JP2008253270A - New polypeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polynucleotide encoding a polypeptide (protein) isolated from PHA-activated lymphocytes, endothelial cells or dendritic cells (DC), and to inhibit or activate the actions of the polynucleotide and/or polypeptide (protein). <P>SOLUTION: There are provided an isolated LLR-J24-stalk nucleotide, a polypeptide, a polynucleotide encoding transmembrane protein LLR-J24, transmembrane protein LLR-J24, a polynucleotide encoding the extracellular domain of LLR-J24, a polypeptide which is the extracellular domain of LLR-J24, their uses in a method for identifying the agonist or antagonist of the polypeptide/protein, the agonist or antagonist of the polypeptide/protein, and its use as a medicine. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a polynucleotide encoding a polypeptide (protein) isolated from PHA-activated lymphocytes, endothelial cells, or dendritic cells (DC). The invention also relates to inhibiting or activating the action of such polynucleotides and / or polypeptides (proteins).

  Endothelial cells (EC) constitute the internal surface of large and small blood vessels and mediate the migration of immune system cells to the underlying tissue. Unstimulated EC represents an anticoagulant surface that ensures unobstructed blood flow. Upon stimulation with inflammatory mediators such as TNFα, IL-1 or lipopolysaccharide (LPS), ECs include cell adhesion molecules (eg, E-selectin, ICAM-1 and VCAM-1), cytokines (IL-1). , IL-6, IL-8), a variety of molecules including components of the coagulation system (tissue factor, PAI-1) and others (iNOS). These expressed molecules mediate chemotaxis, immune cell adhesion and translocation, and several other functions (de Martin et al. Arterioscler. Thromb. Vasc. Biol. 20 [2000] e83-e88. ). Thus, the immune response is a common basic mechanism for a variety of diseases including, for example, inflammation, arteriosclerosis, restenosis, reperfusion injury, inflammatory bowel disease, psoriasis and atopic dermatitis.

  Most of the expressed molecules are upregulated in an inducible and transient manner involving the transcription factor NF-kB. Inhibition of NF-kB by pharmacological or genetic inhibitors has been shown to block other aspects of inflammation and EC activation (Wrighton et al. J. Exp. Med. 183 [1996] 1013- 1022; Oitzinger et al. Blood 97 [2001] 1611-1617). Thus, inhibitors of NF-kB have broad therapeutic potential.

  The current understanding of the regulatory steps that lead to activation of NF-kB includes the following: after stimulation, NF-kB (eg, p65 / p50 heterodimer) inhibits the inhibitory subunit IkBa, -b or -e. Released from the inactive cytoplasmic complex. The IkB subunit, when bound to NF-kB, masks the NF-kB nuclear localization signal and thus prevents its nuclear translocation. NF-kB release is initiated by IkB phosphorylation of two serine residues Ser32 and Ser36 in IkBa, followed by a corresponding site in IkBb that is a b-TrCP-like component of the E3 ubiquitin ligase complex ( It is recognized by the Skp1 / Cul1 / ROC1 / F-box protein FWD1), ubiquitinated, and degraded via the 26S proteasome.

  A major breakthrough in the field is the identification of two kinases (IKK1 / IKKa and IKK2 / IKKb) that specifically phosphorylate IkBa. These kinases form homo- and heterodimers through their leucine zipper domains and bind to a third protein (NEMO / IKKg). This IkB kinase complex (IKC), consisting of IKK1, IKK2 and NEMO, is contained within a high molecular weight (500-700 kD) complex called the signalosome, which contains the catalytic subunit of PKA (csPKA) and phospho-tyrosine Other proteins such as proteins can be included. Genetic evidence using knockout mice revealed a prominent role for IKK2 in inflammatory signaling, but IKK1 appears to be involved in the differentiation process.

  In addition to IKK, pp90rsk and DNA-PK have been shown to be able to directly phosphorylate and activate IkB. In addition, two novel IKKs are described, one is inducible by LPS at the mRNA level in macrophages (IKK-i), and one (TBK1) forms a complex with TRAF2 and TANK, Thus, it represents an alternative signaling pathway for TNFa that leads to NF-kB activation. Several groups have described modulators that affect IKK activity either positively or negatively (eg, TAK1; Hofer-Warbinek et al. J. Biol. Chem. 275 [2000] 22064-22068) Shows an additional important level of modulation of NF-kB activity.

  In various cell types, multiple upstream activators of IKC have been described. These include the MAP3K type kinases NIK (NF-kB inducible kinase) and MEKK1 (MAPK / ERK kinase kinase 1), TGFb inducible kinase TAK1 and Akt and PKC isoforms zeta and theta, which are heterogeneous and different It reflects both a certain degree of specificity of the NF-kB signaling pathway with respect to cell type and stimulation. These upstream activators act on different components of the receptor-specific cytoplasmic signaling complex recruited to the receptor by stimuli such as TRAF1, TRAF2 and TRADD for TNF-R, and TRAF6 for IL-1R and Toll family receptors. Join. Thus, IKK2 appears to be centrally important in NF-kB signaling.

  This time, from the cDNA library of isolated PHA-activated lymphocytes, for example, the nucleotide sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 or a sequence having at least 80% identity thereto is obtained. It was shown that the IKK2-55 gene encoding a polypeptide having can be isolated.

  Endothelial cells (EC) are localized in the vasculature. They are connected by a junction complex and form the endothelium, which lines the entire vasculature. They control the flow of substances between the circulation and specific organs and are the main site of the inflammatory response. In addition, they are sites where interactions occur between the circulating cells of the body's own immune defense system and the modified cells of a particular organ. The exchange of substances, peptides and immune cells between the bloodstream and various tissues of the body is controlled by the EC barrier. This selectivity of the EC barrier is very important for the formation of several so-called “blood-tissue” barriers that are essential for maintaining the integrity of each organ.

  In response to various pathological stimuli, ECs can regulate their constitutive function, resulting in several types of reversible changes in their functional state called endothelial dysfunction . The EC must respond in a balanced manner to a number of physiological signals essential for maintaining proper blood flow, which provides oxygen and nutrients to all tissues, and microorganisms, viruses And fight against parasitic infections. In addition, EC is also involved in damage repair by inducing the growth and regression of new blood vessels, cancer progression, and atherosclerosis progression (see, eg, Cines et al. Blood 91 [1998] 3527-3561). ).

  Capillary ECs can be isolated from human tissue by immunomagnetic beads using specific monoclonal antibodies or by Ulex europaeus lectin type 1 coating magnetic beads (eg, Manconi et al. Meth. Cell Science [ 2000] 22 89-99). EC cDNA libraries can be generated and EC gene expression patterns can be determined by various hybridization techniques such as oligonucleotide fingerprinting, subtractive hybridization or RNA profiling, and by sequencing conventional methods, Can be obtained.

  This time, from the isolated EC cDNA library, for example, it encodes the amino acid sequence shown in SEQ ID NO: 4 or the DINO polypeptide having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4. It has also been found that the DINO gene can be isolated.

  Dendritic cells (DCs) are specialized antigen-presenting leukocytes that play a central role in the induction of early immune responses and tolerance. In an undifferentiated state, DCs can be present in various tissues of the body that are made to capture antigens of invading pathogens. After antigen capture, DCs mature into antigen-presenting cells and migrate to lymphoid organs to activate T cells (Banchereau, M. and RM Steinman Nature 329 [1998] 245-252). For example, Langerhans cells (LC) present in the skin have been shown to present various antigens that can be produced in or penetrate the skin. In contact hypersensitivity, topical administration of reactive hapten can activate LC and move out of the epidermis and into the draining lymph node, where LC is selected T An antigen can be presented to a cell. During contact between the LC and the T cell, the LC may provide a signal to the T cell that induces proliferation and differentiation into an effector cell.

  Depending on the type of T cell and the type of interacting molecule, the cytotoxic, regulatory and helper T cells involved can be formed. DC have also been shown to phagocytose all types of apoptotic cells and thus play a critical role in maintaining tolerance to self-antigens (Steinman RM and K. Inaba J. Leukoc. Biol. 66 [ 1999] 205-208). Diseases in which DC plays a causal or consequential role as a key regulator of the immune response are associated with DC-specific pharmaceutical or iatrogenic practice, for example in chronic inflammatory diseases, autoimmune diseases or transplant rejection As well as diseases including inflammatory skin diseases such as contact hypersensitivity or atopic dermatitis, and diseases or syndromes where significant pathological components are immunosuppressive, such as in AIDS and cancer.

  DCs are monocytes (CD14 +), T cells (CD3 +), B cells (CD19 +) and NK cells by negative selection, ie by capturing on specific mAB coated magnetic beads or panning according to conventional methods. It can be isolated from peripheral blood by isolation from (CD16 +). DC cDNA libraries can be generated, and DC gene expression patterns can be sequenced by various hybridization techniques such as oligonucleotide fingerprinting, subtractive hybridization or RNA profiling, and according to conventional methods Can be obtained.

  The transmembrane receptor for DC is important. Because, in part, they are involved in the uptake of parasites and apoptotic cells that lead to antigen presentation by DC. On the other hand, these receptors interact with soluble ligands and surface-bound ligands on T cells and other immune cells. These interactions are involved in the regulation of DC function as well as the function of interacting cells such as T cells. These regulatory events, depending on the differentiation state of the cell and the interacting ligand, include activation and inhibition of DCs, T cells and other immune cells.

  Receptors with type II transmembrane orientation and a C-type lectin-like domain in the extracellular part have been found to be frequently expressed on various hematopoietic cells, and some are important in the initial immune response (Weis WI, Taylor ME and Drickamer K. Immunol. Rev. 163 [1998] 19-34). Proteins such as CD69 and AICL (Santis AG et al. Eur. J. Immunol. 24 [1994] 1692-7; Hamann et al., Immunogenetics 45 [1997] 295-300) that are widely expressed in hematopoietic cells. Apart from that, some are further restricted in cell type expression, such as lectin-like NK cell receptors, which have been intensively studied recently.

  The lectin-like NKR-P1 and Ly49 genes (Yokoyama WM et al. J. Immunol. 147 [1991] 3229-3236) first detected in rodent NK cells, and CD94 first described from human NK cells And NKG2 gene (Chang C. et al. Eur. J. Immunol 25 [1995] 2433-2437; Houchins JP et al. J. Exp. Med. 173 [1991] 1017-1020; Hofer E. et al. Immunol. Today 13 [1992] 429-430) is a mouse chromosome 6 (Brown MG et al. Genomics 42 [1997] 16-25), rat chromosome 4 and human 12p12.3-p13.2 (Sobanov Y. et al. al. Immunogenetics 49 [1999] 99-105). These regions are therefore called NK gene complexes. Mediates resistance to viral infection or mediates allogeneic lymphocyte lysis, consistent with the concept of a chromosomal area that contains several genes important for natural immune cell function Loci that map to this region in rodents.

  Today, it is well established that lectin-like NK receptors have an important role in monitoring the expression of proper MHC class I (Lopez-Botet M. et al. Semin. Immunol. 12 [2000] 109-119; Hoglund P. Immunol. Rev. 155 [1997] 11-28). In human NK cells, the CD94 chain forms a heterodimeric receptor with different NKG2 isoforms, which can bind to the nonclassical MHC class Ib molecule HLA-E. CD94 / NKG2A functions as an inhibitory receptor, and CD94 / NKG2C receptor can activate NK cells. HLA-E presents peptides from the leader sequence of other class Ia molecules and is only transported onto the surface when loaded with the appropriate peptide.

  Thus, recognition of HLA-E by the CD94 / NKG2A receptor is a highly susceptible mechanism for monitoring the proper biosynthesis of MHC class I molecules, which can be down-regulated by many viruses (Alcami A. and UH Koszinowski Immunol. Today 21 [2000] 447-55). The function of activating the CD94 / NKG2C receptor in this context is not yet understood. Activated NKG2D receptors that are only slightly associated with other NKG2 molecules form heterodimers and recognize the class I-like molecule MICA, which is upregulated by stress, eg viral infection, and on some tumor cells (Bauer S. et al., Science 285 [1999] 727-729). The corresponding CD94 and NKG2 receptors in mice appear to have similar functions.

  Genomic structure and transcription of human NK receptor genes (Glienke J. et al. Immunogenetics 48 [1998] 163-173) and their protein product functions (Duchler M. et al. Eur. J. Immunol. 25 [1995] 2923 -31) has been studied in the past. In an attempt to discover genes related to evolution and function for the NKG2 and CD94 receptor genes, these studies have been extended to encompass regions of the NK gene complex flanking these genes on both sides of about 1 Mb. . In addition to the Ly49L pseudogene, no additional human Ly49 gene could be detected on the centromere side (Bull C. et al. Genes and Immunity 1 [2000] 280-287). A novel cluster of lectin-like telomeric genes has been found. This cluster consists of LOX-1 (Sawamura T. et al. Nature 386 [1997] 73-77), a novel human lectin-like receptor (LLR-J24) gene and the recently reported Clec-1 and Clec-2 genes (Colonna M ., Samaridis J., Angman L. Eur. J. Immunol. 30 [2000] 697-704). Their chromosomal localization, expression pattern, and homology to the NK receptor gene suggest important intrinsic immune functions in DCs, monocytes and endothelial cells.

  DC is an antigen-presenting cell population in lymphoid and nonlymphoid organs (Hart D.N. Blood 90 [1997] 3245-87). They play an important role in the initiation of antigen-specific T cell proliferation. Immature DCs appear in peripheral nonlymphoid organs, where their primary function is to filter surrounding tissues for foreign antigens and pathogens. These immature DCs are highly differentiated in antigen uptake and processing and stay in non-lymphoid tissues for long periods. After antigen uptake, these DCs can migrate to T-cell rich areas of secondary lymphoid organs such as lymph nodes or spleen. This is closely related to a phenotype and functional change known as DC maturation. In this process, DC upregulates MHC class I and II molecules and T cell costimulatory molecules such as CD80 and CD86.

  Mature DCs are potent stimulators of helper and cytotoxic T cells. Understanding the molecular signals that lead to the differentiation of DCs and then program the differentiation of lymphocytes allows for more effective manipulation of the immune response with respect to the vaccination strategy or damage It is extremely important to be able to switch off the immune response being received. Based on current knowledge about the specific expression of LLR-J24 in DC and its downregulation during the maturation process, this receptor is thought to play a role during DC maturation or T cell stimulation.

  The LLR-J24 gene was now found, isolated and selectively expressed in monocyte-derived and cord blood progenitor cell-derived DCs (MoDC and CBDC). The LLR-J24-1 gene represented by nucleotide SEQ ID NO: 5 and its splice variant LLR-J24-2 represented by nucleotide SEQ ID NO: 7 are related but not identical to NKG2 and CD94 NK receptor chains (Houchins JP et al. , J. Exp. Med. 173 [1991] 1017-1020; Chang C. et al., Eur. J. Immunol. 25 [1995] 2433-7; GenBank accession numbers: NKG2A [X54867], NKG2C [X54869], NKG2E [L14542], NKG2H [AF078550], NKG2D [54780]), oxLDL receptor LOX-1 (Sawamura T. et al. Nature 386 [1997] 73-77; GenBank accession number AF035776) and two other receptor genes It has recently been found to be selectively expressed in DC, CLEC-1 and CLEC-2 (Colonna M. et al. Eur. J. Immunol. 30 [2000] 697-704; GenBank accession number CLEC-1 [ AF200949], CLEC-2 [AF124841]).

