EP0808366A1 - Identifizierung von dec-205, (dentritischen und epithelzellen, 205 kda), rezeptor mit c-typ lektin regionen, dafür kodierende nukleinsäure und verwendungen davon - Google Patents

Identifizierung von dec-205, (dentritischen und epithelzellen, 205 kda), rezeptor mit c-typ lektin regionen, dafür kodierende nukleinsäure und verwendungen davon

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Publication number
EP0808366A1
EP0808366A1 EP96906258A EP96906258A EP0808366A1 EP 0808366 A1 EP0808366 A1 EP 0808366A1 EP 96906258 A EP96906258 A EP 96906258A EP 96906258 A EP96906258 A EP 96906258A EP 0808366 A1 EP0808366 A1 EP 0808366A1
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European Patent Office
Prior art keywords
dec
ser
leu
glu
lys
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English (en)
French (fr)
Inventor
Ralph M. Steinman
Michel C. Nussenzweig
William J. Swiggard
Wanping Jiang
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Rockefeller University
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Rockefeller University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to the identification and characterization of a receptor associated with antigen presentation in immune responses, endocytosis, and trans- epithelial transport. Identification of the receptor, its characterization as having ten lectin-binding domains, and evidence of its role in the uptake and processing of oligosaccharides and oligosaccharide-decorated molecules, e.g. , glycoproteins, has important ramifications for modifying immune response, and for trans-epithelial transport of molecules.
  • Dendritic cells are a unique class of leukocytes whose primary function is to capture, process, and present antigens to T cells (Steinman, 1991 , Annu. Rev. Immunol. 9:271-96). Interaction between dendritic cells and specific T cells in the peripheral immune system leads to the induction of immune responses, whereas in the thymus presentation by dendritic cells leads to negative selection (Tanaka et al. , 1993, Eur. J. Immunol. 23:2614-2621 ; Matzinger et al. , 1989, Nature 338:74- 76).
  • thymic epithelial cells Like dendritic cells, thymic epithelial cells present MHC-bound peptides to T cells, but instead of inducing T cell activation or negative selection, thymic epithelial cells direct positive selection (Hugo et al. , 1993. Immunol. Rev. 135: 133-35; Elliott. Immunol. Rev. 135:215-25). Consistent with the known requirements for interactions with T cells, both dendritic cells and thymic epithelial cells express a number of cell surface proteins that facilitate cell-cell contact and mediate T cell activation (Steinman, 1991 , Annu. Rev Immunol. 9:271-96; Hugo et al. , 1993, Immunol Rev.
  • NLDC-145 The antigen bound by NLDC-145 is also abundant on thymic cortical epithelium. However, cloning and characterization of the NLDC-145 antigen has proved elusive. For one thing, dendritic cell cDNA libraries have not been readily prepared. Dendritic cells themselves are rare, making their RNA extremely rare. Moreover, monoclonal antibodies are not usually effective reagents for screening expression libraries, e.g. , a ⁇ gt-11 expression library.
  • the present invention relates to an integral membrane protein, termed herein "DEC,” found primarily on dendritic cells, but also found on B cells, brain capillaries, bone marrow stroma, epithelia of intestinal villi and pulmonary airways, as well as cortical epithelium of the thymus and the dendritic cells in the T cell areas of peripheral lymphoid organs. In addition, trace amounts of this protein are found in organs like the liver, heart, and kidney.
  • the murine and human counterparts of DEC have an apparent molecular mass of 205 kDa, the murine counterpart has an isoelectric point at pH 7.5, and carbohydrates comprise about 7 kDa of the total mass of murine DEC.
  • the carbohydrates appear to consist of eight distinct but related biantennary N-linked glycans, with no O-linked glycans present. Because the protein has been found predominantly on Dendritic cells and thymic Epithelial Cells, and has a molecular weight of 205 kDa, it is termed DEC-205.
  • the invention relates to isolation and cloning of human DEC, which is further characterized by having a carboxyl- terminal sequence RHRLHLAGFSSVRYAQGVNEDEIMLPSFHD (SEQ ID NO: 1), and characterized by binding to a rabbit polyclonal antibody raised against full length murine DEC-205, but not reacting with monoclonal antibody NLDC-145.
  • human DEC has the amino acid sequence depicted in SEQ ID NO: 8; it may be encoded by the nucleotide sequence depicted in SEQ ID NO: 7.
  • Another characteristic of DEC based on the deduced amino acid sequence information obtained from cloning the dec cDNA is a unique coated pit localization consensus domain or motif on the cytoplasmic tail of the protein, which, as shown in Figure 9, has regions of homology and regions of dissimilarity between the two counte ⁇ art proteins. It has been further discovered that DEC- 205 is rapidly internalized via coated vesicles, and delivers bound substances to a multivesicular endosomal compartment that resembles the MHC class-II containing vesicles implicated in antigen processing.
  • the present invention is directed to identification of additional ligands of the DEC-205 receptor, which can be advantageously targeted to dendritic cells and other cells that bear DEC-205.
  • Targeting antigens for presentation by dendritic cells can provide for tolerance when the dendritic cells are quiescent, or for immune stimulation ( . e. , vaccination) when the dendritic cells are activated, e.g. , by stimulation with a cytokine or lymphokine, such as colony stimulating factor (CSF).
  • CSF colony stimulating factor
  • the presence of DEC on epithelial cells suggests an important role in trans-epithelial transport of molecules, e.g. , from the basolateral surface of the lung respiratory epithelium into lung airways or the basolateral surface of the intestinal epithelium into the lumen of the intestines, and from the apical (lumenal) surface of these epithelial to the pulmonary or intestinal circulation, respectively.
  • the invention provides for parenteral delivery of pharmacological agents, e.g.
  • antibiotics to infections of the lung or the intestines, by targeting the pharmacological agent with a ligand to DEC, and for systemic delivery of pharmacological agents by aerosolization and inhalation via the lungs, or ingestion and absorption via the intestines.
  • the invention provides for targeting pharmacological agents to cross the blood brain barrier via DEC located on the brain capillaries.
  • the invention provides a method for identifying a ligand for DEC, comprising contacting a protein comprising at least one DEC lectin domain with a candidate ligand; and detecting binding of the candidate ligand with the DEC lectin domain. Binding of the candidate ligand and the DEC lectin domain indicates that the ligand candidate is a ligand for DEC.
  • the ligand is a saccharide, which binds to one or more of the lectin domains on DEC.
  • the protein comprising at least one DEC lectin domain is expressed by cells as an integral membrane protein, and the candidate ligand is labeled, such that binding of the candidate ligand with the DEC lectin domain is detected by detecting association of the label with the cells.
  • the protein comprising at least one DEC lectin domain is solubilized, and the candidate ligand is irreversibly associated with a solid phase support, such that binding of the candidate ligand with the DEC lectin domain is detected by detecting binding of the protein with the solid phase support.
  • the protein comprising at least one DEC lectin domain is irreversibly associated with a solid phase support, and the candidate ligand is labeled, such that binding of the candidate ligand with the DEC lectin domain is detected by detecting association of label with the solid phase support.
  • die protein comprising at least one DEC lectin domain is a truncated DEC protein; in another embodiment, the protein comprising at least one DEC lectin domain is a full length DEC protein.
  • the present invention advantageously provides a nucleic acid encoding at least a portion of a DEC protein.
  • the invention provides for expression of DEC proteins, or truncated fragments thereof, including chimeric proteins, which can be used for identifying a DEC ligand.
  • the nucleic acid of the invention comprises at least fifteen base pairs, thus, the nucleic acids of the invention provide useful probes for detecting expression of mRNA for DEC, PCR primers for reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA, or for cloning DEC, and probes for the presence of DEC cDNA or genomic DNA, e.g. , in a library or cell.
  • the nucleic acid encodes a human DEC protein.
  • a nucleic acid encoding human DEC is provided.
  • the present invention further provides an expression vector comprising the nucleic acid encoding DEC.
  • the nucleic acid is a DNA molecule encoding at least a lectin domain of DEC, operatively associated with an expression control sequence.
  • the invention provides a recombinant host cell comprising the expression vector.
  • the host cell is a mammalian cell selected from the group consisting of a Chinese hamster ovary cell, an African Green Monkey COS cell, a Madin-Darby canine kidney cell, and an NIH-3T3 fibroblast cell.
  • the invention further provides an antibody reactive with a human DEC-205 protein, in particular a monoclonal antibody and a polyclonal antibody.
  • the present invention advantageously provides for identifying ligands of DEC, which ligands are capable of targeting a molecule to which they are attached, i.e. , conjugated, to a cell bearing DEC in vitro or in vivo.
  • the ability to target cells that express DEC in vivo has important implications from the perspective of specifically targeting dendritic cells, epithelial cells, e.g. , of the thymus, small intestine, and lung.
  • the invention is naturally directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a molecule targeted to a tissue selected from the group consisting of pulmonary circulation, intestinal circulation, pulmonary *airways, lumen of the small intestine, dendritic cells in the skin and T cell areas of lymphoid organs, thymus, and brain, which molecule is conjugated to a DEC-ligand.
  • the DEC-ligand is selected from the group consisting of a carbohydrate that binds DEC and an anti-DEC antibody, and a pharmaceutically acceptable carrier.
  • the molecule is selected from the group consisting of an anti-cancer drug, an ami- viral drug, an antibiotic, an anti-parasitic drug, and an anti- inflammatory drug.
  • a recombinant vector for introduction of a gene into cells selected from the group consisting of dendritic cells, thymic epithelial cells, lung epithelial cells, small intestine epithelial cells, and brain capillary cells comprising a DNA vector conjugated to a DEC-ligand
  • the DEC-ligand is selected from the group consisting of a carbohydrate that binds DEC and an anti-DEC antibody.
  • the DNA vector is selected from the group consisting of a viral vector, a liposome vector, and a naked DNA vector.
  • the present invention provides a vaccine comprising an antigen from a pathogen conjugated to a DEC-ligand, wherein the DEC-ligand is selected from the group consisting of a carbohydrate that binds DEC and an anti-DEC antibody, and an immune stimulator.
  • pathogens include, but are not limited to, a virus, a bacterium, a parasite, and a tumor.
  • the immune stimulator may be selected from the group consisting of a cytokine, a lymphokine, and an adjuvant.
  • the invention advantageously provides for targeting a molecule that is either a poor immunogen, or that is not immunogenic at all, to dendritic cells for efficient processing (as DEC is shown herein to be associated with antigen processing mechanisms of dendritic cells) and presentation to responsive T lymphocytes.
  • the invention provides a composition to induce immune suppression comprising an autoantigen or an allergen conjugated to a DEC-ligand, wherein the DEC ligand is selected from the group consisting of a carbohydrate that binds DEC and an anti-DEC antibody, with the proviso that the composition lack immune stimulatory agents.
  • the quiescent dendritic cells can process and present antigen. Presentation of antigen by quiescent dendritic cells is believed to induce antigen- specific T cell anergy or immune tolerance.
  • the autoantigen may be selected from the group consisting of myelin basic protein, collagen or a fragment thereof. DNA, a nuclear protein, a nucleolar protein, a mitochondrial protein, and a pancreatic 0-cell protein.
  • an important corolly object of the invention is to identify ligands that specifically bind DEC.
  • a related object is to express nucleic acids encoding DEC, or a portion thereof comprising a carbohydrate binding portion of a DEC lectin domain.
  • FIGURE 1 The apparent mass of the antigen bound by NLDC-145 is 205 kDa.
  • A Immunoprecipitation of ( 5 S)methionine-cysteine-labeled bone marrow DC extracts with immobilized NLDC-145 (right) reveals an actively synthesized antigen with an apparent mass > 200 kDa. This antigen is not precipitated by immobilized nonspecific rat IgG2a (left).
  • B NLDC-145 binds an antigen of 205 kDa in non-reducing Western blots of crude thymic detergent extract.
  • FIGURE 2 Summary: purification of DEC-205 from thymi. All steps were performed at 0-4°C. Leupeptin and PMSF were added to ice-cold buffers just before use.
  • A Reducing 8% acrylamide SDS-PAGE analysis of 5 ⁇ g of purified protein, stained first with Coomassie Brilliant Blue R-250 (left), then counterstained with silver (right).
  • B Isovolumic Western blot of key fractions from the purification, stained with 10 ⁇ g/ml of NLDC-145 IgG.
  • Lane 10 intentional five-fold increase in antigen concentration, to demonstrate a "ladder" of minor mAb-reactive bands ranging down to about 80 kDa in apparent mass.
  • FIGURE 4 DEC-205 is an integral membrane protein with a pi of 7.5.
  • A Immunoblot of thymic membrane proteins solubilized with detergent, 1 M KC1 or 100 mM Na 2 CO 3 , pH 1 1.5 (lanes 1, 2, 3), and proteins initially insoluble in the high salt and high-pH buffers, but then released from membranes with detergent (lanes 4 and 5). The filter was stained with 10 ⁇ g/ml of NLDC-145 IgG.
  • B Isoelectric focusing of 10 ⁇ g of purified DEC-205 under denaturing conditions. A single lane from a silver-stained slab gel is shown, with pH values assigned after elution of ampholytes from a neighboring unstained lane.
  • FIGURE 5 Studies of the carbohydrates bound to DEC-205.
