoxic agents include, but are not limited to, agents such as bacterial or plant toxins, drugs, e.g., cyclophosphamide (CTX; Cytoxan), chlorambucil (CHL; leukeran), cisplatin (CisP; CDDP; platinol), busulfan (myleran), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and other alkylating agents; methotrexate (MTX), etoposide (VP- 16; vepesid), 6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5FU), dacarbazine (DTIC), 2-chlorodeoxyadenosine (2-CdA), and other antimetabolites; antibiotics including actinomycin D, doxorubicin (DXR; a
  • radioisotopes and chemocytotoxic agents that can be coupled to compounds of the invention by well known techniques, and delivered to specifically destroy dendritic cells (see, e.g., U.S. 4,542,225).
  • photo-activated toxins include dihydropyridine-and omega- conotoxin.
  • cytotoxic reagents that can be used include ,25 I, ,31 I, m In, m l, "mTc, and 32 P.
  • the antibody can be labeled with such reagents using techniques known in the art. For example, see Wenzel and Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, N.Y.
  • the linker-chelator tiuexutan is conjugated to the compound by a stable thiourea covalent bond to provide a high-affinity chelation site for Indium- 111 or Yttrium-90.
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a polypeptide complex of the invention, or actin or a fragment thereof, wherein the complex or actin is conjugated to a cytotoxic agent.
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is decreased upon the administration of a compound that binds a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein.
  • Polypeptide complexes, conjugates or compounds useful for the methods of the invention may also be conjugated to an "antigen”.
  • antigen is further intended to encompass peptide or. protein analogs of known or wild-type antigens such as those described above.
  • the analogs may be more soluble or more stable than wild type antigen, and may also contain mutations or modifications rendering the antigen more immunologically active.
  • Also useful in the present invention are peptides or proteins which have amino acid sequences homologous with a desired antigen's amino acid sequence, where the homologous antigen induces an immune response to the respective tumor or organism.
  • a “cancer antigen,” as used herein is a molecule or compound (e.g., a protein, peptide, polypeptide, lipid, glycolipid, carbohydrate and/or DNA) associated with a tumor or cancer cell and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule.
  • Cancer antigens include self-antigens, as well as other antigens that may not be specifically associated with a cancer, but nonetheless induce and/or enhance an immune response to and/or reduce the growth of a tumor or cancer cell when administered to an animal.
  • an "antigen from a pathogenic and/or infectious organism” as used herein, is an antigen of any organism and includes, but is not limited to, infectious virus, infectious bacteria, infectious parasites including protozoa (such as Plasmodium sp.) and worms and infectious fungi.
  • the antigen is a protein or antigenic fragment thereof from the organism, or a synthetic compound which is identical to or similar to naturally-occurring antigen which induces an immune response specific for the corresponding organism.
  • Compounds or antigens that are similar to a naturally-occurring organism antigens are well known to those of ordinary skill in the art.
  • a non-limiting example of a compound that is similar to a naturally- occurring organism antigen is a peptide mimic of a polysaccharide antigen.
  • cancer antigens include, e.g., mutated antigens such as the protein products of the Ras p21 protooncogenes, tumor suppressor p53 and HER- 2/neu and BCR-abl oncogenes, as well as CD 4, MUM1, Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, Aldolase A, FRAME, Her2/neu, ErbB-2 and KSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionic gonadotropin (hCG); self-antigens such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens such as Mart 1 Melan A, gplOO, gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such as PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryo
  • Cancer antigens and their respective tumor cell targets include, e.g., cytokeratins, particularly cytokeratin 8, 18 and 19, as antigens for carcinoma.
  • Epithelial membrane antigen (EM A) human embryonic antigen (HEA-125), human milk fat globules, MBrl, MBr8, Ber-EP4, 17-1 A, C26 and T16 are also known carcinoma antigens.
  • Desmin and muscle-specific actin are antigens of myogenic sarcomas.
  • Placental alkaline phosphatase, beta-human chorionic, gonadotropin, and alpha- fetoprotein are antigens of trophoblastic and germ cell tumors.
  • Prostate specific antigen is an antigen of prostatic carcinomas, carcinoembryonic antigen of colon adenocarcinomas.
  • HMB-45 is an antigen of melanomas.
  • useful antigens could be encoded by human papilloma virus.
  • Chromagranin-A and synaptophysin are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid tumor masses having necrotic areas. Antigens derived from pathogens known to predispose to certain cancers may also be advantageously used in the present invention.
  • Pathogens of particular interest for use in the cancer vaccines provided herein include the hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptomas), Epstein Barr virus (EBV) (Burkitt lymphoma, nasopharynx cancer, PTLD in immunosuppressed individuals), HTLVL (adult T-cell leukemia), oncogenic human papilloma viruses types 16, 18, 33, 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B-cell gastric lymphoma).
  • EBV Epstein Barr virus
  • HTLVL adult T-cell leukemia
  • HTLVL adult T-cell leukemia
  • oncogenic human papilloma viruses types 16, 18, 33, 45 adult cervical cancer
  • Helicobacter pylori B-cell gastric lymphoma
  • Other medically relevant microorganisms that may serve as antigens in mammals and more particularly humans are described extensively in the literature, e.g.,
  • Exemplary viral pathogens include, but are not limited to, infectious virus that infect mammals, and more particularly humans.
  • infectious virus include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-IH, LAV or HTLV-III LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g.
  • Flaviridae e.g. dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronoviridae e.g. coronaviruses such as the SARS coronavirus
  • Rhabdoviradae e.g. vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g. ebola viruses
  • Paramyxoviridae e.g. parairifluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g.
  • African swine fever virus African swine fever virus
  • gram negative and gram-positive bacteria may be targeted by the subject compositions and methods in vertebrate animals.
  • Such gram-positive bacteria include, but are not limited to Pasteurella sp, Staphylococci sp., and Streptococcus sp.
  • Gram- negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas sp., and Salmonella sp.
  • infectious bacteria include but are not limited to: Helicobacter pyloris, Borella burgdorferi, Legionella pneumophilia, Mycobacteria sp. (e.g. M. tuberculosis, M. avium, M: intracellular , M. kansaii, M.
