EP2542573A1 - Nouvelle protéine avec une homologie structurelle par rapport aux protéines comportant une pliure de type chimiokinique il-8, et utilisations de celle-ci - Google Patents

Nouvelle protéine avec une homologie structurelle par rapport aux protéines comportant une pliure de type chimiokinique il-8, et utilisations de celle-ci

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Publication number
EP2542573A1
EP2542573A1 EP11705893A EP11705893A EP2542573A1 EP 2542573 A1 EP2542573 A1 EP 2542573A1 EP 11705893 A EP11705893 A EP 11705893A EP 11705893 A EP11705893 A EP 11705893A EP 2542573 A1 EP2542573 A1 EP 2542573A1
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European Patent Office
Prior art keywords
protein
seq
acid sequence
amino acid
cells
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EP11705893A
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German (de)
English (en)
Inventor
Maria Teresa Pisabarro
Aurelie Tomczak
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Technische Universitaet Dresden
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Technische Universitaet Dresden
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Publication of EP2542573A1 publication Critical patent/EP2542573A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5421—IL-8

Definitions

  • Novel protein with structural homology to proteins with IL-8-like chemokine fold and uses thereof
  • the present invention relates to the identification and uses of a protein having structural homology to proteins with IL-8-like chemokine fold.
  • the invention has various industrial applications in medical, pharmaceutical and biotechnological applications, such as pharmaceuticals, diagnostics, biosensors and bioreactors.
  • Chemokines are a large family of small proteins with similar structure secreted by cells. Proteins are classified as chemokines according to shared structural characteristics such as their small size, which comprising a molecular mass between 8 and 10 kilodaltons (kDa) containing an IL8-like chemokine fold, and the presence of in most cases four cysteine residues that interact with each other forming intramolecular disulfide bonds essential for their function.
  • kDa kilodaltons
  • the disulfide bonds are typically formed between the first and third and the second and fourth cysteine residue forming a "Greek key" shaped characteristic tertiary structure so-called IL-8 fold, which consists of an N-terminal loop of approximately ten amino acids, a single turn helix, three beta-strands and a C-terminal alpha-helix.
  • Known chemokines show a wide range of sequence similarity, ranging from 10 % up to more than 90 % sequence identity. Chemokines can be categorized into four groups, depending on the spacing of the first two cysteine residues.
  • CXC chemokines possess two adjacent cysteines, in CXC chemokines the first two cysteines are separated by one amino acid, CX 3 C chemokines have three amino acids between the two cysteines and XC chemokines only possess two cysteine residues at all.
  • Chemokines induce directed chemotaxis in nearby responsive cells by binding to their respective receptors on the cells and thus promoting signaling cascades that generate responses like chemotaxis, degranulation, release of superoxide anions or changes in the avidity of cell adhesion molecules. Attracted chemotactic cells thereby follow a signal of increasing chemokine concentration towards the source of the chemokine, which may be for example infected or damaged cells.
  • Some chemokines, like truncation regulated MCP2-4 can inhibit migration instead of inducing it.
  • chemokines are considered pro-inflammatory and can be induced during an immune response to promote cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development.
  • IL-8 Interleukin-8
  • macrophages and other cells for example epithelial cells. It is known to play a role in acute inflammatory responses. These responses are mediated primarily by TNF- alpha, IL-1 and IL-6. Localized effects include increased adherence of circulating white blood cells to vascular endothelial cells and their extravasation into tissue spaces.
  • TNF- alpha TNF- alpha
  • IL-1 and IL-6 Localized effects include increased adherence of circulating white blood cells to vascular endothelial cells and their extravasation into tissue spaces.
  • CAMs cell-adhesion molecules
  • IL-8 chemotactically attracts neutrophils and promotes their adherence to endothelial cells. Specifically, IL-8 chemoattracts monocytes and dendritic cells. Both cell types play an important role in the initiation of an immune response.
  • Drugs that alter cell migration represent a particularly promising class of new anti-inflammatory drugs that appear to show potential in clinical trials and in many animal models of inflammatory diseases.
  • Cell migration inhibitors not only interfere with migration of cells to a tissue, but also can affect other necessary processes such as mediator release and angiogenesis.
  • Angiogenic chemokines CXCL 1 , 2, 3, 5, 6, 7, 8, 12 and the viral chemokines vMIP-I and vMIP-II (Boshoff 1997) promote tumor growth by stimulation proliferation and chemotaxis of endothelial cells of the blood vessels that serve the tumor whereas angiostatic chemokines (CXCL4, 9, 10, 11, 13, 14) promote tumor necrosis by inhibition of endothelial cell proliferation and chemotaxis.
  • chemokines and their receptors were shown to be involved in HIV-1 infections (Boshoff 1997).
  • Two CC chemokines that are encoded in Kaposi sarcoma herpes virus (KSHV), vMIP-I and vMIP-II act as inhibitors for HIV-1 entry into CD4 T cells.
  • Viral MIP-I has been shown to function as a specific agonist for the CC-Chemokine receptor CCR8, a chemokine receptor expressed by Th2 lymphocytes and cultured monocytes. As already discussed, it has also been shown to be highly angiogenic. In primary effusion lymphoma- derived cell lines viral MIP-1-alpha inhibits chemically induced cell death by apoptosis in these cells (Liu 2001). Louahed et al. (Louahed 2003) have reported that viral MIP-1 has anti-apoptotic activities.
  • Protein threading is a method of computational protein structure prediction based on statistical analysis of available protein structures used for characterization of protein sequences, which do not show any statistically significant sequence homology to any known characterized protein.
  • chemokines may contribute to a number of important medical conditions related to immune function and cell division. Given the potential of chemokine related molecules to occupy important roles in the control of immune function, there is an interest in the identification of other members of this family and the receptors that direct the actions of these molecules through particular target cell populations.
  • the present invention describes the identification, characterization and use of novel proteins that are structurally similar to proteins with an IL-8-like chemokine fold, and active variants thereof.
  • the object of the invention is to identify, characterize and use novel secreted proteins with a structural homology to proteins with an IL-8-like chemokine fold which are involved in immune- mediated and inflammatory diseases or neoplastic malignancies.
  • the object is solved by providing a novel protein, named and further referred to herein as "B42", with structural similarity to proteins of the IL-8-like chemokine fold, in particular to the known chemokines vMIP-I and vMIP-II, which is characterized by its amino acid sequence as presented in SEQ ID No. 1 and its nucleic acid sequence as presented in SEQ ID No. 2 and SEQ ID No. 3.
  • the invention relates to an isolated protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence of the protein shown in one of the amino acid sequences presented in SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No . 7, preferably SEQ ID No. 1.
  • the invention relates to an isolated protein comprising an amino acid sequence that is identical to one of the amino acid sequences presented in SEQ ID No. l, SEQ ID No. 6 and SEQ ID No. 7.
  • the proteins with SEQ ID No. 6 and SEQ ID No. 7 are preferred examples for proteins with at least 80 % amino acid sequence identity to SEQ ID No. 1.
  • the inventors used the fold recognition algorithm ProHit for threading the uncharacterized sequences against two fold libraries built up for this purpose; a fold library containing all known structures of the members of the IL-8-like fold family (consisting of 217 entries) and a fold library obtained from filtering the Brookhaven Protein Data Bank (PDB) structures at 95 % sequence identity (consisting of 22009 representative three-dimensional protein structures).
  • a fold library containing all known structures of the members of the IL-8-like fold family (consisting of 217 entries) and a fold library obtained from filtering the Brookhaven Protein Data Bank (PDB) structures at 95 % sequence identity (consisting of 22009 representative three-dimensional protein structures).
  • B42 protein according to the invention, referred herein also as "B42" was identified to have an IL-8-like three-dimensional structure, and therefore to be a novel member of the chemokine family. Furthermore, B42 exhibits a strong structural similarity to vMIP-I and vMIP-II, members of the IL-8-like chemokine fold family, which are involved in blockade of viral infiltration into host cells.
  • the sequence identity between B42 and vMIP-II is 26.2 %.
  • the sequence identity between B42 and vMIP-I is 22.6 %.
  • B42 shares 11.9 % sequence identity with IL-8.
  • the identified polypeptide sequence of B42 was not yet characterized as a protein in sequence databases (ENSEMBL) since it was wrongly annotated as being located in an intron structure.
  • ENSEMBL release 55 it is labelled as "processed transcript”.
  • the inventors cloned this gene product and obtained B42 protein by in vitro expression.
  • isolated protein means that a subject protein
  • a) is preferably free of at least some other proteins with which it would normally be found
  • b) is preferably essentially free of other proteins from the same source, e. g. from the same species, c) is preferably expressed by a cell from a different species,
  • d) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature,
  • e is preferably not associated (by covalent or noncovalent interaction) with portions of a protein with which the "isolated protein" is associated in nature, or
  • f is preferably operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature.
  • Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof.
  • the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • amino acid sequence identity with respect to a specific amino acid sequence it is referred to the percentage of amino acid residues in a candidate sequence that is identical with the amino acid sequence in the specific amino acid sequence.
  • the B42 protein according to the invention has structural homology to the chemokine protein family that share a common 3D structure, the IL-8-like chemokine fold.
  • the B42 protein according to the invention in particular possesses a high structural homology to vMIP-I and vMIP-II.
  • the 3-dimensional structure of the B42 protein according to the invention features an IL-8 like chemokine fold. More specifically, it comprises at least two cysteine residues that form an intramolecular disulfide bond, three ⁇ -strands and an a-helix.
  • the characteristic 3-dimensional structure of the proteins according to the invention is formed by cysteines that interact with each other in pairs by the formation of disulfide bonds to create a characteristic Greek key shape (which is named for its resemblance to the Greek key meander pattern in art).
  • the IL8-like chemokine fold more specifically comprises an N-terminal loop (N- loop) followed by a single-turn helix or a 3io-helix (3-turn helix), three anti-parallel ⁇ -strands and a C- terminal ⁇ -helix (4-turn helix). These helices and strands are connected by turns preferably so called 30s, 40s and 50s loops.
  • the amino acid sequence of the B42 protein according to the invention comprises at least two cysteine residues, preferably four cysteine residues, more preferably six cysteine residues.
  • four cysteine residues are involved in formation of intramolecular disulfide bonds, which preferably join the first to third, and the second to fourth cysteine residues, numbered as they appear in the protein sequence of the chemokine. More preferably, six cysteine residues are involved in intramolecular disulfide bonds.
  • chimeric molecules which comprise a B42 polypeptide according to the invention fused to a heterologous amino acid sequence.
  • this heterologous amino acid sequence is an epitope tag sequence or an Fc region of an immunoglobulin.
  • the invention further comprises proteins that are circular permutations of the B42 protein according to the invention, further referred to as circularly permutated variants.
  • the invention comprises circularly permutated variants of the B42 protein with the amino acid sequence according to SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7 (or a protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence according to SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7).
  • circular permutation in coherence with the invention is meant a genetic operation in which part of the C-terminus of a protein is moved to its N-terminus by maintaining the three-dimensional structure and biological function of said protein.
  • a circular permutation is built from one or more sets of structural elements in cyclic order.
  • An engineered circular permutation of a protein can be envisioned as the result of taking a linear protein sequence, joining its ends to create a circle and then cleaving the circle at another site to generate a new linear monomeric protein sequence, only altered at the sites of joining and cleavage and with a resulting different sequential order of the secondary structure elements but same 3D structure. This approach of engineering a new protein sequence by circularly permuting was pioneered by Goldenberg in 1983 (Goldenberg 1983).
  • the newly engineered protein maintains the original function either totally (preferred) or partially.
  • the B42 protein according to the invention preferably contains loops (amino acids counted) at the following amino acid positions: amino acids 8 - 20 (N-loop), amino acids 27 - 33 (30S-loop), amino acids 38 - 42 (40S-loop), amino acids 47 - 57 (50S-loop).
  • Circular permutated variants are obtained by connecting the C-terminus with the N-Terminus and cutting (genetic operation) within one of said mentioned loops (to generate a new C-terminus and N-Terminus).
  • the cutting site is not positioned in the N-loop of the protein.
  • Circularly permutated variants of a B42 protein according to the invention preferably comprise the amino acid sequence according to SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7 (or a protein having at least 80 %>, preferably at least 90 %>, preferably at least 95 %>, preferably at least 99 % amino acid sequence identity to the amino acid sequence according to SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7) in rearranged order.
  • beta sheet a) beta sheet, beta sheet, alpha helix, beta sheet,
  • beta sheet alpha helix, beta sheet, beta sheet, or
  • alpha helix beta sheet, beta sheet, beta sheet.
  • structural elements of circular permutated variants of the B42 protein according to the invention comprise the following amino acid sequences (each one comprising at least 80%, preferably 90%), more preferably 95%, even more preferably 99% amino acid sequence identity to the respective amino acid sequence according to SEQ ID No. 1).
  • beta sheet according to amino acids 34 to 37 in SEQ ID No. 1 (S2) beta sheet according to amino acids 43 to 46 in SEQ ID No. 1 (S3), alpha helix according to amino acids 58 to at least 65 in SEQ ID No. 1, beta sheet according to amino acids 23 to 26 in SEQ ID No. 1 (SI);
  • beta sheet according to amino acids 43 to 46 in SEQ ID No. 1 (S3) alpha helix according to amino acids 58 to at least 65 in SEQ ID No. 1, beta sheet according to amino acids 23 to 26 in SEQ ID No. 1 (SI); beta sheet according to amino acids 34 to 37 in SEQ ID No. 1 (S2), c) alpha helix according to amino acids 58 to at least 65 in SEQ ID No. 1, beta sheet according to amino acids 23 to 26 in SEQ ID No. 1 (SI); beta sheet according to amino acids 34 to 37 in SEQ ID No. 1 (S2), beta sheet according to amino acids 43 to 46 in SEQ ID No. 1 (S3).
  • Chemokine receptor activation is a two step process (Baggiolini 2001). First, a chemokine binds the N- terminal region of the receptor by interaction mainly through basic surface residues and second, the receptor signaling is triggered by interaction of the flexible N-terminus of the chemokine with a second site on the receptor. Often the receptor affinity is modulated by proteolytic cleavage of the chemokine N- terminus (Wolf 2008). By this, the chemokine can be either turned into an inhibitor of the receptor or the receptor activation is increased or reduced.