  • DEC-205 is a glycoprotein. Purified 205 kDa protein, transferrin (Tf, positive control), and creatinase (cre, negative control) were electroblotted to nitrocellulose, and the filter was oxidized with NaIO 4 . converting v/ ' c-diols within sugars to immobilized aldehydes. Reaction with a digoxigenin (DIG)-labeled hydrazide. followed by an anti-DIG antibody conjugated to alkaline phosphatase. revealed the positions of glycoproteins on the blot. Like transferrin. but unlike creatinase. DEC-205 stains for sugar.
  • DIG digoxigenin
  • the glycans on DEC-205 comprise about 7 kDa of its apparent molecular mass.
  • Apotransferrin (aTf) and DEC-205 were either treated (+) or not (-) with anhydrous trifluoromethanesulfonic acid (TFMSA), to nonselectively hydrolyze protein-bound carbohydrates. Both treated proteins exhibited increased electrophoretic mobility, corresponding to a 5 kDa loss of apparent mass by apotransferrin, and a 7 kDa loss by DEC-205.
  • TFMSA trifluoromethanesulfonic acid
  • (Glc) 5 , (Glc) ]0 positions of selected bands in a standard oligo-glucose ladder.
  • (D) Exoglycosidase digestions and FACE analysis of the mixture of N-linked glycans released from DEC-205. Lane 1 : Undigested N-linked oligosaccharides (the dark band at (Glc)- in lanes 1 -5 is a detergent artifact). Lane 2: digested with ⁇ -galactosidase. Lane 3: digested with ⁇ -galactosidase plus NANase III. Lane 4: digested with the previous 2 enzymes plus ⁇ -galactosidase.
  • Lane 5 digested with the previous 3 enzymes plus ⁇ -N- acetylhexoseaminidase.
  • Lane 6 as for lane 5, but 2-fold higher concentration of ⁇ -N-acetylhexoseaminidase.
  • Lane 7 digested with the previous 4 enzymes plus ⁇ - mannosidase.
  • Lane 8 as for lane 7, but 2-fold higher concentration of ⁇ - mannosidase plus ⁇ -fucosidase.
  • Lane 9 mannosylchitobiose core standards: FC, fucosylated core; C, non-fucosylated core.
  • E Summary of findings from lectin staining and FACE analysis. Two fucosylated core structures are present, with and without bisecting GlcNAc. Further heterogeneity at the termini produces the 8 glycan variants observed in (C).
  • FIGURE 6 N-terminal amino acid sequence of DEC-205, and blotting by polyclonal antibodies.
  • A The amino-terminal sequence (SEQ ID NO: 2), as determined by two different core facilities. A peptide spanning the first 19 residues was synthesized and coupled to KLH for use as an immunogen.
  • B Preclearing study: NLDC-145 specifically depletes the 205 kDa bands detected by both polyclonal antibodies. Immunoblots of crude thymic membrane extract, a depleted fraction produced by passing the same extract over the NLDC-145 immunoaffinity column twice, and material eluted from the column.
  • FIGURE 7 Schematic representation of DEC-205.
  • FIGURE 8 Sequence of murine DEC-205 and related proteins.
  • N N, E or Q.
  • the two missing cvsteines in CRD 8 are highlighted with a *.
  • Peptide sequences determined by automated Edman degradation from purified DEC-205 protein are overlined and numbered (N indicates amino terminal, T indicates peptides generated with Trypsin, and L indicates peptides generated with endoproteinase lys-C).
  • FIGURE 9 Comparison of carboxyl-terminal cytoplasmic domain sequences of human (top) (SEQ ID NO: l) and murine (bottom) (SEQ ID NO:6) DEC-205. Regions of identity are underlined; regions of similarity are italicized.
  • FIGURE 10 DEC-205 Expression. Expression of DEC-205 in mouse tissues and transfected Cos-7 cells.
  • A Northern blot of poly-A-i- A extracted from the indicated tissues. Symbols: Br. brain mRNA; DC. dendritic cell mRna; Ht, heart mRNA; Kd, kidney mRNA; Lv, liver mRNA; LN, lymph node mRNA; Sk, skin mRNA; Thm thymus mRNA; tg. tongue mRNA.
  • FIGURE 11 Endocytosis of DEC-205.
  • FIGURE 12 Antigen Presentation. Antigen presentation by Dendritic cells incubated with rabbit anti-DEC-205 antibodies or non-reactive rabbit antibody controls. IL-2 production by the 2R.50 cells is plotted against the concentration of antibody in the cultures on a log scale. The error bars indicate the standard deviation from the mean. Symbols: anti-DEC-205, cultures that received the indicated amount of rabbit anti-DEC-205 polyclonal IgG; anti-IgG2a. cultures that received the indicated amount of IgG2a specific polyclonal rabbit antibodies; IgG, cultures that received the indicated amount of non-immune rabbit IgG.
  • FIGURE 13 Selective staining of Langerhans cells with monoclonal and polyclonal antibodies to DEC-205.
  • Cultured epidermal cells were double-labeled with a PE-tagged mAb to class II MHC proteins (y axis) and multiple antibodies to leukocyte antigens, followed by FITC-anti-Ig (x axis).
  • the mean FITC fluorescence intensity for the MHC-I1 (+) DCs (e.g., arrows in E and H) is shown in the upper right corner of each panel.
  • A-D Specificity: Langerhans cells stain for DEC-205, but not for macrophage, B cell or T cell antigens. Rat IgG2a hybridoma supernatants were applied.
  • FIGURE 14 Trypsin sensitivity and resynthesis of DEC-205 epitopes.
  • Langerhans cells (A-F) or lymph node B cells (G-L) that had been cultured overnight were either exposed to 0.25% trypsin for 30 min on ice. or were not treated.
  • the lymph node B cells had been stimulated with LPS to sustain viability. Cells were either stained immediately (dl ) or after an additional day of culture (d2).
  • the antibodies were: 30 ⁇ g/ml anti-DEC-205 or nonimmune F(ab') 2 fragments (A, C, E, G, I, and K); 2 ⁇ g/ml NLDC-145 or nonimmune rat IgG2a; or anti-CD45 (clone Ml/9, rat IgG2a) hybridoma supernatant (B, D, F, H, J, and L).
  • FIGURE 15 Expression of DEC-205 by fresh and cultured dendritic cells (arrows) from spleen and skin.
  • Spleen DCs enriched in the low-density fraction of spleen cells, were identified with anti-CDl lc (y axis. A-H) and counterstained with: NLDC-145; F(ab') 2 fragments of the anti-DEC-205 polyclonal; and corresponding nonimmune controls. Staining was performed either immediately after flotation (fresh), or after overnight culture.
  • Fresh and cultured Langerhans cells identified in an epidermal suspension with a mAb to class II MHC proteins (y axis, I-P), are shown for comparison.
  • FIGURE 16 Expression of class II MHC proteins and DEC-205 by bone marrow DCs grown from progenitors in the presence of GM-CSF.
  • FIGURE 17 Expression of DEC-205 on peritoneal cells.
  • Peritoneal cells either resident or in exudates elicited with the indicated proinflammatory agents, were stained with 30 ⁇ g/ml of anti-DEC-205 or nonimmune F(ab') 2 fragments and FITC-anti-rabbit F(ab') 2 . The cells were then counterstained with PE-tagged mAbs to macrophages.
  • B cells and T cells Shown here is staining by PE-anti-Mac- 1/CDl lb (mAb Ml/70, y axis).
  • the Mac-l br ' Bht cells are macrophages (arrowheads), the Mac-l d ⁇ m cells are B cells (arrows) and the Mac-1 negative cells, T cells.
  • Con A concanavalin A
  • TGC thioglycollate
  • BCG Mycobacterium bovis Bacille Calmette-Guerin.
  • FIGURE 18 Expression of DEC-205 by leukocytes in fresh cell suspensions from three organs. B cells are arrowed. Cells from spleen (A-J), bone marrow (K-T), and peripheral blood (U- ⁇ ) were stained with PE-tagged antibodies to subsets of leukocytes (y axis), and counterstained with 30 ⁇ g/ml of nonimmune (A- E, K-O, and U-Y) or anti-DEC-205 (F-J, P-T, and Z- ⁇ ) F(ab') 2 fragments (x axis, FITC).
  • A-J spleen
  • K-T bone marrow
  • U- ⁇ peripheral blood
  • the PE-labeled mAbs reacted with granulocytes (RB6-8C5, anti Gr-1), the integrin CDl lb, abundant on granulocytes and macrophages (Ml/70. anti-Mac-1), B cells (RA3-6B2, anti-B220/CD45RB), T cells (53-2.1, anti-Thy-1.2/CD90), and class II MHC proteins (AMS-32.1, anti- I-A d ).
  • FIGURE 19 Immunoblot.
  • Graded doses of whole-cell NP-40 extracts of bone marrow dendritic cells (BMDC), bulk splenic leukocytes (SPL, ca. 65% B cells) and resident peritoneal cells (PC, ca. 70% B cells, 30% macrophages) were transferred to a filter.
  • the filter was stained with 10 ⁇ g/ml of NLDC-145 IgG.
  • BMDCs express roughly 10 times more DEC-205 per cell than splenic B cells, and roughly 50 times more than peritoneal B cells.
  • FIGURE 20 Inability of antibodies to DEC-205 to block dendritic cell stimulatory activity in vitro.
  • a mixed leukocyte reaction where graded doses of mitomycin C-treated spleen dendritic cells were added to 3 x 10 5 allogeneic lymph node cells in the continuous presence of 10 ⁇ g/ml of each of the indicated antibodies, except for anti-DEC-205, which was used at 30 ⁇ g/ml.
  • Anti- Ig ⁇ negative control polyclonal to a surface Ig-associated signalling protein on B cells (Sanchez et al., 1993. J. Exp. Med.
  • FIGURE 21 Expression of DEC-205 in the thymus and in lymph nodes, (a- c): Low power of thymus cortex and medulla (M), stained with: monoclonal NLDC-145 (a); polyclonal anti-DEC-205 F(ab') 2 fragments (b); and polyclonal anti-DEC-205 IgG (c), all at 10 ⁇ g/ml, and counterstained with hematoxylin.
  • M monoclonal NLDC-145
  • b polyclonal anti-DEC-205 F(ab') 2 fragments
  • c polyclonal anti-DEC-205 IgG
  • Presumptive dendritic cells are scattered throughout the medulla, but the strongest thymic staining is on cortical epithelium, (d-f): Low power views of a mesenteric lymph node, showing a B cell follicle (B), the T cell area of the deep cortex (T), and the medulla (M), stained with: mAb NLDC-145 (d), polyclonal anti-DEC-205 F(ab') 2 fragments (e), and polyclonal anti-DEC-205 IgG (/).
  • FIGURE 22 Expression of DEC-205 in the spleen, (a-c): Low power views of a splenic white pulp nodule, stained with antibodies to: B cells (rabbit anti-Ig ⁇ , a); DEC-205 (polyclonal anti-DEC-205 IgG, b), and class II MHC proteins (mAb M5/1 14, c).
  • B cells rabbit anti-Ig ⁇ , a
  • DEC-205 polyclonal anti-DEC-205 IgG, b
  • class II MHC proteins mAb M5/1 14, c.
  • the central arteries within the T cell areas are arrowed.
  • the T cell areas contain few B cells (a, anti-Ig ⁇ ), but numerous scattered DEC-205- and class II MHC-positive dendritic cells (b-c).
  • B cell follicles are denoted with a "B", and the marginal sinus by arrowheads, (d-e): Higher power views of splenic T cell areas (periarterial sheaths, central arteries are arrowed) stained with: mAb NLDC- 145 (d), polyclonal anti-DEC-205 (e), and anti-class II MHC (/). Staining for DEC-205 has a punctate quality, in addition to the more prominent staining of dendritic cell bodies.
  • FIGURE 23 Expression of DEC-205 in several nonlymphoid organs,
  • (a-d) Brain capillaries (arrows, a-c) and small arteries (arrow, d). stained with: mAb NLDC-145 (a), polyclonal anti-DEC-205 F(ab ' ) 2 fragments (b). and polyclonal anti-DEC-205 IgG (c-d).
  • Class II MHC proteins are not evident within airway epithelium, but there are many positive profiles surrounding the airways (arrowheads, f).
  • FIGURE 24 Tissue distribution of DEC-205 by immunoblotting. Lysates of the indicated organs were blotted to compare relative levels of expression of DEC- 205 protein (A, filter stained with mAb NLDC-145) and the LAMP-1 lysosomal membrane antigen (B, filter stained with mAb 1D4B). Fifty ⁇ g of total protein were loaded in each lane.
  • FIGURE 25 Amino acid and nucleotide sequence comparisons for human and murine DEC-205.
  • A Matrix plot (pam 250 matrix) of translated murine (y- axis) and human (x-axis) DEC-205 amino acid sequences. The window size for this plot was 60. the minimum percent score was 60. and the hash value is 2.
  • B Matrix plot (DNA identity matrix) of murine (y-axis) and human (x-axis) DNA sequences. The window size was 60, the minimum percent score was 65, the hash value was 6, and the jump value was 1. Both strands were evaluated.
  • the present invention relates to an integral membrane protein, termed herein "DEC,” found primarily on dendritic cells, but also found on B cells, brain capillaries, bone marrow stroma, epithelia of intestinal villi and pulmonary airways. as well as cortical epithelium of the thymus and the dendritic cells in the T cell areas of peripheral lymphoid organs. In addition, trace amounts of this protein are found in organs like the liver, heart, and kidney.
  • the murine and human counterparts of DEC have an apparent molecular mass of 205 kDa, the murine counterpart has an isoelectric point at pH 7.5, and carbohydrates comprise about 7 kDa of the total mass of murine DEC.