  • antigens can be isolated or prepared recombinantly or by any other means known in the art.
  • pathogens further include, but are not limited to, infectious fungi and parasites that infect mammals, and more particularly humans.
  • infectious fungi include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.
  • Examples of parasites include intracellular parasites and obligate intracellular parasites.
  • Examples of parasites include but are not limited to Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmdodium vivax, Plasmodium knowlesi, Babesia microti, Babesia divergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense, Wuchereria bancrofti, Brugia malayi, Brugia timori, Ascaris lumbricoides, Onchocerca volvulus and Schistosoma mansoni.
  • compositions and methods of the present invention are useful for treating infections of nonhuman mammals.
  • non-human pathogens include, but are not limited to, mouse mammary tumor virus (“MMTV”), Rous sarcoma virus (“RSV”), avian leukemia virus (“ALV”), avian myeloblastosis virus (“AMV”), murine leukemia virus (“MLV”), feline leukemia virus (“FeLV”), murine sarcoma virus (“MSV”), gibbon ape leukemia virus (“GALV”), spleen necrosis virus (“SNV”), reticuloendotheliosis virus (“RV”), simian sarcoma virus (“SSV”), Mason-Pfizer monkey virus (“MPMV”), simian retrovirus type 1 (“SRV-l”), lentiviruses such as HIV-1, HIV-2, SIV, Visna virus, feline immunodeficiency virus (“FIV”), and equine infectious anemia virus (“EIAV”), T-cell leukemia viruses such as HTLV-1, HT
  • the uptake and/or clearance of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen.
  • antigen recognition, processing and/or presentation of material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells, or dead cells, or a portion thereof, or surrounding cells is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen.
  • the immune responses to material derived from cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, or surrounding cells is increased upon the administration of a polypeptide complex of the invention, or that binds actin or a fragment thereof as defined herein, conjugated to an antigen.
  • Polypeptide complexes, conjugates or compounds useful for the invention may be employed in a range of detection systems.
  • the polypeptide complex, conjugate or compound may be used in methods for imaging an internal region of a subject and/or diagnosing the presence or absence of a disease in a subject.
  • diagnostic, prognostic and/or monitoring methods of the present invention involve a degree of quantification to determine levels of Clec9A, Clec9A expressing cells, ligand and/or ligand expressing cells present in patient samples. Such quantification is readily provided by the inclusion of appropriate control samples.
  • internal controls are included in the methods of the present invention.
  • a preferred internal control is one or more samples taken from one or more healthy individuals.
  • Polypeptide complexes, conjugates or compounds useful for the present invention when used diagnosticall may be linked to a diagnostic reagent such as a detectable label to allow easy detection of binding events in vitro or in vivo.
  • a diagnostic reagent such as a detectable label to allow easy detection of binding events in vitro or in vivo.
  • Suitable labels include radioisotopes, or non-radioactive labels such as biotin, enzymes, chemiluminescent molecules, fluorophores, dye markers or other imaging reagents for detection and or localisation of target molecules.
  • a second labelled molecule such as an antibody which binds the polypeptide complex, actin or fragment thereof, conjugate or compound can be used for detection.
  • an enzyme can be conjugated to the second molecule, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above.
  • fluorescent compounds such as but not limited to fluorecein and rhodamine amongst others, may be chemically coupled to, for examples, antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the compounds coupled to imaging agents can be used in the detection of Clec9A in histochemical tissue sections.
  • the polypeptie complexes, actin, or compound (for example) may be covalently or non- covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, echogenic, radioactive, or non-radioactive labels such as biotin or avidin.
  • Cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells can be detected in a sample by a variety of techniques well known in the art, including cell sorting, especially fluorescence-activated cell sorting (FACS), by using an affinity reagent bound to a substrate (e.g., a plastic surface, as in panning), or by using an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles).
  • FACS fluorescence-activated cell sorting
  • any detectable substance which has the appropriate characteristics for the cell sorter may be used (e.g., in the case of a fluorescent dye, a dye which can be excited by the sorter's light source, and an emission spectra which can be detected by the cell sorter's detectors).
  • a beam of laser light is projected through a liquid stream that contains cells, or other particles, which when struck by the focussed light give out signals which are picked up by detectors. These signals are then converted for computer storage and data analysis, and can provide information about various cellular properties.
  • Cells labelled with a suitable dye are excited by the laser beam, and emit light at characteristic wavelengths. This emitted light is picked up by detectors, and these analogue signals are converted to digital signals, allowing for their storage, analysis and display.
  • FACS fluorescence- activated cell sorters
  • the instruments electronics interprets the signals collected for each cell as it is interrogated by the laser beam and compares the signal with sorting criteria set on the computer. If the cell meets the required criteria, an electrical charge is applied to the liquid stream which is being accurately broken into droplets containing the cells. This charge is applied to the stream at the precise moment the cell of interest is about to break off from the stream, then removed when the charged droplet has broken from the stream. As the droplets fall, they pass between two metal plates, which are strongly positively or negatively charged. Charged droplets get drawn towards the metal plate of the opposite polarity, and deposited in the collection vessel, or onto a microscope slide, for further examination.
  • the cells can automatically be deposited in collection vessels as single cells or as a plurality of cells, e.g. using a laser, e.g. an argon laser (488 run) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA).
  • a laser e.g. an argon laser (488 run)
  • a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA).
  • FACS machines include, but are not limited to, MoFloTM High-speed cell sorter (Dako-Cytomation ltd), FACS AriaTM (Becton Dickinson), FACS Diva (Becton Dickinson), ALTRA Hyper sort (Beckman Coulter) and CyFlow sorting system (Partec GmbH).
  • any particle with the desired properties may be utilized.
  • large particles e.g., greater than about 90-100 ⁇ in diameter
  • the particles are "magnetic particles" (i.e., particles which can be collected using a magnetic field). Labelled cells are retained in the column (held by the magnetic field), whilst unlabelled cells pass straight through and are eluted at the other end.
  • Magnetic particles are now commonly available from a variety of manufacturers including Dynal Biotech (Oslo, Norway) and Milteni Biotech GmbH (Germany).