  There is a somewhat further relationship to other members of the so-called NK receptor gene complex on human chromosome 12 (Sobanov Y. et al. Immunogenetics 49 [1999] 99-105). The LLR-J24-1 gene is isolated as a splice variant LLR-J24-2 consisting of the nucleotide sequence shown in SEQ ID NO: 5 and an additional exon encoding a stalk peptide inserted between nucleotides 357-358. Can appear inside. Thus, the splice variant shown in SEQ ID NO: 5 is the same as SEQ ID NO: 7 except that it lacks the spliced out nucleotides 358-495.

  The LLR-J24 gene encodes a novel member of a subfamily of type II transmembrane receptors with a C-type lectin-like domain (CTLD) in the extracellular part (Weis WI et al. Immunol Rev. 163 [1998] 19-34 ). Its closest analogs are mouse DECTIN-1 (59% identity, Ariizumi et al. J. Biol. Chem. 275 [2000] 20157-20167, accession number AF262985), human LOX-1 (44% homology in CTLD) Sawamura T. Nature 386 [1997] 73-77; accession number AF035776), CLEC-1 (37% homology in CTLD, Colonna M. et al. Eur. J. Immunol. 30 [2000] 697-704, Accession No. AF200949), CLEC-2 (37% homology in CTLD, Colonna M. et al. Eur. J. Immunol. 30 [2000] 697-704, Accession No. AF124841), NKG2-D (33% homology in CTLD) Sex, Houchins JP et al. J. Exp. Med. 173 [1991] 1017-1020, accession number X54870), NKG2-A (30% homology in CTLD, accession number X54867), NGK2-C (30% in CTLD) Homology, accession number X54869) and CD94 receptor (31% homology in CTLD, Chang C. et al. Eur. J. Immunol. 25 [1995] 2433-7).

  The LLR-J24 gene is located on chromosome 12 in the NK receptor gene complex, which also includes the above homologues and some further related genes. The LLR-J24-1 gene shown in SEQ ID NO: 5 and SEQ ID NO: 7 encode a typical CTLD and ITAM-like sequence at its cytoplasmic end. The sequence of CTLD is clearly homologous to all CTLDs of other members of the superfamily, and the cytoplasmic termini have little or no relationship to members of other families, but mouse DECTTIN-1 Clearly homologous to the sequence. The LLR-J24-2 gene shown in SEQ ID NO: 7 is identical to SEQ ID NO: 5 but includes an additional exon encoding a stalk peptide. The corresponding protein with the stalk peptide is shown in SEQ ID NO: 8. The stalk exon nucleotide sequence is represented by SEQ ID NO: 9 and the corresponding stalk peptide is represented by SEQ ID NO: 10.

  To date, it has been found that the stalk peptide is required for expression of the receptor on the cell surface, and that the receptor without stalk is expressed in the cell. CTLD binds to surface peptide structures on other immune cells such as T cells. This may provide a regulatory, eg costimulatory signal, for T cell activation. During DC maturation, the receptor is downregulated. This may also reduce the inhibitory signal for T cell activation. The cytoplasmic domain of the receptor controls DC maturation by binding of CTLD to the ligand. CTLD binds to lipoprotein structures such as parasites, oxLDL and the membrane structure of apoptotic cell membranes. Cells and compounds containing these structures are internalized resulting in efficient antigen presentation. If DC phagocytosis is reduced, the receptor is downregulated during DC maturation. The cytoplasmic domain of the receptor mediates internalization and activation of DC maturation by binding of CTLD to the ligand.

  The intracellular portion of the receptor without the stalk peptide is involved in the monitoring of abnormal intracellular structures and parasites and thus has a different function than the portion containing the stalk peptide shown in SEQ ID NO: 10.

Accordingly, the present invention provides an isolated
An IKK2-55 gene comprising a nucleotide sequence encoding the IKK2-55 polypeptide set forth in SEQ ID NO: 2;
A DINO gene comprising a nucleotide sequence encoding a DINO polypeptide represented by SEQ ID NO: 4; or an LLR-J24-stalk comprising a nucleotide sequence encoding an LLR-J24-stock peptide represented by SEQ ID NO: 10 Nucleotides are provided.

Furthermore, the present invention provides an isolated
-An IKK2-55 gene encoding the IKK2-55 polypeptide set forth in SEQ ID NO: 2, eg comprising the nucleotide sequence set forth in SEQ ID NO: 1;
Encodes a DINO polypeptide represented by SEQ ID NO: 4, eg a DINO gene comprising the nucleotide sequence represented by SEQ ID NO: 3; or encodes a stalk peptide represented by SEQ ID NO: 10, eg SEQ ID NO 9 An LLR-J24-stalk nucleotide comprising the nucleotide sequence shown in FIG.

Furthermore, the present invention provides an isolated
The IKK2-55 polypeptide shown in SEQ ID NO: 2;
A DINO polypeptide represented by SEQ ID NO: 4; or an LLR-J24-stalk peptide represented by SEQ ID NO: 10.

  An isolated polynucleotide encoding the IKK2-55 gene, DINO gene, LLR-J24-stalk gene, transmembrane protein LLR-J24 and the extracellular domain of LLR-J24, This is referred to as “gene (s) of the invention”.

  An IKK2-55 polypeptide, DINO polypeptide, LLR-J24-stalk peptide, an isolated polypeptide that is an extracellular domain of LLR-J24 and an isolated polypeptide that is a transmembrane protein LLR-J24 It is referred to as “polypeptide (s) of the invention”.

  Polypeptides of the present invention include polypeptides having the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 10, and corresponding amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10, respectively Amino acid sequences having at least 80% identity and the same biological activity as the corresponding polypeptide of the invention are included. Polypeptides of the present invention include polypeptides encoded by corresponding genes of the present invention, including: for example, as a result of duplication (degeneration) of the genetic code, and corresponding Sequences that encode the polypeptides; or polynucleotides that hybridize to the nucleotide sequences of the genes of the invention, including, for example, allelic variants and / or complements of the genes of the invention.

  The polypeptides of the present invention may be in the form of a “mature” polypeptide, such as a protein, or may be part of a larger polypeptide, such as a protein, such as a fusion protein; It is advantageous to include sequences, prosequences, sequences useful in purification, such as multiple histidine residues, or additional sequences for stability during recombinant production into the polypeptides of the invention. obtain.

  The polypeptides of the present invention also include polypeptide fragments of the polypeptides of the present invention. Such a polypeptide fragment means a polypeptide having an amino acid sequence that is partially identical to the amino acid sequence of the polypeptide of the present invention, but not entirely the same. Such polypeptide fragments may be “free-standing” or such polypeptide fragments form a part or region, most preferably as a single continuous region. It may be part of a larger polypeptide. Preferably, such polypeptide fragments retain the biological activity of the corresponding polypeptide of the invention.

  Variants of defined polypeptide (fragment) sequences of the present invention also form part of the present invention. Preferred variants are those that have been changed from an indicator by substitution of conservative amino acids, eg, substitution of a residue with another of similar characteristics. Typically, such substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln; the basic residues Lys and Arg. Between; or between the aromatic residues Phe and Thr. Particularly preferred are variants in which several, 5-10, 1-5, or 1 or 2 amino acids are substituted, deleted or added in any combination.

The polypeptide of the present invention, or a fragment thereof, is an isolated natural polypeptide of the present invention, a recombinantly produced polypeptide, a synthetically produced polypeptide, and a combination of such polypeptides,
As well as fragments thereof.

  Thus, the polypeptides of the invention or fragments thereof can be suitably prepared, eg, by conventional methods. Unless otherwise specified herein, “isolated” includes the meaning of “isolated from the coexisting material”, eg, “modified by the hand of man” from the natural state.

  The genes of the present invention include the corresponding nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 9, respectively, and their allelic variants and their complements, and their splice variants; For example, polynucleotides that hybridize under stringent conditions, e.g., to the nucleotide sequence of the gene of the present invention, e.g., individual nucleotide sequences of the gene of the present invention, e.g., as a result of genetic code redundancy (degeneration). Different from the gene sequence of the present invention, but encoding the corresponding polypeptide of the present invention, or at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10, respectively. Which encodes a polypeptide of the present invention having a unique amino acid sequence, and It comprises a sequence having the same biological activity as de. “Stringent conditions” means at least 80%, such as 90%, such as 95%, 97% or 99% identity between the nucleotide sequence of the gene of the invention and the corresponding polynucleotide sequence that hybridizes thereto. Hybridization occurs only when is present.

The gene of the present invention encodes a corresponding polypeptide, or a portion (fragment) of the corresponding polypeptide of the present invention, or a corresponding encoded polypeptide of the present invention and, for example, the full length of the corresponding amino acid sequence. Polypeptide of amino acid sequence or portion of a polypeptide having at least 80% identity; eg, 80% -100%, eg 90%, eg 95%, eg 97%, eg 99% or 100% identity The identity is
Where n _a is the number of amino acid changes, x _a is the total number of amino acids in the corresponding amino acid sequence, and y is the percent identity divided by 100. ]
For example, a polypeptide encoded by an allelic variant of the gene, or an isoform of the corresponding amino acid sequence made by alternative splicing of a transcript from the corresponding gene. “Identity” is a measure of the identity of a nucleotide or amino acid sequence and can be calculated by routine techniques, eg, using commercially available computer programs.

  Unless otherwise specified herein, “polypeptide” includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. Unless otherwise specified herein, “polynucleotide” includes any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or may be modified RNA or DNA. , RNA, which is a mixture of single and double stranded RNA, and single and double stranded regions; and RNA hybrid molecules.

  The gene of the present invention encoding the corresponding polypeptide of the present invention can be obtained by, for example, reverse transcription PCR using DC mRNA, for example, using expression sequence tag (EST) analysis, and PHA-activated lymphocytes or endothelial cells. , Or from cDNA libraries derived from mRNA in DCs using standard cloning and screening methods (Adams MD et al. Science 252 [1991] 1651-1656; Adams MD et al. Nature 355 [1992] 632-634; Adams MD et al. Nature 377 Suppl. 3 [1995] 174). The genes of the present invention can be obtained from natural sources, such as genomic DNA libraries, or synthesized according to conventional methods.

  The gene of the present invention is useful for the recombinant production of the corresponding polypeptide (fragment) of the present invention, and when used for such recombinant production, the gene sequence itself is a mature polypeptide ( Fragment); or other coding sequences, such as leader or secretory sequences, pre- or pro- or prepro-protein sequences, or sequences that encode portions of other fusion proteins A coding sequence for the mature polypeptide (fragment). For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. The marker sequence is provided in a suitable conventional marker sequence, such as the pQE vector (Qiagen, Inc.) and as described in Gentz et al. Proc. Natl Acad. Sci. USA 86 [1989] 821-824. It can be a FLAG-tag (Sigma) or a hexa-histidine peptide, or an HA tag. Any gene of the present invention may also include non-coding 5 ′ and 3 ′ sequences, such as transcribed and untranslated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

  Nucleotide sequences that are identical or sufficiently identical to the nucleotide sequence of the gene of the invention, such as those containing fragments or splice variants thereof, are full-length cDNAs encoding the corresponding polynucleotide (fragment) of the invention and To isolate genomic clones; and, for example, cDNAs and genomic clones of other genes having high sequence homology with the gene of the invention (including genes encoding homologs and orthologs from non-human species) ) Can be used as hybridization probes for cDNA and genomic DNA.

  Hybridization can be performed, for example, according to conventional methods. Typically, a sequence similar to a gene sequence is 80% identical, preferably 90% identical, more preferably 95% identical to the gene (fragment) sequence of the invention. A hybridization probe comprises, for example, at least 15 nucleotides, such as at least 30 nucleotides, such as at least 50 nucleotides, preferably from about 30 to about 50 nucleotides; or a cDNA sequence corresponding to the coding region of the entire cDNA sequence or any portion thereof. obtain. In order to obtain a polynucleotide encoding a polypeptide of the invention, including homologs or orthologs from species other than human, a suitable hybridization technique, eg, a suitable library, is prepared under stringent hybridization conditions. A technique comprising screening with a labeled probe having a polynucleotide sequence or a splice variant or fragment thereof sequence, and isolating full-length cDNA and genomic clones containing the polynucleotide sequence may be used. .

  For example, stringent hybridization techniques are well known. Stringent hybridization conditions are, for example, as defined above, or alternatively, a suitable solution such as a solution comprising formamide, SSC, sodium phosphate, Denhardt's solution, dextran and salmon sperm DNA, For example, 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, cleaved salmon sperm The condition is that the solution is incubated overnight at around 40 ° C. in a solution containing DNA, and then the filter is washed at about 65 ° C. in 0.1 × SSC.

  In a further aspect, the present invention provides a vector comprising the gene of the present invention, eg, a polynucleotide.

  Such vectors can suitably be produced, for example, by conventional methods. Appropriate vectors can be provided according to conventional methods. A vector comprising a gene of the present invention is an expression system capable of recombinantly producing, for example, a polypeptide encoded by the gene of the present invention in a host cell, eg, a compatible host cell. Can be useful to obtain. For the recombinant production of a polypeptide of the invention, a host cell is treated, for example, by genetic engineering, eg by the use of a vector comprising the gene of the invention, or a part thereof, with the polypeptide (fragment of the invention ) Can be expressed in a host cell. Cell-free translation systems can also be used to produce the genes of the present invention, eg, in a conventional manner, for example using RNA from the DNA construct of the present invention.

  In a further aspect, the present invention provides an expression system comprising a DNA or RNA molecule, eg isolated from the natural environment, eg an expression system comprising a gene of the invention prior to isolation, the expression system Alternatively, if the portion is present in a compatible host cell, the expression system or portion thereof can produce the polypeptide of the invention.

In a further aspect, the present invention provides:
An isolated host cell comprising the expression system of the invention;
Culturing an isolated host cell comprising the expression system of the invention under conditions sufficient for the production of the polypeptide of the invention in culture and recovering the polypeptide of the invention from the culture A process for producing the polypeptide of the present invention comprising:
Producing a polypeptide of the invention comprising transforming or transfecting a host cell with an expression system of the invention such that the host cell produces the polypeptide of the invention under suitable culture conditions A method for producing a recombinant host cell to be produced; and • by transforming or transfecting the host cell with the expression system of the invention such that the host cell produces the polypeptide of the invention under suitable culture conditions. A recombinant host cell,
I will provide a.