  • the carbohydrates appear to consist of eight distinct but related biantennary N-linked glycans, with no O-linked glycans present. Because the protein has been found predominantly on Dendritic cells and thymic Epithelial Cells, and has a molecular weight of 205 kDa, it is termed DEC- 205. Although characterization of the murine and human counterparts of DEC demonstrates the presence of ten carbohydrate binding domains, with a high degree of homology, it is possible that DEC from other species may have more or fewer such domains. Similarly, DEC may be expressed by other cell types, such as epithelial cells from other tissues or organs.
  • the invention further relates to cloning of the gene encoding DEC-205, and characterization of the encoded protein.
  • the sequence information indicates that DEC-205 is a receptor with ten C-type lectin domains, which is homologous, or similar, to the macrophage mannose receptor and other related receptors that bind carbohydrates and mediate endocytosis.
  • the human counte ⁇ art also appears to have lectin domains. Accordingly, DEC is believed to have a corresponding number of lectin domains, and to be involved in antigen processing by dendritic cells.
  • Still another aspect of the present invention is the identification of a human DEC protein and gene encoding it.
  • DEC deduced amino acid sequence information obtained from cloning the dec cDNA
  • Another characteristic of DEC is a unique coated pit localization consensus domain or motif on the cytoplasmic tail of the protein, which, as shown in Figure 9, has regions of homology and regions of dissimilarity between the two counte ⁇ art proteins.
  • DEC-205 is rapidly internalized via coated vesicles, and delivers bound substances to a multivesicular endosomal compartment that resembles the MHC class-II containing vesicles implicated in antigen processing.
  • the invention is also based, in part, on the further discovery that rabbit antibody specific for DEC-205 was efficiently processed by dendritic cells and presented to rabbit-specific T cell clones.
  • the present invention is directed to identification of additional ligands of the DEC-205 receptor, which can be advantageously targeted to dendritic cells and other cells that bear DEC-205.
  • Targeting antigens for presentation by dendritic cells can provide for tolerance when the dendritic cells are quiescent, or for immune stimulation (i.e., vaccination) when the dendritic cells are activated, e.g., by stimulation with a cytokine or lymphokine, such as colony stimulating factor (CSF).
  • CSF colony stimulating factor
  • the presence of DEC on epithelial cells suggests an important role in trans-epithelial transport of molecules, e.g., from the basolateral surface of the lung respiratory epithelium into lung airways or the basolateral surface of the intestinal epithelium into the lumen of the intestines, and from the apical (lumenal) surface of these epithelial to the pulmonary or intestinal circulation, respectively.
  • the invention provides for parenteral delivery of pharmacological agents, e.g. , antibiotics, to infections of the lung or the intestines, by targeting the pharmacological agent with a ligand to DEC. and for systemic delivery of pharmacological agents by aerosolization and inhalation via the lungs, or ingestion and absorption via the intestines.
  • the invention provides for targeting pharmacological agents to cross the blood brain barrier via DEC located on the brain capillaries. Accordingly, various terms are used throughout this specification, which have the meanings as defined below.
  • candidate ligand is used herein to refer to a molecule under consideration of test for its ability to specifically bind to DEC.
  • candidate ligands include, but are by no means limited to, saccharides (i.e., sugars, carbohydrates, or glycans).
  • ligand as used herein can also refer to an antibody reactive with DEC.
  • a molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization.
  • a molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.
  • a composition comprising "A” (where "A” is a single protein, DNA molecule, vector, recombinant host cell, etc.) is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A".
  • "A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al.. Immunology. Second Ed., 1984, Benjamin/Cummings: Menlo Park. California, p. 384).
  • Adjuvants include, but are not limited to.
  • complete Freund"s adjuvant is pharmaceutically acceptable.
  • incomplete Freund ' s adjuvant is saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueriri) and Corynebacterium parvum.
  • the adjuvant is pharmaceutically acceptable.
  • PBS phosphate- buffered saline
  • mAb monoclonal antibody
  • SPF specific pathogen-free
  • PMSF phenylmethylsulfonyl fluoride
  • DIFP diisopropyl fluorophosphonate
  • FACE fluorophore-assisted carbohydrate electrophoresis.
  • the present invention contemplates isolation of a gene encoding a functional portion of a DEC receptor of the invention, including a full length, or naturally occurring form of DEC, and any antigenic fragments thereof from any animal, particularly mammalian or avian, and more particularly human, source.
  • the term "gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.
  • a specific nucleotide sequence of a human DEC-encoding DNA is provided (SEQ ID NO:8); also provided are deduced coding sequences for both murine and human DEC polypeptides having the amino acid sequences depicted in SEQ ID NO:3 and SEQ ID NO:8, respectively, are provided.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”) in either single stranded form, or a double- stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g. , restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA. genomic DNA, or RNA. when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al.. supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T m of 55°. can be used. e.g.. 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS).
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • RNA:RNA, DNA:RNA, DNA:DNA The relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al., supra, 9.50-0.51).
  • the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 1 1.7-1 1.8).
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; more preferably at least about 15 nucleotides; most preferably the length is at least about 20 nucleotides.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g. , mammalian) DNA. and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Expression control sequences e.g., transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control of or “operatively associated with” a transcriptional and translational control sequence in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • a “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.
  • the term "translocation signal sequence” is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.
  • sequence homology in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667).
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that do not share a common evolutionary origin (see Reeck et al., supra).
  • Two DNA sequences are "substantially homologous” or “substantially similar” when at least about 75% (preferably at least about 80%. and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al. supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • amino acid sequences are "substantially homologous" or
  • substantially similar when greater than 70% of the amino acids are identical, or functionally identical.
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison. Wisconsin) pileup program.
  • a gene encoding DEC can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining DEC gene are well known in the art, as described above (see, e.g., Sambrook et al., 1989, supra). In specific embodiment, infra, a cDNA encoding murine DEC-205 is isolated from a dendritic cell library. In addition, probes derived from the murine gene were used to isolate the corresponding human dec cDNA and the murine genomic dec gene.
  • any animal cell potentially can serve as the nucleic acid source for the molecular cloning of a dec gene.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g. , a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein (e.g., a dendritic cell cDNA or thymic epithelial cDNA library, since these are the cells that evidence highest levels of expression of DEC), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example.
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • DNA fragments are generated, some of which will encode the desired gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA. or the DNA can be physically sheared, as for example, by sonication.
  • the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography. Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired dec gene may be accomplished in a number of ways.
  • the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).
  • a set of oligonucleotides corresponding to the partial amino acid sequence information obtained for the DEC protein can be prepared and used as probes for DNA encoding DEC, as was done in a specific example, infra, or as primers for cDNA or mRNA (e.g., in combination with a poly-T primer for RT-PCR).
  • a fragment is selected that is highly unique to DEC of the invention. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used.
  • the human dec cDNA was cloned using a 300 base-pair probe derived from the 3' coding sequence of murine dec cDNA.
  • the human cDNA was obtained from a B lymphoma library, using high stringency hybridization conditions (0.1 SSC, 65°C). Thus, high stringency hybridization conditions are favored to identify a homologous dec gene from other species.
  • cDNA clones, or DNA clones which hybrid-select the proper mRNAs can be selected which produce a protein that, e.g.. has similar or identical electrophoretic migration, isoelectric focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for DEC.
  • the rabbit polyclonal antibody to murine DEC. described in detail infra can be used to confirm expression of DEC. both murine 28 and human counte ⁇ arts.
  • a protein that has an apparent molecular weight of 205 kDa, and which is specifically digested to form a defined ladder (rather than a smear) of lower molecular weight bands is a good candidate for DEC.
  • a dec gene of the invention can also be identified by mRNA selection, i.e., by nucleic acid hybridization followed by in vitro translation.
  • nucleotide fragments are used to isolate complementary mRNAs by hybridization.
  • DNA fragments may represent available, purified dec DNA, or may be synthetic oligonucleotides designed from the partial amino acid sequence information.
  • Immunoprecipitation analysis or functional assays e.g. , tyrosine phosphatase activity
  • specific mRNAs may be selected by adso ⁇ tion of polysomes isolated from cells to immobilized antibodies specifically directed against DEC. such as the rabbit polyclonal anti-murine DEC antibody described herein.
  • a radiolabeled dec cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template.
  • the radiolabeled mRNA or cDNA may then be used as a probe to identify homologous dec DNA fragments from among other genomic DNA fragments.
  • the present invention also relates to cloning vectors containing genes encoding analogs and derivatives of DEC of the invention, that have the same or homologous functional activity as DEC, and homologs thereof from other species.
  • the production and use of derivatives and analogs related to DEC are within the scope of the present invention.
  • the derivative or analog is functionally active, i.e.. capable of exhibiting one or more functional activities associated with a full-length, wild-type DEC.
  • a DEC protein of the invention can be prepared by substituting a lectin domain or domains from another protein, such as the mannose receptor of macrophages or the phospholipase receptor on muscle, for those found in DEC 205.
  • DEC derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Preferably, derivatives are made that have enhanced or increased functional activity relative to native DEC. Alternatively, such derivatives may encode soluble fragments of DEC extracellular domain that have the same or greater affinity for the natural ligand of DEC of the invention. Such soluble derivatives may be potent inhibitors of ligand binding to DEC.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as a dec gene may be used in the practice of the present invention. These include but are not limited to allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of dec genes which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • the DEC derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a DEC protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine. leucine, isoleucine. valine. proline. phenylalanine. tryptophan and methionine.
  • the polar neutral amino acids include glycine. serine, threonine. cysteine, tyrosine, asparagine. and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis. or isoelectric point.
  • the genes encoding DEC derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned DEC gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the DEC-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • mutations enhance the functional activity of the mutated DEC gene product.
  • deletion mutants can be produced that encode fragments of DEC, e.g., one or a few of the lectin domains (see Taylor et al., 1992. J. Biol. Chem. 267:1719). Any technique for mutagenesis known in the art can be used, including but not limited to. in vitro site-directed mutagenesis (Hutchinson. C. et al.. 1978. J.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can. for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid. which provides for expansion in a cloning cell, e.g., E.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2 ⁇ plasmid.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation. can be done before insertion into the cloning vector.
  • Expression of DEC Polvpeptides The nucleotide sequence coding for DEC, or antigenic fragment, derivative or analog thereof, or a functionally active derivative, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, . e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • nucleic acid encoding DEC of the invention is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding DEC and/or its flanking regions.
  • Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g.. baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA. plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g.. baculovirus
  • microorganisms such as yeast containing yeast vectors
  • bacteria transformed with bacteriophage DNA. plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant DEC protein of the invention, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).
  • the cell into which the recombinant vector comprising the nucleic acid encoding DEC is cultured in an appropriate cell culture medium under conditions that provide for expression of DEC by the cell.
  • Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).
  • DEC protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used to control DEC gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon. 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al.. 1980. Cell 22:787-797), the he ⁇ es thymidine kinase promoter (Wagner et al.. 1981. Proc. Natl. Acad. Sci.
  • prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci.
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter.
  • PGK phosphoglycerol kinase
  • alkaline phosphatase promoter promoter
  • animal transcriptional control regions which exhibit tissue specificity and have been utilized in transgenic animals.
  • Expression vectors containing a nucleic acid encoding a DEC of the invention can be identified by four general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, and (d) expression of inserted sequences.
  • the nucleic acids can be amplified by PCR to provide for detection of the amplified product.
  • the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., ⁇ -galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector.
  • selection marker e.g., ⁇ -galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
  • recombinants containing the DEC insert can be identified by the absence of the DEC gene function.
  • recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation.
  • the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g. , lambda), and plasmid and cosmid DNA vectors, to name but a few.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification ⁇ e.g.. glycosylation. cleavage [e.g.. of signal sequence]) of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system can be used to produce an nonglycosylated core protein product.
  • the transmembrane DEC protein expressed in bacteria may not be properly folded.
  • Expression in yeast can produce a glycosylated product, although the pattern of glycosylation will likely differ from that obtained by expression in a mammalian cell.
  • Expression in eukaryotic cells can increase the likelihood of "native" glycosylation and folding of a heterologous protein.
  • expression in mammalian cells such as Chinese hamster ovary (CHO), African Green Monkey COS cells, and fibroblast NIH-3T3 cells (e.g., 293 cells), can provide a tool for reconstituting, or constituting, DEC activity.
  • different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.
  • DEC is introduced into model epithelial cells, such as Madin-Darby canine kidney (MDCK) cells, for investigation of the efficacy and rate of trans-epithelial migration of ligands or molecules targeted to DEC.
  • model epithelial cells such as Madin-Darby canine kidney (MDCK) cells
  • the dec gene can be introduced into epithelial or dendritic cells for gene therapy, either by in vivo or ex vivo gene transfer.
  • Vectors are introduced into the desired host cells by methods known in the art. e.g. , transfection, electroporation, microinjection, transduction. cell fusion. DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g. , Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988. J. Biol. Chem. 263: 14621-14624; Hartmut et al.. Canadian Patent Application No. 2.012.31 1, filed March 15, 1990).
  • a recombinant DEC protein expressed as an integral membrane protein can be isolated and purified by standard methods.
  • the integral membrane protein can be obtained by lysing the membrane with detergents, such as but not limited to, sodium dodecyl sulfate (SDS), Triton X-100, Nonidet P-40 (NP-40), digoxin, sodium deoxycholate, and the like, including mixtures thereof. Solubilization can be enhanced by sonication of the suspension.