  • the three dimensional IL8-like structure is preferably maintained by circular permutated variants according to the invention.
  • the circularly permutated variants according to the invention are able to bind to the receptor (as inhibitors or activators).
  • the biological function of a circularly permutated variant according to the invention is maintained in activating variants with structures according to a) or c).
  • an additional extension comprising an amino acid sequence from the original N-terminus is appended to the N-terminus of a circularly permutated variant according to the invention. Otherwise, only binding ability (inhibition) is maintained, whereas signaling ability is not.
  • a circularly permutated variant according to a structure described in b) is preferably an inhibitor of the natural receptor. Said variant is able to bind the receptor but does not have the flexible N-terminus at the right position to induce receptor signaling.
  • the present invention relates to an isolated nucleic acid encoding for the B42 polypeptide according to SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7 (also herein referred to as "B42 nucleic acid").
  • the invention relates to an isolated nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence encoding the B42 protein shown in SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7.
  • the isolated nucleic acid has at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence presented in SEQ ID No. 2 or to the nucleotide sequence presented in SEQ ID No. 3.
  • nucleic acids as referred to herein mean single-stranded or double- stranded nucleic acid polymers of at least 10 nucleotides in length.
  • the nucleotides comprising the nucleic acids can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2', 3 '-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphor odiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromouridine, ribose modifications such as arabinoside and 2', 3 '-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphor odiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • nucleic acid specifically includes single and double stranded forms of DNA.
  • nucleic acid sequence identity refers to the percentage of nucleic acid residues in a candidate sequence that are identical with the nucleic acid sequence in the specific nucleic acid sequence.
  • aspects of the invention are directed to an isolated nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence comprising the coding sequence of the full length B42 polypeptide cDNA as presented in SEQ ID No. 2 or the full transcript sequence as presented in SEQ ID No. 3.
  • Another aspect of the invention relates to a vector comprising a B42 nucleic acid according to the invention (also herein referred to as "B42 vector").
  • the vector according to the invention comprises a B42 nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence encoding the B42 protein shown in SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7.
  • the vector according to the invention comprises a nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence shown in SEQ ID No. 2.
  • the vector according to the invention comprises a nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence shown in SEQ ID No. 3.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e. g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e. g. non-episomal mammalian vectors
  • the vector comprising the nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.
  • operably linked means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
  • a control sequence "operably linked" to a protein coding sequence, as comprised in the vector according to the invention is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.
  • the term "host cell” is used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest.
  • the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.
  • the invention refers to a host cell comprising the B42 vector according to the invention.
  • the host cells are CHO cells, HEK-293T cells, HEK-293A cells, HeLa cells, E. coli cells, yeast cells or baculovirus infected insect cells.
  • a multi-cellular organism according to the invention is a naturally occurring or otherwise genetically modified organism, preferably an invertebrate or vertebrate organism, like Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis, Medaka or Zebrafish or Mus musculus, or an embryo thereof.
  • the invention also relates to a process for producing a B42 polypeptide as presented in SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7 or a polypeptide with at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % sequence identity to the polypeptide presented in SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7.
  • the process comprises the steps of:
  • Suitable conditions for culturing a prokaryotic or eukaryotic host are well known to a person skilled in the art. In general, the skilled person is also aware that these conditions may have to be adapted to the needs of the host and the requirements of the polypeptide expressed.
  • an inducible promoter controls the nucleic acid of the invention in the vector present in the host cell
  • expression of the polypeptide can be induced by addition of an appropriate inducing agent.
  • Suitable expression protocols and strategies are known to a person skilled in the art.
  • the proteins according to the invention are recovered from the cells or the culture supernatant by methods well-known in the art. These methods comprise without limitation method steps such as ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, disc gel electrophoresis or immunoprecipitation.
  • the B42 protein according to the invention displays a target protein for therapeutic and diagnostic applications related to immune related disorders.
  • the invention further comprises an antibody, which specifically binds to a B42 protein according to the invention.
  • antibody refers to an intact antibody, a binding fragment thereof or derivatives thereof that are capable of specifically binding to the protein according to the invention. Binding fragments and derivatives comprise the epitope-binding fragments of the intact antibody and include, but are not limited to, F (ab), F (ab'), F (ab') 2, Fv, and single-chain antibodies.
  • the antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody or a single chain antibody.
  • the B42 protein according of the invention or an antagonist or agonist thereof or an antibody according to the invention are composed in combination with a carrier.
  • the invention refers to a composition of matter comprising a) a B42 protein according to the invention, b) an agonist of said B42 protein, c) an antagonist of said B42 protein or d) an antibody the specifically binds to said B42 protein in combination with a carrier.
  • Suitable carriers are well known to a person skilled in the art.
  • the carrier is a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • Suitable pharmaceutically acceptable carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc. Compositions comprising such carriers can be formulated by well known conventional methods.
  • An agonist as referred to herein includes any molecule that mimics a biological activity of the protein according to the invention.
  • An antagonist according to the invention includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of the protein according to the invention.
  • Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of polypeptides according to the invention, peptides, chemokine mutants or small organic molecules.
  • Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, chemokine mutants, fragments of chemokines, fragments of chemokine mutants, antisense oligonucleotides or small organic molecules.
  • Biological activity as used herein refers to a biological function, which may be either inhibitory or stimulatory, that is caused by a native or naturally- occurring protein according to the invention and which is other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally- occurring protein according to the invention.
  • Antagonists or agonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the protein according to the invention.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
  • a small molecule according to the invention has a molecular weight below about 500 Daltons.
  • the invention relates to the use of a composition of matter according to the invention that comprises a B42 protein according to the invention, an agonist of said B42 protein, an antagonist of said B42 protein or an antibody the specifically binds to said B42 protein in combination with a carrier to treat an immune related disorder in a mammal or human.
  • a B42 protein according to the invention, an agonist thereof, an antagonist thereof or an antibody that specifically binds to said protein is capable of increasing the proliferation of T-lymphocytes in a mammal, inhibiting the proliferation of T-lymphocytes in a mammal, increasing infiltration of inflammatory cells into a tissue of a mammal, or decreasing the infiltration of inflammatory cells into a tissue of a mammal.
  • composition of matter according to the invention comprises a therapeutically effective amount of a) a B42 protein according to the invention, b) an agonist of said B42 protein, c) an antagonist of said B42 protein or d) an antibody the specifically binds to said B42 protein.
  • the therapeutically effective amount for a given situation or a given immune related disorder will readily be determined by routine experimentation and is within the skills and judgment of the ordinary clinician or physician.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 5 g units per day.
  • a preferred dosage might be in the range of 0.01 mg to 100 mg, preferably 0.01 mg to 50 mg and preferably 0.01 mg to 10 mg per day.
  • the invention further comprises an article of manufacture, comprising a container, a label on said container, and a composition of matter comprising a) a B42 protein according to the invention, b) an agonist of said B42 protein, c) an antagonist of said B42 protein or d) an antibody that specifically binds to said B42 protein, contained within said container, wherein a label on said container indicates that said composition of matter can be used for treating an immune related disease.
  • the invention further comprises a method of treating an immune related disorder in a mammal or human in need thereof comprising administering to said mammal or human a therapeutically effective amount of a) a B42 protein according to the invention, b) an agonist of said B42 protein, c) an antagonist of said B42 protein or d) an antibody that specifically binds to said B42 protein.
  • Immune related disorders include without limitations systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, a spondyloarthropathy, systemic sclerosis, an idiopathic inflammatory myopathy, Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus type I, immune-mediated renal disease, a demyelinating disease of the central or peripheral nervous system, idiopathic demyelinating polyneuropathy, Guillain-Barre syndrome, a chronic inflammatory demyelinating polyneuropathy, a hepatobiliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, Whipple'
  • the B42 protein according to the invention is a target for diagnostic applications, e.g. for diagnosis of an immune related disorder in a patient.
  • the invention refers to a method for determining the presence of a B42 protein according to the invention or a naturally- occuring B42 protein with an amino acid sequence according to SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7 in a test sample suspected of containing said B42 protein.
  • Said method according to the invention comprises the steps of a) exposing said sample to an antibody according to the invention, that specifically binds to said B42 polypeptide and b) determining binding of said antibody by detecting the formation of a complex between the antibody and the B42 protein according to the invention.
  • the detection is qualitative or quantitative, and is preferentially performed in comparison with monitoring the complex formation in a control sample.
  • a larger quantity of complexes formed in the test sample indicates the presence or absence of an immune disease in the mammal from which the test tissue cells were obtained.
  • the antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, ELISA, immunohistochemistry or other techniques known in the art.
  • the detection methods of antibody binding are well known to an ordinary person skilled in the art.
  • Further aspects of the invention refer to methods for diagnosis of immune related diseases in a mammal or human.
  • the invention is related to a method of diagnosing an immune related disease in a mammal, said method comprising detecting the level of expression of a gene encoding the B42 amino acid sequence shown in SEQ ID No. 1, SEQ ID No. 6 or SEQ ID No. 7, in a test sample of tissue cells obtained from the mammal or human, and in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower level of expression of said gene in the test sample as compared to the control sample is indicative of the presence of an immune related disease in the mammal from which the test tissue cells were obtained.
  • the invention relates to a method of diagnosing an immune related disease in a mammal or human, said method comprising
  • formation of said complex is indicative of the presence of an immune related disease in the mammal from which the test tissue cells were obtained.
  • Further aspects of the invention relate to methods of identifying a compound that modulates the activity of a B42 protein according to the invention, said method comprising contacting cells which normally respond to said protein with said B42 protein and a candidate compound, and determining the lack of responsiveness by said cell to a).
  • modulation of protein activity in that context is meant a compound that either stimulates or inhibits the biological activity of the said protein.
  • the invention encompasses methods of screening compounds to identify those that mimic the B42 protein of the invention (agonists) or prevent the effect of said B42 protein (antagonists).
  • Methods for identifying agonists or antagonists of a protein according to the invention may comprise contacting said protein with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with said protein.
  • the protein according to the invention is preferably added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of said protein indicates that the compound is an antagonist to said protein.
  • antagonists to the protein according to the invention are detected by combining said protein and a potential antagonist with membrane-bound receptors binding to a protein according to the invention or recombinant receptors under appropriate conditions for a competitive inhibition assay.
  • the protein according to the invention can be labeled, such as by radioactivity, such that the number of said protein molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist.
  • the invention concerns a method of identifying agonists of or antagonists to a B42 protein according to the invention which comprises contacting said protein with a candidate molecule and monitoring a biological activity mediated by said protein.
  • the agonist or antagonist is an anti-B42 antibody.
  • Assays for identification of antagonists or agonists are designed to identify compounds that bind or complex with the proteins according to the invention, or otherwise modulate the interaction of the encoded proteins with other cellular proteins.
  • Such screening assays will include assays amenable to high- throughput screening of chemical libraries, making them particularly suitable for identifying small molecule candidates for agonists or antagonists.
  • Small molecules contemplated include synthetic organic or inorganic compounds.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • Another aspect of the invention refers to a method of identifying a compound that inhibits the expression of a gene encoding the B42 amino acid sequence shown in SEQ ID No. l, SEQ ID No. 6 or SEQ ID No. 7, said method comprising contacting cells which normally express said B42 protein with a candidate compound, and determining the lack of expression of said gene.
  • the decrease in the expression level of B42 gene expression can be detected through measurement of B42 RNA level in the cell, or through measurement of B42 protein level in the cell.
  • the candidate compound is an antisense nucleic acid.
  • the object is also solved by providing a novel protein, named and further referred to herein as "N73", with structural similarity to proteins of the IL-8-like chemokine fold, in particular to the known chemokines vMIP-II and vMIP-I, which is characterized by its amino acid sequence as presented in SEQ ID No. 9 or SEQ ID No. 10, and its nucleic acid sequence as presented in SEQ ID No. 11 or SEQ ID No. 12.
  • the protein according to SEQ ID No. 10 corresponds to the protein according to SEQ ID No . 9 additionally comprising its associated signal peptide.
  • the invention relates to an isolated N73 protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence of the N73 protein shown in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
  • the invention relates to an isolated N73 protein comprising an amino acid sequence that is identical to one of the amino acid sequences presented in SEQ ID No. 9, SEQ ID No. 13 and SEQ ID No. 14.
  • the N73 protein according to the invention contains at least 2, preferably at least 6, more preferably 7, 8 or 12 cysteine residues.
  • the amino acid sequence of the N73 protein according to the invention possesses two adjacent cysteine residues (CC-motif), which are preferably located at position 54 and 55 in the amino acid sequence presented in SEQ ID No. 9.
  • the N73 protein according to the invention was identified by fold recognition analysis and shows structural similarity with proteins belonging to the IL-8-like chemokine fold family, in particular with vMIP-II and vMIP-I.
  • N73 protein according to the invention, referred herein also as "N73" was identified to have an IL-8-like three- dimensional structure, and therefore to be a novel member of the chemokine family. Furthermore, N73 exhibits a strong structural similarity to vMIP-II and vMIP-I, members of the IL-8-like chemokine fold family, which are involved in blockade of viral infiltration into host cells.
  • the sequence identity between N73 and vMIP-II is 23.2 %.
  • the sequence identity between N73 and vMIP-I is 18.7 %.
  • N73 shares 17,2 % sequence identity with IL-8.
  • the amino acid sequence of N73 comprises 208 amino acids, of which 12 are cysteines, the amino acid sequence includes a CC motif.
  • the full N73 amino acid sequence is presented in SEQ ID No. 10 and comprises a signal peptide, which consists of 23 amino acids.
  • the signal peptide comprises 4 cysteine residues.
  • the N73 amino acid sequence without signal peptide is presented in SEQ ID No. 9.
  • the nucleic acid sequence of N73 was first identified as a gene related to cancer development and progression (Wan 2004) in a large-scale cDNA transfection screening.
  • the amino acid sequence of the N73 protein according to the invention comprises at least two cysteine residues, preferably 7 cysteine residues, more preferably 8 cysteine residues, more preferably 12 cysteine residues.
  • six cysteine residues are involved in formation of intramolecular disulfide bonds.
  • chimeric molecules which comprise a N73 polypeptide according to the invention fused to a heterologous amino acid sequence.
  • this heterologous amino acid sequence is an epitope tag sequence or an Fc region of an immunoglobulin.