  For recombinant production, the host cell can be genetically engineered to incorporate an expression system for the gene of the invention, or a portion thereof.

  Introduction of the polynucleotide into the host cell is suitably performed according to, for example, conventional methods, for example, Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY (1986); Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold According to Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation Transduction, scrape loading, ballistic introduction or infection, or protein transduction. Suitable host cells can be easily found. Examples of suitable host cells include, for example, bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces, Bacillus subtilis; yeast Fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; CHO, COS, HeLa, C127, CCL39, 3T3, BHK, HEK 293, And isolated animal cells such as Bowes melanoma cells; and plant cells.

  Suitable expression systems include, for example, chromosomal, episomal, and virus-derived systems such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papoviruses such as SV40 Vectors derived from vaccinia virus, adenovirus, fallox virus, pseudorabies virus and retrovirus, and combinations thereof, such as vectors derived from plasmids and bacteriophage genetic elements such as cosmids and phagemids. Expression systems include control regions that define and generate expression. In general, any system or vector suitable for maintaining, growing, or expressing a polynucleotide for producing a polypeptide in a host cell can be used. Appropriate nucleotide sequences can be suitably inserted into the expression system, eg according to conventional methods, eg according to Sambrook et al., MOLECULAR CLONING A LABORATORY MANUAL (supra).

  An appropriate secretion signal may be incorporated into the desired polypeptide for secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment. These can be heterogeneous signals.

  Generally, a polypeptide can be made on the cell surface if the polypeptide of the invention is expressed for use in a screening assay. In this case, the cells can be harvested prior to use in the screening assay. Alternatively, the polypeptide may be made intracellularly.

  The polypeptides of the invention are suitably obtained from recombinant cell culture medium, for example, detergent extraction, ultracentrifugation, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography. Can be recovered and purified according to conventional methods, including hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, such as high performance liquid chromatography. If the polypeptide of the present invention is denatured during isolation and / or purification, regeneration of the active conformation, eg, refolding of the denatured polypeptide of the present invention, can be accomplished by any suitable method, eg, conventional methods. Can be done according to

  If the polypeptide of the invention is produced intracellularly, the cell can be lysed, for example, prior to recovery of the polypeptide.

  The gene (fragment) of the present invention or the polypeptide (fragment) of the present invention can be used as a test reagent and a test substance for discovery and diagnosis of treatment for animal and human diseases.

  The transcription factor NF-kB plays an important regulatory role in regulating the expression of immune and inflammatory genes. One central component of the signal transduction pathway that leads to activation of NF-kB is IKK2. A protein that interacts with IKK2 may have a regulatory function for IKK2 activity. IKK2-55 was found to interact with IKK2 in a yeast two-hybrid screen and co-immunoprecipitation. It has been found to regulate NF-kB activity as well as the activity of certain promoters. Thus, IKK2-55 and its pharmacological agonists or antagonists include, for example, inflammation, arteriosclerosis, restenosis, reperfusion injury, inflammatory bowel disease, psoriasis, atopic dermatitis, transplant rejection, autoimmune diseases, allergies and It can be used to treat a variety of immune or inflammation-related diseases including cancer.

  The DINO gene of the present invention has been found to belong to a HERC protein family that typically contains a C-terminal HECT domain of about 350 amino acids and various numbers of RCC1 repeats at the N-terminus. HECT domain proteins constitute a subfamily of E3 ubiquitin ligases that move ubiquitin from E2-linked enzymes to specific substrates destined for degradation by the proteasome. Substrates for HERC family members have been shown to be involved in the regulation of cell cycle (cyclin E, p21 and p53), cancer (E6-AP) and regulation of BMP signaling in TGF-β family members. (Mitsui et al. BBRC 266 [1999] 115-122; Cruz et al. FEBS letters 488 [2001] 74-80; Scheffner et al. Cell 75 [1993] 495-505; Zhu et al. Nature 400 [1999] 687-693).

  It was found by yeast two-hybrid screening that DINO can interact with the TSG101 gene. TSG101 regulates MDM2 degradation and MDM2 / p53 feedback control by forming a TSG101 / MDM2 regulatory loop (Li et al. PNAS 98 [2000] 1619-1624). Furthermore, using a reporter gene assay, the expression of DINO was found to increase p53 transcriptional activity. DINO is deeply involved in the functional interaction between TSG101 and the MDM2 / p53 loop and is therefore considered to be a mediator of cell proliferation, apoptosis and inflammatory responses.

  DINO has 5 RCC1 repeats at the N-terminus and is different from Cep1 with 3 full and half repeats. RCC1 is a low molecular weight GTPase Ran guanine nucleotide exchange factor (GEF) (Bischoff and Ponstingl Nature 354 [1991] 80-82). DINO can now be thought to exert GEF activity to identify low molecular weight GTPases and thus may be involved in the regulation of cell proliferation, cytoskeletal morphology and intracellular trafficking.

  DINO may be located on chromosome 4.21-23 as a cluster containing Herc3 and Cep1.

The LLR-J24-1 and -2 genes were found to belong to the superfamily of type II transmembrane receptors with CTLD in the extracellular part (Weis WI et al. Immunol. Rev. 163 [1998] 19- 34). An important part of the gene for these receptors is located in the NK receptor complex on human chromosome 12 (Sobanov, Y. et al. Immunogenetics 49 [1999] 99-105). The CTLD of the receptor is typically encoded by three exons. CTLD evolved from a carbohydrate recognition domain in the process of evolution. This contained a lectin fold-bound Ca ⁺⁺ and had a sugar ligand such as the liver asialoglycoprotein receptor.

The binding domain of the LLR-J24 gene has modified features of CTLD, for example it can fold independently of Ca ⁺⁺ and can bind to non-sugar ligands. In order to establish the relationship between other CTLD family genes and the LLR-J24 gene, the predicted amino acid sequences of CTLD were compared. The data obtained indicate that LLR-J24, together with LOX-1, CLEC-1 and CLEC-2, forms a more closely related lectin-like receptor subfamily of NKC. The highest homology with LLR-J24 is found in LOX-1 (44%), with CLEC-1 and CLEC-2 being somewhat more distantly related (37%). The CTLD NK receptor subfamily consisting of NKG2 and CD94 proteins is the next most closely related protein (28% for NKG2-E and 33% for NKG2-D). The AICL / LLT-1 / CD69 group and the KLRF1, MAFA and NKR-P1 receptors are somewhat more distant and are shown in separate branches in multiple alignments.

  LOX-1 was found to be expressed by vascular endothelial cells and macrophages. It is kinetically regulated in vivo by inflammatory stimuli such as TNF-α, lipopolysaccharide and oxLDL, but in hypertension TGF-β, angiotensin II and fluid shear stress It is also adjusted by. Importantly, LOX-1 was found to be upregulated in atherosclerotic lesions believed to contribute to foam cell formation and endothelial dysfunction. It has been described to link oxLDL and anionic phospholipids and aging red blood cells and apoptotic cells (Sawamura T. et al. Nature 386 [1997] 73-77). One of the first events in aging or apoptotic cells is the exposure of phosphatidylserine outside the cell membrane, which is recognized and bound by LOX-1. Therefore, LOX-1 has been proposed to be involved in removing those cells from the bloodstream. In contrast to signaling events initiated by ligand binding of other lectin-like receptors such as the CD94 / NKG2 receptor, molecules and cell fragments bound to LOX-1 are internalized by endocytosis.

  Like LOX-1, the LLR-J24 gene may be thought to bind to oxidized lipoproteins such as oxLDL, parasite surface structures and apoptotic cell fragments.

  The other two closely related genes are called CLEC-1 and CLEC-2, and corresponding cDNAs have recently been identified and described in the EST database (Colonna M. et al. Eur. J. Immunol. 30. [2000] 697-704). CLEC-1 mRNA has been shown to be selectively expressed in DC and is also found in placenta, lung and thymus. CLEC-2 mRNA was also detected in DCs, but also showed more extensive expression in the hematopoietic system. Furthermore, CLEC-2 mRNA levels are particularly high in the liver. CLEC-1 and CLEC-2 ligands have not yet been identified.

  The next most closely related gene is the NKG2 NK receptor gene. Today, it is well established that lectin-like NK receptors have an important role in monitoring proper MHC class I expression according to the missing-self hypothesis (Hoglund P. et al. al. Immunol. Rev. 155 [1997] 11-28). In human NK cells, the CD94 chain forms a heterodimeric receptor with different NKG2 isoforms, which can bind to the nonclassical MHC class Ib molecule HLA-E (Lopez-Botet M. et al. Semin. Immunol. 12 [2000] 109-119). CD94 / NKG2A functions as an inhibitory receptor, and CD94 / NKG2C receptor can activate NK cells. HLA-E predominantly presents peptides from the leader sequence of other class I molecules and is only transported onto the surface when loaded with the appropriate peptide. Thus, recognition of HLA-E by the CD94 / NKG2A receptor is a sensitive mechanism for monitoring the biosynthesis of MHC class I molecules, which can be downregulated by many viruses. The function of activated CD94 / NKG2C receptor in this context is not yet fully understood.

  Activated NKG2D receptors, slightly apart from other NKG2 molecules, form homodimers and recognize the class I-like molecules MICA and MICB, which are due to stress, eg viral infection, and in some tumor cells Is up-regulated (Bauer S. et al. Science 285 [1999] 727-729). An additional ligand is the so-called UL16 binding protein, which binds to the CMV encoded UL16 protein and is also upregulated in tumor cells. Thus, NKG2-D is an activating receptor that is directly associated with NK-mediated killing of virus-infected and transformed cells. It should be noted that among the NKG2 proteins, LLR-J24 is most closely related to NKG2-D. Corresponding CD94 and NKG2 proteins in mice appear to have comparable functions. Furthermore, in mice, more than 14 isoforms of the Ly49 receptor are implicated in monitoring the expression of various MHC class I molecules.

  Similar to the NKG2 receptor, LLR-J24 is thought to be able to bind to certain MHC class I / peptide complexes or to class I related molecules that are up-regulated in viral infection and tumor cells.

  The sequence of the LLR-J24 gene shows high homology with the recently described murine DECTIN-1 cDNA except that the stalk exon is missing from the initially isolated human cDNA. This can be measured when it is found to be correctly spliced in a subfraction of about 5-10% transcripts (FIGS. 9 and 10) and corresponding predicted full-length proteins of mouse and human DECTIN-1 The sequences were compared. Both sequences show 59% identity (69% homology). High homology is conserved in the cytoplasm and in the extracellular domain including the stalk domain. Near the N-terminus of the cytoplasmic domain, an ITAM-like sequence is present in human as well as that of mice.

This sequence is slightly different from the classical ITAM motif found in the CD3-γ chain or DAP12 molecule (Lanier LL et al. Nature 391 [1998] 703-707). The first tyrosine (residue 3) in the motif is located 4 amino acids before leucine (residue 7), but this distance is at the 3 amino acid position in the classical Yx ₂ Lx _6-8 Yx ₂ L motif. is there. The next residue of the motif is positioned correctly. Both proteins further exhibit a consensus site for N-glycosylation. Human and murine lectin-like domains lack most of the residues described to be involved in Ca ⁺⁺ binding presenting non-sugar ligands. The transmembrane domain does not show charged amino acids, indicating that no association with ITAM-containing adapters such as DAP molecules has occurred.

The expression of LLR-J24 mRNA in primary human DCs, monocytes, T and B lymphocytes and endothelial cells was also examined. Based on the data obtained, human LLR-J24 mRNA is expressed in large amounts and selectively in monocyte-derived DC (MoDC) and in CD34 ⁺ hematopoietic progenitor cells (HPDC). A small amount of LLR-J24 mRNA is present in primary monocytes, but primary T and B lymphocytes and endothelial cells treated or not treated with various stimuli expressed detectable LLR-J24 transcripts. do not do. Expression is not seen in several additional cell lines, namely Northern blots including Jurkat, RPMI 8866, NKL and NK92. The size of RNA in DC is estimated to be 3.0-3.5 kb from Northern blots, which is the first or some detected in genes in the region 1.6-1.9 kb downstream from the stop codon This corresponds to the expected transcript size assuming the use of a continuous polyadenylation signal.

  DC activation and final maturation are associated with altered expression of genes that regulate their migration and function. Therefore, it was tested whether DC activation by inflammatory mediators, CD40-ligand or zymosan A resulted in a change in the expression level of LLR-J24 mRNA. As shown in FIG. 8, treatment of MoDC in combination with IL-1β and TNF-α for 18-24 hours resulted in a 3-5 fold reduction in reproducible LLR-J24 mRNA. Instead, HPDC was activated by a mAb specific for the CD40 receptor in the absence or presence of zymosan A that stimulates phagocytosis of yeast particles. Based on these results, LLR-J24 mRNA downregulation is specific in cultures treated with CD40 ligation combined with potent inducers of maturation such as TNF-α and IL-1β or phagocytosis stimuli. It is expressed in Since MDC chemokine (CCL22) is known to be strongly induced after maturation in DC, the same Northern blot was used as a specific probe for MDC to confirm the successful activation of MoDC or HPDC. And hybridized. As seen in FIG. 8, the strong induction of MDC parallels the downregulation of the LLR-J24 transcript in both types of DC.

  Since LLR-J24-1 lacks the stalk exon region, the presence of the stalk exon in the LLR-J24 transcript from DC was examined. Northern blot analysis failed to distinguish transcripts that were expected to differ by only 141 bases. Therefore, reverse transcription PCR was performed using a primer pair to amplify the fragment between the second and fourth exons. Two products of 505 bp and 367 bp were obtained, corresponding to the size of the expected fragment with and without stalk exons, respectively (FIGS. 9 and 10). It is estimated that about 5-10% of the product is sized with stalk exons.

  This upper band was scraped from the gel and subcloned, and the two resulting clones were sequenced. Both clones displayed correctly spliced stalk exons as expected from the genomic sequence. The PCR fragments obtained with the primer pairs were further analyzed, which amplify mRNA fragments from the 5′-untranslated region to the stalk exon and from the stalk exon to the sixth exon, respectively. In both cases, the main product obtained corresponded to the expected size for a correctly spliced exon. These data indicate that the LLR-J24 transcript contains a stalk exon and is correctly spliced at other exon locations.

  Type II lectin-like receptors have usually been shown to form dimers and either homodimers or heterodimers that combine two different receptor chains. In certain cases, dimer formation with heterologous lectin-like receptor chains has been shown to be necessary for surface expression. This is true, for example, for the NKG2 / CD94 receptor (Carretero M. et al. Eur J Immunol. 27 [1997] 563-567). Therefore, surface expression of FLAG-tagged recombinant LLR-J24 in 293 cells and HUVEC was evaluated after transient transfection with each expression construct. To assess surface expression, staining of non-permeable cells with anti-FLAG antibody was compared to parallel with a sample of cells permeated with Triton X-100 prior to the staining procedure. For LLR-J24, a construct with and without a stalk exon was used.