  • detergents such as but not limited to, sodium dodecyl sulfate (SDS), Triton X-100, Nonidet P-40 (NP-40), digoxin, sodium deoxycholate, and the like, including mixtures thereof. Solubilization can be enhanced by sonication of the suspension.
  • infra, DEC-205 is solubilized from thymic membrane pellets in a buffer containing 0.5% NP-40.
  • Soluble forms of the protein can be obtained by collecting culture fluid, or solubilizing inclusion bodies, e.g., by treatment with detergent, and if desired sonication or other mechanical processes, as described above.
  • the solubilized or soluble protein can be isolated using various techniques, such as polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2- dimensional gel electrophoresis, chromatography (e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography), centrifugation, differential solubility, immunoprecipitation, or by any other standard technique for the purification of proteins.
  • PAGE polyacrylamide gel electrophoresis
  • isoelectric focusing e.g., isoelectric focusing
  • 2- dimensional gel electrophoresis e.g., ion exchange, affinity, immunoaffinity, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, immunoaffinity, and sizing column
  • the recombinant DEC product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis. immunoassay, etc.
  • the ability of the expressed protein, or a fragment comprising the cytoplasmic domain thereof, to mediate endocytosis and targeting to coated pits, and thence to endocytic vesicles associated with Class II MHC processing can be determined.
  • endocytosis was evaluated by electron microscopy, using an anti-DEC antibody and a gold-labeled secondary antibody reactive with the anti-DEC antibody.
  • the ability to process and present antigen is evaluated by assaying antibody-specific T cell proliferation in response to processing of anti-DEC antibody and a control non-specific antibody.
  • the structure of DEC of the invention can be analyzed by various methods known in the art.
  • the structure of the various domains, particularly the lectin binding and cytoplasmic domains is analyzed.
  • Structural analysis can be performed by identifying sequence similarity with other known proteins. The degree of similarity (or homology) can provide a basis for predicting structure and function of DEC, or a domain thereof.
  • sequence comparisons can be performed with sequences found in GenBank. using, for example, the FASTA and FASTP programs (Pearson and Lipman. 1988. Proc. Natl. Acad. Sci. USA 85:2444-48).
  • the protein sequence can be further characterized by a hydrophilicity analysis (e.g., Hopp and Woods, 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824).
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the DEC protein.
  • Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art.
  • the present invention enables quantitative structural determination of DEC. or domains thereof.
  • enough material is provided for nuclear magnetic resonance (NMR). infrared (IR). Raman, and ultraviolet (UV). especially circular dichroism (CD), spectroscopic analysis.
  • NMR nuclear magnetic resonance
  • IR infrared
  • UV ultraviolet
  • CD circular dichroism
  • NMR provides very powerful structural analysis of molecules in solution, which more closely approximates their native environment (Marion et al., 1983, Biochem. Biophys. Res. Comm. 1 13:967-974; Bar et al., 1985, J. Magn. Reson. 65:355-360; Kimura et al., 1980, Proc. Natl. Acad. Sci. U.S.A. 77:1681-1685).
  • Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, A., 1974, Biochem. Exp. Biol. 1 1 :7-13).
  • co-crystals of DEC and a DEC-specific ligand can be studied. Analysis of co-crystals provides detailed information about binding, which in turn allows for rational design of ligand agonists and antagonists.
  • Computer modeling can also be used, especially in connection with NMR or X-ray methods (Fletterick, R. and Zoller, M. (eds.), 1986. Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • a putative DEC of the invention can be tested to determine whether it cross-reacts with an antibody specific for murine DEC-205.
  • the putative DEC can be reacted with a rabbit polyclonal antibody, as described in the Example, infra, to determine whether it binds.
  • a DEC protein can be used to generate antibodies, which can be tested for cross reactivity with DEC-205 from mice or human sources. The degree of cross reactivity provides information about structural homology or similarity of proteins.
  • the carbohydrate composition of DEC can be studied by various means known in the art, including but not limited to, lectin binding, chemical analysis, immunoassay.
  • immunochemical analysis e.g.. by converting glycoconjugates to digoxigenin-labeled hydrazones after periodate oxidation of v c-diols
  • chemical deglycosylation e.g.. by converting glycoconjugates to digoxigenin-labeled hydrazones after periodate oxidation of v c-diols
  • chemical deglycosylation e.g. by converting glycoconjugates to digoxigenin-labeled hydrazones after periodate oxidation of v c-diols
  • enzymatic deglycosylation e.glycosylation
  • exoglycosidase digestions followed by FACE (fluorophore-assisted carbohydrate electrophoresis) analysis e.g. by
  • Ligands for DEC Most importantly, the present invention advantageously provides for identifying ligands of DEC, e.g., carbohydrate ligands that bind to one or more of the lectin domains of DEC. Such ligands are especially useful for targeting binding to DEC.
  • ligand has its ordinary meaning, i.e., a molecule capable of specifically binding to a receptor, in this case DEC.
  • carbohydrate ligand refers to a carbohydrate or sugar that is capable of specifically binding DEC.
  • such carbohydrates alternatively termed herein “glycans,” “saccharides,” or “oligosaccharides,” are the carbohydrate portion of a glycoprotein.
  • Identification and isolation of a gene encoding DEC of the invention provides for expression of the receptor, or truncated portions thereof, in quantities greater than can be isolated from natural sources, in recombinant cells for classical receptor binding experiments, or in indicator cells that are specially engineered to indicate the activity of a receptor expressed after transfection or transformation of the cells. According, the present invention contemplates identifying specific ligands DEC using various screening assays known in the art.
  • the recombinantly expressed protein can comprise one or more DEC lectin domains, and may be a truncated form of the native protein or portions of the native protien expressed as a chimeric construct with another protein.
  • the present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to DEC in vivo.
  • the present invention provides for identification of carbohydrate groups that bind DEC, and more specifically, identification of carbohydrate groups that bind DEC with high affinity and specificity.
  • detection of DEC ligands is accomplished by binding solubilized DEC or DEC fragments to columns prepared from sugars or glycans conjugated to a solid phase support, such as SEPHAROSE (Taylor et al., 1992, J. Biol. Chem. 267:1719).
  • a solid phase support such as SEPHAROSE (Taylor et al., 1992, J. Biol. Chem. 267:1719).
  • the invention contemplates dissecting the ligand specificity of various of the lectin domains by expressing truncated mutant DEC proteins comprising only one or a few of the domains.
  • candidate glycans can be conjugated to a carrier protein, such as bovine serum albumin, which is labelled, e.g., with 125 I, and binding detected to DEC or DEC fragments expressed by a cell, such as a recombinant cell as described supra (Taylor et al., supra).
  • a carrier protein such as bovine serum albumin
  • binding detected to DEC or DEC fragments expressed by a cell such as a recombinant cell as described supra (Taylor et al., supra).
  • binding of labeled glycan-carrier protein is evaluated in microtiter assays, as described (Taylor and Drickamer, 1993. J. Biol. Chem. 268:399).
  • Candidate carbohydrate ligands include, but are not limited to, mannose, fucose, N-acetyl-glucosamine, glucose, galactose, N-acetyl-galactosamine, to mention but a few such carbohydrates.
  • Other ligand candidates include disaccharides, and larger order polysaccharides, e.g., such as are recognized by various lectins.
  • the term "detection of binding” refers to any of the miriad techniques commonly employed to detect the association of one molecule with another, i.e., DEC-ligand with DEC. These techniques include the immunoassay techniques discussed infra, or modifications thereof, and generally depend on detecting association of a label conjugated with one of the binding entities, either the DEC-lectin containing polypeptide or the candidate ligand, with the other entity, which may be found on a solid phase support or a cell. However, detection of binding can be accomplished indirectly, by detecting the absence of a labeled binding entity, e.g.. from supernatant.
  • binding can be detected by first removing unbound substances, followed by removing the labeled entity (e.g. , using a chaotropic agent) from the bound pair.
  • labeled entity e.g. , using a chaotropic agent
  • one entity of the binding pair will be irreversibly associated with a solid phase support, such as a bead (e.g..
  • latex particle latex particle, chromatographic support, magnetic particle, silica particle, silicon wafer, or a plastic microtiter plate.
  • the term "irreversibly associated” refers to covalent or non-covalent binding, characterized by no dissociation, or a rate of dissociation that is so low in comparison to the assay time that it is virtually undetectable.
  • identification of carbohydrate ligands for DEC will be accomplished by attaching known glycans to a protein such as the classic neo- glycoprotein. bovine serum albumin, or ovalbumin. or creatinase.
  • a protein such as the classic neo- glycoprotein. bovine serum albumin, or ovalbumin. or creatinase.
  • the binding assay may comprise a classical binding assay, as described above, or may involve an antigen processing assay, by evaluating stimulation of antigen-specific T lymphocytes.
  • T cell lines and clones specific for BSA and ovalbumin are available: the ability of neo-glycosylated BSA or ovalbumin to efficiently stimulate specific T cell proliferation is indicative of the ability of the glycan conjugated to the BSA or ovalbumin to bind to DEC.
  • the heavily glycosylated protein fetuin. present in fetal calf serum can be used to evaluate glycan ligands.
  • a fetuin binding system based on T cell activation or endocytosis of a marker, can be developed.
  • Specific glycosidases can be used to specifically "knock-out" glycans. and the ability of the modified fetuin to function in the binding system evaluated. Diminishment of functional activity would indicate that the enzymatically modified sugar residue was involved in binding to DEC.
  • the ninth and tenth lectin domains of DEC may be involved in membrane associated antibody-mediated antigen presentation, by "chaperoning" antibody into endosomes, suggests that these domains are specific for binding carbohydrates found on cell surface immunoglobulin molecules.
  • synthetic libraries (Needels et al., 1993, "Generation and screening of an oligonucleotide encoded synthetic peptide library," Proc. Natl. Acad. Sci. USA 90: 10700-4; Lam et al., International Patent Publication No. WO 92/00252 and U.S. Patent No. 5,382,513. issued January 17, 1995. each of which is incorporated herein by reference in its entirety), and the like can be used to screen for ligands according to the present invention.
  • assays for binding of soluble ligand to cells that express recombinant forms of a ligand binding domain or domains (preferably domains) of DEC can be performed.
  • a ligand binding domain or domains (preferably domains) of DEC can be performed.
  • the presence of multiple lectin domains on DEC may contribute to the affinity and specificity of binding to glycans.
  • the screening can be performed with recombinant cells that express the DEC, or alternatively, using purified receptor protein, e.g., produced recombinantly, as described above.
  • purified receptor protein e.g., produced recombinantly
  • the ability of labeled, soluble or solubilized DEC that includes the ligand-binding portion of the molecule, to bind ligand can be used to screen libraries, as described in the foregoing references.
  • DEC produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins may be used as an immunogen to generate antibodies that recognize DEC.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • a rabbit polyclonal antibody is prepared against the N-terminal amino acid sequence of DEC-205.
  • a polyclonal antibody against intact, purified, DEC-205 was generated.
  • DEC non-allogeneic DEC
  • a derivative e.g.. fragment or fusion protein
  • the DEC or fragment thereof can be conjugated to an immunogenic carrier, e.g.. bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • Various adjuvants be used to increase the immunological response, depending on the host species, as described above.
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983. Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT US90/02545).
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026- 2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).
  • human hybridomas Cote et al., 1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026- 2030
  • transforming human B cells with EBV virus in vitro Cold-e et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96.
  • techniques developed for the production of "chimeric antibodies” (Morrison et al., 1984, J. Bac
  • Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab')-, fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays.
  • immunoradiometric assays gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of DEC. one may assay generated hybridomas for a product which binds to a DEC fragment containing such epitope. For selection of an antibody specific to DEC from a particular species of animal, one can select on the basis of positive binding with DEC expressed by or isolated from cells of that species of animal, and the absence of binding to DEC from other species. Binding to DEC may be detected as binding to dendritic cells that express DEC.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the DEC, e.g., for Western blotting, imaging DEC in situ, measuring levels thereof in appropriate physiological samples, etc.
  • the antibodies of the present invention advantageous provide for detecting and enumerating human dendritic cells.
  • such antibodies can be used to isolate human dendritic cells, e.g. , by panning.
  • the antibodies of the invention can be used to target molecules to human dendritic cells. It will be recognized that this is a significant advantage, since the prior art antibody of Kraal et al. failed to recognize human DEC.
  • Antibodies that are targeted to DEC and participate in the activity of DEC can be generated. Such antibodies can be tested using the assays described supra for identifying ligands.
  • a rabbit polyclonal anti-DEC antibody targets binding of DEC, is endocytosed. and is efficiently presented to immunoglobulin-specific T cells.
  • Targeting Molecules to DEC advantageously provides for targeting molecules to DEC for immune modulation, e.g., stimulation of T cell immunity, suppression immunity or induction of T cell anergy, and clonal deletion mechanism; trans-epithelial transport, with delivery of a molecule across epithelium into the pulmonary circulation or intestinal circulation, or from the bloodstream into the pulmonary or intestinal lumen; and crossing the blood brain barrier.
  • a ligand for DEC as described supra, or an antibody reactive with DEC (or a DEC-binding portion thereof), as described supra, is conjugated to a molecule which is to be targeted to DEC.
  • Immunomodulation With respect to immunomodulation. the present invention provides for both stimulating T cell-mediated immune responses, particularly for vaccination, and inducing tolerance, particularly with respect to autoimmunity.