  • the invention further comprises proteins that are circular permutations of the N73 protein according to the invention, further referred to as circularly permutated variants.
  • the invention comprises circularly permutated variants of the N73 protein with the amino acid sequence according to SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14 (or a protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence according to SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14).
  • circular permutation in coherence with the invention is meant a genetic operation in which part of the C-terminus of a protein is moved to its N-terminus by maintaining the three-dimensional structure and biological function of said protein.
  • a circular permutation is built from one or more sets of structural elements in cyclic order.
  • An engineered circular permutation of a protein can be envisioned as the result of taking a linear protein sequence, joining its ends to create a circle and then cleaving the circle at another site to generate a new linear monomeric protein sequence, only altered at the sites of joining and cleavage and with a resulting different sequential order of the secondary structure elements but same 3D structure. This approach of engineering a new protein sequence by circularly permuting was pioneered by Goldenberg in 1983 (Goldenberg 1983).
  • the newly engineered protein maintains the original function either totally (preferred) or partially.
  • the N73 protein according to the invention preferably contains loops (amino acids counted without signal peptide) at the following amino acid positions: amino acids 8 - 20 (N-loop), amino acids 74 - 82 (30S-loop), amino acids 86 - 99 (40S-loop), amino acids 103 - 107 (50S-loop).
  • Circularly permutated variants of a N73 protein according to the invention preferably comprise the amino acid sequence according to SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14 (or a protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence according to SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14) in rearranged order.
  • structural elements of circular permutated variants of the N73 protein according to the invention comprise the following amino acid sequences (each one comprising at least 80%, preferably 90%, more preferably 95%, even more preferably 99% amino acid sequence identity to the respective amino acid sequence according to SEQ ID No. 9).
  • beta sheet according to amino acids 83 to 85 in SEQ ID No. 9 (S2) beta sheet according to amino acids 100 to 102 in SEQ ID No. 9 (S3), alpha helix according to amino acids 67 to at least 72 in SEQ ID No. 9, beta sheet according to amino acids 66 to 73 in SEQ ID No. 9 (SI);
  • an additional extension comprising an amino acid sequence from the original N-terminus is appended to the N-terminus of a circularly permutated variant according to the invention. Otherwise, only binding ability (inhibition) is maintained, whereas signaling ability is not.
  • a circularly permutated variant according to a structure described in b) is preferably an inhibitor of the natural receptor. Said variant is able to bind the receptor but does not have the flexible N-terminus at the right position to induce receptor signaling.
  • the present invention relates to an isolated nucleic acid encoding for a N73 polypeptide according to SEQ ID No. 9 or SEQ ID No. 10 (herein also referred to as "N73 nucleic acid").
  • N73 nucleic acid a nucleic acid encoding for a N73 polypeptide according to SEQ ID No. 9 or SEQ ID No. 10 (herein also referred to as "N73 nucleic acid").
  • the invention relates to an isolated N73 nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence encoding the N73 protein shown in SEQ ID No. 9 or SEQ ID No. 10.
  • the isolated nucleic acid has at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence presented in SEQ ID No. 11 or to the nucleotide sequence presented in SEQ ID No. 12.
  • nucleic acids as referred to herein means single-stranded or double- stranded nucleic acid polymers of at least 10 nucleotides in length.
  • the nucleotides comprising the nucleic acids can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2', 3 '-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromouridine, ribose modifications such as arabinoside and 2', 3 '-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • nucleic acid specifically includes single and double stranded forms of DNA.
  • N73 nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence comprising the coding sequence of the full length N73 polypeptide cDNA as presented in SEQ ID No. 11 or the full transcript sequence as presented in SEQ ID No. 12.
  • N73 vector a vector comprising a nucleic acid according to the invention
  • the N73 vector according to the invention comprises a nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence encoding the N73 protein shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 13 or SEQ ID No. 14.
  • the N73 vector according to the invention comprises a nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence shown in SEQ ID No. 1 1.
  • the N73 vector according to the invention comprises a nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence shown in SEQ ID No. 12.
  • the N73 vector comprising the nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.
  • the invention refers to a host cell comprising the N73 vector according to the invention.
  • the host cells are CHO cells, HEK-293T cells, HEK-293A cells, HeLa cells, E. coli cells, yeast cells or baculovirus infected insect cells.
  • the invention also relates to a process for producing a N73 polypeptide as presented in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 13 or SEQ ID No. 14 or a polypeptide with at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % sequence identity to the polypeptide presented in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 13 or SEQ ID No. 14.
  • the process comprises the steps of: culturing the host cells or multi cellular organisms according to the invention under conditions suitable for the expression of said N73 polypeptide and
  • the N73 protein according to the invention displays a target protein for therapeutic and diagnostic applications related to immune related disorders or cancer.
  • the invention further comprises an antibody, which specifically binds to a N73 protein according to the invention.
  • the antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody or a single chain antibody.
  • the N73 protein according of the invention or an antagonist or agonist thereof or an antibody according to the invention are composed in combination with a carrier.
  • the invention refers to a composition of matter comprising a) a N73 protein according to the invention, b) an agonist of said N73 protein, c) an antagonist of said N73 protein or d) an antibody the specifically binds to said N73 protein in combination with a carrier.
  • the invention relates to the use of a composition of matter that comprises a) a N73 protein according to the invention, b) an agonist of said N73 protein, c) an antagonist of said N73 protein or d) an antibody the specifically binds to said N73 protein in combination with a carrier to treat a disease in a mammal or human.
  • diseases treated with a composition of matter comprising a) a N73 protein according to the invention, b) an agonist of said N73 protein, c) an antagonist of said N73 protein or d) an antibody the specifically binds to said N73 protein in combination with a carrier are immune related disorders, preferably the aforementioned immune related disorders.
  • the immune related disorder is AIDS.
  • a N73 protein according to the invention an agonist thereof, an antagonist thereof or an antibody that specifically binds to said N73 protein is capable of increasing the proliferation of T- lymphocytes in a mammal, inhibiting the proliferation of T-lymphocytes in a mammal, increasing infiltration of inflammatory cells into a tissue of a mammal, or decreasing the infiltration of inflammatory cells into a tissue of a mammal.
  • said diseases treated by a composition of matter comprising a) a N73 protein according to the invention, b) an agonist of said N73 protein, c) an antagonist of said N73 protein or d) an antibody the specifically binds to said N73 protein in combination with a carrier are malignant neoplasms, such as tumor diseases or other types of cancer.
  • the therapeutically effective amount for a given situation or a given disease will readily be determined by routine experimentation and is within the skills and judgment of the ordinary clinician or physician.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 5 g units per day.
  • a preferred dosage might be in the range of 0.01 mg to 100 mg, preferably 0.01 mg to 50 mg and preferably 0.01 mg to 10 mg per day.
  • the invention further comprises an article of manufacture, comprising a container, a label on said container, and a composition of matter comprising a) a N73 protein according to the invention, b) an agonist of said N73 protein, c) an antagonist of said N73 protein or d) an antibody that specifically binds to said N73 protein, contained within said container, wherein a label on said container indicates that said composition of matter can be used for treating a disease.
  • Preferred diseases treated are immune related disorders or malignant neoplasm.
  • the invention further comprises a method of treating a disease in a mammal or human in need thereof comprising administering to said mammal or human a therapeutically effective amount of a) a N73 protein according to the invention, b) an agonist of said N73 protein, c) an antagonist of said N73 protein or d) an antibody that specifically binds to said N73 protein.
  • Diseases treated with a method according to the invention include the above-mentioned diseases.
  • the treated disease is AIDS.
  • the N73 protein according to the invention is a target for diagnostic applications, e.g. for diagnosis of a disease in a patient, like immune related disorders or cancer.
  • the invention refers to a method for determining the presence of a N73 protein according to the invention in a test sample suspected of containing said N73 protein.
  • Said method according to the invention comprises the steps of a) exposing said sample to an antibody according to the invention, that specifically binds to said N73 protein and b) determining binding of said anti-N73 antibody by detecting the formation of a complex between the antibody and the N73 protein according to the invention.
  • the detection is qualitative or quantitative, and is preferentially performed in comparison with monitoring the complex formation in a control sample.
  • a larger quantity of complexes formed in the test sample indicates the presence or absence of a disease, like immune related disorders or cancer, in the mammal from which the test tissue cells were obtained.
  • the anti-N73 antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, ELISA, immunohistochemistry or other techniques known in the art.
  • the detection methods of antibody binding are well known to an ordinary person skilled in the art.
  • Diseases to be diagnosed with a method according to the invention include all above-mentioned diseases.
  • diseases diagnosed are cancer diseases.
  • the method of diagnosis according to the invention is used to diagnose immune related disorders, preferably above mentioned immune related disorders, more preferably AIDS.
  • One aspect of the invention is related to a method of diagnosing a disease, preferably an immune related disorder or cancer as mentioned above, in a mammal, said method comprising detecting the level of expression of a gene encoding the N73 amino acid sequence shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 13 or SEQ ID No. 14 in a test sample of tissue cells obtained from the mammal or human, and in a control sample of known normal tissue cells of the same cell type. A higher or lower level of expression of said gene in the test sample as compared to the control sample is indicative of the presence of said disease in the mammal from which the test tissue cells were obtained.
  • Another embodiment of the invention relates to a method of diagnosing a disease as mentioned above for N73 in a mammal or human, said method comprising
  • Further aspects of the invention relate to methods of identifying a compound that modulates the activity of a N73 protein according to the invention, said method comprising contacting cells which normally respond to said N73 protein with said N73 protein and a candidate compound, and determining the lack of responsiveness by said cell to a).
  • the invention encompasses methods of screening compounds to identify those that mimic the N73 protein of the invention (agonists) or prevent the effect of said N73 protein (antagonists).
  • Methods for identifying agonists or antagonists of a protein according to the invention may comprise contacting said protein with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with said protein.
  • the protein according to the invention is preferably added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of said protein indicates that the compound is an antagonist to said protein.
  • antagonists to the protein according to the invention are detected by combining said protein and a potential antagonist with membrane-bound N73 protein receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay.
  • the protein according to the invention can be labeled, such as by radioactivity, such that the number of said protein molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist.
  • the invention concerns a method of identifying agonists of or antagonists to a protein according to the invention which comprises contacting said protein with a candidate molecule and monitoring a biological activity mediated by said protein.
  • the agonist or antagonist is an anti-N73 antibody.
  • Another aspect of the invention refers to a method of identifying a compound that inhibits the expression of a gene encoding the N73 amino acid sequence shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 13 or SEQ ID No. 14, said method comprising contacting cells which normally express said N73 protein with a candidate compound, and determining the lack of expression of said gene.
  • the decrease in the expression level of N73 gene expression can be detected through measurement of N73 RNA level in the cell, or through measurement of N73 protein level in the cell.
  • the candidate compound is an antisense nucleic acid.
  • the object is also solved by providing a protein, named and further referred to herein as "F10", with structural similarity to proteins of the IL-8 like chemokine fold, in particular to the known chemokines viral MIP-I (vMIP-I) and CXCL10, which is characterized by its amino acid sequence as presented in SEQ ID No. 16, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37.
  • the invention relates to an isolated protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence of the protein shown in SEQ ID No. 16, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, further referred to herein as "F10".
  • the invention relates to an isolated F10 protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence of the protein shown in SEQ ID No. 16.
  • said F10 protein comprises cysteine residues at position 20 and 47, which preferentially form a disulfide bridge.
  • said F10 protein further comprises a proline residue at position 30 of SEQ ID No. 16.
  • the F10 protein according to SEQ ID No. 17 corresponds to the F10 protein according to SEQ ID No. 16 additionally comprising its associated signal peptide, which is cleaved during protein release from the cells.
  • the nucleic acid sequence according to SEQ ID No. 18 corresponds to the F10 protein coding region, whereas the nucleic acid sequence according to SEQ ID No. 19 additionally comprises regions of the full spliced gene transcript.
  • Another aspect of the invention relates to an isolated F10 protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence of the protein shown in SEQ ID No. 17.
  • the F10 protein according to the invention contains at least 2, preferably at least 4, more preferably 5 cysteine residues.
  • the F10 protein according to the invention was identified by fold recognition analysis and shows structural similarity with proteins belonging to the IL-8-like chemokine fold family, in particular with CXCL10.
  • the protein according to the invention referred herein also as "F10” or "F10 protein”
  • F10 exhibits a strong structural similarity to CXCL10, a member of the IL-8-like chemokine fold family, which is an inhibitor of angiogenesis.
  • the sequence identity between F10 and vMIP-I is 20 %.
  • the sequence identity between F10 and CXCL10 is 17.8 %.
  • F10 shares 16.7 % sequence identity with IL-8.
  • the amino acid sequence of F10 is also known as C4orf26 and comprises 130 amino acids, of which 5 are cysteines that could potentially be involved in disulfide bond formation.
  • the full F 10 amino acid sequence is presented in SEQ ID No. 17 and comprises a signal peptide which consists of 23 amino acids.
  • the signal peptide comprises 2 cysteine residues.
  • the F10 amino acid sequence without signal peptide is presented in SEQ ID No. 16.
  • Orthologues of the human F10 protein could be found in other mammals comprising the following amino acid sequences: flying fox SEQ ID No. 23, dog SEQ ID No. 24, microbat SEQ ID No. 25, hyrax SEQ ID No. 26, tenrec SEQ ID No. 27, bushbaby SEQ ID No. 28, alpaca SEQ ID No. 29, dolphin SEQ ID No. 30, chimpanzee SEQ ID No. 31, orangutan SEQ ID No. 32, mouse lemur SEQ ID No. 33, tree shrew SEQ ID No. 34, horse SEQ ID No. 35, squirrel SEQ ID No. 36, rabbit SEQ ID No. 37.
  • chemokines all bind the same receptors: CXCR3 as agonist with angiostatic function that is expressed by TH1 cells and NK cells and CCR3 as antagonist/inhibitor.
  • a putative signaling function of the protein according to the invention is via said receptors, CXCR3 and/or CCR3.
  • CXCR3 Chemokine binding to CXCR3 induces a variety of cellular responses, such as integrin activation, cytoskeletal changes and chemotactic migration.
  • CXCR3 has been implicated in diseases, such as atherosclerosis, multiple sclerosis, pulmonary fibrosis, type 1 diabetes, autoimmune myasthenia gravis, nephrotoxic nephritis, acute cardiac allograft rejection and Celiac Disease.