  Full-length LLR-J24 accumulated efficiently on the cell surface of HUVEC as shown by intense staining of non-permeating cells (FIG. 6A). In contrast, no detectable level of LLR-J24 isoform without a stalk was observed on non-permeant cells, but significant intracellular staining of permeate cells was obtained. Through more precise analysis of permeabilized cells at higher magnification, cells transfected with the full-length LLR-J24-2 expression construct can be observed for perinuclear cell components as well as surface cells with strong surface expression staining (FIG. 6B). LLR-J24-1 without stalk is observed to be uniformly distributed throughout the cytoplasm but not on the surface. Similar results were obtained for 293 cells (data not shown).

In a further aspect, the present invention provides:
An isolated polynucleotide encoding the transmembrane protein LLR-J24;
An isolated polynucleotide encoding the extracellular domain of LLR-J24;
An isolated polypeptide that is an extracellular domain of LLR-J24; and an isolated polypeptide that is a transmembrane protein LLR-J24.

The present invention also provides the use of the gene of the present invention as a diagnostic reagent.
Detection of mutant forms of the gene of the invention associated with dysfunction can be, for example, under-expressed, over-expressed, or modified of the corresponding gene or variant thereof and / or the polypeptide of the invention. A diagnostic tool is provided, eg, in a diagnostic assay, that can define the diagnosis of a disease due to differential expression or the susceptability of the disease. Individuals with mutations in the corresponding gene can be detected at the DNA level according to conventional methods.

  Nucleic acids for diagnosis can be obtained from cells of interest, such as blood, urine, saliva, cytological tissue, or autopsy samples. Genomic DNA can be used directly for detection or can be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA can also be used in the analysis. Deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled gene nucleotide sequences of the invention.

  Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting points. DNA sequence differences may also be due to changes in electrophoretic mobility of DNA fragments in gels, with or without denaturing reagents, or according to eg Myers et al. Science 230 (1985) 1242. It can be detected by direct DNA sequencing. Sequence changes at specific positions can also be determined by nuclease protection assays, such as RNase and S1 protection methods, or chemical cleavage methods such as Cotton et al. Proc. Natl. Acad. Sci. USA 85 (1985). It can be revealed according to 4397-4401. An array of oligonucleotide probes comprising the gene nucleotide sequences of the invention or fragments thereof can be configured to perform effective screening, eg, gene mutations. Array technology methods can be used to address various problems in molecular genetics, including gene expression, gene binding, and gene variability, for example according to M. Chee et al. Science 274 (1996) 610-613 .

  In the case of the IKK2-55 gene of the present invention, diagnostic assays can detect diseases such as inflammation, arteriosclerosis, restenosis, reperfusion injury, inflammatory bowel disease, psoriasis, atopy through detection of mutations in the gene of the present invention. Methods for diagnosing or measuring the susceptibility of, for example, immune or inflammatory diseases, including atopic dermatitis, transplant rejection, autoimmune diseases, allergies and cancer.

  In the case of the DINO gene of the present invention, diagnostic assays can be used to determine the susceptibility of diseases including, for example, inflammatory diseases such as acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection, cancer, atherosclerosis and restenosis. Provide a method for diagnosing or measuring.

  In the case of the LLR-J24 gene of the present invention, diagnostic assays may include inflammatory skin diseases such as chronic inflammatory diseases, autoimmune diseases, transplant rejection, contact hypersensitivity and atopic dermatitis, or virus-induced immunity. Provide a method for diagnosing or measuring the susceptibility of diseases associated with inhibition, eg, disproportionate, increased or decreased DC function, including AIDS, eg, susceptibility of all forms of viruses, bacteria, parasitic infections and cancer To do.

  The disease is diagnosed, for example, according to conventional methods, for example, by a method comprising measuring abnormally reduced or increased levels of a gene or polypeptide or gene mRNA of the invention from a sample from a subject. obtain. Decreased or increased expression is measured at the RNA level, e.g. according to conventional methods for quantification of polynucleotides, e.g. according to PCR, RT-PCR, RNase protection methods, Northern blotting and other hybridization methods. obtain. Assay techniques that can be used to measure levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host can be performed, for example, according to conventional methods. Such assay techniques include, for example, radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays.

Accordingly, in a further aspect, the present invention provides a diagnostic kit for a disease or disease susceptibility, such as those described above;
a) a gene of the invention (eg including an allelic variant thereof, or a fragment thereof, or a splice variant);
b) a sequence complementary to the nucleotide sequence of (a);
c) a polypeptide of the invention (eg comprising a polypeptide with an amino acid sequence having at least 80% identity thereto, eg, a fragment or variant of the polypeptide of the invention, or at least 80 with the polypeptide of the invention) A polypeptide fragment or variant of the amino acid sequence having% identity); or
d) an antibody against the polypeptide of the present invention;
Providing a kit comprising
For example, any of the kits (a), (b), (c), or (d) can be used, for example, in the appropriate environment of the sample to be tested, eg, a), b), c in the sample to be tested. ) Or d) may include essential components including methods suitable for measuring the effect of either.

  The gene of the present invention is also useful for chromosome identification. The sequences are specifically targeted and can hybridize at specific locations on individual human chromosomes. Mapping of related sequences of the present invention to chromosomes is an important first step in correlating those sequences with gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the natural position of the sequence on the chromosome can be correlated to genetic map data. Corresponding data is disclosed, for example, in V. McKusick Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library). The relationship between a gene mapped to the same chromosomal region and the disease can be identified by linkage analysis (simultaneous inheritance of physically adjacent genes). Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be measured. If the mutation is observed in some or all affected individuals, but not at all in normal individuals, the mutation may be responsible for causing the disease.

  Thus, for example, IKK2-55 is located on chromosome 12q23.1 in close proximity to the long form of APAF1 (apoptotic protease activator 1) and the LLR-J24 gene of the present invention is an NK receptor gene complex. Used for chromosome identification on the short arm of the body's chromosome 12; viral disease and arthritis susceptibility have been mapped to similar synthetic regions in rodents.

  The polypeptide of the present invention or a fragment thereof, or a cell expressing such a polypeptide of the present invention or a fragment thereof is also used as an immunogen for producing an antibody immunospecific for the polypeptide of the present invention. Can be used. The term “immunospecific” means that the antibody has a substantially greater affinity for a polypeptide of the invention than its affinity for another related or unrelated polypeptide. To do.

  An antibody raised against a polypeptide of the invention can be administered, for example, by administering such a polypeptide, or epitope-bearing fragment-analog or cell, to an animal, preferably a non-human, using conventional procedures. Can be obtained. For the preparation of monoclonal antibodies, suitable techniques for providing antibodies, such as those produced by continuous cell line culture, such as hybridoma technology (Kohler, G. and C. Milstein Nature 256 [1975 495-497), trioma technology, human B cell hybridoma technology (Kozbor et al. Immunology Today 4 [1983] 72) and EBV-hybridoma technology (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. [1985] 77-96) can be used.

  Also, techniques for producing single chain antibodies (eg, US Pat. No. 4,946,778) can be applied to produce single chain antibodies to the polypeptides of the invention. In addition, other organisms including transgenic mice or other mammals can be used to express humanized antibodies. The above antibodies can be used, for example, in the isolation or identification of clones expressing the polypeptide of the invention, or for the purification of the polypeptide (fragment) of the invention by affinity chromatography.

  The antibodies against the polypeptides of the present invention may also be used in the treatment of diseases, for example in the case of antibodies to the DINO gene, inflammatory diseases such as acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection, cancer, atherosclerosis. In diseases such as arteriosclerosis and restenosis; or in the case of antibodies to the LLR-J24 gene, chronic inflammatory diseases, autoimmunity, including for example inflammatory skin diseases such as contact hypersensitivity, atopic dermatitis It may be useful in disease, transplant rejection or in any form of susceptibility to virus-induced immunosuppression, eg AIDS, eg viruses, bacteria, parasitic infection diseases and cancer.

In a further aspect, the present invention provides antibodies against the polypeptides of the present invention. The antibodies of the present invention include LLR-J24 polypeptides and bispecific antibodies that bind to viruses, bacteria or eukaryotic parasites or cancer cells.
In a further aspect, the invention provides LLR-J24 polypeptides and bispecific antibodies that bind to viruses, bacteria or eukaryotic parasites.

In a further aspect, the present invention provides bispecific antibodies that bind to LLR-J24 polypeptides and cancer cells.
Instead of bispecific antibodies, LLR-J24 polypeptides and other bispecific reagents capable of binding to viruses, bacteria or eukaryotic parasites or cancer cells can also be used.

  In a further aspect, the present invention provides bispecific reagents that are not antibodies and have the ability to bind to LLR-J24 polypeptides and viruses, bacteria or eukaryotic parasites or cancer cells.

In a further aspect, the present invention is a method for inducing an immune response in a mammal, comprising delivering the polypeptide of the present invention via a vector, producing an antibody to protect the animal from disease Directing the expression of the corresponding gene of the present invention in vivo;
In a still further aspect, an immune / vaccine formulation (composition) that, when introduced into a mammalian host, induces an immune response against the polypeptide of the invention in the mammal, comprising the polypeptide or the present invention. A composition comprising the gene of the invention;
I will provide a.

  In a further aspect, the present invention is a method of inducing an immune response in a mammal, each comprising an antibody and / or a T cell immune response to protect the animal from a disease, eg, the disease described above. A method comprising inoculating a DINO polypeptide of the present invention or a fragment thereof, or an LLR-J24 polypeptide of the present invention or a fragment thereof, suitable for production, wherein the LLR- The J24 polypeptide is preferably the one represented by SEQ ID NO: 6 or 8 and more preferably the one represented by SEQ ID NO: 8.

  The vaccine formulation may further comprise a suitable carrier. Since the polypeptides of the present invention can be degraded in the stomach, they can preferably be administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Immuno / vaccine formulations suitable for parenteral administration include, for example, aqueous and non-aqueous sterile injections that may contain antioxidants, buffers, bacteriostats and solutes that provide isotonic formulations with the recipient's blood. Solutions; and aqueous and non-aqueous sterile suspensions that may include suspending or thickening agents. The formulations can be provided in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in lyophilized conditions requiring only the addition of a sterile liquid carrier just prior to use. Vaccine formulations may also include adjuvant systems that increase the immunogenicity of the formulation, such as oil-in-water systems and other suitable systems. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

  The polypeptides of the present invention may be involved in many biological functions, such as those underlying many pathological conditions. Thus, for example, biologically compatible, e.g., transcriptionally, activated and / or stimulates the activity of a polypeptide of the present invention, or alternatively stimulates expression of a gene of the present invention (agonist), And on the other hand, it is desirable to find compounds and drugs that can inhibit the function of the polypeptides of the invention or the expression of the genes of the invention (antagonists). Accordingly, the polypeptides of the present invention or functional mimetics thereof, such as those described in Coligan et al., Current Protocols in Immunology 1 (2) Chapter 5 (1991), for example, cells, are free of cells. It can be used to evaluate the binding of agonists or antagonists of the receptor polypeptides of the invention in formulations, chemical libraries, and natural product mixtures, eg, in screening assays.

  Agonists and antagonists of the IKK2-55 polypeptide of the present invention may include, for example, diseases including immune or inflammation related diseases such as inflammation, arteriosclerosis, restenosis, reperfusion injury, inflammatory bowel disease, psoriasis, atopic dermatitis, It can be used in the treatment of transplant rejection, autoimmune diseases, allergies and cancer.

  Agonists and antagonists of DINO polypeptides of the invention can be used, for example, in the treatment of inflammatory diseases, acute or chronic, autoimmune diseases, transplant rejection, cancer, atherosclerosis and restenosis.

  Agonists and antagonists of the LLR-J24 polypeptides of the present invention include, for example, chronic inflammatory diseases, autoimmune diseases, diseases including transplant rejection, such as inflammatory skin diseases such as contact hypersensitivity, atopic dermatitis, or virus-induced It can be used in the treatment of immunosuppression, such as AIDS, such as any form of susceptibility to viruses, bacteria, parasitic infections, and cancer.

  Stimulation or inhibition of the expression of a gene of the invention, for example by a low molecular weight compound (LMW) or an antisense oligonucleotide, preferably corresponds to the corresponding polypeptide of the invention and the physiology / function of EC or DC, respectively, and It can modulate the effect of the ligand.

  The screening procedure may involve the generation of suitable cells expressing, in the case of IKK2-55 or LLR-J24, the receptor for the polypeptide of the invention on its surface. Suitable cells include, for example, cells from mammals, yeast and Drosophila. In the case of IKK2-55, the cell expressing IKK2-55; and in the case of LLR-J24, the cell expressing the receptor (or the cell membrane containing the expressed receptor) binds or has a functional response. In order to observe the stimulation or inhibition of, a test (candidate) compound may be contacted.

  A screening assay can be used to test the binding of a candidate compound, where the cell carrying the IKK2-55 or LLR-J24 receptor, respectively, is labeled with a label that binds directly or indirectly to the candidate compound. Alternatively, it can be detected in assays involving competition with a labeled competitor. The screening assay further tests whether the candidate compound results in a signal produced by activation of the receptor, using a detection system suitable for cells carrying the IKK2-55 or LLR-J24 receptor on the surface, respectively. Can be used to Inhibitors of activation can be assayed in the presence of a known agonist and the effect on activation by the agonist can be observed in the presence of the candidate compound.

  The screening assay standardizes the step of mixing candidate compounds with a solution containing a polypeptide of the invention, measuring the activity of the polypeptide in the mixture, and the activity of the mixture to form a mixture. Comparing with the activity of the substance may be included.

  The gene (cDNA) of the present invention, the polypeptide of the present invention and the antibody against the polypeptide of the present invention are also used in screening assays for detecting the effect of candidate compounds on the production of the gene (mRNA) and the polypeptide in cells. Can be used to provide For example, an ELISA can be made according to conventional methods, eg, using monoclonal and polyclonal antibodies, to measure cell binding levels of the polypeptide, and the ELISA can be performed in appropriately treated cells or tissues. And can be used to discover agents (antagonists or agonists) that can inhibit or increase the production or activity of the polypeptide. Assays for screening can be performed, for example, according to conventional methods.

  Examples of agonist (antagonist) candidates for the gene of the present invention are oligonucleotides or proteins closely related to antibodies, or in some cases, ligands of the gene (antagonists that bind to the polypeptide of the gene). (Polypeptide), for example, a fragment of the ligand, or a small molecule that binds to the receptor but does not elicit a response, thereby interfering with the activity of the receptor.

  Examples of agonist (antagonist) candidates of the present invention include compounds that bind to a polypeptide of the present invention, including, for example, oligopeptides, polypeptides, proteins, antibodies, mimetics, small molecules such as low molecular weight (LMW) compounds. Is mentioned.