  • Stimulation of T cell immunity can be effected by introducing an antigen, e.g.. ma weak or poorly immunogenic antigen, conjugated to a DEC-binding moiety (ligand or antibody) into a subject, along with a factor that activates the dendritic cells that initially present antigen to the T cells.
  • Dendritic cell activation can be accomplished by use of an adjuvant, such as an ' adjuvant as described above, which has the ability to induce a generalized immune response.
  • the "vaccine" of the invention may comprise the antigen conjugated to the DEC- binding moiety and a cytokine or a lymphokine, such as granulocyte-macrophage colony stimulating factor (GM-CSF), or some other CSF.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • Suitable antigens for use in such a vaccine include bacterial, viral, parasite, and tumor antigens.
  • the present invention provides for inducing tolerance.
  • Tolerance is desirable to avoid detrimental immune responses, in particular, autoimmunity and allograft rejection.
  • tolerance is induced by administering an antigen modified by conjugation with a DEC-binding moiety under conditions that promote dendritic cell quiescence, e.g. , in the absence of an infection, without adjuvant, using pyrogen-free pharmaceutical carriers, and in the absence of additional lymphokines or cytokines.
  • the invention further relates to introducing recombinant dendritic cells, or cell recombinantly modified to express both DEC and MHC Class II, into a subject, along with antigen conjugated to a DEC-binding moiety.
  • the dec gene can be targeted to appropriate cells in vivo, for gene therapy.
  • tolerance can be induced through the clonal deletion mechanism.
  • antigen conjugated with a DEC-binding moiety can be introduce into a subject, preferably directly into the thymus, either by targeting or physical injection, for processing and presentation by the thymic epithelium and medullary dendritic cells. This processing and presentation step is believed to be involved in the selection process to eliminate autoreactive T cells, i.e.. clonal deletion.
  • the level of expression of DEC may be manipulated. e.g., by introducing additional dec genes into the thymic epithelium and medullary dendritic cells.
  • Attractive candidates for conjugation with a DEC-ligand to induce tolerance, T cell anergy, or clonal deletion include, but by no means are limited to, allergenic substances, autoantigens such as myelin basic protein, collagen or fragments thereof, DNA, nuclear and nucleolar proteins, mitochondrial proteins, pancreatic ⁇ - cell proteins, and the like (see Schwarz, 1993, In Fundamental Immunology, Third Edition, W.E. Paul (Ed.), Raven Press, Ltd.: New York, pp. 1033-1097).
  • a molecule can be targeted for trans-epithelial migration by conjugating it with a DEC-binding moiety.
  • the invention provides for targeting a therapeutic molecule for abso ⁇ tion across lung or intestinal epithelium.
  • the invention provides for delivering an aerosolized therapeutic agent by inhalation, i.e., by pulmonary administration of the drug.
  • the invention provides for delivery of a therapeutic agent by DEC-mediated abso ⁇ tion across the small intestine.
  • the invention advantageously provides for abso ⁇ tion, or more accurately, trans-mucosal migration, of hydrophilic molecules, which are usually not as easily absorbed as hydrophobic molecules. This aspect of the invention takes advantage of the presence of DEC on the apical (or lumenal) surface of the epithelial cells.
  • the presence of DEC on the basolateral surface of the epithelial cells provides a route for transport of a molecule conjugated to a DEC-ligand from the bloodstream into the lumen of the lung or the small intestine.
  • This delivery route can be very important for administration of an acid labile, hydrophilic therapeutic agent to the intestines.
  • Such a drug cannot be ingested, as the acid conditions present in the stomach would result in its destruction; transport of such a drug from the bloodstream to the lumen of the intestines would not readily occur spontaneously, since a hydrophilic agent ' does not have a significant partition coefficient across cell membranes.
  • the present invention provides for administration of chemotherapeutic agents and antibiotics, particularly anti-parasite drugs, by conjugating them to a ligand for DEC. administering the agent parenterally, preferably intravenously, such that the drug is targeted for transport from the basolateral surface of the intestinal epithelium to the lumenal surface.
  • a therapeutic agent may be targeted for delivery from the bloodstream to the airways of the lung by targeting the DEC receptor on the basolateral surface of the lung epithelium.
  • a delivery system would be particularly advantageous for delivery of drugs to individuals with impaired lung capacity, e.g., who cannot inhale adequately, and thus, for whom administration via the bloodstream is indicated.
  • lung impairments include, but are not limited to, pneumonia, emphysema, lung cancer, adult respirator distress syndrome, dyspnea, hemoptysis, chronic obstructive pulmonary disease (COPD), fibrogenic dust diseases, pulmonary fibrosis, organic dust diseases, chemical injury, smoke injury, thermal injury (burn or freeze), asthma (allergy, bronchoconstriction.
  • the invention provides for administration of antibiotics, anti-inflammatory agents, complement inhibitors (e.g., complement receptor 1 [CD35]), and the like for trans-epithelial migration into the lumen of the lung.
  • complement inhibitors e.g., complement receptor 1 [CD35]
  • a molecule targeted for the brain can be conjugated to a DEC-binding moiety. The molecule would then bind to DEC found in the capillaries of the brain, which are believed to promote trans-blood brain barrier transport or migration.
  • Such molecules for transport across the blood brain barrier include, but are not limited to. neurotrophic factors (brain-derived neurotrophic factor. NT- 3. NT-4. ciliary neurotrophic factor), growth factors (e . nerve growth factor), and the like: antibiotic or antiviral agents, for incipient infections of the brain: and vectors for gene therapy. 50
  • the present invention provides ligands for targeting DNA vectors to cells that express DEC, in particular, dendritic cells, epithelial cell of the thymus, small intestine, and lung, and brain capillaries.
  • a DNA vector such as a viral vector
  • DNA virus vectors include, but are not limited to, a defective he ⁇ es virus 1 (HSV1) vector (Kaplitt et al., 1991 , Molec. Cell. Neurosci.
  • an attenuated adenovirus vector such as the vector described by Stratford-Perricaudet et al. (1992, J. Clin. Invest. 90:626-630).
  • a defective adeno-associted virus vector (Samulski et al., 1987, J. Virol. 61 :3096-3101 ; Samulski et al.. 1989, J. Virol. 63:3822-3828), as well as a papillomavirus vector, Epstein Barr virus (EBV) vector, and the like.
  • the viral particles can be modified to include a ligand for DEC, e.g., by chemically cross-linking a DEC ligand to the virus.
  • the vector can be introduced in vivo by lipofection.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding DEC (Feigner, et. al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417; see Mackey, et al.. 1988. Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031)).
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, 1989. Science 337:387-388).
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. Accordingly, the present invention advantageously provides for targeting a gene for dendritic cells and thymic epithelium by conjugating a DEC-ligand to a liposome vector.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (.see Mackey. et. al., 1988. supra).
  • Targeted antibodies or glycans could be coupled to liposomes chemically. It is also possible to introduce the vector in vivo as a naked DNA plasmid, preferably by using a DEC ligand as a vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988. J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,31 1, filed March 15, 1990).
  • a DEC ligand as a vector transporter
  • EXAMPLE 1 DEC-205, A 205 kDa PROTEIN ABUNDANT ON MOUSE
  • This Example describes the purification and biochemical characterization of the antigen recognized by monoclonal antibody NLDC-145 (Krall et al., 1986, J. Exp. Med. 163:981).
  • the protein has been purified at a scale that permits direct biochemical study.
  • the antigen proves to be an integral membrane glycoprotein with a mildly alkaline (pi 7.5) and an electrophoretic molecular mass of 205 kDa, not 145 kDa. as originally reported (Kraal et al., supra).
  • NLDC-145 rat IgG2a ascitic fluids were prepared in normal, 6-8 week old. non-SPF, CD2 (BALB/c x DBA/2 FI ) female mice (Trudeau Institute) as described (North and Izzo, 1993, J. Exp. Med. 177: 1723).
  • the monoclonal was purified by sequential chromatography on immobilized Protein A and Protein G (Pierce). Both the monoclonal and nonspecific rat IgG2a (Zymed) were coupled to resins by reductive animation (AminoLink, Pierce).
  • BMDC Bone marrow dendritic cells
  • BMDC BMDC were lysed by resuspending them in 700 ml of ice-cold lysis buffer (50 mM Tris-HCl, pH 6.8, 1% Nonidet P-40 (Calbiochem), 50 mg/ml BSA (Intergen), with a mixture of protease inhibitors: 5 mM EDTA, 0.5 mg/ml Pefabloc SC (Boehringer Mannheim), 100 mg/ml PMSF. 5 mg/ml aprotinin, 5 mg/ml pepstatin A and 10 mg/ml leupeptin (the latter 4 inhibitors from Sigma)). Lysates were precleared with 20 mg of rat IgG (Jackson ImmunoResearch).
  • Immunoblotting SDS-PAGE was performed in 8% acrylamide minigels, 1.5 mm thick. Transfer to nitrocellulose (BA-85, Schleicher and Schuell) was performed at 30 constant volts overnight at 4°C. Filters were blocked in PBS containing 3% (w/v) nonfat dry milk and 0.1 % Tween 20 for 1 h at room temperature with shaking. Incubation with primary antibodies (0.1-10 ⁇ g/ml of purified IgG, ascites or serum diluted 1 :1000, or hybridoma supernatant diluted 1 :1) was performed in heat-sealed bags for 1 h at room temperature.
  • primary antibodies 0.1-10 ⁇ g/ml of purified IgG, ascites or serum diluted 1 :1000, or hybridoma supernatant diluted 1 :1 was performed in heat-sealed bags for 1 h at room temperature.
  • Thymi were removed from 50 outbred CD-I Swiss mice (Taconic) per preparation. Thymi were placed into 50 ml of ice-cold PBS containing 200 mg/ml PMSF and 5 mM EDTA to remove blood, and washed once with the same buffer. Washed organs could be frozen at -20°C. All subsequent purification steps were performed at 0-4°C. Thymi were transferred to a 40 ml Dounce homogenizer
  • hypotonic lysis buffer 10 mM Tris- HCl, pH 6.8. with a mixture of protease inhibitors: 5 mM EDTA. 100 mg/ml PMSF, 4 mg/ml aprotinin, 0.5 mg/ml Pefabloc SC, 4 mg/ml pepstatin A, 10 ⁇ g/ml leupeptin).
  • Organs were homogenized with 20 strokes of the loose (0.2 mm clearance) pestle, then 20 strokes of the tight (0.1 mm clearance) pestle. The suspension was left on ice for 20 min, then re-homogenized with an additional 20 strokes of the tight pestle.
  • Clarified membrane extract was precleared by passage over a nonspecific rat IgG column. Nonadsorbed fractions from the preclearing column were pooled, then applied to the NLDC-145 affinity column. Washes were performed in 2 steps: 6 ml (3 bed volumes) of wash- 1 (hypotonic lysis buffer with 0.5 M NaCl, without NP-40, substituting 0.5% (17 mM) n-octyl glucoside (Boehringer Mannheim)), then 10 ml of wash- 2 (wash-1 without added NaCl).
  • wash- 1 hypertonic lysis buffer with 0.5 M NaCl, without NP-40, substituting 0.5% (17 mM) n-octyl glucoside (Boehringer Mannheim)
  • the column was eluted with at least 5 bed volumes of 50 mM glycine-NaOH, pH 1 1, 0.5% n-octyl glucoside, reducing the maximum flow rate to 10 ml cm "2 hr ' 1 .
  • the pH of eluted fractions (1 ml) was adjusted to 7 with 20-30 ml of 2 M glycine-HCl, pH 2. Peak eluates were pooled and concentrated to ⁇ 1 ml by ultrafiltration in Centricon- 100 units (Amicon) that had been pre-coated with 0.1% SDS, to reduce nonspecific losses to the plastic.
  • 70-150 ⁇ g of DEC-205 could be obtained from 50 thymi.
  • Isoelectric focusing Isoelectric focusing was performed in thin (0.75 mm) slab gels, under denaturing conditions (5.5% acrylamide gels containing 8 M urea. 4% total Ampholine (2:1 ratio of pH 3.5-10 and 5-7, Pharmacia), 0.67% NP-40, 10% glycerol). Samples were focused at 400 constant volts overnight, for a minimum of 6000 volt-hours, at which time the current was less than 1 mA. Lanes were either silver-stained or cut into 0.5 cm sections and eluted into degassed dH 2 O for pH gradient measurement.
  • glycans DEC-205, transferrin (positive control) and creatinase (negative control) were blotted onto nitrocellulose as before. Glycoconjugates were converted to digoxigenin (DIG)-labeled hydrazones after mild nonselective periodate oxidation of v/c-diols to aldehydes. Staining patterns were visualized with an anti-DIG antibody conjugated to alkaline phosphatase (First CHOice, Boehringer Mannheim).
  • DIG digoxigenin
  • PNGase F Flavobacterium meningosepticum
  • PNGase F Boehringer Mannheim
  • Lectin blotting Several digoxigenin-labeled plant lectins (Boehringer Mannheim) were used to stain electroblotted DEC-205 and appropriate positive and negative control glycoproteins. The lectins, their specificities and the concentrations used for staining are summarized in Table 1.
  • Exoglycosidase digestions and FACE analysis N-linked oligosaccharides were released from DEC-205 with PNGase F and labeled with the fluorophore ANTS (8- aminonaphthalene-l,3.6-trisulfonic acid) (Jackson. 1990, supra; Jackson and Williams, supra; Jackson, 1993, supra; Jackson, 1994, supra). Recombinant exoglycosidases were from Glyko. Electrofluorograms were visualized on an SE1000 FACE workstation (Glyko).