  • CCR3 is highly expressed in eosinophils and basophils. It can also be detected in TH1 and TH2 cells, as well as in airway epithelial cells. This receptor may contribute to the accumulation and activation of eosinophils and other inflammatory cells in the allergic airway. It is also known to be an entry co-receptor for HIV-1.
  • the analysis of the F10 candidate sequence revealed several chemokine- like properties like the presence of a signal peptide for secretion, chromosomal proximity to the angiostatic chemokines CXCL9 - 11. In addition the formation of one disulfide bond is possible (classifying as C motif-containing chemokine). Two FIO isoforms are expressed, which have 2 and 3 exons like most chemokines.
  • the FI O amino acid sequence is conserved across many mammalian species.
  • Isoform A corresponds to the FIO amino acid sequence as presented in SEQ ID No. 17 (with signal peptide), which is encoded by a nucleic acid as presented in SEQ ID No. 18 (protein coding region only) or SEQ ID No. 19 (full spliced gene transcript region).
  • Isoform B corresponds to the FIO amino acid sequence as presented in SEQ ID No. 20 (with signal peptide), which is encoded by a nucleic acid as presented in SEQ ID No. 21 (protein coding region only) or SEQ ID No. 20 (full spliced gene transcript region).
  • FI O (C4orf26) was used as one of 51 marker genes to correctly distinguish between high-risk and low- risk lymph-node-negative breast cancer patients (Karlsson 2008). Significantly lower expression of F10 was found in the tumors from deceased patients compared to the tumors from 10-year survivors. Moreover, gene expression of F10 was found to be 17 fold unregulated in response to cyclic mechanical stress in trabecular meshwork cells (Luna 2009). It was accompanied by changes in genes related to cytoskeleton, cell adhesion as well as genes involved in protection against stress like heat shock proteins and the chemokine CCL7.
  • Kittler et al, 2007 showed a cell cycle arrest / cell division defect phenotype in an RNAi screen similar to known angiostatic chemokines (CXCL10, 13, 14) suggesting a similar role of F10 in cancer.
  • CXCL10 angiostatic chemokines
  • F10 presents most necessary CXCR3 receptor binding residues known from CXCL10 in sequence and 3D.
  • F10 has been found to be expressed mainly in placenta it could be a placenta specific CXCL10 analog performing similar functional roles by binding to CXCR3, which is known to be expressed in placenta, too.
  • amino acid sequence with 99% sequence identity to SEQ ID No. 16 has been annotated previously and is included in EP 2107127-A1. There, the amino acid sequence is included in a diagnostic screening assay for somatic and ovarian cancers with a variety of other amino acid sequences. Structural and functional information about the previously known sequence are not provided in EP 2107127-A1.
  • the F10 protein according to the invention differs from the previously known protein in a proline residue at position 30 of SEQ ID No. 16.
  • the previously known sequence comprises a leucine residue at this position.
  • the F10 protein according to the invention preferably comprises a proline residue at this position (position 30 of SEQ ID No. 16). More preferably, the F10 protein according to the invention does not comprise a leucine residue at position 30 of SEQ ID No. 16.
  • the F10 protein according to the invention in particular possesses a high structural homology to vMIP-I and CXCLlO.
  • the amino acid sequence of the F10 protein according to the invention comprises at least two cysteine residues, preferably three cysteine residues, more preferably five cysteine residues.
  • two cysteine residues are involved in formation of intramolecular disulfide bonds.
  • the IL8-like chemokine fold more specifically comprises an N-terminal loop (N-loop) followed by a single-turn helix or a 3 io-helix (3-turn helix), three anti-parallel ⁇ -strands and a C-terminal a-helix (4-turn helix).
  • N-loop N-terminal loop
  • 3-turn helix 3-turn helix
  • three anti-parallel ⁇ -strands and a C-terminal a-helix (4-turn helix).
  • These helices and strands are connected by turns preferably so called 30s, 40s and 50s loops.
  • inventions are also chimeric molecules which comprise an F10 protein according to the invention fused to a heterologous amino acid sequence.
  • this heterologous amino acid sequence is an epitope tag sequence or an Fc region of an immunoglobulin.
  • the invention further comprises proteins that are circular permutations of the F10 protein according to the invention, further referred to as circularly permutated variants.
  • the invention comprises circularly permutated variants of the protein with the amino acid sequence according to SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31 , SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably to SEQ ID No.
  • SEQ ID No. 16 (or a protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence according to SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31 , SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably to SEQ ID No. 16).
  • circular permutation in coherence with the invention is meant a genetic operation in which part of the C-terminus of a protein is moved to its N-terminus by maintaining the three-dimensional structure and biological function of said protein.
  • a circular permutation is built from one or more sets of structural elements in cyclic order.
  • An engineered circular permutation of a protein can be envisioned as the result of taking a linear protein sequence, joining its ends to create a circle and then cleaving the circle at another site to generate a new linear monomeric protein sequence, only altered at the sites of joining and cleavage and with a resulting different sequential order of the secondary structure elements but same 3D structure. This approach of engineering a new protein sequence by circularly permuting was pioneered by Goldenberg in 1983 (Goldenberg 1983).
  • the newly engineered protein maintains the original function either totally (preferred) or partially.
  • the F10 protein according to the invention preferably contains loops (amino acids counted without signal peptide) at the following amino acid positions: amino acids 20 - 29 (N-loop), amino acids 38 - 48 (30S-loop), amino acids 52 - 58 (40S-loop), amino acids 61 - 66 (50S-loop).
  • Circularly permutated variants of a F10 protein according to the invention preferably comprise the amino acid sequence according to SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31 , SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably to SEQ ID No.
  • SEQ ID No. 16 (or a protein having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % amino acid sequence identity to the amino acid sequence according to SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably to SEQ ID No. 16) in rearranged order.
  • structural elements of circular permutated variants of the FIO protein according to the invention comprise the following amino acid sequences (each one comprising at least 80%, preferably 90%, more preferably 95%, even more preferably 99% amino acid sequence identity to the respective amino acid sequence according to SEQ ID No. 16).
  • S2 alpha helix according to amino acids 67 to at least 72 in SEQ ID No. 16, beta sheet according to amino acids 35 to 35 in SEQ ID No. 16 (SI); beta sheet according to amino acids 49 to 51 in SEQ ID No. 16 (S2), beta sheet according to amino acids 59 to 60 in SEQ ID No. 16 (S3).
  • an additional extension comprising an amino acid sequence from the original N-terminus is appended to the N-terminus of a circularly permutated variant of F10 protein according to the invention. Otherwise, only binding ability (inhibition) is maintained, whereas signaling ability is not.
  • a circularly permutated variant according to a structure described in b) is preferably an inhibitor of the natural receptor. Said variant is able to bind the receptor but does not have the flexible N-terminus at the right position to induce receptor signaling.
  • the present invention relates to an isolated nucleic acid encoding for the F10 protein according to the invention (herein also referred to as "F10 nucleic acid").
  • F10 nucleic acid encodes for a protein according to SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably to SEQ ID No. 16 or SEQ ID No.
  • the invention relates to an isolated F10 nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence encoding the F10 protein shown in SEQ ID No. 18.
  • the isolated F10 nucleic acid has at least 80 %, preferably at least 90 %, more preferably at least 95 %, even more preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence presented in SEQ ID No. 19.
  • FIG. 1 Further aspects of the invention are directed to an isolated FIO nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence comprising the coding sequence of the full length F10 polypeptide cDNA as presented in SEQ ID No. 18 or the full transcript sequence as presented in SEQ ID No. 19.
  • the F10 vector according to the invention comprises an F10 nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to a nucleotide sequence encoding the protein shown in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No.
  • the F10 vector according to the invention comprises a nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence shown in SEQ ID No. 18.
  • the F10 vector according to the invention comprises a nucleic acid having at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % nucleic acid sequence identity to the nucleotide sequence shown in SEQ ID No. 19.
  • the F10 vector comprising the nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.
  • the invention refers to a host cell comprising the F10 vector according to the invention.
  • the host cells are CHO cells, HEK-293T cells, HEK-293A cells, HeLa cells, E. coli cells, yeast cells or baculovirus infected insect cells.
  • the invention also relates to a process for producing a F10 polypeptide as presented in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably in SEQ ID No.
  • SEQ ID No. 16 or a polypeptide with at least 80 %, preferably at least 90 %, preferably at least 95 %, preferably at least 99 % sequence identity to the polypeptide presented in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably in SEQ ID No. 16.
  • the process comprises the steps of: culturing the host cells or multi cellular organisms according to the invention under conditions suitable for the expression of said FIO polypeptide and
  • the FIO protein according to the invention displays a target protein for therapeutic and diagnostic applications related to diseases, preferably immune related disorders or cancer.
  • the invention further comprises an antibody, which specifically binds to a FIO protein according to the invention (herein also referred to as "anti-FlO antibody").
  • the anti-FlO antibody is a monoclonal antibody, a polyclonal antibody, a humanized antibody or a single chain antibody.
  • the FIO protein according of the invention or an antagonist or agonist thereof or an anti-FlO antibody according to the invention are composed in combination with a carrier.
  • the invention refers to a composition of matter comprising a) a FIO protein according to the invention, b) an agonist of said FIO protein, c) an antagonist of said FIO protein or d) an antibody the specifically binds to said FIO protein in combination with a carrier.
  • the invention relates to the use of a composition of matter comprising a) a FI O protein according to the invention, b) an agonist of said FIO protein, c) an antagonist of said FIO protein or d) an antibody the specifically binds to said FIO protein in combination with a carrier to treat a disease in a mammal or human.
  • diseases treated with a composition of matter comprising a) a FI O protein according to the invention, b) an agonist of said FIO protein, c) an antagonist of said FIO protein or d) an antibody the specifically binds to said FI O protein in combination with a carrier are immune related disorders, more preferably the above mentioned immune related disorders.
  • a FIO protein according to the invention an agonist thereof, an antagonist thereof or an antibody that specifically binds to said FIO protein is capable of increasing the proliferation of T- lymphocytes in a mammal, inhibiting the proliferation of T-lymphocytes in a mammal, increasing infiltration of inflammatory cells into a tissue of a mammal, or decreasing the infiltration of inflammatory cells into a tissue of a mammal.
  • said diseases treated by a composition of matter comprising a) a FI O protein according to the invention, b) an agonist of said FIO protein, c) an antagonist of said FIO protein or d) an antibody the specifically binds to said FIO protein in combination with a carrier are malignant neoplasms, such as tumor diseases or other types of cancer.
  • composition of matter according to the invention comprises a therapeutically effective amount of a) a FIO protein according to the invention, b) an agonist of said FIO protein, c) an antagonist of said FIO protein or d) an antibody the specifically binds to said FIO protein.
  • the therapeutically effective amount for a given situation or a given disease will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 5 g units per day.
  • a preferred dosage might be in the range of 0.01 mg to 100 mg, preferably 0.01 mg to 50 mg and preferably 0.01 mg to 10 mg per day.
  • the invention further comprises an article of manufacture, comprising a container, a label on said container, and a composition of matter comprising a) a F10 protein according to the invention, b) an agonist of said F10 protein, c) an antagonist of said F10 protein or d) an antibody that specifically binds to said F10 protein, contained within said container, wherein a label on said container indicates that said composition of matter can be used for treating a disease.
  • Preferred diseases treated are immune related disorders or malignant neoplasm.
  • the invention further comprises a method of treating a disease in a mammal or human in need thereof comprising administering to said mammal or human a therapeutically effective amount of a) a F10 protein according to the invention, b) an agonist of said F10 protein, c) an antagonist of said F10 protein or d) an antibody that specifically binds to said F10 protein.
  • Diseases treated with a method according to the invention include the above-mentioned diseases.
  • the treated disease is AIDS.
  • the F10 protein according to the invention is a target for diagnostic applications, e.g. for diagnosis of a disease in a patient, like immune related disorders or cancer.
  • the invention refers to a method for determining the presence of a F10 protein according to the invention in a test sample suspected of containing said F10 protein.
  • Said method according to the invention comprises the steps of a) exposing said sample to an antibody according to the invention, that specifically binds to said F10 protein and b) determining binding of said antibody by detecting the formation of a complex between the antibody and the F10 protein according to the invention.
  • the detection is qualitative or quantitative, and is preferentially performed in comparison with monitoring the complex formation in a control sample.
  • a larger quantity of complexes formed in the test sample indicates the presence or absence of a disease, like immune related disorders or cancer, in the mammal from which the test tissue cells were obtained.
  • the antibody preferably carries a detectable label. Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluorimetry, ELISA, immunohistochemistry or other techniques known in the art.
  • the detection methods of antibody binding are well known to an ordinary person skilled in the art.
  • Diseases to be diagnosed with a method according to the invention include all above-mentioned diseases.
  • diseases diagnosed are cancer diseases.
  • the method of diagnosis according to the invention is used to diagnose immune related disorders, preferably above mentioned immune related disorders, more preferably AIDS.
  • One aspect of the invention is related to a method of diagnosing a disease, preferably an immune related disorder or cancer as mentioned above, in a mammal, said method comprising detecting the level of expression of a gene encoding the F10 amino acid sequence shown in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably in SEQ ID No. 16 or SEQ ID No.
  • Another embodiment of the invention relates to a method of diagnosing a disease as mentioned above in a mammal or human, said method comprising
  • Further aspects of the invention relate to methods of identifying a compound that modulates the activity of a F10 protein according to the invention, said method comprising contacting cells which normally respond to said F10 protein with said protein and a candidate compound, and determining the lack of responsiveness by said cell to a).
  • the invention encompasses methods of screening compounds to identify those that mimic the F10 protein of the invention (agonists) or prevent the effect of said F10 protein (antagonists).
  • Methods for identifying agonists or antagonists of a F10 protein according to the invention may comprise contacting said F10 protein with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with said F10 protein.
  • antagonists to the F10 protein according to the invention are detected by combining said protein and a potential antagonist with membrane-bound F10 protein receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay.
  • the F10 protein according to the invention can be labeled, such as by radioactivity, such that the number of said protein molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist.
  • the invention concerns a method of identifying agonists of or antagonists to a FIO protein according to the invention which comprises contacting said FIO protein with a candidate molecule and monitoring a biological activity mediated by said FIO protein.