Accordingly, in a further aspect, the present invention provides a screening assay for identifying agonists or antagonists of the polypeptides of the present invention, the assay comprising as a major component:
a) a polypeptide of the invention;
b) a recombinant cell expressing the polypeptide of the invention;
c) a cell membrane expressing a polypeptide of the invention; or d) an antibody against the polypeptide of the invention;
And, for example, means for contacting the candidate compound; for example, means for measuring the effect of the candidate compound on any of a), b), c) or d);
For example, means for determining whether the production and / or biological activity of a polypeptide of the invention is diminished or increased in the presence of a candidate compound, such as in the presence and absence of a candidate compound, a means by comparing the activity of any of a), b), c) or d); and in another aspect, reducing the production and / or biological activity of the polypeptides of the invention A method of identifying an agonist or antagonist, preferably an agonist, comprising, for example, a ligand, receptor, antibody, or LMW molecule comprising:
A) a candidate compound,
a) a polypeptide of the invention;
b) a recombinant cell expressing the polypeptide of the invention;
c) a cell membrane expressing a polypeptide of the invention; or d) an antibody against the polypeptide of the invention;
Bringing into contact;
B) measuring the effect of the candidate compound on any of a), b), c) or d); for example, in the presence of the candidate compound, the production and / or biological activity of the polypeptide of the invention is Determining whether it is diminished or increased, eg, by comparing the activity of any of a), b), c) or d) in the presence and absence of the candidate compound; and C) selecting the agonist or antagonist measured in step B)
For example, selecting appropriate candidate compounds whose agonist / antagonist effect is positively measured in step B),
Comprising that.

  In any such screening assay, a), b), c) or d) may comprise essential components. Candidate compounds include compounds (libraries) whose effects on any of a), b), c) or d) are unknown. The compound (library) includes the compounds shown above as agonists (antagonists) for the polypeptides of the present invention. An agonist (antagonist) is a candidate compound that is found to have an effect on any of a), b), c) or d) in a screening assay or in a method for identifying an agonist (antagonist) as described above. is there. An agonist (antagonist) may reduce or increase the production and / or biological activity of the polypeptides of the invention.

In a further aspect, the invention provides an antagonist or agonist, preferably an antagonist, of the polypeptide of the invention, wherein the antagonist or agonist comprises the following steps:
A) a candidate compound,
a) a polypeptide of the invention;
b) a recombinant cell expressing the polypeptide of the invention;
c) a cell membrane expressing a polypeptide of the invention; or d) an antibody against the polypeptide of the invention;
Contacting with;
B) determining the effect of the candidate compound on any of a), b), c) or d); for example, production and / or biological activity of a polypeptide of the invention in the presence of the candidate compound Measuring, for example, by reducing the activity of any of a), b), c) or d) in the presence and absence of the candidate compound; And C) selecting the agonist or antagonist measured in step B),
For example, selecting appropriate candidate compounds whose agonist / antagonist effect is positively measured in step B),
It can be provided by.

  An agonist (antagonist) of IKK2-55 (polypeptide) of the present invention is used for example in immune or inflammation-related diseases such as inflammation, arteriosclerosis, restenosis, reperfusion injury, inflammatory bowel disease, psoriasis, atopic skin It can be used in the treatment of diseases including inflammation, transplant rejection, autoimmune diseases, allergies and cancer.

  An agonist (antagonist) of DINO (polypeptide) of the present invention may have immunomodulatory activity, and for example, inflammatory diseases such as acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection, cancer, atheroma It can be used in the treatment of diseases including atherosclerosis and restenosis.

  An agonist (antagonist) of the LLR-J24 (polypeptide) of the present invention may have immunomodulatory activity and, for example, chronic inflammatory disease, autoimmune disease, transplant rejection, eg inflammatory skin disease, eg contact It can be used in the treatment of diseases including hypersensitivity, atopic dermatitis, or virus-induced immunosuppression, such as AIDS, eg, all forms of susceptibility to viruses, bacteria, parasitic infections and cancer. Therefore, the agonist (antagonist) of the polypeptide of the present invention may be useful as a medicament.

Several methods are available for such use:
If the activity of the gene and / or polypeptide of the invention is excessive, one method is to block the binding of the ligand to the polypeptide or by inhibiting a second signal, the gene of the invention And / or administering to the subject an antagonist of a polypeptide of the invention in an amount effective to inhibit activation of the polypeptide, eg, in combination with a pharmaceutically acceptable excipient, and, as a result, For example, alleviating abnormal conditions caused by over-, under- or altered expression of the gene (or variants thereof).

  In another method, for example in the case of DINO or LLR-J24, a soluble form of the corresponding polypeptide of the present invention capable of binding to the ligand competitively with the endogenous polypeptide can be administered. Exemplary embodiments of such competitors can include fragments of the polypeptide of the invention.

  In a further aspect, the present invention provides soluble LLR-J24 polypeptides of the present invention that can bind to a ligand on T cells or other immune cells and activate immune function and eg allergies.

  In yet a further method, the present invention includes inhibition of expression of a gene encoding an endogenous polypeptide of the present invention using an expression blocking technique.

  Known such techniques include those produced internally according to, for example, O'Connor, J. Neurochem. 56 (1991) 560, Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Or the use of antisense sequences administered separately. Alternatively, oligomers, such as oligonucleotides that form a triplex with the gene of the present invention, are described in, for example, Lee et al. Nucleic Acids Res. 6 (1979) 3073; Cooney et al. Science 241 (1988) 456; Dervan et al. Science 251 (1991) 1360. Such oligomers can be administered as such or expressed in vivo.

  For the treatment of underexpression of the polypeptide of the present invention and abnormal conditions related to its activity, one method is to therapeutically activate the gene of the present invention in a subject in need of increasing the expression of the polypeptide of the present invention. An effective amount of a compound (agonist) is administered, for example, in combination with a pharmaceutically acceptable excipient, thereby alleviating the abnormal condition.

  Alternatively, gene therapy can be performed and the endogenous production of the polypeptides of the invention can be influenced by the relevant cells in the subject. For example, the genes of the present invention can be engineered for expression in a replication defective retroviral vector, eg, according to the methods described above. The resulting retroviral expression construct can be isolated and introduced into packaging cells transformed with a retroviral plasmid vector containing RNA encoding a polypeptide corresponding to the gene of the invention, As a result, the packaging cell produces infectious viral particles containing the gene of interest. These producer cells can be administered to a subject to manipulate the cells in vivo and to express the polypeptide in vivo. For an overview of gene therapy, see, for example, Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches (and references cited therein) in Human Molecular Genetics, T Strachan and AP Read, BIOS Scientific Publishers Ltd. (1996).

In another aspect, the present invention provides an agonist or antagonist, preferably an antagonist, of the polypeptide of the present invention for use as a medicament, eg for the treatment or prevention of the diseases indicated above, and In terms of:
-The use of an agonist or antagonist of the invention in the manufacture of a medicament for the treatment or prevention of the diseases indicated above; and-for use as a medicament, for example, IKK2-55 or LLR-J24, respectively, Soluble forms of the IKK2-55 polypeptide or LLR-J24 polypeptide of the present invention are provided, respectively, for the treatment of the same disease for which an agonist (antagonist) is appropriate.

  The (ant) antagonists of the polypeptides of the invention may be administered in the form of a pharmaceutical composition.

In another aspect, the present invention comprises an agonist or antagonist of the present invention as an active ingredient, preferably an antagonist, in combination with a pharmaceutically acceptable excipient (s) / carrier (s). For example, the antagonist or agonist comprises the following steps:
A) a candidate compound,
a) a polypeptide of the invention;
b) a recombinant cell expressing the polypeptide of the invention;
c) a cell membrane expressing a polypeptide of the invention; or d) an antibody against the polypeptide of the invention;
Contacting,
B) measuring the effect of the candidate compound on any of a), b), c) or d); for example, in the presence of the candidate compound, the production and / or biological activity of the polypeptide of the invention is Measuring whether it decreases or increases; for example, by comparing the activity of any of a), b), c) or d) in the presence and absence of a candidate compound; and C) selecting an agonist or antagonist measured in step B); for example selecting an appropriate candidate compound whose agonist / antagonist effect is positively measured in step B);
The pharmaceutical composition which can be provided by is provided.

  Such a pharmaceutical composition suitably comprises the agonist (antagonist) provided by eg steps A), B) and C), eg according to conventional methods, excipient (s) / carrier (s) And the like, and further processing the resulting mixture to obtain a pharmaceutical composition for appropriate administration.

  In a further aspect, the present invention relates to both an excess or insufficient level, preferably excess, of the gene of the invention; or an excess and insufficient, preferably excess, of a polypeptide of the invention, Methods of treating abnormal conditions related to activity are provided. For example, in the case of IKK2-55, immune or inflammation related diseases such as inflammation, arteriosclerosis, restenosis, reperfusion injury, inflammatory bowel disease, psoriasis, atopic dermatitis, transplant rejection, autoimmune disease, allergy And methods of treating diseases including cancer; for example, in the case of DINO, diseases including inflammatory diseases such as acute or chronic inflammatory diseases, autoimmune diseases, transplant rejection, cancer, atherosclerosis and restenosis In the case of LLR-J24, chronic inflammatory disease, autoimmune disease, transplant rejection, eg inflammatory skin disease, eg contact hypersensitivity, atopic dermatitis, or virus-induced immunosuppression, eg AIDS Provided are methods for treating diseases including all forms of susceptibility to, for example, viruses, bacteria, parasitic infection diseases and cancerThe method comprises, for example, a therapeutically effective amount of an agonist or antagonist of the present invention, which can be provided by the steps of A), B) or C) indicated above, for example, a pharmaceutically acceptable excipient ( Administration in combination with carrier (s); for example, in the case of IKK2-55 or LLR-J24, respectively, in a therapeutically effective amount of each soluble form, IKK2-55 or LLR-J24, a polypeptide of the invention, eg, in combination with a pharmaceutically acceptable excipient (s) / carrier (s); a subject in need of such treatment Administering.

  In another aspect, the present invention is a method of treating an infection or cancer that can increase antigen uptake and T cell presentation and activation by DCs in a subject in need of such treatment. Methods are provided by administering an effective amount of a bispecific antibody or reagent of the invention.

  In another aspect, the invention provides a method of treating an infection or cancer wherein a subject in need of such treatment is administered an effective amount of a soluble LLR-J24 protein capable of activating immune function. To provide a method comprising:

  IKK2-55 is an intracellular protein. Suitable forms for systemic administration of the pharmaceutical composition of the present invention include injection, typically intravenous injection. Other injection routes such as subcutaneous injection, intramuscular injection, or intraperitoneal injection may also be used. Other means for systemic administration include transmucosal and transdermal administration using, for example, penetrants such as bile salts or fusidic acid or other detergents. Furthermore, oral administration may also be possible if properly formulated with enteric or capsule formulations. Administration of the composition of the invention can also be topical and / or localized, eg in the form of a cream, paste, gel, etc.

  The required dosage range may depend on the polypeptide of the invention, or agonist or antagonist, route of administration, nature of the pharmaceutical composition, nature of the patient's condition, and the judgment of the physician examined. However, suitable dosages can range from about 0.1 mg / kg to about 10 mg / kg, such as from about 0.1 mg / kg to about 1 mg / kg (subject weight). However, the required dosage variation can be expected considering the variety of compounds utilized and the different efficiencies of the various routes of administration. For example, oral administration is expected to require a higher dose than administration by intravenous injection. These dosage changes can be adjusted, for example, according to standard rules of thumb for optimization.

  Polypeptides used for treatment can also be produced endogenously, eg, in a subject in need of such treatment, eg in a treatment modality often described as “gene therapy” as described above. Thus, for example, cells from a subject can be manipulated ex vivo with a polynucleotide, such as DNA or RNA, that encodes an IKK2-55 polypeptide, eg, by using a retroviral plasmid vector. Engineered cells can be introduced into a subject in need of such treatment, for example.

  In accordance with the present invention, there is provided a genetically modified DC that expresses an LLR-J24 construct that can improve antigen uptake and presentation, such as improved T cell and immune system activation. obtain.

  In another aspect, the invention is genetically modified to express an LLR-J24 construct that can improve antigen uptake and presentation, such as improved T cell and immune system activation. Provide dendritic cells.

An antibody, ligand or reagent that binds to an LLR-J24 polypeptide can inhibit DC formation / maturation and, as a result, can improve immune function and allergies, for example.
In another aspect, the invention provides an antibody, ligand or reagent that binds to an LLR-J24 polypeptide of the invention that inhibits DC formation / maturation.

Figure 1 (IKK2-55)
Interaction of IKK2 CoIP with IKK2-55. Flag-tagged IKK2-55 and myc-tagged IKK2 were transfected into Hela cells as indicated. Lysates were prepared and IKK2-55 was precipitated with anti-Flag antibody, and the precipitate was analyzed by Western blotting with anti-Myc antibody (upper panel). The presence of Flag-IKK2-55 and myc-IKK2 in the cell extract was confirmed by Western blotting (lower panel).

Figure 2 (IKK2-55)
Subcellular localization of IKK2-55. HUVEC were transfected with the YFP-IKK2-55 fusion construct along with the CFP fusion gene for the different cell compartments and observed under a fluorescence microscope. The left column shows the localization of IKK2-55 in the yellow channel, and the right column shows the localization of the marker in the cyan channel. Upper panel: endoplasmic reticulum (ER); middle panel: mitochondria (lower); lower panel: Golgi apparatus.

Figure 3 (IKK2-55)
Effect of IKK2-55 on NF-kB dependent promoter. HUVECs were transfected with 5xNF-kB-Luc construct with different amounts of IKK2-55 and ubiquitin-bgal as internal standard and stimulated with 100 U / ml TNFα for 6 hours after 2 days. Luciferase values (relative light intensity units) were normalized for b-gal expression.
prom = promoter; unstim = unstimulated; stim = stimulated.

Figure 4 (IKK2-55)
Effect of IKK2-55 (in combination with different kinases) on the NF-kB dependent promoter. HUVECs were transfected with the 5xNF-kB-Luc construct, along with IKK2-55, with the different kinases indicated (IKK2, NIK, TAK1, p38) as well as ubiquitin-bgal as an internal standard. In one case, cells were stimulated with 100 U / ml TNFα for 6 hours. Luciferase values were expressed as relative luminosity units and normalized for β-gal expression.
Dark column: 500 ng IKK2-55; shaded column: 0 ng IKK2-55
RLU-βgal: Relative luminosity unit per β-gal.

FIG. 5 (LLR-J24)
A phylogenetic tree containing all known lectin-like genes of the human NK receptor complex:
The CTLD sequences of the represented genes were aligned and a phylogenetic tree was generated as described in the examples. All sequences were chosen to start with the amino acid two before the first cysteine that is always conserved in the domain. NKG2E and H are two separately spliced transcripts from the same gene. For LY49L, a hypothetical sequence was selected that contained all three CTLD exons that were spliced in a manner comparable to other genes, but only aberrantly spliced transcripts were detected for this gene. This gene was found to be more closely related to each other consistently with different parameter settings for alignment, and the tree structure is indicated by the right parenthesis. The lower scale is a measure of replacement events. The selective expression of subfamilies in NK cells, T cells, endothelial cells (EC), monocytes (MO), dendritic cells (DC) or more broadly in leukocytes (LEUK) is shown.