  • Amino acid sequencing— DEC-205 was electrophoresed in multiple lanes of 1.5 mm thick 4% minigels prepared using Duracryl (Millipore). Gels were blotted onto polyvinylidene difluoride (PVDF, Bio-Rad). After transfer, filters were soaked for 1 min in 1% acetic acid, stained for 2 min in 0.1% Ponceau S, then were destained briefly in dH 2 O. Bands at 205 kDa were excised and submitted for analysis. The N-terminal sequence was aligned to all current databases on the BLAST Internet servers (NCBI, National Library of Medicine, NIH), running the program BLASTP (Altschul et al., 1990, J. Med. Biol. 215:403).
  • NCBI National Library of Medicine
  • Polyclonal antibodies to intact DEC-205 Two New Zealand White rabbits (Hazelton) were injected 6 times with the 205 kDa bands cut from Coomassie- stained, 1.5 mm thick, 4% Duracryl SDS-PAGE gels. Doses ranged from 40-70 ⁇ g of stained protein per animal, per injection (4-6 slices), and were given every 3 weeks, with test bleeds (about 15 ml of serum) taken 2 weeks post-injection. For the first injection, slices were emulsified in Complete Freund's adjuvant (CFA) and injected intradermally into multiple sites on the back. Incomplete Freund's (IFA) was the adjuvant for boosts.
  • CFA Complete Freund's adjuvant
  • IFA Incomplete Freund's
  • the KLH-peptide conjugate was divided into aliquots of 400-500 ⁇ g each, and was injected eight times into two New Zealand White rabbits (200-250 ⁇ g per injection), again emulsifying into CFA for the initial immunization and IFA for boosts. To remove any anti-KLH reactivity from the sera, they were precleared on a KLH-cysteine column. Anti- peptide antibodies were isolated on a peptide-OVA column, where the peptide was coupled to an irrelevant carrier.
  • NLDC-145 was used as the probe in a Western blot.
  • Five thymi from 8 week-old BALB/c mice were homogenized in 2 ml of the same lysis buffer used for immunoprecipitation.
  • Graded doses of clarified thymic extract were electrophoresed under nonreducing and reducing conditions, and blotted to nitrocellulose.
  • NLDC-145 bound a single major band (Figure I B) that co-migrated with the prestained myosin marker at 205 kDa. confirming the estimate made by immunoprecipitation (above). Under nonreducing conditions (Figure I B.
  • DEC-205 is an integral membrane protein with an isoelectric point of 7.5— To determine whether the 205 kDa protein was an integral membrane protein, thymic membrane pellets (prepared as in Figure 2) were resuspended in: (1 ) hypotonic lysis buffer containing 0.5% NP-40 (as usual); (2) the same buffer containing 1 M KC1 instead of detergent; or (3) 100 mM Na,CO 3 pH 1 1.5, containing all 6 protease inhibitors used in routine purifications. After one hour of gentle mixing, suspensions were clarified (100,000 x g, 60 min, 4°C), and supernatants were collected.
  • DEC-205 is a glycoprotein, bearing heterogeneous N-linked glycans — To determine whether DEC-205 was glycosylated, purified 205 kDa protein was electrophoresed on a gel which also contained samples of transferrin (a known glycoprotein) and creatinase (a known nonglycoprotein), and was electroblotted to nitrocellulose.
  • blotted purified material was probed with a panel of digoxigenin-labeled plant lectins. Asparagine-linked glycans were removed from an aliquot of DEC-205 by treating it with peptide N-glycosidase F (PNGase F). Treated and untreated protein was blotted to nitrocellulose along with positive and negative control glycoproteins. After confirming transfer by staining the filters with Ponceau S (not shown), membranes were blocked and stained with DIG-lectins, used at the concentrations listed in Table 1.
  • PNGase F peptide N-glycosidase F
  • Table 1 Staining of electroblots with digoxig enin-Jabeled plant lectins
  • AAA Aleuria a ⁇ rantia L-Fuc ⁇ l-6GlcNAc 1 (-) strong (+)
  • PNA did not bind. Pretreatment of the protein with neuraminidase did not render it stainable with PNA (not shown), so any O-glycans present were not capped with sialic acid. If present, they are few in number, since selective removal of N-linked
  • N-linked glycans on DEC-205 were further defined by fluorophore-assisted carbohydrate electrophoresis (FACE).
  • FACE fluorophore-assisted carbohydrate electrophoresis
  • PNGase F released 8 different N-linked glycan structures from DEC-205, with electrophoretic migrations ranging from 5.1 to 10.1 glucose units (Figure 5C).
  • the glycan yield was too low to permit excision and sequencing of each of the 8 individual bands, so the mixture was subjected to analysis with exoglycosidases (Figure 5D).
  • Digestion with ⁇ - galactosidase (lane 2) simplified the pattern, indicating that some of the glycans terminate with Gal ⁇ l -(1 or 2)Gal. Addition of NANase III (lane 3.
  • the bisecting GlcNac was revealed by the fact that the upper band could be at least partially digested (2-fold decrease in fluorescence intensity) when the enzyme concentration doubled (lane 6). Further digestion with ⁇ -mannosidase (lane 7) was incomplete, mostly releasing a single mannose (major band), but showed the beginning of release of a band that co-migrated with the fucosylated mannosylchitobiose core structure (lane 9, "FC").
  • DEC-205 contains biantennary N-linked glycans with two kinds of fucosylated core structures, one with a bisecting GlcNAc, one without ( Figure 5E). Further heterogeneity is introduced at the outer ends of these structures, which terminate with either ⁇ -linked galactose, ⁇ l-4 linked galactose or ⁇ 2-8-linked sialic acid, in a total of 8 different permutations.
  • Peptide- reactive antibodies from hyperimmune sera were purified on an affinity resin prepared by coupling the peptide to the irrelevant carrier ovalbumin.
  • purified DEC-205 was injected into a second pair of rabbits, and hyperimmune IgG was purified by Protein A chromatography.
  • both polyclonals stained a 205 kDa band in crude extracts, like NLDC-145 ( Figure 6B. "extract' lanes). The affinities of both polyclonals for the blotted 205 kDa protein were roughly 100 times higher than the monoclonal.
  • a purification method based on immunoaffinity chromatography was used to isolate the antigen bound by the mAb NLDC-145, an antigen which was reported to be expressed at high levels by murine dendritic cells and thymic epithelium (Kraal et al., supra).
  • whole murine thymi were lysed rather than attempting the daunting task of purifying large numbers of DCs.
  • the isolated protein was about 95% pure, and was obtained with a yield in the hundred- microgram range, sufficient for N-terminal amino acid sequencing and basic biochemical studies. It would have required approximately 10 9 -10 l ⁇ DCs to provide a comparable amount of protein.
  • the electrophoretic molecular mass of the protein was consistently 205 kDa.
  • NLDC-145 was used to immunoprecipitate detergent extracts from surface-iodinated low-density lymph node cells. Only a single serine protease inhibitor, 1 mM PMSF, was present in the lysis buffer. A single predominant labeled protein was bound, with an apparent molecular mass of 145 kDa under both reducing and nonreducing electrophoresis conditions, leading the authors to append the number "145" to the clone's name.
  • the 205 kDa purified protein was the antigen detected by NLDC-145
  • polyclonal antibodies to the N- terminal peptide and to the intact purified protein were prepared. Both polyclonals stained a 205 kDa band on immunoblots. This staining could be eliminated by pretreating extracts with NLDC-145, demonstrating that the correct protein had been purified and sequenced.
  • the 205 kDa protein purified here is the authentic antigen recognized by the NLDC-145 monoclonal antibody.
  • DEC-205 is an integral membrane glycoprotein, bearing 2-3 biantennary complex-type N-linked glycans that comprise about 7 kDa of the overall electrophoretic molecular mass. These glycans are built on two different core structures, and vary further at their termini, to produce 8 variants, some of which contain sialic acid. Nevertheless, on electrofocusing, the isoelectric point of DEC-205 is slightly alkaline (pH 7.5), suggesting that the protein may be relatively rich in basic residues.
  • the pi is fairly homogeneous: a faint-staining, narrow "fringe” of protein surrounds the main pi, but extends only from pH 7.4 to 7.6, reflecting limited heterogeneity of charge. Considering the large overall mass of the protein and the relative paucity of bound carbohydrates, the sialylated glycan variants detected should not perturb the pi of DEC-205 excessively.
  • DEC-205 is very sensitive to proteolytic degradation. Precautions had to be taken to inhibit a broad range of proteases during the purification, and to remove the cytosolic fraction after hypotonic lysis, or else the yield was very low. Proteolysis appears to proceed by a distinctly nonrandom pathway. Despite the continuous presence of high concentrations of six protease inhibitors in our buffers, we invariably observed a "ladder" of 6-8 discrete, minor, lower molecular mass bands, ranging down to about 80 kDa and containing the NLDC-145 epitope, whenever the antigen was blotted at high levels.
  • EXAMPLE 2 DEC-205, A RECEPTOR EXPRESSED BY DENDRITIC
  • DEC-205 is a receptor with ten C-type lectin domains which is homologous to the macrophage mannose receptor (MMR), and other related receptors that bind carbohydrates and mediate endocytosis.
  • MMR macrophage mannose receptor
  • the function of DEC-205 was investigated with monoclonal and polyclonal anti-DEC-205 antibodies. It was determined that DEC-205 on dendritic cells is rapidly internalized via coated vesicles, and delivered to a multivesicular endosomal compartment that resembles the MHC class-II containing vesicles implicated in antigen processing. Furthermore, rabbit anti-DEC-205 antibodies were efficiently processed by dendritic cells and presented to rabbit IgG specific T cell clones. These experiments suggest that DEC-205 is a novel endocytic receptor that can be used by dendritic cells and thymic epithelial cells to direct captured antigens from the extracellular space to a processing compartment.
  • Dendritic cells from 7 day bone marrow cultures were treated with polyclonal rabbit anti-DEC-205 F(ab)'2 fragments and 10 nm gold-labeled goat anti-rabbit IgG as described in Figure 1 1 and processed for electron microscopy. For each time point 10 grids were examined, and all cells that were labeled with gold were photographed, gold particles were counted and scored by a blinded observer based, on standard mo ⁇ hological criteria. The numbers in parentheses represent the percentage of total gold particles scored in each compartment.
  • Northern blotting For Northern blots, 2 ⁇ g of mRNA were electrophoresed in 0.8% agarose formaldehyde gels. Samples were transferred to nylon membranes and probed with an anti-sense RNA probe that spanned nucleotide positions 3688- 5200 in the DEC-205 cDNA. The blot was subsequently stripped and re- hybridized with glyceralaldehyde-3 -phosphate dehydrogenase probe as a loading control.
  • Electron microscopy Dendritic cells harvested from 7 day mouse bone marrow cultures were incubated with 10 ⁇ g/ml of either: polyclonal anti-DEC-205, Fab'2 fragments of polyclonal anti-DEC-205; or biotinylated monoclonal NLDC-145, on ice for 30'. Excess primary antibody was removed by washing cells 3 times with RPMI-1640, 10% FCS, 0.02% NaN3. The cells were then incubated for 30' on ice with either: a 1 :5 dilution of 10 nm gold labeled goat anti-rabbit IgG; or a 1 :5 dilution of 10 nm gold labeled streptavidin respectively.
  • Dendritic cells were then either fixed with 2.5% glutaraldehyde for a time-zero point, or incubated for the stated times at 37°C before fixation and processing for electron microscopy.
  • Antigen presentaion 105 BALB/c mouse Dendritic cells obtained from day 7 bone marrow cultures were co-cultured with 105 2R.50 rabbit IgG specific T hybridoma cells for 48 hours in triplicate (Boom et al., J Exp Med 1988 167: 1350- 64). The supernatants were assayed for IL-2 concentration using the HT-2 indicator cell line. IL-2 production by the 2R.50 cells is plotted against the concentration of antibody in the cultures on a log scale. The error bars indicate the standard deviation from the mean.
  • Example 2 reports the molecular characterization of the 205 kDa cell surface protein described in Example 1.
  • fourteen cDNA clones were obtained from three separate thymic and dendritic cell cDNA libraries. All of the cDNA clones were derived from the same mRNA. Clones containing the putative 5' end of the DEC-205 cDNA encoded the N-terminal peptide of the DEC-205 antigen, and this was preceded by a hydrophobic leader consistent with a signal sequence.
  • the protein contains ten C- type lectin domains, a transmembrane domain, and a cytoplasmic domain ( Figure 7).
  • the composite cDNA had a single 5.2 Kb open reading frame encoding a protein of 1,722 a.a. with a predicted molecular weight of 195 Kda that included all 29 unambiguous DEC-205 peptide sequences (Figure 8).
  • Figure 8 the high degree of sequence identity and similarity of the cytoplasmic domains of both murine and human DEC ( Figure 9).
  • Figure 10 A 7.5 Kb mRNA that corresponds to this cDNA was expressed at high levels in dendritic cells, thymus. and lymph nodes, a pattern that corresponds to that which was obtained by staining tissues with the NLDC-145 monoclonal antibody (Kraal et al.. supra) ( Figure 10).