  • the agonist or antagonist is an anti-FlO antibody.
  • Another aspect of the invention refers to a method of identifying a compound that inhibits the expression of a gene encoding the amino acid sequence shown in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 37, preferably to SEQ ID No. 16 or SEQ ID No.
  • said method comprising contacting cells which normally express said FIO protein with a candidate compound, and determining the lack of expression of said gene.
  • the decrease in the expression level of FI O gene expression can be detected through measurement of FI O RNA level in the cell, or through measurement of FIO protein level in the cell.
  • the candidate compound is an antisense nucleic acid.
  • Fig. 1 shows the 3D-structure of the B42 protein as determined by threading and 3D-modelling and a known structure of vMIP-II, a chemokine from the IL-8-like chemokine fold family. The two 3D-structures in superposition are shown in the middle.
  • Fig. 2 shows a sequence-to-structure alignment of B42 with the vMIP-I template structure
  • Fig. 3 shows the comparison of stability and energetic properties of B42 (lower graph) with known chemokines (CXCL17, upper graph) as determined by molecular dynamics simulation.
  • Fig. 4 shows a sequence alignment of orthologous B42 proteins in human (B42_Hum), chimpanzee (B42_chimp, SEQ ID No. 6) and orangutan (B42_orang, SEQ ID No. 7). conserveed regions are highlighted.
  • Fig. 5 shows ENSEMBL identifiers for B42. 1) ENSEMBL version 54; 2) ENSEMBL version
  • Figure shows chromosomal location of B42 protein with surrounding genes and exon structure.
  • Fig. 6 demonstrates splicing of B42.
  • Two possible isoforms exist for B42 which are referred herein as "isoform A” and "isoform B".
  • Isoform B exhibits a stop codon within the additional longer exon 3 sequence and, if protein coding, would only encode a short peptide of 9 residues. Thus, only isoform A is coding for the full length protein.
  • Fig. 7 shows the in vivo expression of B42 isoforms in human tissues as determined by PCR analyses.
  • Protein coding isoform A is expressed in placenta, heart, lung, liver, pancreas, skeletal muscle and weakly in brain.
  • Fig. 8 shows the construct used for B42 expression. Cleavage position is indicated by vertical line (I). GA-addition remains at the N-terminus of B42-protein after cleavage with TEV protease.
  • Fig. 9 shows results of an SDS gel electrophoresis of eluted fractions of B42 protein. Peak fractions from the eluate were pooled (line 3) and concentrated over a 3 kDa cutoff membrane (line 2: 2 ⁇ sample; line 1 : 20 ⁇ 1 sample) and analyzed by SDS gel electrophoresis to check protein recovery.
  • Fig. 10 shows the region of interest of the deconvoluted B42 mass spectrum (scale Dalton).
  • Fig. 11 shows the region of interest of the deconvoluted B42 mass spectrum (scale Dalton).
  • Fig. 12 shows the secondary structure composition of the in vitro expressed B42 protein (right side graph) and the known chemokine vMIP-II (left side graph) as determined by circular dichroism spectroscopy.
  • Fig. 13 shows the Westernblot analysis of the N-terminal GFP-tagged B42 transgenic cell pool using antibody to GFP.
  • a band of the expected size 38.8kD (GFP-tag: 31kD + B42 8.8kD) was detected (marked with *).
  • Fig. 14 shows the localization of the N-terminal GFP-tagged B42 protein in the cytoplasm of
  • HeLa cells with antibody to GFP in A Localization of antibody to alpha-tubulin is shown in B and localization of DNA is shown in C.
  • the cell cycle phase is interphase.
  • Fig. 15 shows a sequence-to-structure alignment of N73 with the vMIP-II template structure
  • Fig. 16 shows the 3D-structure of the N73 protein as determined by threading and 3D-modelling and a known structure of vMIP-II (pdb:2fj2, SEQ ID No. 15), a chemokine from the IL-8- like chemokine fold family. The two 3D-structures in superposition are shown in the middle.
  • Fig. 17 shows the comparison of stability and energetic properties of N73 (lower graph) with known chemokines (CXCLl 7, upper graph) as determined by molecular dynamics simulation.
  • Fig. 18 shows ENSEMBL identifiers for N73. Chromosomal location of the N73 protein with surrounding genes and exon structure is shown. The open reading frame is located on chromosome 8q23.3 (142,513,827-142,517,232 forward strand) and in close proximity to the N73 gene an orphan G protein coupled receptor (GPR20) can be found.
  • GPR20 G protein coupled receptor
  • Fig. 19 shows a sequence alignment of orthologous N73 proteins in human (N73_Hum/l-211,
  • Fig. 20 shows the in vivo expression of N73 isoforms in human tissues, taken from a GeneNote expression study. N73 is expressed in placenta, heart, lung, liver, pancreas, skeletal muscle and weakly in brain.
  • Fig. 21 shows the sequence-structure alignment of the F10 amino acid sequence with vMIP-I
  • Fig. 22 shows the 3D-structure of the F10 polypeptide as determined by threading and 3D- modelling and a known structure of CXCLl 0 (pdb: 1 o7z), a chemokine from the IL-8 fold family, The two 3D-structures in superposition are presented in the middle of Fig. 22.
  • Fig. 23 shows 3D-structures of F10 (left) and CXCLI O (right) with receptor binding amino acid residues indicated in sticks.
  • Fig. 24 shows a structure-based sequence alignment of F10 (starting at amino acid #9 of SEQ ID
  • sequence is also presented in SEQ ID No. 42) with CXCR3 binding chemokines CXCL9 (SEQ ID No. 40), CXCLI O (SEQ ID No. 39) and CXCLl 1 (SEQ ID No. 41).
  • CXCR3 binding residues indicated.
  • the secondary structure of CXCLI O is indicated as follows: SI -S3 ... beta-sheets, a ... a-helix.
  • Fig. 25 shows the comparison of stability and energetic properties of F10 (lower graph) with known chemokines (CXCLl 7, upper graph) as determined by molecular dynamics simulation.
  • Fig. 26 shows genetic localization of F10.
  • the open reading frame is located on chromosome
  • Fig. 28 shows the in vivo expression of F10 isoforms in human tissues, taken from a GeneNote expression study. F10 is widely expressed in all tissues tested namely placenta, bone marrow, brain, heart, kidney, liver, lung, pancreas, prostate, skeletal muscle, spinal cord, spleen and thymus.
  • Fig. 29 demonstrates splicing of F10. Five possible F10 isoforms were identified in HeLa cells and placenta.
  • Fig. 30 shows expression of F10 isoforms in human cDNA of placenta.
  • isoform A is the most abundant form showing the biggest band the in vivo expression.
  • Example 1 Overview of the workflow and results obtained in the computational chemokine screening
  • the computational chemokine screening protocol consists of the following steps:
  • the subsequent sequence-based pre-filtering step includes prediction and removal of signal peptides (SP) and transmembrane (TM) regions as they might produce noise due to variant amino acid composition or a lack of structural information about them in our representative fold libraries. Then, a BLAST search against the Protein Databank (PDB) is performed to identify and remove regions that can be already annotated by sequence similarity to a characterized protein of known structure. From the remaining sequences/ sequence parts only those longer than 54 amino acids were selected that at the same time contain more than one cysteine. In total 2141 protein sequences passed this pre-filtering step successfully.
  • SP signal peptides
  • TM transmembrane
  • the 760 best ranked sequence-structure alignments covering the full chemokine fold were automatically modeled and evaluated for disulfide bond formation possibilities by measuring distances between the cysteines in the chemokine- like 3D models. In 454 sequences the cysteines came close enough together to form at least one disulfide bond in the obtained 3D model.
  • a control threading run was performed to assess the quality and ranking of the chemokine structure alignment within the structural templates of the whole PDB.
  • the pdb95 was used as fold library, which comprises all to date known 3D structures with less than 95% sequences identity and, furthermore, all known chemokine structures were added to that library.
  • a sequence hit was further analyzed in case the chemokine template was ranked high in the pdb95 fold library and by manual inspection no other template was considered to be superior compared to the chemokine template.
  • the first approach model obtained from the threading alignment was refined by building up the amino acid side chains including cysteines forming disulfide bonds. Then, the structure was energy minimized and side chain rotamers were manually adjusted to improve the packing of the core.
  • chemokines were analyzed for conservation across different species as well as additional chemokine features like similar exon organization, chromosomal location and proximity to known chemokines, expression profiles, presence of a PolyA site (ENSEMBL, PolyA), common transcription factor binding sites of chemokines within their promoter region.
  • the initial sequence dataset was extracted from the Uniprot knowledgebase (SwissProt and TrEMBL) by filtering all human protein sequences labeled as "unknown”, “or/” (open reading frame), "hypothetical”, “uncharacterized' or “putative” that contain at least two cysteine.
  • SP Signal peptides
  • TM transmembrane regions
  • the fold recognition algorithm ProHit ProCeryon Biosciences (Sippl 1992, Sippl 1993) and a fold library containing the three-dimensional (3D) protein structures of all members of the chemokine fold family (SCOP: d.9.1.1, 107 PDB entries with in total 217 protein chains) from the Protein Data Bank was used to generate sequence-structure alignments.
  • ⁇ is the standard deviation of the population.
  • the distance between the raw score and the population mean in units of the standard deviation is represented by the quantity z.
  • z-pair which is the pairwise (residue-residue) potential component of the alignment.
  • z-surf which is the surface (residue-solvent) potential component of the alignment.
  • the z-combined score (z-comb) is a weighted combination of the pairwise and surface z-scores.
  • z-seq which is the sequence substitution (sequence similarity) component of the alignment.
  • Threading index is a combination of sequence similarity (z-seq), residue-residue and residue-solvent interactions (z-comb) normalized by the query sequence length.
  • HC High confidence alignments covering more than 75% of the template structure have fl/pl values in the range (Pisabarro 2006): 0.6 ⁇ fl/pl ⁇ 1.3
  • the quality of an alignment is only given by the overall ranking within all structural templates in the pdb95 fold library (95 Rank and the fl/pl filtered Rank) but not by a certain score alone.
  • a redundancy-reduced summary of the results obtained for the novel protein B42 using the fold library containing all known structures of the members of the IL-8-like chemokine fold family is shown in table 1.
  • the "95 rank" of those B42 alignments with chemokine template structures corresponds to the overall rank in the pdb95 library.
  • the novel protein B42 according to SEQ ID No.
  • vMIP-I rank #1 ; ID: 22,6) and v-MIP-II (rank #4; ID: 26,2) both encoded by kaposi's sarcoma-associated herpesvirus.
  • CCL14 pdb: 2Q8R was found as best scoring human chemokine template (ID: 18%) then the human chemokines CCL5 (ID: 20,6%) and CCL3 (ID: 17,2%).
  • z-score corresponding to the surface (residue-solvent) potential component of the alignment z sec ... z score corresponding to sequence identity , %>ID ... percentage of amino acid sequence identity, pi ... alignment path length, fl ... template fold length, Scop ... SCOP family identifier, pdb ... template identifier in the Protein Data Bank, chain ... template chain (author chain label), %>D_cov ... percentage of domain coverage (folds with coverage lower than 70%> are filtered out), descr ... description of PDB template, Uniprot ... Uniprot identifier, Rec. agonist ... Chemokine receptors for that the template protein is an agonist (activator), Rec. antagonist ... Chemokine receptors for that the template protein is an antagonist (inhibitor).
  • the MODELLER package was used to generate 3D models of the query sequences with the sequence to structure alignments calculated by ProHit. In those 3D models, the distances between cysteine were measured and analyzed for possibilities of disulfide bond formation. To refine the candidate's model generated from the threading alignment, the cysteines were connected, the structure energy minimized and side chain rotamers were manually adjusted using MOE2008.10.
  • the sequence-to-structure alignment of B42 with its best ranked template structures shows a good matching of the structurally important cysteines involved in disulfide bridges (Fig. 2).
  • Most conserved residues are located in the core of the protein (L16, Y20, W24, V34, L37, L48, V57, W59, 160, M61 , A63, A64, L68 and 4 cysteines) that is necessary for stability of the fold.
  • the 3D model of B42 built on the vMIP-I template structure (pdb: lzxt) has 2 to 3 disulfide bonds.
  • the first model has disulfide bonds between C7 - C29 and C8 - C45, which would correspond to the CC-motif.
  • FIG. 1 A detailed 3D-model of the B42 protein with the amino acid sequence according to SEQ ID No. 1 and the experimental structure ofvMIP-II (pdb: lcm9) are shown in Fig. 1. Superposition of both structures is also shown in Fig. 1. Disulfide bonds in the tertiary structure of the protein with the amino acid sequence according to SEQ ID No. 1 are formed at equivalent positions in 3D.
  • a control threading run is performed to assess the quality and ranking of the chemokine structure alignment within the structural templates of the whole PDB.
  • the pdb95 is used as fold library (22009 entries), which comprises all to date known 3D structures with less than 95 % sequences identity and, furthermore, all chemokine and cytokine structures were added to that library.
  • a sequence hit was further analyzed in case the chemokine alignment was within the top 10 folds in the pdb95 fold library and by manual inspection none of the better ranked alignments was considered to be superior compared to the chemokine template in terms of gap content and disulfide bonding possibilities.
  • a redundancy-reduced summary of the pdb95 control results of the B42 protein sequence are shown in table 2.
  • Ranking with this fold library confirms that the structure of the novel protein resembles an IL-8- like chemokine fold.
  • the novel protein shows high structural similarity to vMIP-I even among all possible protein folds (rank #1 , table 2). Ergotoxin (rank #11), Metallothionein (rank #15), Zinc finger (rank #23) and Leupaxin (rank #33) are considered false positive because of too high gap content.
  • Ranks #12, #16, #26, #31 are all alpha helical proteins, which contradict the CD spectroscopy results to be protein containing both alpha helices and beta sheets (see example 10).
  • the alignments with the structures at ranks #10, #20, #24, #25, #34 and #35 are not covering the full length of the template folds and are therefore also considered false positive.
  • the structure of the known chemokine CXCL17 with low sequence identity to solved chemokine structures was predicted by threading using the same methodology previously described.
  • the threading alignment of CXCL17 was generated with the best compatible 3D structure (pdb:lNR4 seq.ID: 21.4 %) of an IL8-like chemokine as template for the MODELLER package.