6 and 7 (LLR-J24)
Expression of recombinant LOX-1, LLR-J24 and CLEC-1 in HUVEC:
C-terminally FLAG-tagged LOX-1, LLR-J24 and CLEC-1 expression plasmids were transfected into HUVEC. Protein expression was analyzed 24 hours after infection after staining with anti-FLAG antibody and secondary Texas Red-labeled antibody.

  Comparison of non-permeating and permeating cell staining. In the left column, non-permeating cells fixed with formaldehyde (fixed) and in the right column osmotic cells (fixed / permeated) are shown. The upper panel shows staining with anti-FLAG antibody, and the lower panel shows staining of DNA with Hoechst 333258 to visualize the total cell number.

  The LLR-J24 construct without the stalk exon is shown in FIG. 2B (ΔLLR-J24-1). Comparison of osmotic cell staining for all four expression constructs at higher magnification.

FIG. 8 (LLR-J24)
Downregulation of LLR-J24 mRNA during dendritic cell maturation:
FIG. 8A:
Northern blot of total RNA isolated from immature MoDC and cultures stimulated with TNF-α and IL-β for 18 hours. As a control, hybridization of LLR-J24 cDNA and β-actin probe is shown.

FIG. 8B:
Northern blots with isolated RNA from immature MoDC and HPDC, from MoDC and HPDC treated with anti-CD40 antibody, or from MoDC treated with a combination of anti-CD40 antibody and zymosan A for 24 hours. The blot was successively hybridized with probes for LLR-J24, MDC and GAPDH as a loading control. The positions of 18s and 28s rRNA are indicated.

9 and 10 (LLR-J24)
The fraction of human LLR-J24 mRNA contains a stalk exon sequence:

  RT-PCR of LLR-J24 mRNA. RNA from MoDC was reverse transcribed with specific LLR-J24 primers and PCR was performed on the resulting cDNA with the indicated different primer pairs. (a) shows amplification of transcripts with and without stalk exons for MoDC RNA, (b) shows 5′-part amplification, and (c) shows B in FIG. 4A. The 3′-part of the stalk containing transcripts for MoDC and HL60 RNA shown in FIG. Lane 6 shows the product obtained from the plasmid containing the full length LLR-J24 cDNA.

Schematic of oligonucleotide primers for use in RT-PCR. The first primer pair (a) was designed to test for the presence of a stal exon fragment that resulted in the expected 505 bp and 367 bp PCR products with and without the stalk exon fragment, respectively. The second primer pair (b) tests the correct splicing of the 5′-part, and the third primer pair (c) corrects the splicing of the 3′-part of the transcript containing the stalk exon.
c = cytoplasmic; TM = transmembrane; CTLD = C-type lectin-like domain 1;
CTLD2 = C-type lectin-like domain 2; CTLD3 = C-type lectin-like domain 3.

In the examples below, all temperatures are in degrees Celsius and are not corrected.

Example 1: IKK2-55 gene A cDNA clone comprising SEQ ID NO: 1 of the IKK2-55 gene was isolated from a library of PHA-activated lymphocyte mRNA and by sequence comparison using the BLAST algorithm with the GenBank database. To do.

  The IKK2-55 gene was found in a yeast two-hybrid screen using the C-terminal HLH domain of IKK2 as a bait. The first clone comprising a C-terminal fragment was ESTs, and two additional IMAGE clones (# 1: clone ID = 2048289; GenBank accession number = AI375687 and # 2: clone ID = 2382861; GenBank accession number = AI759984) Stretch using an array. Nucleic acid and amino acid sequences are given in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The first ATG of nucleotides 58-60 is preceded by an in-frame stop codon at positions 16-18. The open reading frame extends to the stop codon at positions 1189-1191. IKK2-55 encodes a 377 amino acid protein that does not show any significant homology with database entries.

  It also does not contain the distinct protein motifs present in the Pfam and Prosite databases, except for the low composition complexity region of amino acids 45-58 and the two coiled-coil regions 116-259 and 287-323. Genomic clones that resemble human genes are present in the database (AC013283.htgs). Investigation of the human draft genome sequence in the EST clone, such as AI375687, revealed that IKK2-55 is located on chromosome 12q23.1. As a result, the IKK2-55 transcription initiation site is located within 2 kb relative to the transcription site of APAF1 (apoptotic protease activator 1), which means the regulatory crosstalk of these two genes. .

Example 2: IKK2-55 gene Y2HS provides initial information on the interaction between two proteins, but these results were confirmed by biochemical means. Expression vectors for IKK2 (with a Flag tag attached) and IKK2-55 (with a myc tag attached) are transfected into HeLa cells. After 2 days, an extract is prepared and IKK2 is precipitated with anti-Flag antibody. The precipitate is analyzed by Western blotting using an anti-myc antibody. The presence of a specific band in lane 3 of FIG. 1 suggests co-immunoprecipitation of IKK2-55 with IKK2. In the lower panel, whole cell extracts are probed for IKK2-55 and IKK2 using anti-Flag and anti-myc antibodies, respectively, to show that the cells express their respective genes.

Example 3: IKK2-55 gene Intracellular localization may provide evidence of protein function. By co-transfection of different specific markers that appear to resemble the endoplasmic reticulum, transfection of the YFP-IKK2-55 fusion gene results in localization to the perinuclear region (FIG. 2) (ER; the construct Contains the target sequence of calreticulin fused to CFP; Clontech Living Colors ER Subcellular Localization Vector).

Example 4: IKK2-55 gene (expression pattern)
MTN (Multiple Tissue Northern Blot; Clontech) was hybridized with the IKK2-55 probe and two bands of 2.4 and 4.3 kb were found in the kidney, liver, placenta, spleen and It is selectively detected in the heart.

Limited searches are found in searches of ESTs using the TIGR human gene index:

  Virtual SAGE is performed (aaaatagcc chain is selected by the program) and high expression is detected in the human breast cancer line MDA-MB-468, which is PTEN − / −. RT-PCR is performed using IKK2-55 specific primers and mRNA from stimulated and controlled EC. Increased IKK2-55 expression is found in ECs stimulated with IL-1, LPS, or VEGF.

Example 5: IKK2-55 gene Since IKK2-55 interacts with IKK2 as indicated by genetic and biochemical means, the effect of IKK2-55 on NF-kB activity was tested. An NF-kB dependent promoter-luciferase reporter construct, 5xNF-kB-Luc, containing 5 copies of NF-kB binding site is used in transient transfection of HUVEC with or without TNFα stimulation. The results are shown in FIG. 3 and depending on the amount used, IKK2-55 is shown to have either stimulatory (low, physiological amount) or inhibitory (in high amounts) activity. Using the 5xNF-kB promoter, cotransfection of IKK2-55 with kinases known to be involved in NF-kB signaling, such as IKK2, TAK1, NIK, p38, further induces expression of the reporter gene (FIG. 4).

Example 6: DINO gene (homology)
From a library of skin microvascular endothelial cell mRNA and by sequence comparison using the BLASTX algorithm with the SWISSPROT Protein database, a cDNA clone comprising SEQ ID NO: 3 of the isolated DINO gene was found to be a HERC protein (E6 binding protein C-terminal Homologous domain and RCC1 domain), in particular HERC3 (accession number D25215) and Cep1 (accession number AB027289).

  The DINO gene of SEQ ID NO: 3 has an overall homology of 37.8% to HERC3 and Cep1, but the homology is significantly higher (up to 70%) in the defined protein domain. There is a characteristic difference between DINO and Cep1 that Cep1 has an N-terminal extension of about 100 amino acids. The HECT domain of the HERC protein is involved in the transfer of ubiquitin to specific cellular target proteins that are to be degraded or processed.

Example 7: DINO gene (pathway profiling system)
The potential of DINO to induce p53 transcriptional activity can be assayed in vivo in a signaling pathway profiling system. The system is based on a cis-acting element upstream from the luciferase reporter gene. If the pathway is activated by overexpression of DINO in transfected HeLa or endothelial cells, luciferase expression can be measured. In this way, the effects of various drugs, stimuli or co-transfected genes can be studied by assaying luciferase expression.

Example 8: LLR-J24 gene (homology)
A cDNA clone comprising the LLR-J24-1 gene of SEQ ID NO: 5 is isolated from a library of DC mRNA and detected in the GenBank expression sequence tagging database (dbEST). The library is examined using short consensus sequences established from NKG2-A, -C, -D and the human Ly49 gene (Houchins JP et al. J. Exp. Med. 173 [1991] 1017-1020; Bull C et al. Genes and Immunity 1 [2000] 280-287). A cDNA clone comprising SEQ ID NO: 7 of splice variant LLR-J24-2 is isolated from monocyte-derived DC mRNA by reverse transcription PCR. The cDNA clone comprising SEQ ID NO: 5 and SEQ ID NO: 7 was found to be homologous to the CTLD receptor superfamily by sequence comparison using the BLASTX algorithm.

  To establish the relationship of LLR-J24 to other known genes of NKC, the predicted amino acid sequence of CTLD is compared. Multiple alignment of CTLD domains of all known lectin-like genes of the NK receptor complex is performed using the Clustal W method of Mac Vector 6.5 software with the following parameters: Open gap penalty -7.0, extend gap penalty for pairwise alignment -2.5, and for multiple alignment -1.0. The accession numbers of the molecules used in the alignment are NKG2A (X54867), NKG2C (X54869), NKG2E (L14542), NKG2H (AF078550), NKG2D (X54870), LOX-1 (AF035776), CLEC-1 (AF200949), CLEC-2 (AF124484), DCIR (AJ133532), AICL (X96719), LLT-1 (AF133299), CD69 (L07555), KLRF1 (AF175206), MAFA (AF081675), and NKR-P1A (U11276).

  A hypothetical LY49L (hypLy49L) CTLD sequence was obtained by combining the corresponding three exons of the gene (AF213453), but transcripts with CRD exons correctly spliced according to other CTLD genes are Has not been detected so far. The phylogenetic tree is constructed by the MEGAlign program of DNASTAR software version 3.0 according to the nearest neighbor method.

  A phylogenetic tree is constructed according to multiple alignment using the ClustalW algorithm (FIG. 5). The data obtained indicate that LLR-J24, together with LOX-1, CLEC-1 and CLEC-2, forms a more closely related NKC lectin-like receptor subfamily. The highest homology of LLR-J24 is seen with LOX-1 (44%), and CLEC-1 and CLEC-2 are in a somewhat separated relationship (37%). The CTLD NK receptor subfamily, consisting of NKG2 and CD94 NK receptors, is the next most closely related protein (28% for NKG2-E and 33% for NKG2-D). The AICL / LLT-1 / CD69 group and the KLRF1, MAFA and NKR-P1 receptors are even more separated and are shown in separate branches in the multiple alignment. The sequence of LLR-J24-1 showed high homology with the recently reported murine DECTIN-1 cDNA except that the stalk exon was lost from the human cDNA. It can be shown that the stalk exon is found to be spliced in about 5-10% transcript subfractions shown for the splice variant LLR-J24-2 in SEQ ID NO: 7 (FIGS. 9 and 10).

Comparison of the corresponding predicted full-length protein sequence of human LLR-J24-2 and mouse DECTIN-1 shows 59% identity (69% homology). High homology is conserved in the cytoplasm and in the extracellular domain including the stalk domain. Near the N-terminus of the cytoplasmic domain, an ITAM-like sequence is present in LLR-J24 as well as murine DECTIN-1. This sequence is slightly different from the classical ITAM motif as found in the CD3-γ chain or DAP12 molecule. The first tyrosine (residue 3) in the motif is the position 4 amino acids before leucine (residue 7), but this distance is the position of 3 amino acids in the classical Yx ₂ Lx _6-8 Yx ₂ L motif It is. The next residue of the motif is positioned correctly. Both proteins further exhibit a consensus site for N-glycosylation. Human and murine lectin-like domains lack most of the residues described to be involved in Ca ⁺⁺ binding presenting non-sugar ligands. The transmembrane domain does not show charged amino acids, indicating that no association with ITAM-containing adapters such as DAP molecules has occurred.

  From the homology of LLR-J24 in CTLD to other lectin-like receptors and its expression profile (see below), LLR-J24 receptor is a lipoprotein, MHC class I homolog or another transmembrane or secreted from cells or parasites. It is thought to have a proteinaceous ligand such as a sex protein.

Example 9: LLR-J24 gene isolation and sequencing:
The complete cDNA of the gene of SEQ ID NO: 5 can be obtained by any of the following methods:
a) Rapid Amplification of cDNA Ends (RACE) method can be used to obtain the 5 ′ end (see Frohman et al. Proc. Nat. Acad. Sci USA 85 [1988] 8998-9002). ). Conveniently, a specific oligonucleotide is annealed to the mRNA and used to initiate the synthesis of the cDNA strand. After degradation of the mRNA with RNase H, a poly C anchor sequence is added to the 3 ′ end of the cDNA and the resulting fragment is amplified using a nested set of antisense and anchor sequence primers. . The amplified fragment is cloned into an appropriate vector and subjected to restriction and sequence analysis.

  b) The polymerase chain reaction can be used to amplify the 5 'end of cDNA from a human cDNA library using a nested PCR sequential trial with two sets of primers. One set of antisense primers is specific to the 5 'end of the partial cDNA, and the other set of primers anneals to vector specific sequences.

To obtain LLR-J24, RT-PCR was performed according to the following protocol: First, 40 pg of LLR-J24 specific oligonucleotide primer (5'-GATCTCCATTCTAGGGATCTAC-3 ') was added to 50 mM Tris-HCl. Annealed with 3 μg total RNA in a volume of 10 μl containing (pH 8.3), 75 mM KCl and 3 mM MgCl ₂ by heating to 85 ° C. followed by cooling to 42 ° C. over 30-60 minutes. First strand synthesis was then performed with 20 μl containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 0.5 mM dNTPs, 20 mM DTT and 100 U Superscript Reverse Transcriptase (GIBCO / BRL, Rockville, MD). To do. The cDNA contained in 1 μl of the first strand reaction mixture is then used as a template in the PCR reaction according to standard procedures. The following primers are used:

  The amplified product is cloned into an appropriate vector and subjected to restriction and sequence analysis. In this way, SEQ ID NO: 5 and SEQ ID NO: 6 are obtained.

RNA isolation for cell culture, cell isolation and RT-PCR:
Cord blood progenitor cell-derived DC (CBDC): Umbilical cord blood (CB) samples were collected from healthy full-term births at maternity (Lainzer Hospital, Vienna, Austria) with informed consent of the mother. Mononuclear cells (MNC) are isolated within 10 hours of collection by Lymphoprep® (Nycomed Pharma AS, Oslo, Norway) density gradient centrifugation. CD34 ⁺ cells are isolated from CB-MNC by positive immunomagnetic cell sorting using the MACS CD34 + cell isolation kit from Mittenyi (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The purity of isolated CD34 + cells is in the range of 95-99%.