  • An example of magnetic cell sorting (MACS) is provided by Al-Mufti et al. (1999).
  • Laser-capture microdissection can also be used to selectively detect labelled cells on a slide using methods of the invention.
  • Methods of using laser-capture microdissection are known in the art (see, for example, U.S. 20030227611 and Bauer et al., 2002).
  • the terms “enriching” and “enriched” are used in their broadest sense to encompass the isolation of dendritic cells or precursors thereof such that the relative concentration of dendritic cells or precursors thereof to non-dendritic cells or precursors thereof in the treated sample is greater than a comparable untreated sample.
  • the enriched dendritic cells and/or precursors thereof are separated from at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% of the non-dendritic cells or precursors thereof in the sample obtained from the original sample.
  • the enriched cell population contains no non-dendritic cells or precursors thereof (namely, pure).
  • the terms "enrich” and variations thereof are used interchangeably herein with the term "isolate” and variations thereof.
  • a population of cells enriched using a method of the invention may only comprise a single dendritic cell or precursor thereof.
  • the enrichment methods of the invention may be used to isolate a single dendritic cell or precursor thereof.
  • Dendritic cells or precursors thereof can be enriched from the sample by a variety of techniques well known in the art, including cell sorting, especially fluorescence-activated cell sorting (FACS), by using an affinity reagent bound to a substrate (e.g., a plastic surface, as in panning), or by using an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles).
  • FACS fluorescence-activated cell sorting
  • a substrate e.g., a plastic surface, as in panning
  • an affinity reagent bound to a solid phase particle which can be isolated on the basis of the properties of the beads (e.g., colored latex beads or magnetic particles).
  • the procedure used to enrich the dendritic cells ' and/or precursors thereof will depend upon how the cells have been labelled.
  • any detectable substance which has the appropriate characteristics for the cell sorter may be used (e.g., in the case of a fluorescent dye, a dye which can be excited by the sorter's light source, and an emission spectra which can be detected by the cell sorter's detectors).
  • a beam of laser light is projected through a liquid stream that contains cells, or other particles, which when struck by the focussed light give out signals which are picked up by detectors. These signals are then converted for computer storage and data analysis, and can provide information about various cellular properties.
  • Cells labelled with a suitable dye are excited by the laser beam, and emit light at characteristic wavelengths. This emitted light is picked up by detectors, and these analogue signals are converted to digital signals, allowing for their storage, analysis and display.
  • FACS fluorescence- activated cell sorters
  • the instruments electronics interprets the signals collected for each cell as it is interrogated by the laser beam and compares the signal with sorting criteria set on the computer. If the cell meets the required criteria, an electrical charge is applied to the liquid stream which is being accurately broken into droplets containing the cells. This charge is applied to "the stream at the precise moment the cell of interest is about to break off from the stream, then removed when the charged droplet has broken from the stream. As the droplets fall, they pass between two metal plates, which are strongly positively or negatively charged. Charged droplets get drawn towards the metal plate of the opposite polarity, and deposited in the collection vessel, or onto a microscope slide, for further examination.
  • the cells can automatically be deposited in collection vessels as single cells or as a plurality of cells, e.g. using a laser, e.g. an argon laser (488 nm) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA).
  • a laser e.g. an argon laser (488 nm) and for example with a Flow Cytometer fitted with an Autoclone unit (Coulter EPICS Altra, Beckman- Coulter, Miami, Fla., USA.
  • FACS machines include, but are not limited to, MoFloTM High-speed cell sorter (Dako-Cytomation ltd), FACS AriaTM (Becton Dickinson), FACS Diva (Becton Dickinson), ALTRATM Hyper sort (Beckman Coulter) and CyFlowTM sorting system (Partec GmbH).
  • any particle with the desired properties may be utilized.
  • large particles e.g., greater than about 90-100 ⁇ in diameter
  • the particles are "magnetic particles" (i.e., particles which can be collected using a magnetic field). Labelled cells are retained in the column (held by the magnetic field), whilst unlabelled cells pass straight through and are eluted at the other end.
  • Magnetic particles are now commonly available from a variety of manufacturers including Dynal Biotech (Oslo, Norway) and Milteni Biotech GmbH (Germany).
  • An example of magnetic cell sorting (MACS) is provided by Al- Mufti et al. (1999).
  • Laser-capture microdissection can also be used to selectively enrich labelled dendritic cells or precursors thereof on a slide using methods of the invention. Methods of using laser-capture microdissection are known in the art (see, for example, U.S. 20030227611 and Bauer et al., 2002).
  • the cells can be used immediately or cultured in vitro to expand dendritic cells and/or precursors thereof numbers using techniques known in the art. Furthermore, dendritic cell precursors can be cultured to produce mature dendritic cells. Identification of Compounds
  • Methods of screening test compounds are described which can identify a compound, for example, that binds to a polypeptide complex defined herein, that binds one or both of the tryptophan residues corresponding to amino acid numbers 131 and 227 of SEQ ID NO:l, that binds Clec9A and which through stearic hindrance reduces the binding of the one or both tryptophan residues of Clec9A to a polypeptide complex defined herein, or which competes with the one or both tryptophan residues of Clec9A for binding to the polypeptide complex, and are thus useful in a method of the invention.
  • Compounds may be screened by resort to assays and techniques useful in identifying molecules capable of binding to the ligand (such as a polypeptide complex of the invention) and thereby inhibiting its biological activity by blocking Clec9A binding.
  • assays include the use of mammalian cell lines (for example, CHO cells or 293T-cells) for phage display system for expressing the polypeptide complex and using a culture of transfected mammalian or E. coli or other microorganism to produce proteins for binding studies of potential binding compounds.
  • a method for identifying compounds which specifically bind to the polypeptide complex can include simply the steps of contacting a selected cell expressing the complex with a test compound to permit binding of the test compound to the complex, and determining the amount of test compound, if any, which is bound to the complex.
  • Such a method involves the incubation of the test compound and the complex immobilized on a solid support.
  • the surface containing the immobilized compound is permitted to come into contact with a solution containing the complex and binding is measured using an appropriate detection system. Suitable detection systems are known in the art, some of which are described herein.