  • DEC-205 The sequence of DEC-205 was aligned with known proteins in the database, and it was determined that it is homologous to the macrophage mannose receptor (MMR) (Taylor et al., 1990. J. Biol. Chem. 265: 12156-62; Ezekowitz et al.. 1990, J. Exp. Med. 172:1785-94) and the phospholipase A2 (PLA2) receptors of rabbit skeletal muscle (Lambeau et al., 1994, J. Biol. Chem. 269: 1575-8), and to bovine pancreas (Ishizaki et al., 1994, J Biol Chem. 269:5897-904) ( Figure 8).
  • MMR macrophage mannose receptor
  • PDA2 phospholipase A2
  • C-type CRDs Ca ++ dependent carbohydrate recognition domains
  • C-type CRDs are carbohydrate-binding domains (reviewed by (Drickamer and Taylor, 1993, Annu. Rev. Cell Biol. 9:237-64)) and both the MMR and rabbit PLA2 receptor bind carbohydrates (Lambeau et al., 1994, J. Biol. Chem. 269:1575-8: Drickamer and Taylor, 1993, Annu. Rev. Cell Biol. 9:237-64).
  • CRDs are found in over one hundred other proteins (Drickamer and Taylor. 1993, Annu. Rev. Cell Biol. 9:237-64). but the CRDs in DEC-205 are most closely related to those found in the bovine and rabbit 70 PLA2 receptors, and the MMR (34.6% identity with bPLA2 receptor and 26.7% identity with hMMR). Indeed there is an ordered correspondence between CRDs 1- 5 and 7-8 in DEC-205 and CRDs 1 -5 and 7-8 in other group VI animal lectins, whereas CRD 6 of DEC-205 most closely resembles the first CRDs in other family members.
  • the unusual CRDs in DEC-205 are most closely related to CRDs 7 and 8 in other group VI lectins, and may have arisen during a gene duplication event. At least two of the CRDs in the MMR are known to bind mannose (Taylor et al., 1992, J. Biol. Chem. 267:1719-26), and grouping of several CRDs increases the affinity of the MMR for carbohydrate ligands (Taylor and Drickamer, 1993, J. Biol. Chem. 268:399-404. The same mechanism may be utilized by DEC-205 to increase both the affinity and diversity of carbohydrates bound by this receptor.
  • the receptor's function was investigated using a combination of monoclonal and polyclonal anti-DEC-205 antibodies.
  • DEC-205 is rapidly internalized via coated vesicles, and antibodies bound to the internalized receptor are delivered to multivesicular endosomal compartment.
  • dendritic cells were treated with rabbit anti-DEC-205 antibodies, and assayed for presentation of rabbit IgG-peptide/MHC complexes to T cell clones (Boom et al., 1988, J. Exp. Med. 167: 1350-64). Negative controls included non-specific rabbit antibodies, and rabbit antibodies to IgG2a that are efficiently presented by B cell lines (Boom et al.. 1988, J. Exp. Med. 167: 1350- 64). It was determined that dendritic cells presented rabbit anti-DEC-205 to the T cells clones two orders of magnitude more efficiently than the non-specific rabbit antibodies or rabbit anti-IgG2a ( Figure 12).
  • DEC-205 resembles membrane immunoglobulin on B cells in that the crosslinked receptor is efficiently internalized and bound ligands are delivered to an intracellular compartments that are active in antigen processing (Chestnut and Gray, 1981, J. Immunol. 126:1075- 79; Rock et al., 1984, J. Exp. Med. 160:1 102-25; Lanzavecchia, 1985, Nature 314:538-39).
  • dendritic cells and thymic epithelial cells express a novel receptor, DEC-205, which contributes to antigen presentation.
  • DEC-205 a novel receptor that contributes to antigen presentation.
  • the multi-Iectin domain structure suggests that this receptor can be used by dendritic cells and thymic epithelial cells to capture and endocytose diverse carbohydrate bearing antigens and direct them to an antigen processing compartment.
  • this mAb recognizes DEC-205, a 205 kDa integral membrane glycoprotein with a unique amino-terminal sequence, and a rabbit polyclonal antibody to purified DEC-205 with higher affinity for the blotted antigen than the original mAb was generated. Both the polyclonal and NLDC-145 antibodies have been used in this Example to reassess the expression and function of DEC-205 on leukocytes.
  • Cytofluorography revealed that DCs derived from the epidermis (Langerhans cells) and from proliferating bone marrow progenitors (BMDCs) expressed high levels (2-3 logs) of DEC-205, while freshly-isolated spleen DCs comprised two subsets, most (80%) staining at low levels ( ⁇ 1 log), the remainder moderately ( 1.5 logs).
  • DEC-205 epitopes were sensitive to trypsin. but were regenerated in culture. Resident and inflammatory peritoneal macrophages did not express the antigen, except for small amounts on thioglycollate-elicited cells.
  • mice Adult (6 to 10 wk old) mice of both sexes were studied, including (C57BL/6 x DBA/2) and (BALB/C x DBA/2) FI mice from the Trudeau Institute (Saranac Lake, NY), and (C57BL/6 x BALB/C) FI and BALB/C mice from Japan SLC (Hamamatsu, Shizuoka, Japan).
  • Cells were studied either immediately after isolation from the animal or following a period of culture in RPMI-1640 medium supplemented with 5% FCS. 50 ⁇ M 2- mercaptoethanol, and 20 ⁇ g/ml gentamicin. Spleens, thymi, and lymph nodes were either teased with forceps, or additionally digested with collagenase (Swiggard et al., 1992, In Current Protocols in Immunology. Coligan et al., (Eds). Green Publishing Associates and Wile Interscience: New York Supplement 3. pp. 3.7 1-1 1 ; Crowley et al. 1989. Cell Immunol. 1 18: 108), with similar results.
  • Bone marrow cells were flushed with a syringe from femurs and tibias. while blood was obtained by cardiac puncture in heparin. All cell suspensions were depleted of red cells by lysis in 0.83% ammonium chloride solution. Dendritic cells were obtained from three sources, each as described: the 74 epidermal sheets of mouse ears (Schuler and Steinman, 1985, J. Exp. Med. 161 :526), the low density plastic adherent population of spleen (Saveggard et al., supra; Crowley et al., supra), and proliferating bone marrow progenitors that were expanded in rGM-CSF (Inaba et al., 1992, J.
  • Peritoneal cells were either resident populations or were elicited by various inflammatory stimuli: proteose peptone 3 days earlier, thioglycollate broth 4 days earlier, 50 ⁇ g concanavalin A 2 days earlier, or 10 7 live Mycobacterium bovis BCG organisms 7 days earlier. Several populations were also studied after a period of 1 -3 days in culture. The B cells in lymph node suspensions were stimulated with the B cell mitogens lipopolysaccharide (10 ⁇ g/ml LPS, from E. Coli 01 1 1 :B4.
  • CD40 ligand CD40L: L cells transfected with CD40L (kind gift of Dr. H. Yagita, Juntendo University School of Medicine. Japan), fixed with 1% paraformaldehyde and washed 3 times in PBS before 1 : 1 coculture with B cells).
  • Dendritic cells in skin were cultured as whole epidermal suspensions and then enriched by flotation on dense bovine albumin (Schuler and Steinman. supra), while dendritic cells in spleen were cultured from low density spleen adherent cells, with or without supplementation in rGM-CSF (200 U/ml) or keratinocyte- conditioned medium, as described (Witner-Pack et al.. 1987. J. Exp. Med. 166: 1484).
  • Two-color labeling methods were used to simultaneously identify a particular subset of leukocytes (PE-labeled antibody) and DEC-205 (NLDC-145 rat mAb or rabbit polyclonal anti-DEC-205 followed by FITC labeled anti-Ig).
  • Nonreactive control antibodies were nonimmune rat IgG2a (Zymed. South San Francisco. CA) and rabbit IgG (Jackson ImmunoResearch; intact IgG or F(ab') : prepared by us).
  • the staining sequence was: (a) primary anti-DEC-205 or nonimmune; (b) secondary anti-Ig (FITC conjugates of mouse anti-rat IgG or goat anti-rabbit
  • F(ab') 2 both from Jackson ImmunoResearch
  • (d) the PE- or biotin-labeled antibody The latter reagents were purchased from PharMingen (La Jolla, CA), and were: biotin conjugates directed to class II MHC (clone AMS-32.1), B220/CD45RB (clone RA3-6B2) and Thy- 1.2/CD90 (clone 53-2.1) antigens; or PE conjugates directed to Mac-1/CDl lb (clone Ml/70) and Gr-1 granulocyte (clone RB6-8C5) antigens. At least 10,000 cells per sample were examined in a FACScan cytofluorograph (Becton Dickinson Immunocytometry Systems, Mountainview CA).
  • Monoclonal (10 and 1 ⁇ g/ml) and polyclonal (30 and 10 ⁇ g/ml) antibodies were applied at doses that were close to or above saturation, continuously, to a one-way allogeneic mixed leukocyte reaction (MLR), wherein 3 x 10 5 nylon wool-passed lymph node T cells were stimulated by graded doses of allogeneic irradiated or mitomycin-treated DCs (Inaba and Steinman, 1984, J. Exp.
  • MLR mixed leukocyte reaction
  • the culture period also provides time for most keratinocytes to adhere to the plastic surface, and for the nonadherent Langerhans cells to acquire a low buoyant density (Crowley et al. supra; Schuler and Steinman. supra).
  • preparations with 20-50% dendritic cells can be obtained by studying nonadherent, overnight cultures of epidermal cells, especially following flotation on columns of dense BSA.
  • Epidermal cells were stained with a phycoerythrin (PE)-tagged mAb to class II MHC proteins, to distinguish Langerhans cells from keratinocytes, and were counterstained with hybridoma supernatants of NLDC-145 and mAbs to other leukocyte lineages ( Figure 13. A-D).
  • PE phycoerythrin
  • FIG. 13 A The specificity of NLDC-145 for dendritic cells ( Figure 13 A) was demonstrated by the fact that isotype-matched IgG2a mAbs to macrophages (SER-4 anti-sialoadhesin (Coocker and Gordon, 1989, J. Exp. Med.
  • the anti-DEC-205 rabbit polyclonal was also applied, both as F(ab') 2 fragments and as intact IgG, and was compared with nonimmune F(ab') 2 and IgG over a broad range of doses (0.3-100 ⁇ g/ml).
  • the rabbit reagents did react with the class II MHC-negative keratinocytes, but this binding was entirely nonspecific, since the staining was comparable with immune and nonimmune reagents (Figure 13, compare panels I-L with M-P, and Q-T with U-X).
  • the second rabbit polyclonal antibody raised to a synthetic peptide spanning the first 19 residues of DEC-205, failed to stain Langerhans cells, instead giving a pattern like that of nonimmune IgG (not shown).
  • Freshly-isolated spleen DCs comprised two phenotypic subsets, as previously described (Crowley et al., supra). Most (roughly 80%) expressed relatively low but detectable levels ( ⁇ 1 log) of the antigen, while a smaller population stained moderately (ca. 1.5 logs: arrows, Figure 15C, D, G, and H). After overnight culture, DEC-205 expression by all of the CDl lc (+) DCs had risen to the moderate (1.5 log) level, but never to the levels observed on epidermal dendritic cells (ca.
  • peritoneal cells consisting of about 30% macrophages and 70% B cells
  • Resident peritoneal cells were compared to inflammatory peritoneal cells in exudates elicited with concanavalin A (Con A), thioglycollate (TGC), or live M. bovis Bacille Calmette-Guerin (BCG) organisms ( Figure 17).
  • Con A concanavalin A
  • TGC thioglycollate
  • BCG live M. bovis Bacille Calmette-Guerin
  • Mac-1 (+) peritoneal macrophages did not express surface DEC-205. but peritoneal B cells expressed measurable levels (roughly 1 log above background: Figure 17 E-H arrows). Macrophages in Con A- and BCG-elicted exudates again showed little or no staining with anti-DEC-205 ( Figure 17 E-H, arrowheads), even though these macrophages were all strongly class II MHC-positive (not shown). The Con A and BCG exudates contained significant numbers of T cells, but these did not stain with anti-DEC-205 (anti-Thy- 1 double label not shown). In contrast to the other peritoneal populations tested, TGC-elicited macrophages did express DEC-205, albeit at low levels (0.5-1 log above background). In each of the resident and elicited populations. B cells stained comparably.
  • DEC-205 by resident leukocytes in multiple tissues, particularly B cells. Given the su ⁇ rising finding that resident peritoneal B cells expressed DEC- 205, we explored the distribution of the antigen further by examining cell suspensions from spleen, bone marrow, peripheral blood, lymph node, and thymus for co-expression of DEC-205 with several leukocyte markers. Results with the first 3 organs are illustrated here ( Figure 18). The results with spleen and lymph node were identical (not shown). Thymocytes stained only marginally ( ⁇ 0.5 log) above background (not shown).
  • B cells B220 and MHC-II (+); Gr-1, Mac-1 and Thy-1 (-): Figure 18 F-J, arro >s
  • B cells B220 and MHC-II (+); Gr-1, Mac-1 and Thy-1 (-): Figure 18 F-J, arro >s
  • T cells Thy-1 (+)
  • granulocytes Gr-1 (+), Mac-1 (+)
  • B cells also stained, but less strongly than B cells.
  • B cells were cultured for up to 2 days in the presence of LPS, CD40 ligand. and the combination of anti-IgM and IL-4. None induced a significant increase, but the latter combination induced a modest (2-fold) decrease in surface levels of DEC- 205, as detected with both monoclonal and polyclonal reagents (not shown).
  • GL-1 inhibited proliferation in this system, but did not abolish it, as expected: blockade of multiple costimulators is required to completely ablate proliferation in an allogeneic MLR (Young et al., 1992, J. Clin. Invest. 90:229).