  • the model was refined and the cysteines were connected to disulfide bonds using MOE2008.10.
  • the MD simulation of this control model was performed using the same conditions as previously described.
  • a putative novel chemokine candidate is evaluated by: 1.) Comparison of the RMSD plot of the control chemokine MD simulation with the candidate's simulation RMSD plot and 2.) by visual inspection of the stability of candidate's 3D model during and after the simulation.
  • the MD simulation of the control model shows good stability of the secondary structure elements with low RMSD values of around 0.2-0.3nm throughout the simulation.
  • the MD simulation of the B42 protein with the amino acid sequence according to SEQ ID No. 1 shows as good stability of the secondary structure elements as the control 3D model of CXCL17 with low RMSD values of around 0.2-0.3 nm (Fig. 3) throughout the simulation. Only the RMSD values of the all backbone curve (solid line) and coils are slightly higher because of the relatively long and flexible C- terminus following the helix, which is not present in the control 3D model (but known also from other CKs like Lymphotactin).
  • the selected candidate genes were manually analyzed for conservation across different species (orthologs), exon organization, chromosomal location and proximity to known chemokines, presence of a PolyA site (using ENSEMBL (Flicek 2008), Polyah.pl (softberry) and Polyadq (Tabaska 1999)), transcription factor binding sites of chemokine regulators (Chong 2000; Genin 2000; Gerber 2004; Roebuck 1999; Ueda 2007; Widmer 1993; Yeruva 2008) and expression profiles (GeneNote expression study (Shmueli 2003)).
  • the protein was checked for structural and functional similarity to known chemokines by analyzing glycosylation sites and other eukaryotic linear motifs with the ELM server (Puntervoll 2003), presence of receptor binding residues in sequence and 3D (with MOE2008.10), if known, and leaderless secretion in case no signal peptide is present using SecretomeP (Bendtsen 2004).
  • the B42 (Q1T7F1, AB091373-6, ENSv55: ZNF578-202) amino acid sequence according to SEQ ID No. 1 was previously identified (Maegawa 2002) as hypothetical novel Kruppel-like zinc finger protein. Sequence analysis using BLAST and Pfam did not identify any statistically significant sequence homology with any previously characterized protein. No signal peptide or transmembrane region was identified in B42 by standard prediction methods. However SecretomeP, a tool to predict leaderless secretion, obtained a high score (0.8 of 0.5 needed), suggesting that leaderless secretion of B42 is possible. Also WoLF PSORT (Horton 2007) predicts extracellular location with a high score for B42.
  • ENSEMBL's automatic annotation pipeline Release 54 identified two uncharacterized orthologous proteins, one in chimpanzee sharing 98% sequence identity (SEQ ID No. 6) and the other in orangutan (SEQ ID No. 7) with 91% sequence identity (Fig. 4, table 3) with B42.
  • the B42 gene lies within the same chromosomal region (19ql 3.41) with two other known chemokines (CXCL17 and CCL25) that can be found at 19ql3.2.
  • the gene of B42 was first automatically annotated as zinc finger protein ZNF528 (ENSEMBL Release 54) because its protein coding region was located within the untranslated region (intron) of this kruppel-like zinc finger gene. Since the ENSEMBL Release 55 it was re-annotated as ZNF578, which, according to the ENSEMBL annotation, does not code for a protein but is a processed transcript.
  • the translated B42 protein does not share sequence identity to any characterized zinc finger protein although in close proximity to the B42 gene many zinc finger genes are located.
  • the B42 coding transcript has 3 exons like many known chemokines. Another common feature of chemokines is that the exon boundaries have between exon 1 and 2 the intron phase 1 (position of the codon within the intron) and phase 2 between exon 2 and 3 (Betts 2001). In B42 the protein coding region starts at the end of exon 2 and ends with exon 3 having also a phase 2 intron boundary between exon 2 and 3 (table 4).
  • a poly-A site was predicted at position +657 downstream the translation end in the human and orangutan genes. Furthermore, the promoter region of B42 was analyzed for transcription factor binding sites known from other chemokines like NF-IL6, NF-kappaB, APl(C-Jun), INF-1 and the CK1 motif (Chong 2000; Genin 2000; Gerber 2004; Roebuck 1999; Ueda 2007; Widmer 1993; Yeruva 2008). Different binding sites were identified and the results are shown in table 5.
  • Example 7 In vivo expression of the B42 nucleic acid sequence
  • the mRNA expression of B42 was analyzed by PCR with templates from 8 different tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) using Human MTC Panel I (Clontech, Catalog No. 636742, LOT No. 8082935A). 43 PCR cycles were done at an annealing temperature of 58°C. The expected size of the PCR product is 272 bp and the following primers were used: forward primer: 5'- CCTAAGGAAGAAGCCTAGAAGAGG-3' (SEQ ID No. 4) and reverse primer: 5'- CAGGC AGTTGTGCACATTAAG-3 ' (SEQ ID No. 5).
  • Isoform B has a stop codon within the additional longer exon 3 sequence and, if protein coding, would only encode a short peptide of 9 residues (Fig. 6). It is expressed mainly in kidney, placenta, pancreas, liver and weakly in lung and muscle. B42 was not expressed in bone marrow (data not shown).
  • Example 8 In vitro expression of the B42 protein
  • the gene encoding the mature B42 protein (Uniprot: Q1T7F1 ; AB091373-6) was synthesized and cloned in an expression plasmid pETMM-60. The expression and purification was done as described in Magistrelli et al. (Magistrelli 2005) using a His-tagged NusA fusion protein to make the B42 as fusion partner soluble and Origami B cells (for disulfide bonds formation in cytoplasm because of a glutathion S transferase mutation).
  • the B42 protein was successfully expressed in E. coli and soluble as fusion protein. Then, the construct was cleaved from NusA with TEV protease and leaving a GA-addition at the N-terminus of the putative chemokine (Fig. 8).
  • the expressed protein from example 8 was purified and concentrated. The expression of the right construct was confirmed by SDS-Page (Fig. 9) and Mass spectrometry (measured Mass: 8931 Da) (Fig. 10).
  • the column was washed with 50 mmol/1 Tris-Cl pH 8, 300 mmol/1 NaCl, 20 mmol/1 imidazole, subsequently with 50 mmol/1 Tris-Cl pH 8, 1305 mmol/1 NaCl, 20 mmol/1 imidazole, and subsequently with 50 mmol/1 Tris-Cl pH 8, 300 mmol/1 NaCl, 20 mmol/1 imidazole. Elution was carried out using 50 mmol/1 Tris-Cl pH 8, 200 mmol/1 NaCl, 400 mmol/1 imidazole collecting 1 ml fractions at a flow rate of 2 ml/min. After checking protein content in the fractions with Bradford reagent, fractions 5 - 14 were pooled and desalted on PD10 columns into 50 mmol/1 Tris-Cl pH 8.
  • the yielded solution comprised 12.02 mg/ml NusA-His-B42 (MW 65,9 kDa).
  • 0.5 ml TEV-protease (1.6 mg/ml) was added to the solution and incubated overnight at 18 °C.
  • 100 mmol/1 Tris-Cl pH 8, 600 mmol/1 NaCl, 40 mmol/1 imidazole was added to a final volume of 40 ml and was loaded on a pre- equilibrated HisTrap HP column and eluted with 50 mmol/1 Tris-Cl pH 8, 300 mmol/1 NaCl, 20 mmol/1 imidazole collection 2 ml fractions.
  • Bound NusA was eluted with 50 mmol/1 Tris-Cl pH 8, 200 mmol/1 NaCl, 400 mmol/1 imidazole from the columns.
  • Fractions 5 - 16 were pooled after checking their protein content with Bradford reagent. The yielded solution contained a protein concentration of 0.467 mg/ml. The solution was diluted to yield a NaCl concentration ⁇ 50 mmol/1. The sample was loaded directly onto a HiTrap Q HP columns pre- equilibrated with anion buffer pH 8.5. After washing the column was eluted with a salt gradient from 0 to 1 mo 1/1 NaCl. Column was washed with 1.5 mol/1 NaCl. SDS-Page was run to identify peak fractions. Peak fractions were pooled and concentrated on a 3 kDa cutoff centrifugal ultrafiltration membrane (Millipore) and desalted into PBS. Concentration was checked and revealed a protein concentration of 0.486 mg/ml. SDS-Page was run to crosscheck for recovery (Fig. 9) ⁇
  • Example 10 Structural comparison of B42 with vMIP-II by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR)
  • CD measurements were carried out in the far UV-range (190-260 nm) using cuvettes of 0,02 cm path length (size 0,2 mm). The results were averaged over ten repetitive scans at a scan rate of 1 nm. Ellipticity was normalized to molarity using mass of 8931 Da as determined by mass spectrometry from example 9. The secondary structure was calculated using CONTINLL and SELCON3 both provided in the CDpro software package.
  • Example 11 Generation of a BAC transgenic HeLa cell line with the B42 gene and tag-based B42 protein localization
  • the following method describes the generation of a cell line expressing an N-terminal GFP-tagged B42 protein and its tag-based protein localization.
  • a transgenic HeLa cell line was generated carrying the tagged B42 gene in a BAC (bacterial artificial chromosome) using the methodology described in (Poser 2008).
  • the GFP-tag was then used for localization experiments and the results (Fig. 14) show localization in the cytoplasm that is not changing during different cell cycle phases.
  • chemokines are almost always secreted proteins
  • B42 is located in the cytoplasm.
  • the GFP-tag affects the protein secretion or an additional stimulus is necessary for induction of the B42 secretion.
  • Example 12 Expression of B42 in Yeast
  • the following method describes recombinant expression of B42 polypeptides in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of B42 from the ADH2/GAPDH promoter.
  • DNA encoding the B42 polypeptide and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the B42 polypeptide.
  • DNA encoding B42 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of B42.
  • yeast cells such as yeast strain AB110
  • yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
  • the transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant B42 polypeptides can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
  • the concentrate containing the B42 polypeptide may further be purified using selected column chromatography resins.
  • the following method describes recombinant expression of B42 polypeptides in Baculovirus-infected insect cells.
  • the sequence coding for B42 is fused upstream of an epitope tag contained within a Baculovirus expression vector.
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen).
  • the sequence encoding B42 or the desired portion of the coding sequence of B42 such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may incorporate flanking (selected) restriction enzyme sites.
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold(TM) virus DNA (Pharmingen) into Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 171 1 ) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28[deg.] C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
  • Expressed poly-His tagged B42 can then be purified, for example, by Ni ⁇ 2+> -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgC12; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice.
  • sonication buffer 25 mL Hepes, pH 7.9; 12.5 mM MgC12; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1
  • the sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%> glycerol, pH 7.8) and filtered through a 0.45 [mu]m filter.
  • loading buffer 50 mM phosphate, 300 mM NaCl, 10%> glycerol, pH 7.8
  • a Ni ⁇ 2+> -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
  • the filtered cell extract is loaded onto the column at 0.5 mL per minute.
  • the column is washed to baseline A280 with loading buffer, at which point fraction collection is started.
  • the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10%> glycerol, pH 6.0), which elutes nonspecifically bound protein.
  • a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10%> glycerol, pH 6.0
  • the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer.
  • One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ⁇ 2+> -NTA-conjugated to alkaline phosphatase (Qiagen).
  • Fractions containing the eluted His l O-tagged B42 are pooled and dialyzed against loading buffer.
  • purification of the IgG tagged (or Fc tagged) B42 can be performed using known chromatography techniques, including for instance, Protein A or Protein G column chromatography.
  • This example illustrates preparation of monoclonal antibodies which can specifically bind B42.
  • Immunogens that may be employed include purified B42 polypeptides, fusion proteins containing B42 polypeptides, and cells expressing recombinant B42 polypeptides on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
  • mice such as BALB/c are immunized with the B42 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms.
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads.
  • MPL-TDM adjuvant Ribi Immunochemical Research, Hamilton, Mont.
  • the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti- B42 antibodies.
  • the animals "positive" for antibodies can be injected with a final intravenous injection of B42.
  • the mice Three to four days later, the mice are sacrificed and the spleen cells are harvested.
  • the spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. 1, available from ATCC, No. CRL 1597.
  • the fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin, and thymidine
  • hybridoma cells will be screened in an ELISA for reactivity against B42. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against B42 is within the skill in the art.
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing the anti-B42 monoclonal antibodies.
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Protein A or Protein G can be employed.
  • Example 15 Purification of B42 Polypeptides Using Specific Antibodies
  • Native or recombinant B42 polypeptides may be purified by a variety of standard techniques in the art of protein purification.
  • pro-B42 polypeptide, mature B42 polypeptide, or pre-B42 polypeptide is purified by immunoaffinity chromatography using antibodies specific for the B42 polypeptide of interest.
  • an immunoaffinity column is constructed by covalently coupling the anti-B42 polypeptide antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE(TM) (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated SEPHAROSE(TM) (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • Such an immunoaffinity column is utilized in the purification of B42 polypeptide by preparing a fraction from cells containing B42 polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble B42 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
  • a soluble B42 polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of B42 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/B42 polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and B42 polypeptide is collected.
  • a low pH buffer such as approximately pH 2-3
  • a chaotrope such as urea or thiocyanate ion
  • the pre-filtering and structure-based chemokine screening of N73 was performed by the same methodology as described in example 2 for B42.
  • a redundancy-reduced summary of the results obtained for the novel protein N73 using the fold library containing all known structures of the members of the IL-8-like chemokine fold family is shown in table 7.
  • the "95 rank" of those N73 alignments with chemokine template structures corresponds to the overall rank in the pdb95 library.
  • the novel protein N73 according to SEQ ID No. 9 shows high structural similarity to the known chemokines vMIP-II (rank #1, ID 23.2%) and v-MIP-I (rank #2, ID 18.7%), both encoded by kaposi's sarcoma-associated herpesvirus.
  • CCL20 (pdb: 1M8A) was found as best scoring human chemokine template (ID: 21 %), then the human chemokines CXCL2 (ID: 18.2 %) and XCL1 (ID: 12.8 %).
  • CCL20 (pdb: 2Q8R) is the only natural ligand of CCR6 (Hoover 2002) that has important functions in the defense response to bacterium by attraction of natural killer and memory T cells to sites of inflammation.