Immunomagnetically purified CD34 ⁺ progenitor cells were seeded in 6-well plates (Costar, Cambridge, MA, USA) at an initial cell density of 5 × 10 ⁴ cells / ml and 10% fetal bovine Culture in RPMI 1640 medium (Life Technologies Ltd., Paisley, UK) supplemented with serum (FCS), 2 mM glutamine and 50 μg / ml streptomycin and penicillin, respectively. The CM is further supplemented with SCF (50 U / ml) and Flt3-L (25 U / ml), both purchased from R & D Systems Inc., Minneapolis, MN, USA. On day 6 the cell culture was split to obtain a cell density of 2 × 10 ⁵ cells / ml and SCF (25 U / ml), Flt3-L (12.5 U / ml), GM Add CM containing CSF (300 U / ml, Novartis, Basel, Switzerland), TNFα (50 U / ml; R & D) and TGFβ (5 ng / ml; R & D).

Monocyte-derived DC (MoDC):
Human monocytes obtained by countercurrent elutriation were supplemented with GM-CSF (300 U / ml) and rIL-4 (200 U / ml) in a Costar 6-well plate (Costar, Cambridge, MA, USA). Seed at 8 × 10 ⁵ cells / ml. Cultures were cultured on day 3 by changing half of the medium for new CM supplemented with GM-CSF and IL-4 to achieve the final concentrations shown above. Is done.

Stimulation of T cells, B cells, monocytes or DCs:
Human T cells, B cells and monocytic cells obtained with countercurrent washed T cells or B cells are cultured in CM and respectively PHA (10 μg / ml, Pharmacia Biotech, Uppsala, Sweden) or PWM ( Stimulate for 24 hours in the presence of 10 μg / ml, Sigma, St. Louis, MO, USA) and rhIL-4 (100 U / ml, Novartis Pharma, Basel, Switzerland). Monocyte, MoDC or HPDC added at 100 ng / ml LPS (Sigma) or 2 μg / ml of F (ab ′) 2-fragment of goat-anti-mouse IgG (Pierce Chemical Corp, Rockford, IL, USA) In the presence of 4 μg / ml anti-CD40 mAb (mAb clone 626.2) further crosslinked in solution at 37 ° C. for 6 hours. In some cases, zymosan A (25 μg / ml, Sigma) is used in conjunction with anti-CD40 mAb cross-linked by F (ab ′) 2-fragment of goat-anti-mouse IgG. Instead, terminal maturation of MoDC was achieved by incubation in the presence of inflammatory cytokines such as TNFα (200 U / ml) and IL-1β (100 U / ml) as shown in the “Brief Description of the Drawings”. Induce for 18-24 hours.

RNA isolation:
Total cellular RNA is isolated from cells after cell lysis in TRIzol reagent (GIBCO / BRL, Rockville, MD, USA), extraction of DNA and proteins with chloroform, and precipitation of RNA with isopropanol.

Example 10: LLR-J24 gene expression in mammalian cells:
The receptors for the genes shown in SEQ ID NO: 5 and SEQ ID NO: 6 are expressed in either human embryonic kidney 293 (HEK293) cells or adherent CCL39 or dhfr CHO cells, and the LLR-J24 gene of the present invention is It is expressed in either human embryonic kidney 293 (HEK293) cells or adherent CCL39 or dhfr CHO cells or recombinant baculovirus infected Sf9 cells. Expression vectors typically contain strong promoters, such as coding regions without the 5 ′ and 3 ′ UTRs downstream of the CMV-IE and Koscak sequences and antibiotic resistance genes such as neomycin or zeocin.

  Cells are transfected with individual receptor cDNAs by lipofectin and selected in the presence of 600 mg / ml G418 (neomycin). After 3 weeks of selection, individual clones are picked and expanded for further analysis. HEK293 or CHO cells transfected with the vector alone serve as a negative control. To isolate cell lines that stably express individual receptors, approximately 96 clones are typically selected and analyzed by RT-PCR analysis. Receptor mRNA is generally detectable in about 50% of the analyzed G418-resistant clones. Recombinant baculovirus for expression of the gene is generated, selected, purified and propagated using standard techniques.

  To test the expression of LLR-J24 on the cell surface or in the intracellular fraction, the expression construct was obtained in a modified pcDNA3.1 / HisA vector (Invitrogen, Groningen, Netherlands) that provides a cytomegalovirus enhancer / promoter. And tested in transient transfection. The vector is modified by insertion of an oligonucleotide containing the sequence encoding the Flag peptide. In the pcDNA3.1 / N-Flag modified vector, the Flag coding sequence is inserted between the HindIII and BamHI sites of the multiple cloning site, and in the pcDNA3.1 / C-Flag modified vector, between the XhoI and ApaI sites. insert.

  Two differently modified vectors are used to provide an N-terminal or C-terminal Flag epitope for the protein by cloning each cDNA into the corresponding restriction cleavage site before or after the Flag sequence, respectively. Can be done. The coding regions of all three genes are then ligated with EcoRI and NotI cleavage sites for insertion into the pcDNA3.1 / N-Flag vector or EcoRI and XhoI cleavage for insertion into the pcDNA3.1 / C-Flag vector. Propagated from cDNA by PCR with specific oligonucleotides providing sites. PCR, restriction enzyme digestion, fragment purification, and ligation to vectors are performed according to established procedures. All constructs are verified by complete sequencing of the inserted DNA fragment.

  Twenty-four hours prior to transfection, seeding 293 cells and HUVEC in 6-well tissue culture plates reaches 80-90% confluence the next morning. 293 cells are transfected by the calcium phosphate method using a mammalian transfection kit (Stratagene, La Jolla, CA, USA). Transient transfection of HUVEC cells is performed by using Lipofectamine ™ reagent (GIBCO / BRL, Rockville, MD).

  The immunofluorescence assay is performed primarily as described below: 24 hours after transfection, cells transfected with the appropriate expression plasmid are washed twice with PBS, 3.7% formaldehyde, 2% sucrose. Fix with (anti-Flag M2 monoclonal antibody, Sigma, St. Louis, MO, USA) for 10 minutes at room temperature, dilute in PBS, 1% BSA and incubate the cells for 1 hour at room temperature. Cells are washed 3 times with PBS for 5 minutes, incubated with Texas Red-labeled donkey anti-mouse IgG (Accurate Chemical, Westbury, NY, USA) for 1 hour at room temperature and washed again with PBS. To visualize cell nuclei, a fluorescent groove binding probe for DNA (Hoechst 333258, Sigma Chemicals, St. Louis, MO, USA) is added to the secondary antibody solution at 500 ng / ml. Results are analyzed on a Nikon Diaphot TMD fluorescence microscope (Nikon Ltd.) connected to a cooled charge coupled device camera (Kappa Gmbh, Gleichen, Germany).

Recombinant expressed LLR-J24-2 is found on the cell surface, while LLR-J24-1 accumulates in the cell:
Type II lectin-like receptors are usually shown to form dimers and heterodimers that combine homodimers or two different receptor chains. In certain cases, dimer formation with heterologous lectin-like receptor chains has been shown to be necessary for surface expression. This is true, for example, for the NKG2 / CD94 receptor. Therefore, after transient transfection with each expression construct, surface expression of FLAG-tagged recombinant LLR-J24 was evaluated in comparison to CLEC-1 and LOX-1 in 293 cells and HUVEC. To assess surface expression, staining of non-permeable cells with anti-FLAG antibody was compared to a parallel sample of cells permeated with Triton X-100 prior to the staining procedure.

  Constructs with LLR-J24-2 and LLR-J24-1 (with or without stalk exons) were used. LOX-1 and full-length LLR-J24 effectively accumulated on the cell surface of HUVEC as shown by intense staining of non-permeable cells (FIG. 6). In contrast, detectable levels of the LLR-J24-1 isoform without the stalk and the full-length CLEC-1 protein are not observed on non-permeable cells, but yield significant intracellular staining of osmotic cells. It is done. With more precise analysis of permeabilized cells at higher magnification, cells transfected with LOX-1 and LLR-J24-2 expression constructs can be observed with strong staining of perinuclear cell components and across whole cells It was revealed to show surface expression (FIG. 7). LLR-J24-1 without stalk is observed to be uniformly distributed throughout the cytoplasm but not on the surface. Similar results are obtained for 293 cells.

  Therefore, these data indicate that the stalk sequence shown in SEQ ID NO: 10 is required for surface expression of LLR-J24. The reason is probably that the sequence contains a signal for transport to the surface and can promote homodimer formation. These data indicate that LLR-J24-2 functions as a surface receptor, while LLR-J24-1 may have an intracellular function.

Example 11: LLR-J24 gene (preparation of recombinant cells and recombinant proteins)
Based on previous experience with NK receptors and in the preparation of recombinant lectin-like receptors, expression constructs can be prepared for expression of full-length transmembrane receptors as well as those that are soluble. Stably transfected cells with full-length form of recombinant expression are used for the production of monoclonal antibodies and for functional studies. Soluble receptors can be obtained more easily and in larger quantities in purified form and are therefore used for immunization and for ligand identification and isolation. In the early stages of the project, no antibody is available to monitor expression, so constructs that supply tags to the N- and C-termini of the molecule can be used.

  For full-length expression, constructs without and with a FLAG-tag in a pCDNA vector (InVitrogen) were prepared. These constructs are used to stably transfect two different cell lines. Stably transfected cells can be used to immunize mice to obtain monoclonal antibodies. A hybrid cell line between murine hybridoma and human DC can then be used. Such hybrid cells do not express endogenous LLR-J24 and can therefore be candidates for functional studies with stable transfectants due to partial dendritic cellular properties from humans.

  In addition, the construct can be designed to prepare larger quantities of soluble LLR-J24 receptor using cDNA encoding the extracellular portion fused to a signal sequence for export to the outside of the cell. For this purpose, the baculovirus system available from InVitrogen can be used. Based on published data, such proteins also have the ability to bind appropriately to the NK receptor HLA-E ligand. An amount of mg of protein is produced, which is advantageous for immunization, as well as for isolation experiments and experiments of expressed clonal latent ligands from cell types that interact with DCs such as T cells. Instead, immunoglobulin fusion proteins are produced in COS cells that have an extracellular domain fused to the C-terminus of the immunoglobulin moiety. This is because the receptor has a type II orientation and therefore may require a free carboxy terminus for proper conjugation.

Example 12: LLR-J24 gene (production of polyclonal and monoclonal antibodies)
Since antibodies against lectin-like receptors can be difficult to produce, synthetic peptides from the C-terminus (last 15 amino acids) of LLR-J24 are used. This is because this is a method for obtaining the function of a polyclonal rabbit antibody in a Western blot which serves to monitor the expression of the recombinant. These antibodies can also be affinity purified with immunizing peptides and used to study cell expression in FACS analysis. In addition, soluble recombinant receptors are used for immunization of rabbits and for the preparation of affinity purified antibodies. This provides an antibody useful for measuring receptor surface expression by flow cytometry and may also exert a neutralizing effect on receptor-ligand interactions.

  In addition, monoclonal antibodies can be obtained to ultimately obtain epitope-specific reagents useful for studying receptor structure-function relationships. Monoclonal antibodies that mimic ligand binding are produced, as well as other monoclonal antibodies that block receptor-ligand interactions. This object can be achieved by using a mouse cell line stably transfected with human genes. Expression in cells such as murine MT cells results in proper surface expression and proper antigenic structure of the molecule, resulting in a reagent with good binding properties for proteins on human dendritic cells. A combined immunization protocol using purified recombinant protein as well as recombinant cells is used.

Example 13: LLR-J24 gene (characterization of a potential binding protein chain)
In some examples, lectin-like receptors have been shown to form non-covalent associations with disulfide-linked heterodimers and signaling adapter molecules with different lectin-like proteins. For example, some isoforms of the NKG2 protein heterodimerize with the CD94 protein to form several variants of inhibitory and active NK cell receptors. Furthermore, the triggering NKG2 / CD94 receptor associates with the signaling adapter DAP12 via their NKG2 moiety (Lanier LL et al. Immunity 8 [1998] 693-701). DAP12 contains an ITAM motif and has a function of giving an activation signal to cells. On the other hand, the inhibitory form of NKG2-A / CD94 contains an ITIM motif on the cytoplasmic tail of NKG2-A, which recruits and triggers inhibitory signals via SHP-1 and -2 phosphatases (Palmieri G. et al., J. Immunol. 162 [1999] 7181-8). Following surface labeling of recombinant cells and primary human DCs with biotin (EZ-Link biotinylation reagent, Pierce), LLR-J24 is immunoprecipitated and the potential co-immunoprecipitated strand is labeled with the corresponding avidin-HRP reagent. Use to score in a Western blot of sediment. Protein chains exposed on the surface can be co-immunoprecipitated and thus LLR-J24 of different sizes can be detected and isolated by this method.

Sufficient to detect the signaling adapter which is not exposed on the surface, after the metabolically resulting labeled DC were used and polyacrylamide gel separation of the precipitated proteins in S ³⁵ methionine co-immunoprecipitated A score can be given for the protein. In addition, known proteins that can interact with receptors such as DAP molecules can be tested. In this way, potential interactions with active and inhibitory signaling molecules are established.

Example 14: LLR-J24 gene (ligand screening with LLR-J24 polynucleotide)
A collection of putative receptor ligands is collected for screening. The collection: acts through various oxLDL compounds and derivatives, carbohydrate compounds, parasite membranes, MHC class I soluble compounds and their homologs, MICA, MICB allelic forms, related LOX-1 and NKG2 Various UL16 binding proteins or other lectin-like receptors known to be; putative agonists of human lectin-like receptors, non-mammalian, biologically active peptides for which no mammalian counterpart has yet been identified Possible natural products; and compounds that activate lectin-like receptors with unknown natural ligands that are not found in nature. This collection uses both functional assays (i.e., calcium and cAMP release, tyrosine and serine kinase activation, phosphatases such as SHP-1 and -2) and binding assays with various labeled compounds, Used to initially screen for receptors for known ligands.

  The search for potential ligands is performed using recombinant cells and isolated soluble receptors. Initially, two potential ligand sources can be investigated. Based on the homology of LLR-J24 with respect to LOX-1 and NK receptors and close chromosomal localization, no glycoligand is likely, but residual glycoligand properties are found in some lectin-like receptors. Has been issued. By analogy with the NK receptor or LOX-1, proteinaceous ligands or lipoproteins are expected. Similar to LOX-1, oxidized LDL can bind. As suggested by preliminary experiments with the relevant murine Dectin-1 (Ariizumi K. et al. J. Biol. Chem. 275 [2000] 20157-20167), LLR-J24 is rather rather a T cell. It is expected to engage with surface molecules.