  • Nonimmune F(ab') 2 1 2.63 13.27 2 2.36 13.83 3 2.89 12.01 4 2.06 12.80
  • DEC-205 expression Differences in DEC-205 expression among different classes of leukocytes were observed. Other than dendritic cells, B cells expressed the most DEC-205, although their levels were 10 to 50 times lower than those on BMDCs ( Figure 19). The DEC-205 detected on B cell surfaces was actively synthesized by the cells themselves, and unlikely to be adsorbed to their surfaces from an extracellular source, since after trypsinization, DEC-205 epitopes were regenerated in culture. Expression of DEC-205 appears to be coordinated with the developmental transition from pre-B cell to surface IgM (+) B cell in the marrow.
  • peripheral B cell stimulation with a variety of mitogens was not accompanied by a significant increase in surface expression of DEC-205.
  • Granulocytes expressed DEC-205 with higher levels on granulocytes in bone marrow than in blood.
  • Thymocytes and mature T cells from spleen and lymph node expressed very low but detectable levels of DEC-205, whereas T cells in blood and peritoneal fluid did not express detecable levels.
  • DEC-205 detected on granulocytes and T cells was adsorbed from surrounding stromal cells that are rich in DEC-205, such as bone marrow stroma, thymic epithelium, and the dendritic cells in the T cell areas of peripheral lymphoid tissues.
  • Most macrophage populations lack DEC-205, although thioglycollate- elicited cells are weakly positive, as previously described (Wiffels et al.. 1991 , Immunobiol. 184:83).
  • dendritic cells express some 10-50 times more of the antigen, as assessed by immunoblotting.
  • DEC-205 expression is regulated on dendritic cells in some way.
  • Freshly isolated splenic DCs have relatively little DEC-205. and the levels increase only modestly in culture.
  • the dendritic cells that express high levels of DEC-205 are those in skin, in the T cell regions of peripheral lymphoid organs, and dendritic cells that are grown from proliferating bone marrow precursors in the presence of high-dose GM-CSF.
  • EXAMPLE 4 EXPRESSION OF THE DEC-205 PROTEIN IN SITU IN LYMPHOID AND NONLYMPHOID TISSUES
  • the monoclonal and polyclonal antibodies to DEC-205 were used to reassess the tissue distribution of DEC-205 by immunohistochemical staining of frozen sections from a variety of organs, and by multiple-organ immunoblotting.
  • DEC-205 Abundant expression of DEC-205 was confirmed histologically on thymic and intestinal epithelia and on dendritic cells in the T cell areas of peripheral lymphoid organs. In addition, DEC-205 was visualized in several other locations: B lymphocytes within B cell follicles, the stroma of the bone marrow, the epithelia of pulmonary airways, and the capillaries of the brain. Immunoblotting confirmed the presence of substantial levels of DEC- 205 protein in lysates prepared from lymphoid tissues and from lung, marrow and intestine. Thus, while DEC-205 is expressed at high levels by dendritic cells, it is also expressed by a number of other cell types in situ.
  • mice Adult (6-12 week old) female mice of three strains were studied: inbred C57BL/6 x DBA/2 (Trudeau Institute, Saranac Lake, NY) and BALB/C, and outbred CD-I Swiss- Webster (the latter 2 strains from Taconic Farms, Germantown, NY).
  • Immunohistology Immediately after organs were removed, they were frozen at - 20°C in O.C.T. tissue embedding medium (Miles, Elkhart, IN), and stored at - 20°C. Tissue sections, usually 10 ⁇ m thick, were cut on a Minotome cryostat (IEC division of Damon, Needham Heights, MA) and applied to 10- well slides (Carlson Scientific. Peotone, IL). The sections were fixed in neat acetone for 10 min at room temperature, and air-dried. Subsequent steps were performed in a humid chamber. Sections were rehydrated in a drop (30-50 ⁇ L) of PBS. then primary antibody was applied.
  • Minotome cryostat IEC division of Damon, Needham Heights, MA
  • Hybridoma supernatants were used either undiluted or diluted 1 :5 in PBS + 1% BSA, depending on their titer.
  • Purified IgGs, ascites fluids and antisera were diluted in the same medium to optimized doses determined by titration, usually 1-10 ⁇ g/ml for purified protein and 1 :3000-1 : 1000 for ascites and hyperimmune sera.
  • the primary antibodies were the NLDC-145 mAb, applied either as a hybridoma culture supernatant or as purified IgG (protein G eluate), or rabbit polyclonal anti-DEC-205, (Example 1 supra), applied either as intact IgG (protein A eluate) or as F(ab') 2 fragments.
  • Extracts were clarified (3000 x g, 10 min, room temperature), and total protein levels were measured (BCA assay, Pierce, Rockford, IL). Immunoblotting was performed as described (Chomczynski, supra), normalizing protein loads to 50 ⁇ g per lane. Filters were stained with either 10 ⁇ g/ml of NLDC-145 IgG or mAb 1D4B (anti- LAMP-1) hybridoma supernatant, diluted 1 :1.
  • Thymus Staining patterns in this organ were identical to those in the original description of the NLDC-145 mAb (Kraal et al., supra). Very strong peroxidase immunolabeling was observed on thymic cortical epithelium, while weaker staining was noted on scattered dendritic profiles in the medulla (M, Figure 21 , panels a-c and ).
  • NLDC-145 and polyclonal anti-DEC-205 reagents produced linear staining along capillaries (arrows, Figure 23 a-c) and small arteries (arrow, Figure 23 d) in the cerebrum and cerebellum. Staining of capillaries was not observed in any other organ studied.
  • DEC-205 was present in the epithelium of all the small airways (arrows, Figure 23 e, h). In contrast. anti-MHC class II did not stain the airway epithelium, but did stain cells surrounding the airways ( Figure 23 arrowheads).
  • Bone marrow When bone marrow was extruded from the femur as an intact plug and sectioned, a lacy pattern of DEC-205 stain was evident throughout the plug, presumably on marrow stromal cells (arrows. Figure 23 ). Most of the dark staining of round cells represented background staining of eosinophils. which express endogenous peroxidases. It was evident in the absence of any antibody (not shown). Upper gastrointestinal tract. The oral epithelium of the tongue served as an example of a stratified squamous epithelium. Some DEC-205 positive profiles, presumably Langerhans cells, were found suprabasally (arrows, Figure 23 j). Anti- MHC class II antibodies stained these intraepithelial dendritic cells more frequently and/or more intensely, and in addition stained many subepithelial profiles in the upper dermis (not shown).
  • Human dec cDNA was cloned using a 300 base-pair probe derived from the 3' coding sequence of murine dec cDNA.
  • the human cDNA was obtained from a B lymphoma library, using high stringency hybridization conditions (0.1 SSC, 65°C).
  • the sequence of the human DEC-205 gene was determined, and is shown in SEQ ID NO:7.
  • the deduced amino acid sequence is shown in SEQ ID NO:8.
  • the deduced sequence in SEQ ID NO: 8 includes putative segments after the stop site at after amino acid number 1743; these are irrelevant and may be ignored.
  • Trp He Ala Leu Gin Asp Gin Asn Asn Thr Gly Glu Tyr Thr Trp Lys 580 585 590
  • Trp He Gly Leu Phe Arg Asn Val Glu Gly Thr Trp Leu Trp He Asn 1300 1305 1310
  • CAAAGTGCCT CTGGGCCCTG ATTACACAGC
  • AATAGCTATC ATAGTTGCCA
  • CACTAAGTAT 510 CTTAGTTCTC ATGGGCGGAC TGATTTGGTT CCTCTTCCAA AGGCACCGTT TGCACCTGGC 5160
  • Trp Glu Lys Asn Glu Gin Phe Gly Ser Cys Tyr Gin Phe Asn Thr Gin 245 250 255

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EP96906258A 1995-01-31 1996-01-31 Identifizierung von dec-205, (dentritischen und epithelzellen, 205 kda), rezeptor mit c-typ lektin regionen, dafür kodierende nukleinsäure und verwendungen davon Ceased EP0808366A1 (de)

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US381528 1982-08-20
US38152895A 1995-01-31 1995-01-31
PCT/US1996/001383 WO1996023882A1 (en) 1995-01-31 1996-01-31 IDENTIFICATION OF DEC, (DENTRITIC AND EPITHELIAL CELLS, 205 kDa), A RECEPTOR WITH C-TYPE LECTIN DOMAINS, NUCLEIC ACIDS ENCODING DEC, AND USES THEREOF

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EP0808366A1 true EP0808366A1 (de) 1997-11-26

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EP (1) EP0808366A1 (de)
JP (1) JPH10513350A (de)
AU (1) AU716056B2 (de)
CA (1) CA2211993A1 (de)
MX (1) MX9705923A (de)
WO (1) WO1996023882A1 (de)

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US9814780B2 (en) 2010-08-10 2017-11-14 Ecole Polytechnique Federale De Lausanne (Epfl) Compositions for inducing antigen-specific tolerance
US9850296B2 (en) 2010-08-10 2017-12-26 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10046056B2 (en) 2014-02-21 2018-08-14 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10392437B2 (en) 2010-08-10 2019-08-27 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10821157B2 (en) 2014-02-21 2020-11-03 Anokion Sa Glycotargeting therapeutics
US10946079B2 (en) 2014-02-21 2021-03-16 Ecole Polytechnique Federale De Lausanne Glycotargeting therapeutics
US10953101B2 (en) 2014-02-21 2021-03-23 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US11253579B2 (en) 2017-06-16 2022-02-22 The University Of Chicago Compositions and methods for inducing immune tolerance

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US7071171B1 (en) 1996-12-20 2006-07-04 Board Of Regents The University Of Texas System Unique dendritic cell-associated c-type lectins, dectin-1 and dectin-2 compositions and uses thereof
AU737727B2 (en) 1997-04-10 2001-08-30 University Of Southern California Modified viral surface proteins for binding to extracellular matrix components
US6004798A (en) * 1997-05-14 1999-12-21 University Of Southern California Retroviral envelopes having modified hypervariable polyproline regions
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EP1046651A1 (de) * 1999-04-19 2000-10-25 Koninklijke Universiteit Nijmegen Zusammensetzungen und Verfahren zur Modulierung des interaktion zwischen dendritischer und T-zellen
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JP5007409B2 (ja) 2000-05-08 2012-08-22 セルデックス リサーチ コーポレーション 樹状細胞に対するヒトモノクローナル抗体
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EP3055331B1 (de) * 2013-10-11 2021-02-17 Oxford Bio Therapeutics Limited Konjugierte antikörper gegen ly75 zur behandlung von krebs
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US10265416B2 (en) 2010-08-10 2019-04-23 École Polytechnique Fédérale de Lausanna (EPFL) Compositions for generation of immune tolerance to specific antigens
US9850296B2 (en) 2010-08-10 2017-12-26 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US9878048B2 (en) 2010-08-10 2018-01-30 Ecole Polytechnique Federale De Lausanne (Epfl) Compositions for generating immune tolerance by targeting erythrocytes
US9901645B2 (en) 2010-08-10 2018-02-27 Ecole Polytechnique Fedrale de Lausanne (EPFL) Methods for reducing immune responses
US9901646B2 (en) 2010-08-10 2018-02-27 Ecole Polytechnique Federale De Lausanne (Epfl) Methods for induction of antigen-specific immune tolerance
US9814780B2 (en) 2010-08-10 2017-11-14 Ecole Polytechnique Federale De Lausanne (Epfl) Compositions for inducing antigen-specific tolerance
US11246943B2 (en) 2010-08-10 2022-02-15 École Polytechnique Fédérale De Lausanne (Epfl) Antigen-specific tolerance and compositions for induction of same
US10265415B2 (en) 2010-08-10 2019-04-23 École Polytechnique Fédérale De Lausanne (Epfl) Compositions for inducing antigen-specific tolerance
US10392437B2 (en) 2010-08-10 2019-08-27 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10471155B2 (en) 2010-08-10 2019-11-12 École Polytechnique Fédérale De Lausanne (Epfl) Antigen-specific tolerance and compositions for induction of same
US10800838B2 (en) 2010-08-10 2020-10-13 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US11884721B2 (en) 2010-08-10 2024-01-30 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10919963B2 (en) 2010-08-10 2021-02-16 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10046056B2 (en) 2014-02-21 2018-08-14 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10946079B2 (en) 2014-02-21 2021-03-16 Ecole Polytechnique Federale De Lausanne Glycotargeting therapeutics
US10953101B2 (en) 2014-02-21 2021-03-23 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10940209B2 (en) 2014-02-21 2021-03-09 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US11654188B2 (en) 2014-02-21 2023-05-23 Ecole Polytechnique Federale De Lausanne (Epfl) Glycotargeting therapeutics
US11666638B2 (en) 2014-02-21 2023-06-06 Ecole Polytechnique Federale De Lausanne (Epfl) Glycotargeting therapeutics
US11793882B2 (en) 2014-02-21 2023-10-24 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US11801305B2 (en) 2014-02-21 2023-10-31 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
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US11253579B2 (en) 2017-06-16 2022-02-22 The University Of Chicago Compositions and methods for inducing immune tolerance

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WO1996023882A1 (en) 1996-08-08
AU4970296A (en) 1996-08-21
CA2211993A1 (en) 1996-08-08
MX9705923A (es) 1998-07-31
JPH10513350A (ja) 1998-12-22

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