  • z-score corresponding to the surface (residue-solvent) potential component of the alignment z sec ... z score corresponding to sequence identity , %ID ... percentage of amino acid sequence identity, pi ... alignment path length, fl ... template fold length, scopid ... SCOP family identifier, pdb id ... template identifier in the Protein Data Bank, chain ... template chain (author chain label), cover ... percentage of domain coverage (folds with coverage lower than 70% are filtered out), descr ... description of PDB template, Uniprot ... Uniprot identifier, Rec. agonist ... Chemokine receptors for that the template protein is an agonist (activator), Rec. antagonist ... Chemokine receptors for that the template protein is an antagonist (inhibitor).
  • the MODELLER package was used to generate 3D models of the query sequences with the sequence to structure alignments calculated by ProHit. In those 3D models, the distances between cysteine were measured and analyzed for possibilities of disulfide bond formation. To refine the candidate's model generated from the threading alignment, the cysteines were connected, the structure energy minimized and side chain rotamers were manually adjusted using MOE2008.10.
  • FIG. 16 A detailed 3D-model of the N73 protein with the amino acid sequence according to SEQ ID No. 9 and the experimental structure of vMIP-II are shown in Fig. 16. Superposition of both structures is shown in Fig. 16 (middle). Disulfide bonds in the tertiary structure of the protein with the amino acid sequence according to SEQ ID No. 9 are equivalent in 3D to the bonds found in the template and other known chemokines.
  • a control threading run is performed to assess the quality and ranking of the chemokine structure alignment within the structural templates of the whole PDB.
  • the pdb95 is used as fold library (22009 entries), which comprises all to date known 3D structures with less than 95 % sequences identity and, furthermore, all chemokine and cytokine structures were added to that library.
  • a sequence hit was further analyzed in case the chemokine alignment was within the top 10 folds in the pdb95 fold library and by manual inspection none of the better ranked alignments was considered to be superior compared to the chemokine template in terms of gap content, cysteine matching and disulfide bonding possibilities.
  • a redundancy-reduced summary of the pdb95 control results of the N73 protein sequence are shown in table 8. Ranking with this fold library confirms that the structure of the novel protein resembles an IL-8- like chemokine fold.
  • novel protein shows high structural similarity to the IL-8-like chemokine vMIP-II even among all possible protein folds (rank #2, table 8).
  • Molecular Dynamics (MD) simulations were performed as described in example 5 with the refined models from example 17 and their stability was evaluated by comparison with a known chemokine used as control.
  • the N73 RMSD plot of the 10ns MD simulation is shown in Fig. 17.
  • the MD simulation of the N73 protein with the amino acid sequence according to SEQ ID No. 9 shows, that the backbone RMSD is even lower than expected from the control chemokine model and stable around 0.5 nm throughout the simulation.
  • the RMSD values fluctuation of the secondary structure elements of the control 3D model of CXCL17 are around 0.2-0.3 nm (Fig. 17) throughout the simulation.
  • the secondary structure elements of N73 are stable and their RMSD is not higher than 0.2 nm. Only the insertion in the 40S loop connecting the 2nd and 3rd sheet is flexible at the beginning of the simulation and packs more tightly to the protein core after refinement. Nevertheless, the N73 model is overall more stable than the control chemokine. After the MD the resulting model has kept all secondary structure elements and has "nativelike" overall appearance (RMSD between start and end is only 0.5nm).
  • N73 was first identified as a genes related to cancer development and progression in a large-scale cDNA transfection screening (Wan 2004). The transfection with N73 cDNA resulted in an inhibition of growth in the Hepatoma 7721 cell line (-87%) and stimulation of growth in the fibroblast cell line NIH3T3 (128%).
  • a signal peptide was identified that is cleaved at the sequence position 23 but no transmembrane region was found.
  • WoLF PSORT predicts extracellular location with a score of 25.
  • the ELM server was queried with the N73 protein sequence using the extracellular filter, and the signal peptide prediction from 1 -23 was confirmed.
  • low complexity regions were predicted from positions 33-53 and 164-182.
  • An N-glycosylation site pos:28-33
  • a glycosaminoglycan attachment site pos: 147-150 was identified.
  • RGD motif found from position 198-200 is noteworthy. This is a receptor binding motif recognized by different members of the integrin family.
  • the RGD motif is known to lie on an exposed flexible loop if functional (Assa-Munt 2001), which is the case for N73, as it is located at the C- terminus after a LCR.
  • RGD motifs in proteins of the extracellular matrix are used by integrins to link the intracellular cytoskeleton of cells with the extracellular matrix. Without attachment to the extracellular matrix, cells normally undergo apoptosis ('anoikis') (Frisch 2001). Small soluble peptides containing the RGD motif inhibit cell attachment and consequently induce apoptosis of the integrin presenting cells (Kim 2003). This makes the RGD peptides and mimetics potential therapeutic agents against tumor- induced angiogenesis, inflammation and cancer mestastasis. Thus, it might be possible that the soluble secreted N73 protein has similar functions through integrin interaction with its RGD motif.
  • CXCL4 chemokine-like kinase
  • RGD motif a chemokine-like kinase inhibitor
  • N73 has similar anti-angiogenic functions through integrin interaction with its RGD motif.
  • N73 is conserved across different primate species.
  • the processed protein contains 8 cysteines that could potentially form disulfide bonds.
  • a BLAST search and ENSEMBL (table 9) identified one close ortholog in gorilla (SEQ ID No. 13) sharing 79% sequence identity having all 8 cysteine residues of the processed protein sequence conserved and another in bushbaby (SEQ ID No. 14) sharing 41% sequence identity (Fig. 19).
  • the N73 gene (ENSG00000184334) comprises 3 exons with the protein coding region on the first one only.
  • the open reading frame is located on chromosome 8q24.3 (142,513,827-142,517,232 forward strand) and in close proximity to the N73 gene an orphan G protein coupled receptor (GPR20) can be found (Fig. 18). At present, no other chemokine is known to be located on this chromosome.
  • a poly-A site was predicted using Polyadq (Tabaska 1999) & Polyah.pl (softberry) at position +534 downstream in the human gene. Furthermore the promoter region of N73 was analyzed for transcription factor binding sites and we identified several binding sites (table 10) that are known from other chemokines like NF-IL6, NF-kappaB and APl (C-Jun) (Chong 2000; Genin 2000; Gerber 2004; Roebuck 1999; Ueda 2007; Widmer 1993; Yeruva 2008).
  • Example 21 Information available in public resources about in vivo expression of the N73 nucleic acid sequence
  • GeneNote Gene Normal Tissue Expression
  • a survey of complete gene expression profiles in 12 healthy human tissues found that the N73 transcript is widely expressed in all tissues tested namely heart, skeletal muscle, bone marrow, brain, kidney, liver, lung, pancreas, prostate, spinal cord, spleen and thymus (Fig. 20).
  • Example 22 In vitro expression and purification of the N73 protein in E. coli
  • the following method describes recombinant expression of N73 polypeptides in E. coli.
  • the gene encoding the mature N73 protein is synthesized and cloned in an expression plasmid pETMM- 60.
  • the expression and purification can be done as described in Magistrelli et al. (Magistrelli 2005) using a His-tagged NusA fusion protein to make the N73 as fusion partner soluble and Origami B cells (for disulfide bonds formation in cytoplasm because of a glutathion S transferase mutation).
  • the N73 protein is expressed in E. coli and soluble as fusion protein.
  • the His-tagged fusion protein can be purified using a HisTrap HP column collecting the HisTrap elution. Then, the construct is cleaved from NusA with TEV protease and leaving a GA-addition at the N-terminus of the N73 protein and the His-tag at the NusA.
  • the N73 protein can be separated from the Nus A and purified by running another HisTrap HP column collecting the flow-through as the His-tagged NusA binds to the column but not the N73 protein.
  • the following method describes recombinant expression of N73 polypeptides in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of N73 from the ADH2/GAPDH promoter.
  • DNA encoding the N73 polypeptide and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the N73 polypeptide.
  • DNA encoding N73 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, native N73 signal peptide or another mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of N73.
  • yeast cells such as yeast strain AB 1 10
  • yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
  • the transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant N73 polypeptides can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
  • the concentrate containing the N73 polypeptide may further be purified using selected column chromatography resins.
  • the following method describes recombinant expression of N73 polypeptides in Baculovirus-infected insect cells.
  • the sequence coding for N73 is fused upstream of an epitope tag contained within a Baculovirus expression vector.
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen).
  • the sequence encoding N73 or the desired portion of the coding sequence of N73 such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions.
  • the 5' primer may incorporate flanking (selected) restriction enzyme sites.
  • the product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold(TM) virus DNA (Pharmingen) into Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 171 1 ) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28[deg.] C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
  • Expressed poly-His tagged N73 can then be purified, for example, by Ni 2+ -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgC12; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice.
  • sonication buffer 25 mL Hepes, pH 7.9; 12.5 mM MgC12; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1
  • the sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%> glycerol, pH 7.8) and filtered through a 0.45 [mu]m filter.
  • loading buffer 50 mM phosphate, 300 mM NaCl, 10%> glycerol, pH 7.8
  • a Ni 2+ -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
  • the filtered cell extract is loaded onto the column at 0.5 mL per minute.
  • the column is washed to baseline A280 with loading buffer, at which point fraction collection is started.
  • the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10%> glycerol, pH 6.0), which elutes nonspecifically bound protein.
  • a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10%> glycerol, pH 6.0
  • the column is developed with a 0 to 500 niM Imidazole gradient in the secondary wash buffer.
  • One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ⁇ 2+> -NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted Hisl O-tagged N73 are pooled and dialyzed against loading buffer.
  • purification of the IgG tagged (or Fc tagged) N73 can be performed using known chromatography techniques, including for instance, Protein A or Protein G column chromatography.
  • N73 polypeptides were successfully expressed as described above.
  • This example illustrates preparation of monoclonal antibodies which can specifically bind N73.
  • Immunogens that may be employed include purified N73 polypeptides, fusion proteins containing N73 polypeptides, and cells expressing recombinant N73 polypeptides on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
  • mice such as BALB/c are immunized with the N73 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms.
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads.
  • MPL-TDM adjuvant Ribi Immunochemical Research, Hamilton, Mont.
  • the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti- N73 antibodies.
  • the animals "positive" for antibodies can be injected with a final intravenous injection of N73.
  • the mice Three to four days later, the mice are sacrificed and the spleen cells are harvested.
  • the spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. 1 , available from ATCC, No. CRL 1597.
  • the fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin, and thymidine
  • hybridoma cells will be screened in an ELISA for reactivity against N73. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against N73 is within the skill in the art.
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing the anti-N73 monoclonal antibodies.
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Protein A or Protein G can be employed.
  • Native or recombinant N73 polypeptides may be purified by a variety of standard techniques in the art of protein purification.
  • pro-N73 polypeptide, mature N73 polypeptide, or pre-N73 polypeptide is purified by immuno affinity chromatography using antibodies specific for the N73 polypeptide of interest.
  • an immunoaffinity column is constructed by covalently coupling the anti-N73 polypeptide antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE(TM) (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated SEPHAROSE(TM) (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • Such an immunoaffinity column is utilized in the purification of N73 polypeptide by preparing a fraction from cells containing N73 polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble N73 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
  • a soluble N73 polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of N73 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/N73 polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and N73 polypeptide is collected.
  • a low pH buffer such as approximately pH 2-3
  • a chaotrope such as urea or thiocyanate ion
  • the pre-filtering and structure-based chemokine screening of F 10 was performed by the same methodology as described in example 2 for B42.
  • a redundancy-reduced summary of the results obtained for the novel protein F10 using the fold library containing all known structures of the members of the IL-8-like chemokine fold family is shown in table 11.
  • the "95 rank" of those F10 alignments with chemokine template structures corresponds to the overall rank in the pdb95 library.
  • the novel protein F10 according to SEQ ID No. 16 shows high structural similarity to the known chemokines vMIP-I (rank #1 , 20.0 %>, SEQ ID No. 38), which is encoded by kaposi's sarcoma-associated herpesvirus and CXCLI O (rank #4, 17.8 %>, SEQ ID No. 39).
  • the best scoring human chemokine template (ID: 16,7%) was CXCL8 (rank #2).
  • z-score corresponding to the surface (residue-solvent) potential component of the alignment z sec ... z score corresponding to sequence identity , %>ID ... percentage of amino acid sequence identity, pi ... alignment path length, fl ... template fold length, scopid ... SCOP family identifier, pdb id ... template identifier in the Protein Data Bank, chain ... template chain (author chain label), cover ... percentage of domain coverage (folds with coverage lower than 70%> are filtered out), descr ... description of PDB template, Uniprot ... Uniprot identifier, Rec. agonist ... Chemokine receptors for that the template protein is an agonist (activator), Rec. antagonist ... Chemokine receptors for that the template protein is an antagonist (inhibitor).
  • Table 11 A redundancy-reduced summary of the in the threading result obtained for the novel protein FIO using the fold library containing all known structures of the members of the IL-8-like chemokine fold family.
  • the MODELLER package was used to generate 3D models of the query sequences with the sequence to structure alignments calculated by ProHit. In those 3D models, the distances between cysteine were measured and analyzed for possibilities of disulfide bond formation. To refine the candidate's model generated from the threading alignment, the cysteines were connected, the structure energy minimized and side chain rotamers were manually adjusted using MOE2008.10.
  • One disulfide bridge can be formed between C20 and C47 in a C-motif.
  • Visual inspection of the lzxt-like 3D model of FI O shows good packing of the protein without any long loops. The sequence identity not high with 20% but conservation of important amino acid properties can be observed.
  • Positions of proline and glycine residues are very well conserved (P27-29, P40, P45, P63) as well as hydrophobic properties of residues forming the core of the protein (pos.: V30, F49, F51, F77, V78, F76).
  • a sequence to structure alignment of FIO with vMIP-I is shown in Fig. 21.
  • Fig. 24 The structure-based sequence alignment of F10 with chemokines known to bind CXCR3 (CXCL9, CXCLIO and CXCL11) is shown in Fig. 24. Conservation is indicated as well as the residues involved in binding to CXCR3. Although, the residues important for receptor binding do not present sequence- position conservation in F10, they are recognizable at equivalent functional positions in space (Fig. 23).
  • a control threading run is performed to assess the quality and ranking of the chemokine structure alignment within the structural templates of the whole PDB.