Activation of LLR-J24 expressing recombinant cells versus LLR-J24 negative parent cell line can be assessed. Activation of Ca ⁺⁺ , NFκB and several MAp kinase pathways is tested. Instead, LLR-J24 recombinants are combined with T cells and T cell membrane fractions to assess activation. On the one hand, the nature of binding to oxLDL also mediates binding to apoptotic cells, and such a function is thought to make sense in the context of DC. Endocytosed apoptotic cellular material is further presented to T cells via MHC molecules, thus recognizing the infection of the cells responsible for apoptosis. Direct binding to lipoproteins on parasites can be tested. Instead, LLR-J24 may be involved in interactions with surface proteins on T cells, such as MHC homologs, and this interaction may have an auxiliary function for activation of T cells.

  Soluble receptors are used to identify, isolate and clone ligands. This is done by using an expression library of T cell cDNA in a retroviral vector, which is available and can be used to express the cDNA library in cells that do not bind soluble receptors. Tagged soluble receptors can be used to isolate transduced cells expressing the appropriate ligand by FACS sorting, and cDNA can be recovered from the sorted cells. If the ligand has a more complex structure, for example if it consists of two chains and these cannot be expressed in one cell using the method described, affinity chromatography with soluble receptors can be performed. The biochemical purification used and standard protein separation techniques can be used.

Example 15: LLR-J24 gene (ligand binding assay)
Ligand binding assays provide a direct method for confirming receptor pharmacology and are applicable to high throughput formats. The purified ligand for the receptor is radiolabeled to a highly specific activity (50-2000 Ci / mmol) for binding studies. Then, in the process of radiolabeling, a measurement is performed without reducing the activity of the ligand for the receptor. Assay conditions for other modulators such as buffers, ions, pH and nucleotides are optimized to establish a viable noise to signal ratio for both membrane and whole cell receptor sources. For these assays, specific receptor binding is defined as total bound radioactivity minus the radioactivity measured in the presence of excess unlabeled competing ligand. Where possible, more than one competing ligand is used to define the remaining non-specific binding.

Example 16: LLR-J24 gene (functional assay)
Potential activation or inhibition of cell signaling in recombinant DC-like DC / Sp20 versus non-recombinant cells that do not express LLR-J24 is assessed. The second method is to use preparations of cultures matured by induction with monocyte-derived and cord blood-derived primary DCs and CD40 ligand, which are active in stimulating MLR. In the first method, antibodies and potential soluble ligands, such as oxidized LDL or apoptotic cell fragments, are scored for potential activation of intracellular signals in recombinant cells versus their parent cells. Free Ca ⁺⁺ , induction of NFκB and increase of the major MAP kinase pathways MEK / ERK, p38 and JNK are tested. Intervention of antibodies with a trigger compound or elimination of the inhibitory function of the receptor can be tested.

  The potential contribution of the receptor to DC maturation and to T cell activation can be assessed as follows. In Assay 1, immature Langerhans-type DCs are prepared and antibodies are tested to see if they inhibit or promote DC maturation / activation during CD40 ligand stimulation. For this purpose, CD80 / CD86 upregulation is measured by flow cytometry. In Assay 2, it is assessed whether the antibody and the prepared soluble receptor alter the ability of DC to stimulate allogeneic T cells.

  Immature as well as mature DCs are used as stimulators of allogeneic T cells after preincubation of dendritic cells with antibodies or of T cells with soluble receptors. A preferred reading of T cell stimulation is thymidine incorporation after 5 days. These experiments detect the influence of the receptor on DC maturation as well as on T cell activation. Thus, an auxiliary function of LLR-J24 in the interaction with T cells is detected. Antibodies and soluble receptors that can block the potential interaction of LLR-J24 with ligands on T cells are important for modulating the immune response.

Example 17: LLR-J24 gene (extraction / cell supernatant screening)
There are numerous lectin-like receptors that no longer have cognate activating ligands (agonists). Therefore, the active ligands of these receptors cannot be included in the ligand banks identified to date. Thus, the CTLD receptors of the present invention are also functionally screened (using calcium, cAMP, kinase functional screens) on tissue extracts to identify natural ligands. Extracts that produce a positive functional response can be subfractionated over time until the activating ligand is isolated and identified.

Example 18: LLR-J24 gene (calcium and cAMP functional assay)
Lectin-like receptors expressed in HEK293 cells are shown to be functionally coupled to activation of PLC and calcium mobilization, cAMP stimulation and / or activation or inhibition of tyrosine and serine / threonine kinases. Basal calcium levels in receptor-transfected HEK293 cells or vector control cells were usually observed to be in the range of 100 nM to 200 nM. HEK293 cells expressing recombinant receptors are loaded with fura2 and more than 150 selected ligands or tissue / cell extracts per day are evaluated for agonist-induced calcium mobilization. Similarly, HEK293 cells expressing recombinant receptors are evaluated for stimulation or inhibition of cAMP production using standard cAMP quantitative assays. Agonists that provide calcium transients or cAMP fluctuations are tested in vector-controlled cells to determine whether the response is unique to transfected cells expressing the receptor.

Example 19: LLR-J24 gene RNA regulation of the LLR-J24 gene of the present invention:
LLR-J24 is selectively expressed in DCs:
Since LOX-1, LLR-J24 and CLEC-1 are members of one subfamily, we examine the expression of LLR-J24 and CLEC-1 mRNA in primary human DC, monocytes, T and B lymphocytes and endothelial cells . The obtained data indicate that LLR-J24 mRNA is abundantly and selectively expressed in monocyte-derived DC (MoDC) and in CD34 ⁺ hematopoietic progenitor cell-derived DC (HPDC). Although small amounts of LLR-J24 mRNA are also present in primary monocytes, primary T and B lymphocytes and endothelial cells treated or not treated with various stimuli have detectable levels of LLR-J24 transcripts. It was not expressed. No expression was seen in several additional cell lines, namely Northern blots including Jurkat, RPMI 8866, NKL and NK92. The size of RNA in DC is estimated to be 3.0-3.5 kb from Northern blots, which is the first or several detected in genes in the region 1.6-1.9 kb downstream from the stop codon. This corresponds to the size of the transcript deduced from the use of a continuous polyadenylation signal.

LLR-J24 expression is down-regulated during functional maturation of DCs:
DC activation and final maturation are associated with altered expression of genes that regulate their migration and function. Therefore, it was tested whether stimulation of DCs with inflammatory mediators, CD40-ligand or zymosan A resulted in a change in the expression level of LLR-J24 mRNA. As shown in FIG. 8A, treatment of MoDC with a combination of IL-1β and TNF-α for 18-24 hours resulted in a 3-5 fold reduction in reproducible LLR-J24 mRNA. Instead, HPDC is activated by a mAb specific for the CD40 receptor in the absence or presence of zymosan A that stimulates phagocytosis of yeast particles (FIG. 8B). Based on these results, down-regulation of LLR-J24 mRNA is particularly evident in cultures treated with CD40 ligation combined with potent maturation inducers such as TNF-α and IL-1β or phagocytosis stimuli. Is done. Since MDC chemokines (CCL22) are known to be strongly induced in DCs after their maturation, the same Northern blot was used to confirm the successful activation of MoDC or HPDC. And hybridized. As shown in FIG. 8, strong induction of MDC parallels the downregulation of LLR-J24 transcripts in both types of DC.

The LLR-J24 transcript is variably spliced:
Northern blot analysis failed to distinguish transcripts that were expected to differ by 141 bases for LLR-J24-1 and -2. Accordingly, reverse transcription PCR is performed using a primer pair to amplify the fragment between the second and fourth exons. Two products of 505 bp and 367 bp were obtained, corresponding to the size of the expected fragment with and without stalk exons, respectively (FIGS. 9 and 10). It is estimated that about 5-10% of the product is sized with stalk exons. This upper band is scraped from the gel, subcloned, and the two resulting clones are sequenced. Both clones represented correctly spliced stalk exons as shown in SEQ ID NO: 7 and SEQ ID NO: 9. The resulting PCR fragments were further analyzed with primer pairs that amplify mRNA fragments from the 5′-untranslated region to the stalk exon and from the stalk exon to the sixth exon, respectively (see FIG. 8B). In both cases, the main product obtained corresponded to the size predicted for a correctly spliced exon. These data indicate that the fraction of the LLR-J24 transcript contains a stalk exon and is also correctly spliced with other exons.

Interaction of IKK2 CoIP with IKK2-55. Flag-tagged IKK2-55 and myc-tagged IKK2 were transfected into Hela cells as indicated. Lysates were prepared and IKK2-55 was precipitated with anti-Flag antibody, and the precipitate was analyzed by Western blotting with anti-Myc antibody (upper panel). The presence of Flag-IKK2-55 and myc-IKK2 in the cell extract was confirmed by Western blotting (lower panel). Subcellular localization of IKK2-55. HUVEC were transfected with the YFP-IKK2-55 fusion construct along with the CFP fusion gene for the different cell compartments and observed under a fluorescence microscope. The left column shows the localization of IKK2-55 in the yellow channel, and the right column shows the localization of the marker in the cyan channel. Upper panel: endoplasmic reticulum (ER); middle panel: mitochondria (lower); lower panel: Golgi apparatus. Effect of IKK2-55 on NF-kB dependent promoter. HUVEC were transfected with 5xNF-kB-Luc construct with different amounts of IKK2-55 as well as ubiquitin-bgal as internal standard and stimulated with 100 U / ml TNFα for 6 hours after 2 days. Luciferase values (relative light intensity units) were normalized for b-gal expression. prom = promoter; unstim = unstimulated; stim = stimulated. Effect of IKK2-55 (in combination with different kinases) on the NF-kB dependent promoter. HUVECs were transfected with the 5xNF-kB-Luc construct, along with IKK2-55, with the different kinases indicated (IKK2, NIK, TAK1, p38) as well as ubiquitin-bgal as an internal standard. In one case, cells were stimulated with 100 U / ml TNFα for 6 hours. Luciferase values were expressed as relative luminosity units and normalized for β-gal expression. Dark column: 500 ng IKK2-55; hatched column: 0 ng IKK2-55 RLU-βgal: relative light intensity units per β-gal. Phylogenetic tree containing all known lectin-like genes of the human NK receptor complex: CTLD sequences of the represented genes were aligned and phylogenetic trees were generated as described in the Examples. All sequences were chosen to start with the amino acid two before the first cysteine that is always conserved in the domain. NKG2E and H are two separately spliced transcripts from the same gene. For LY49L, a hypothetical sequence was selected that contained all three CTLD exons that were spliced in a manner comparable to other genes, but only aberrantly spliced transcripts were detected for this gene. This gene was found to be more closely related to each other consistently with different parameter settings for alignment, and the tree structure is indicated by the right parenthesis. The lower scale is a measure of replacement events. The selective expression of subfamilies in NK cells, T cells, endothelial cells (EC), monocytes (MO), dendritic cells (DC) or more broadly in leukocytes (LEUK) is shown. Comparison of non-permeating and permeating cell staining. In the left column, non-permeating cells fixed with formaldehyde (fixed) and in the right column osmotic cells (fixed / permeated) are shown. The upper panel shows staining with anti-FLAG antibody, and the lower panel shows staining of DNA with Hoechst 333258 to visualize the total cell number. The LLR-J24 construct without the stalk exon is shown in FIG. 2B (ΔLLR-J24-1). Comparison of osmotic cell staining for all four expression constructs at higher magnification. Downregulation of LLR-J24 mRNA during dendritic cell maturation: RT-PCR of LLR-J24 mRNA. RNA from MoDC was reverse transcribed with specific LLR-J24 primers and PCR was performed on the resulting cDNA with the indicated different primer pairs. (a) shows amplification of transcripts with and without stalk exons for MoDC RNA, (b) shows 5′-part amplification, and (c) shows B in FIG. 4A. The 3′-part of the stalk containing transcripts for MoDC and HL60 RNA shown in FIG. Lane 6 shows the product obtained from the plasmid containing the full length LLR-J24 cDNA. Schematic of oligonucleotide primers for use in RT-PCR. The first primer pair (a) was designed to test for the presence of a stal exon fragment that resulted in the expected 505 bp and 367 bp PCR products with and without the stalk exon fragment, respectively. The second primer pair (b) tests the correct splicing of the 5′-part, and the third primer pair (c) corrects the splicing of the 3′-part of the transcript containing the stalk exon. c = cytoplasmic; TM = transmembrane; CTLD = C-type lectin-like domain 1; CTLD2 = C-type lectin-like domain 2; CTLD3 = C-type lectin-like domain 3.

Claims (14)

  LLR-J24- comprising an isolated LLR-J24-stalk peptide represented by SEQ ID NO: 10, or a nucleotide sequence encoding a polypeptide having at least 90% identity to the polypeptide represented by SEQ ID NO: 10 stalk nucleotide.   An isolated LLR-J24-stalk nucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 9, or a polynucleotide that hybridizes with the nucleotide sequence set forth in SEQ ID NO: 9 under stringent conditions.   An isolated polynucleotide encoding the transmembrane protein LLR-J24.   An isolated polynucleotide encoding the extracellular domain of LLR-J24.   An isolated LLR-J24-stalk peptide represented by SEQ ID NO: 10, or a polypeptide having at least 90% identity with the polypeptide represented by SEQ ID NO: 10.   An isolated polypeptide that is an extracellular domain of LLR-J24.   An isolated polypeptide which is a transmembrane protein LLR-J24.   A vector comprising the gene according to any one of claims 1 to 4.   8. An expression system comprising a DNA or RNA molecule, wherein the expression system or part thereof is present in a compatible host cell, the expression system or part thereof being a polypeptide according to claim 6 or 7. An expression system capable of producing   A set produced by transforming or transfecting a host cell with the expression system of claim 9 such that the host cell produces the polypeptide of claim 6 or 7 under suitable culture conditions. Replacement host cell.   An isolated host cell comprising the expression system of claim 9.   An antibody against the polypeptide according to claim 6 or 7. A screening assay for identifying agonists or antagonists of the polypeptide of claim 6 or 7, comprising as a major component:
a) the polypeptide of claim 6 or 7,
b) a recombinant cell expressing the polypeptide of claim 6 or 7,
c) a cell membrane expressing the polypeptide of claim 6 or 7, or d) an antibody against the polypeptide of claim 6 or 7,
An assay comprising. A method for identifying an agonist or antagonist of the polypeptide of claim 6 or 7, comprising
A) a) the polypeptide according to claim 6 or 7,
b) a recombinant cell expressing the polypeptide of claim 6 or 7,
c) a cell membrane that expresses the polypeptide of claim 6 or 7, or d) an antibody against the polypeptide of claim 6 or 7,
Contacting with a candidate compound,
B) measuring the effect of the candidate compound on any of a), b), c) or d);
C) selecting the agonist or antagonist measured in step B),
Comprising a method.