  • crystal means a structure (such as a three dimensional
  • crystal refers in particular to a solid physical crystal form such as an experimentally prepared crystal.
  • any reference herein to the atomic coordinates or subset of the atomic coordinates shown in Appendix I shall include, unless specified otherwise, atomic coordinates having a root mean square deviation of backbone atoms of not more than 1.5 A, preferably not more than 1 A, when superimposed on the corresponding backbone atoms described.by the atomic coordinates shown in Appendix I.
  • the following defines what is intended by the term "root mean square deviation (RMSD)" between two data sets. For each element in the first data set, its deviation from the corresponding item in the second data set is computed. The squared deviation is the square of that deviation, and the mean squared deviation is the mean of all these squared deviations.
  • the root mean square deviation is the square root of the mean squared deviation.
  • Preferred variants are those in which the RMSD of the x, y and z coordinates for all backbone atoms other than hydrogen is less than 1.5 A (preferably less than 1 A, 0.7 A or less than 0.3 A) compared with the coordinates given in Appendix I. It will be readily appreciated by those skilled in the art that a 3D rigid body rotation and/or translation of the atomic coordinates does not alter the structure of the molecule concerned.
  • a "subset" of the atomic coordinates provided in Appendix I refers to a group of the co-ordinates which can be used in a method of the invention such as a computer-assisted method of identifying a compound that binds Clec9A, or a computer-assisted method for identifying a compound which binds a polypeptide complex as defined herein.
  • the subset at least comprises a structure defined by the atomic coordinates provided in Appendix II.
  • Clec9A defined, herein can be used to identify antagonists or agonists through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack et al., 1997).
  • Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of a candidate compound to the polypeptide.
  • the tighter the fit e.g., the lower the steric hindrance, and/or the greater the attractive force
  • the more potent the potential agonist or antagonist will be since these properties are consistent with a tighter binding constant.
  • the more specificity in the design of a potential agonist or antagonist the more likely that it will not interfere with other proteins.
  • a potential compound could be obtained, for example, using methods of the invention such as by screening a random peptide library produced by a recombinant bacteriophage or a chemical library. A compound selected in this manner could then be systematically modified by computer modeling programs until one or more promising potential compounds are identified.
  • Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, and of which any one might lead to a useful agonist or antagonist.
  • Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized.
  • a large number of these compounds can be rapidly screened on the computer monitor screen, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • Exemplary forcefields that are known in the art and can be used in such methods include, but are not limited to, the Constant Valence Force Field (CVFF), the AMBER force field and the CHARM force field.
  • CVFF Constant Valence Force Field
  • AMBER AMBER force field
  • CHARM CHARM force field
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behaviour of molecules with each other. c Diseases Associated with Cells with a Disrupted Cell Membrane. Cells Infected with a Pathogen. Dying Cells or Dead Cells
  • diseases associated with cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells include, but are not necessarily limited to, the following:
  • GVHD graft versus host disease
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • Sjogren's syndrome multiple sclerosis
  • insulin dependent diabetes mellitus ulcerative colitis
  • VAHS virus associated hemophagocytic syndrome
  • HCV virus associated hemophagocytic syndrome
  • Leukemia for example, acute lymphatic leukemia.
  • SIRS Systemic inflammatory reaction syndrome
  • the invention is useful for GVHD, human immunodeficiency virus (HIV), hemophagocytic syndrome (HPS), especially virus associated hemophagocytic syndrome (VAHS), acute lymphatic leukemia, influenzal encephalitis, encephalopathy, and malaria.
  • HIV human immunodeficiency virus
  • HPS hemophagocytic syndrome
  • VAHS virus associated hemophagocytic syndrome
  • polypeptide and protein are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors.
  • proteins and polypeptides as used herein may also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides described herein.
  • the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 200 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 200 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
  • biologically active fragment is a portion of a polypeptide as described herein which maintains a defined activity of the full-length polypeptide.
  • Biologically active fragments can be any size as long as they maintain the defined activity.
  • biologically active fragments are at least 100 amino acids in length.
  • soluble fragment refers to a portion of Clec9A which lacks the membrane spanning region.
  • the soluble fragment does not comprise at least about 40, at least about 50, at least about 55, or at least about 100, N-terminal residues of any one of SEQ ID NOs 1 to 8.
  • the soluble fragment comprises the C-type lectin-like domain of a polypeptide which comprises:
  • the soluble fragment comprises:
  • the soluble fragment does not comprise at least the about 40 N-terminal residues of any one of SEQ ID NOs 1 to 8.
  • the polypeptide comprises an amino acid sequence which is at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nomin
  • Amino acid sequence mutants of a polypeptide described herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide.
  • Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.
  • a combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.
  • Mutant (altered) polypeptides can be prepared using any technique known in the art.
  • a polynucleotide described herein can be subjected to in vitro mutagenesis.
  • in vitro mutagenesis techniques may include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations.
  • the polynucleotides defined herein are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they are able to confer the desired phenotype.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
  • Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical, and/or those in which particular residues obtained from related proteins are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1.
  • polypeptides which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide.
  • Polypeptides described herein can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides.
  • an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide.
  • a preferred cell to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production.
  • An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, tissue culture flasks, shake flasks, test tubes, microtiter dishes, and Petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • polynucleotide is used interchangeably herein with the term “nucleic acid”.
  • monomers of a polynucleotide are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units.
  • Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate and phosphoramidate.
  • Recombinant vectors useful for the invention can include at least one polynucleotide molecule described herein, and/or a polynucleotide encoding a polypeptide as described herein, inserted into any vector capable of delivering the polynucleotide molecule into a host cell.
  • a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid.
  • One type of recombinant vector comprises the polynucleotide(s) operably linked to an expression vector.
  • the phrase operably linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors include any vectors that function (i.e., direct gene expression) in recombinant cells, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells. Vectors can also be used to produce the polypeptide in a cell-free expression system, such systems are well known in the art.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element to a transcribed sequence.
  • a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell and/or in a cell-free expression system.
  • promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cw-acting.
  • some transcriptional regulatory elements, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance .
  • expression vectors contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, nematode, plant or animal cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma
  • a recombinant cell comprising a host cell transformed with one or more recombinant molecules described herein or progeny cells thereof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention.