  • the pdb95 is used as fold library (22009 entries), which comprises all to date known 3D structures with less than 95 % sequences identity and, furthermore, all chemokine and cytokine structures were added to that library.
  • a sequence hit was further analyzed in case the chemokine alignment was within the top 10 folds in the pdb95 fold library and by manual inspection none of the better ranked alignments was considered to be superior compared to the chemokine template in terms of gap content, cysteine matching and disulfide bonding possibilities.
  • Molecular Dynamics (MD) simulations were performed as described in example 5 with the refined models from example 28 and their stability was evaluated by comparison with a known chemokine used as control.
  • the MD simulation of the F10 protein with the amino acid sequence according to SEQ ID No. 16 shows as good stability of the secondary structure elements as the control 3D model of CXCL17 with low RMSD values throughout the simulation (Fig. 25: lower graph).
  • the backbone RMSD stays around 0.3-0.4nm and thus much lower than expected from the control CXCL17 chemokine model with 0.6- 0.8nm (Fig. 25: upper graph).
  • the RMSD is stable throughout the simulation as well as the secondary structure elements. Their RMSD is not higher than 0.2-0.3nm and no long flexible loops are present.
  • the resulting model after the MD has "native-like" conformation containing all secondary structure elements and suggesting a good model resolution of about 3-4A.
  • Orthologues of the F10 protein comprise the following amino acid sequences: flying fox SEQ ID No. 23, dog SEQ ID No. 24, microbat SEQ ID No. 25, hyrax SEQ ID No. 26, tenrec SEQ ID No. 27, bushbaby SEQ ID No. 28, alpaca SEQ ID No. 29, dolphin SEQ ID No. 30, chimpanzee SEQ ID No. 31, orangutan SEQ ID No. 32, mouse lemur SEQ ID No. 33, tree shrew SEQ ID No. 34, horse SEQ ID No. 35, squirrel SEQ ID No. 36, rabbit SEQ ID No. 37.
  • the F10 protein sequence is also known as C4orf26 was first identified within a the large-scale cDNA identification within the Mammalian Gene Collection (MGC) project (Gerhard 2004).
  • MMC Mammalian Gene Collection
  • F10 is 130 amino acids long and contains 5 cysteineSi No transmembrane region was identified in the F10 protein sequence but a signal peptide that is cleaved after residue 23.
  • WoLF PSORT predicts extracellular location with a score 30.
  • the ELM server was queried with the F10 protein sequence using the extracellular filter and low complexity regions (LCR) are predicted from position 68 to 90.
  • GlobPlot predicts many disordered regions from 24-74, 83-101 and 123-130.
  • cleavage sites for different enzymes are predicted at positions 77-79 and two overlapping sites from 114-121.
  • the F10 gene (ENSG00000174792) is an alternatively spliced gene encoding iso-forms with 2 and 3 Exons (Fig. 29).
  • the open reading frame is located on chromosome 4q21.1 (4:76,481,273-76,491,081 forward strand, Fig. 26), which is in very close proximity of the chemokine mini cluster with CXCL9, CXCL10 and CXCL1 1.
  • Those chemokines (CXCL9, CXCL10 and CXCL 1 1 ) bind all the same Receptors: 1) CXCR3 as agonist with angiostatic function that is expressed by TH1 cells and NK cells and 2) CCR3 as antagonist/inhibitor.
  • a poly-A site was predicted at position +124 downstream in the human gene. Furthermore the promoter region of F10 was analyzed for transcription factor binding sites and we identified several binding sites (Table 13) that are known from other chemokines like NF-IL6, NF-kappaB, APl(C-Jun) and the CKl motif (Chong 2000; Genin 2000; Gerber 2004; Roebuck 1999; Ueda 2007; Widmer 1993; Yeruva 2008).
  • Example 32 Information available in public resources about in vivo expression of the F10 nucleic acid sequence
  • Example 33 In vivo expression of the F10 nucleic acid sequence in human tissues
  • the mRNA expression of F10 was analyzed by PCR with templates from 8 different tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) using Human MTC Panel I (Clontech, Catalog No. 636742, LOT No. 8082935A). 43 PCR cycles were done at an annealing temperature of 58°C. The expected size of the PCR products of F10 (isoform A/ Transcript 1) is 354bp. The following primers were used for F10: forward primer: 5'-TGGTGGTAACTGTGGCAGAA-3 ' (SEQ ID No. 43) and reverse primer: 5'-TTCCCTCTCAGCTTTCCTCA-3' (SEQ ID No. 44).
  • isoform A is F10 protein coding and pre-dominantly expressed in placenta and weaker in the HeLa cell line.
  • F10 isoA
  • Isoform B has an additional exon in them middle of the intron that shifts the reading frame and thus in the translated protein only the signal sequence would be similar to Iso A. But this Isoform B was not found in placenta or HeLa (Fig. 29 bottom, Fig. 30).
  • RNAi knockdown experiments were performed as described in (Kittler 2007).
  • F10 (c4orf26) results in comparison with results of known chemokines are shown in table 14.
  • F10 (c4orf26) shows a cell division defect phenotype with cell cycle arrest in the G2M and 8N cell cycle phase similar to known angiostatic chemokines (CXCL10, 13, 14, underlined) suggesting a functional similarity of F10 to known angiostatic chemokines.
  • CXCL10, 13, 14, underlined known angiostatic chemokines
  • Abbreviations used in table are as follows: Gl ... 2N DNA content; S ... 2N...4N DNA content; G2M ... 4N DNA content; 8N ... 8N DNA content; CN: cell number; AVn ... averaged normalised value of a parameter; The cell number for DNA content analysis assay for calculating nCN was determined as the sum of cells with Gl, S, G2M and 8N DNA content.
  • Example 35 In vitro expression and purification of the F10 protein in E. coli
  • the following method describes recombinant expression of F10 polypeptides in E. coli.
  • the gene encoding the mature F10 protein (without signal peptide) is synthesized and cloned in an expression plasmid pETMM-60.
  • the expression and purification can be done as described in Magistrelli et al. (Magistrelli 2005) using a His-tagged NusA fusion protein to make the F10 as fusion partner soluble and Origami B cells (for disulfide bonds formation in cytoplasm because of a glutathion S transferase mutation).
  • the F10 protein is expressed in E. coli and soluble as fusion protein.
  • the His-tagged fusion protein can be purified using a HisTrap HP column collecting the HisTrap elution. Then, the construct is cleaved from NusA with TEV protease and leaving a GA-addition at the N-terminus of the
  • the F10 protein can be separated from the Nus A and purified by running another HisTrap HP column collecting the flow-through as the His-tagged NusA binds to the column.
  • Example 36 Expression of F10 in Yeast
  • the following method describes recombinant expression of F10 polypeptides in yeast.
  • yeast expression vectors are constructed for intracellular production or secretion of F10 from the ADH2/GAPDH promoter.
  • DNA encoding the F10 polypeptide and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of the F10 polypeptide.
  • DNA encoding F10 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, native F10 signal peptide or another mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of F10.
  • Yeast cells such as yeast strain ABl 10
  • yeast cells can then be transformed with the expression plasmids described above and cultured in selected fermentation media.
  • the transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
  • Recombinant F10 polypeptides can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters.
  • the concentrate containing the F10 polypeptide may further be purified using selected column chromatography resins.
  • the following method describes recombinant expression of F10 polypeptides in Baculovirus-infected insect cells.
  • the sequence coding for F 10 is fused upstream of an epitope tag contained within a Baculovirus expression vector.
  • epitope tags include poly-His tags and immunoglobulin tags (like Fc regions of IgG).
  • a variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding F10 or the desired portion of the coding sequence of F10 such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.
  • Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold(TM) virus DNA (Pharmingen) into Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 171 1 ) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28[deg.] C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).
  • Expressed poly-His tagged F 10 can then be purified, for example, by Ni ⁇ 2+> -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgC12; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice.
  • sonication buffer 25 mL Hepes, pH 7.9; 12.5 mM MgC12; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1
  • the sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%> glycerol, pH 7.8) and filtered through a 0.45 [mu]m filter.
  • loading buffer 50 mM phosphate, 300 mM NaCl, 10%> glycerol, pH 7.8
  • a Ni ⁇ 2+> -NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
  • the filtered cell extract is loaded onto the column at 0.5 mL per minute.
  • the column is washed to baseline A280 with loading buffer, at which point fraction collection is started.
  • the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10%> glycerol, pH 6.0), which elutes nonspecifically bound protein.
  • a secondary wash buffer 50 mM phosphate; 300 mM NaCl, 10%> glycerol, pH 6.0
  • the column is developed with a 0 to 500 niM Imidazole gradient in the secondary wash buffer.
  • One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ⁇ 2+> -NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His l O-tagged F 10 are pooled and dialyzed against loading buffer.
  • purification of the IgG tagged (or Fc tagged) F 10 can be performed using known chromatography techniques, including for instance, Protein A or Protein G column chromatography.
  • F10 polypeptides were successfully expressed as described above.
  • This example illustrates preparation of monoclonal antibodies which can specifically bind F10.
  • Immunogens that may be employed include purified F10 polypeptides, fusion proteins containing F10 polypeptides, and cells expressing recombinant F10 polypeptides on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.
  • mice such as BALB/c are immunized with the F 10 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms.
  • the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads.
  • MPL-TDM adjuvant Ribi Immunochemical Research, Hamilton, Mont.
  • the immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti- F10 antibodies.
  • the animals "positive" for antibodies can be injected with a final intravenous injection of F10.
  • the mice Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. 1 , available from ATCC, No. CRL 1597.
  • the fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
  • HAT hyperxanthine, aminopterin, and thymidine
  • hybridoma cells will be screened in an ELISA for reactivity against F10. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against F10 is within the skill in the art.
  • the positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing the anti-FlO monoclonal antibodies.
  • the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to Protein A or Protein G can be employed.
  • Native or recombinant F10 polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-FlO polypeptide, mature F10 polypeptide, or pre-FlO polypeptide is purified by immunoaffmity chromatography using antibodies specific for the F10 polypeptide of interest. In general, an immuno affinity column is constructed by covalently coupling the anti-Fl O polypeptide antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE(TM) (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated SEPHAROSE(TM) (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • Such an immunoaffmity column is utilized in the purification of F10 polypeptide by preparing a fraction from cells containing F10 polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble F10 polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.
  • a soluble F10 polypeptide-containing preparation is passed over the immunoaffmity column, and the column is washed under conditions that allow the preferential absorbance of F10 polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/FlO polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and F10 polypeptide is collected. Aidoudi S, Bujakowska K, Kieffer N, Bikfalvi A. The CXC-chemokine CXCL4 interacts with integrins implicated in angiogenesis. PLoS One 2008, 3:e2657
  • Boshoff C Endo Y, Collins PD, Takeuchi Y, Reeves JD, Schweickart VL, Siani MA, Sasaki T, Williams TJ, Gray PW et al: Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines. Science 1997, 278(5336):290-294.
  • Genin 2000 Genin P, Algarte M, Roof P, Lin R, Hiscott J: Regulation of RANTES chemokine gene expression requires cooperativity between NF-kappa B and IFN-regulatory factor transcription factors. J Immunol 2000, 164(10):5352- 5361.
  • Gerber 2004 Gerber A, Heimburg A, Reisenauer A, Wille A, Welte T, Buhling F: Proteasome inhibitors modulate chemokine production in lung epithelial and monocytic cells. Eur Respir J 2004, 24(l):40-48.
  • WoLF PSORT protein localization predictor. Nucleic Acids Res. 2007 Jul;35(WebServer issue):W585-7.
  • Hoover 2002 Hoover DM, Boulegue C, Yang D, Oppenheim JJ, Tucker K, et al. The structure of human macrophage inflammatory protein-3alpha /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding activities with human beta-defensins. J Biol Chem 2002, 277:37647-54
  • Karlsson 2008 Karlsson E, Delle U, Danielsson A, Olsson B, Abel F, et al. Gene expression variation to predict 10-year survival in lymph- node- negative breast cancer.
  • Luttichau HR Johnsen AH, Jurlander J, Rosenkilde MM, Schwartz TW: Kaposi sarcoma-associated herpes virus targets the lymphotactin receptor with both a broad spectrum antagonist vCCL2 and a highly selective and potent agonist vCCL3. J Biol Chem. 2007 Jun 15;282(24): 17794-805.
  • Sippl 1992 Sippl MJ, Weitckus S: Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations. Proteins 1992, 13(3):258-271.
  • Sippl 1993 Sippl MJ: Boltzmann's principle, knowledge-based mean fields and protein folding. An approach to the computational determination of protein structures. J Comput Aided Mol Des 1993, 7(4):473-501.
  • Widmer 1993 Widmer U, Manogue KR, Cerami A, Sherry B: Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines. J Immunol 1993, 150(11):4996-5012.
  • MIP macrophage inflammatory protein

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Abstract

La présente invention se rapporte à une protéine ayant une homologie structurelle par rapport aux protéines comportant une pliure de type chimiokinique IL-8, et à ses utilisations. L'invention a diverses applications industrielles sous forme d'applications médicales, pharmaceutiques et biotechnologiques, comme des produits pharmaceutiques, de diagnostic, des biocapteurs et des bioréacteurs. L'objet de l'invention est la production et l'utilisation de protéines nouvellement secrétées ayant une homologie structurelle par rapport aux protéines comportant une pliure de type chimiokinique IL-8 qui sont impliquées dans des maladies à médiation immunitaire, des maladies inflammatoires ou des maladies malignes néoplasiques. L'invention se rapporte à une protéine isolée ayant au moins 80% d'identité de sa séquence d'acides aminés par rapport à une séquence d'acides aminés de la protéine indiquée dans la SEQ ID n°1. La protéine selon l'invention est structurellement similaire aux protéines comportant une pliure de type chimiokinique IL-8. L'invention se rapporte également à une protéine isolée ayant au moins 80% d'identité de sa séquence d'acides aminés par rapport à une séquence d'acides aminés de la protéine indiquée dans la SEQ ID n°9. L'invention se rapporte, en outre, à une protéine isolée ayant au moins 80% d'identité de sa séquence d'acides aminés par rapport à une séquence d'acides aminés de la protéine indiquée dans la SEQ ID n°16, avec des résidus cystéine aux positions 20 et 47 et avec au moins un pont disulfure entre deux résidus cystéine.
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