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides described herein or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule as described herein.
  • Host cells of the present invention can be any cell capable of producing at least one protein defined herein, and include bacterial, fungal (including yeast), parasite, nematode, arthropod, animal and plant cells.
  • host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, CHO cells, 293 cells, EL4 cells, MDCK cells, CRFK cells,. CV-1 cells, COS (e.g., COS-7) cells, and Vero cells.
  • E. coli including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are. translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
  • transcription control signals e.g., promoters, operators, enhancers
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • compositions comprising the polypeptide complex, actin or fragment thereof, conjugate or compound together with an acceptable carrier or diluent are useful in the methods of the present invention.
  • Therapeutic compositions can be prepared by mixing the desired component having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, A.ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions.
  • Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine,
  • Such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, and cellulose-based substances.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes
  • protamine sulfate disodium hydrogen phosphate
  • potassium hydrogen phosphate sodium chloride
  • colloidal silica magnesium trisilicate
  • compositions to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • the composition may be stored in lyophilized form or in solution if administered systemically. If in lyophilized form, it is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use.
  • An example of a liquid formulation is a sterile, clear, colorless unpreserved solution filled in a single-dose vial for subcutaneous injection.
  • compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the compositions are preferably administered subcutaneously, intramuscularly or parenterally, for example, as intravenous injections or infusions or administered into a body cavity.
  • the polypeptide complex, actin or fragment thereof, conjugate or compound may be administered in an amount of about 0.001 to 2000 mg kg body weight per dose, and more preferably about O.pi to 500 mg/kg body weight per dose. Repeated doses may be administered as prescribed by the treating physician.
  • compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease or immune response required, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference, 56 th ed. (2002). Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.,
  • a polypeptide complex, actin or fragment thereof, conjugate or compound useful for the methods of the invention comprises an antigen, such as a cancer antigen or an antigen of a pathogen or infectious organism, and can be delivered by intramuscular, subcutaneous or intravenous injection, or orally, as a vaccine to enhance humoral and/or T-cell mediated immune responses.
  • the antigen is a self-antigen or allergenic antigen which can used to diminish immune responses similar to that described for 33D1 and DEC-205 (Bonifaz et al., 2002; Finkelman et al., 1996).
  • a radiolabeled form of the polypeptide complex, actin or fragment thereof, conjugate or compound is delivered by intravenous injection as a therapeutic agent to target cells that express Clec9A.
  • radiolabeled antibodies and the methods for their administration to patients as therapeutics are known to those skilled in the art. Examples include Iodine 131 labeled Lym-1, against the ⁇ subunit of HLA-DR and the anti-CD20 Indium 111 and Yttrium 90 labeled Ibritumomab Tiuxetan (IDEC-Y2B8, ZEVALIN ® ) and Iodine 1 131 Tositumomab (BEXXAR ® ).
  • the composition does not comprise an adjuvant.
  • the composition does comprise an adjuvant.
  • adjuvants include, but are not limited to, aluminium hydroxide, aluminium phosphate, aluminium potassium sulphate (alum), muramyl dipeptide, bacterial endotoxin, lipid X, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • the composition comprises liposomes or membrane vesicles.
  • liposomes are described in US 2007/0026057, Leserman (2004) and van Broekhoven et al. (2004).
  • the polypeptide complex, conjugate or compound can be used to target the liposome to enhance the delivery of an agent of interest.
  • processes for the preparation of membrane vesicles for use in the invention are described in WO 00/64471.
  • compositions for detection of cells with a disrupted cell membrane, cells infected with a pathogen, dying cells or dead cells, or a portion thereof, modulating an immune response, and/or antigen recognition, processing and/or presentation are conventionally administered parenterally, by injection, for example, subcutaneously, intramuscularly or intravenously.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
  • Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
  • the therapeutic composition may be administered to a patient either singly or in a cocktail containing other therapeutic agents, compositions, or the like.
  • the immune response is modulated by using a DNA vaccine encoding a polypeptide complex, actin or fragment thereof, conjugate or compound of the invention conjugated to an antigen.
  • DNA vaccination involves the direct in vivo introduction of DNA encoding the antigen into tissues of a subject for expression of the antigen by the cells of the subject's tissue. Such vaccines are termed herein "DNA vaccines” or "nucleic acid-based vaccines”. DNA vaccines are described in US 5,939,400, US 6,110,898, WO 95/20660, WO 93/19183, Deband et al. (2005) and Nchinda et al. (2008).
  • CMV cytomegalovirus
  • Vectors containing the nucleic acid-based vaccine of the invention may also be introduced into the desired host by other methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), or a DNA vector transporter.
  • Clec9A-ecto; stalk and CTLD Clec9A-ecto; stalk and CTLD
  • Clec9A-CTLD Clec9A CTLD only
  • Clec9A-stalk Clec9A stalk only
  • cDNA containing the required ectodomain region was amplified from the original Clec9A cDNA using Advantage high fidelity 2 polymerase (Clontech, MountainView, CA) or HotStar HiFidelity polymerase (Qiagen, Victoria, Australia) and the listed primers (Table 2).
  • Recombinant proteins were expressed in mammalian 293T or FreeStyle 293F cells by transient transfection, followed by culture in protein-free/serum-free media: X- Vivo- 10 (BioWhittaker, Walkersville, MD) or FreeStyle Expression Media (Invitrogen, Victoria, Australia) respectively.
  • Media containing the secreted recombinant protein was assayed for the presence of soluble mClec9A by reactivity with anti-mClec9A mAb (24/04- 10B4), concentrated 100-fold using a 10,000 molecular weight cutoff centrifugal device (Millipore, Billerica, MA) and either used directly or en2ymatically biotinylated using BirA enzyme (Avidity).
  • Clec9A soluble proteins were purified by affinity chromomatography using an anti-FLAG M2 agarose resin (Sigma, Castle Hill, Australia) and elution with 100 g/ml FLAG peptide (Auspep, Victoria, Australia), and further purified by size-exclusion chromatography using a pre-packed Superdex 200 column (GE Healthcare, Rydalmere, Australia). Table 2 - Construct s nthesis.
  • the resulting fusion constructs for hCLEC9A-CTLD (S225D) thus included (in order of N-terminus): the IL-3 signal sequence (to ensure secretion), a FLAG-tag, and hCLEC9A-CTLD (S225D).
  • the resulting fusion constructs for hCLEC9A-ecto included the IL-3 signal sequence, the biotinylation consensus peptide sequence, a FLAG-tag, and hCLEC9A-ecto (WT31A; W227A).
  • Recombinant proteins were expressed in FreeStyle 293F cells and hCLEC9A soluble proteins purified from the secreted media by affinity chromomatography using an anti-FLAG M2 agarose resin, and further purified by size- exclusion chromatography using a pre-packed Superdex 200 column.
  • Circular Dichroism was performed to compare the structural profiles of the
  • Platelets and red blood cells were purified from mouse blood. Binding studies were performed by incubating cells with purified or biotinylated Clec ectodomains and binding detected using fluorescently conjugated secondary reagents and flow cytometric analysis.
  • Isolation of cellular spectrin complexes was performed essentially as in Ungewickell and Gratzer (1978) and Gratzer (1982), and further purified by SEC.
  • erythrocytes were lysed for 30 min at 4°C in a hypotonic lysis buffer (5mM sodium phosphate, pH7.6, lmM PMSF, 1 x EDTA-free protease cocktail (Roche)) and erythrocyte membranes washed repeatedly using the lysis buffer.
  • a hypotonic lysis buffer 5mM sodium phosphate, pH7.6, lmM PMSF, 1 x EDTA-free protease cocktail (Roche)
  • Spectrin was extracted by resuspension of erythrocyte ghosts in a spectrin extraction buffer (0.3mM sodium phosphate, 0.1 mM EDTA, 0.1 mM PMSF) and overnight dialysis at 4°C against spectrin extraction buffer. Membrane fragments were removed by ultracentrifugation (90000g, 30 min, 4°C) and the supernatant containing spectrin harvested for further analysis. Isolation of non-erythroid spectrin from frozen pellets of human 293F cells (1 x 10 8 cells) was performed as for erythrocytic spectrin, with the addition of 50U/ml benzonase (Novagen) in the lysis buffer.
  • a spectrin extraction buffer 0.1% sodium phosphate, 0.1 mM EDTA, 0.1 mM PMSF
  • Spectrin samples were further purified by size exclusion chromatography using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min in PBS containing 10% glycerol, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF. Column fractions of 0.4 mL were collected for analysis.
  • Reassociation of cellular spectrins with actin was performed by collecting fractions of dissociated spectrin following SEC, and concentrating them in the presence of ⁇ of either bovine muscle actin or a control protein BSA, in PBS containing 10% glycerol, 0.1 mM PMSF, 1 mM EDTA, 1 mM DTT.
  • the spectrin-actin BSA samples were then incubated at 30°C for 2.5 h, then further purified by SEC using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min as above, and 0.4 ml fractions collected for analysis.
  • spectrin ⁇ Recombinant GST-tagged fragments of erythrocytic spectrin (spectrin ⁇ ) were expressed as previously described (Pei et al., 2005).
  • the Actin binding domains (ABD) of human Spectrin beta II (spectrin beta chain, brain 1 isoform 1; beta-fodrin, NM_003128.2) and of human alpha-actinin I (alpha-actinin-1 isoform a; alpha-actinin cytoskeletal isoform, NM_001130004.1) were expressed as glutathione S-transferase (GST) fusion proteins using E. coli BL21 (DE3).
  • cDNA containing the ABD regions were amplified from 293F cell cDNA using HotStar HiFidelity polymerase (Qiagen) using the primers listed below (Table 3), and subcloned into a modified pGex2T vector (GE Healthcare).
  • IPTG isopropyl- ⁇ - thiogalactopyranoside
  • GST-tagged proteins were purified by affinity chromatography using Glutathione-Sepharose 4B resin (GE Healthcare) and elution with 50mM Tris-HCl, pH8.0, 150mM NaCl containing 50mM reduced glutathione (Sigma), and further purified by size-exclusion chromatography using a Superose 6 column (300 x 10 mm) at a flow rate of 0.4 mL/min in 50mM Tris-HCl, pH 8.0 containing 150 mM NaCl.
  • Reassociation studies were performed by incubating GST-tagged spectrin and actinin proteins in the presence or absence of bovine muscle actin (Sigma) or platelet actin (>99% purity, Cytoskeleton) in PBS, for 2.5 h (30°C) at a concentration of 0.2mg/ml per protein. Proteins were diluted to ⁇ ⁇ in PBS for coating onto ELISA plates.
  • ELISA plates (Costar) were coated overnight at 4°C with either commercially available erythrocytic spectrin (Sigma), bovine muscle actin (Sigma) or platelet actin (>99% pure, Cytoskeleton) at ⁇ , or with cellular spectrins (K ⁇ g/ml) or complexes containing GST-tagged spectrin fragments, (l( ⁇ g spectrin fragment/ml) or with SEC fractions of spectrin (50 ⁇ 1). Bovine muscle actin and platelet actin appeared to have comparable purity on SDS-PAGE analysis. Unbound proteins were then washed away (PBS-0.05%Tween 20).
  • ELISA plates were blocked (1% BSA in PBS), then incubated with FLAG-tagged Clec9A ectodomain fragments or controls (mClecl2A-ecto , mCire DC-Sign-ecto). Binding was detected by incubation with an anti-FLAG antibody M2-conjugated to HRP (Sigma-Aldrich), and visualized by the addition of the HRP substrate ABTS, followed by measurement of absorbance at 405- 490 nm (Vmax Kinetic Microplate Reader, Molecular Devices).
  • Thermofluor assays were performed to determine protein stability (Phillips and de la Pena, 2011).
  • hCLEC9A-CTLD (S225D) was incubated in Tris buffered saline in the presence or absence of 6mM EDTA, 1.24mM MgS0 4 or 2.47mM CaCl 2 for lh at 4°C.
  • Thermofluor assays were performed using 18.4 ⁇ hCLEC9A-CTLD and Sypro Orange (1 :1250 dilution, Invitrogen) in a total volume of 25 ⁇ .
  • Thermal denaturation curves were measured on a CI 000 thermocycler equipped with a CFX384 Real-Time System fluorescence reader (BIORAD).
  • Tm melting temperature
  • Binding assays were performed in binding buffer (PBS containing 0.2%BSA and 0.02% sodium azide), on ice. Cells were washed 3 times with PBS to remove serum proteins, then resuspended in binding buffer. Cells were incubated with either (1) biotinylated soluble Clec9A and controls, and detected with Streptavidin (SA)-PE, or (2) soluble FLAG-tagged Clec9A and detected either with FITC conjugated rat anti- FLAG mAb 9H1, or with biotinylated rat anti-FLAG mAb 9H1 and SA-PE.
  • SA Streptavidin
  • Live cells were gated on forward and side scatter, or by PI exclusion, whereas dead cells were gated on forward and side scatter, or by PI inclusion.
  • Analysis of soluble Clec9 A binding was performed by flow cytometry using a FACScan (Becton Dickinson). The specificity of the binding was demonstrated by comparison to binding to other soluble FLAG-tagged C-type lectins, mouse Cire/mDCSign (Caminschi et al., 2001) and Clecl2A (Pyz et al., 2008).
  • Venous blood was obtained from C57B16 mice by cardiac puncture into 0.1 volume of Aster Jandl citrate-based anticoagulant (Aster and Jandl, 1964) (85 mM sodium citrate, 69 mM citric acid, 20 mg/ml glucose, pH 4.6). Platelet rich plasma was obtained by centrifugation of the murine blood at 125 x g for 8 min, followed by centrifugation of the supernatant buffy coat at 125 x g for 8 min.
  • Aster Jandl citrate-based anticoagulant Aster and Jandl, 1964
  • Platelet rich plasma was obtained by centrifugation of the murine blood at 125 x g for 8 min, followed by centrifugation of the supernatant buffy coat at 125 x g for 8 min.
  • Platelets were washed by two sequential centrifugations at 860 x g for 5 min in 140 mM NaCl, 5 mM C1, 12 mM trisodium citrate, 10 mM glucose, 12.5 mM sucrose, pH 6.0 the platelet pellet resuspended in 10 mM HEPES, 140 Mm NaCl, 3 mM KC1, 0.5 mM MgCl 2 , 10 mM glucose and 0.5 mM NaHCC>3, pH 7.4. Washed platelets were treated with 0.5 ⁇ ABT-737 for 90 min at 37°C.
  • Control or ABT-737 treated platelets were incubated with 5 ⁇ g/ml of Flag-tagged recombinant proteins mClec9A or hClec9A for 30 min at room temperature.
  • Anti-Flag-FITC secondary antibodies were added to the platelets and incubated for 30 min at room temperature. Samples were diluted out in PBS containing 0.2% BSA and analyzed on a FACSCalibur flow cytometer.
  • ABT-737 treated platelets were confirmed to be AnnexinV-APC positive, and resting control platelets AnnexinV-APC negative.
  • Mouse fibroblasts (NIH 3T3) cells were cultured on glass coverslips, fixed, permeabilised using PBS containing 0.3% Triton X-100 and incubated either with biotinylated mClec9A-ecto, or biotinylated Cire-ecto as a background control and binding detected using SA-Alexa594. Fibroblasts were counterstained with DAPI, and analysed by confocal microscopy using a Leica SP2 (Wilson et al., 2003). Mass Spectrometry Analysis
  • Protein bands were excised from Simply blue (Invitrogen) stained gels and subjected to manual in-gel digestion.
  • the gel bands were reduced with lOmM DTT (Calbiochem) for 30 min, alkykated for 30 min with 50mM iodoacetic acid (Fluka) and digested with 375ng trypsin (Worthington) for 16hrs at 37°C.
  • the extracted peptide solutions (0.1% formic acid) were then concentrated to approximately ⁇ by centrifugal lyophilisation using a SpeedVac AES 1010 (Savant). Digests were then subjected to MS/MS analysis on the LTQ-Orbitrap (Thermo Fischer Scientific) mass spectrometer.
  • Extracted peptides were injected and fractionated by nanoflow reversed-phase liquid chromatography on a nano LC system (1200 series, Agilent, USA) using a nano flow reversed-phase-HPLC (Model 1200, Agilent). Fractionation was performed using a nano-Acquity (CI 8) 150 mm x 0.15 mm I.D. RP-UPLC column (Waters) developed with a linear 60 min gradient from 0-100% Buffer B (0.1% (v/v) aqueous formic acid / 60% (v/v) acetonitrile) with a flow rate of 0.8 ⁇ 7 ⁇ at 45°C, where Buffer A was 0.1% (v/v) aqueous formic acid.
  • the capillary HPLC was coupled on-line to the LTQ- Orbitrap mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific). Positive ion mode was used for data-dependent acquisition.
  • Survey MS scans were acquired with the resolution set to 30,000. Each scan was recalibrated in real time by co-injecting an internal standard from ambient air into the C-trap (Olsen et al., 2005). The five most intense ions per cycle were fragmented and analysed in the linear trap. Target ions already selected for MS/MS were dynamically excluded for 180s.
  • Peak lists were searched against the LudwigNR protein sequence database (10753 sequence entries) (version Q409ml - www.ludwig.edu.au/archiveAudwigNR/ludwigNR.pdf) using the Mascot search algorithm (v2.2.04, Matrix Science, U.K.) (Perkins et al., 1 99).
  • the search parameters were as follows: carboxymethylation of cysteine as a . fixed modification (+58 Da) as well as variable modifications consisting of N3 ⁇ 4- terminal acetylation (+42 Da) and oxidation of methionine (+16 Da), and the allowance for up to three missed tryptic cleavage sites (trypsin P).
  • Precursor and fragment ion mass tolerance values were +/- 20ppm and 0.8Da respectively.
  • bacterial BL21 E. coli were electroporated with plasmid DNA and incubated at 37°C overnight on LB Agar plates containing Ampicillin (50 ⁇ g/ml). Bacterial culture broth containing ampicillin (100 ⁇ g ml) was inoculated with a single colony and incubated with shaking overnight at 37°C. Overnight bacterial cultures were diluted into Superbroth containing ampicillin (100 ⁇ g/ml), at 1:20 dilution and incubated in a shaking incubator at 30°C degrees until the cell density reached an ODioo of 0.8. Isopropyl-P-D- thiogalactopyranoside ( ⁇ ) was added to cultures to induce expression of proteins and cultures incubated at 19°C for 5 hours. Cells were harvested by centrifugation and stored at -80°C.
  • lysozyme lysis buffer phosphate buffered saline (PBS) containing 0.2mg/ml lysozyme, 30 ⁇ g/ml DNase I, 1 mM PMSF
  • Total lysates were subjected to sonication using the Bandelin sonoplus (50% duty cycle, 10 second pulse, 22 second cycle x5) and subsequently clarified by centrifugation.
  • the supernatant was incubated with 50% Giutathione-Sepharose resin slurry for 1 hour at 4°C on a rotating wheel and resin recovered in column (Bio-Rad Laboratories). Resin was washed extensively with PBS, and proteins eluted with 20mM reduced glutathione pH 7.0 in PBS.
  • ELISA plates (Costar) were coated with GST-RNF41 fusion proteins ( ⁇ g/ml) in tris buffered saline or phosphate buffered saline overnight at 4°C. Plates were washed with wash buffer (PBS/ 0.05% Tween20) to remove unbound proteins and then blocked for 1 hour in 5% skim milk in PBS at room temperature. Dilutions of Clec proteins in 5% skim milk in PBS were added and the plates incubated at RT for 2 hours. Plates were washed with wash buffer to remove unbound Clec proteins, and plates incubated with anti-FLAG 2 antibody conjugated to Horseradish Peroxidase (Sigma) for 2 hours at RT.
  • wash buffer PBS/ 0.05% Tween20
  • ELISA to measure enhancement of Clec9A binding to RNF41 by ABD-actin complexes were performed as follows. GST-tagged RNF41 C-terminal domain and control GST were coated on ELISA plates at ⁇ overnight at 4°C. Unbound proteins were washed away and plates blocked with 1% BSA in PBS. ABD-actin complexes of platelet actin with eythrocytic spectrin (spectrin ⁇ N), non-erthyroctic spectrin (spectrin ⁇ N) and a-actinin-1 (a-actinin-1 N) were pre-associated at 50 ⁇ g ml for 2.5 hours at 30°C, before incubation with Clec proteins for 1 hour at room temperature.
  • Clec9A mouse Clec9A
  • human CLEC9A human CLEC9A
  • mClec9A mouse Clec9A
  • hCLEC9A human CLEC9A
  • the inventors then investigated various stages of cell death, by following thymocytes undergoing apoptosis induced by ⁇ -irradiation or mouse embryonic fibroblasts (MEFs) undergoing apoptosis induced by BH3-only ligands (van Delft et al., 2006). Cells were stained with Annexin V, an early marker of apoptosis, and with PI, a late marker, which stains nuclei once the cell membrane is damaged.
  • MEFs mouse embryonic fibroblasts
  • Both mClec9A and hCLEC9A ectodomains bound to all dead mouse or human nucleated cells tested, including cultured cell lines and primary cells ( Figure 2). They also bound to freeze/thawed Chinese hamster (CHO) and African green monkey (Vero) cells (data not shown), and to freeze/thawed insect (SF21) cells, but not to freeze/thawed bacteria or yeast ( Figure 2F). Thus, recognition of the dead cell ligand was conserved across a wide evolutionary range.
  • Clec9A intracellular ligand for Clec9A could be a cytoskeletal component.
  • spectrin spectrin
  • filamin a suspension-adapted subline of human embryonic kidney cells
  • Clec9A interacting proteins isolated from mouse thymocytes and human 293 F cells.
  • Clec9A interacting proteins were isolated by incubation of FLAG-tagged Clec9A-ecto with lysates of mouse thymocytes or a suspension adapted subline of human 293 cells (Freestyle human 293 F) and affinity purification using anti-FLAG resin. Protein complexes were analysed by SDS-PAGE under reducing conditions, and a protein band of 220-300 kDa was excised and subjected to mass spectrometry analysis. The five most abundant proteins are described below.
  • Mass spectroscopic analysis revealed a majority of peptides corresponding to the expected spectrin al and spectrin ⁇ , but also peptides corresponding to spectrin-associated proteins including actin, band 4.1, adducins and tropomodulins (Table 5).
  • Example 8 - Clec9A binds to a higher order complex of erythrocyte spectrin
  • Spectrin extractions from mouse erythrocyte ghosts prepared by osmotic lysis were performed at 4°C, which enables the extraction of spectrin in the form of tetramers and higher order complexes, or at 37°C, which produces mainly spectrin dimers (Ungewickell and Gratzer, 1978; Gratzer et al., 1982).
  • the isolated spectrin was coated onto ELISA plates and investigated for Clec9A binding. Significant binding of mClec9A-ecto was obtained to spectrin isolated if at 4°C, but only poor binding to spectrin isolated at 37°C ( Figure 5A).

Abstract

La présente invention concerne l'identification de molécules qui lient le marqueur de cellule dendritique désigné Clec9A. La présente invention concerne de nouveaux composés utiles pour le ciblage d'agents thérapeutiques tels que des antigènes de cellules dendritiques. L'invention porte également sur des procédés de modulation d'une réponse immunitaire humorale et/ou à médiation par lymphocytes T audit antigène, sur des procédés d'administration d'un agent cytotoxique à des cellules dendritiques de celui-ci impliquées dans des états maladifs, sur des procédés de modulation de captage et/ou de clairance de cellules présentant une membrane cellulaire brisée, de cellules infectées par un pathogène, de cellules moribondes ou mortes, ou une partie de celles-ci, et sur des procédés de modulation de reconnaissance d'antigène, de traitement et/ou de présentation, ainsi que sur des réponses immunitaires à des substances dérivées de cellules présentant une membrane cellulaire brisée, de cellules infectées par un pathogène, de cellules moribondes ou mortes, ou une partie de celles-ci.
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