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Dendritic cell receptor

a dendritic cell and receptor technology, applied in the field of dendritic cell receptors, can solve the problem of not disclosing a receptor on human dendritic cells

Inactive Publication Date: 2005-08-25
THE OF THE TRUSTEES OF THE SISTERS OF MERCY & QUEENSLAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they do not disclose a receptor on human dendritic cells.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0045] Langerhans cells were prepared from human skin. Epidermal cell suspensions were prepared from split thickness normal human breast skin by 30 min dispase (Boehringer-Mannheim, Mannheim, Germany; 0.5% in PBS) treatment at 37° C., followed by 10 min disaggregation in the presence of trypsin (0.25% in PBS), DNase I (5 U / ml in PBS) and 5 mM EDTA at room temperature. Langerhans cells were then enriched by Ficoll / Metrizoate gradient separation (d=1.077 g / cm3). Final cell suspensions contained 3-15% Langerhans cells as determined by HLA-DR positivity. Total RNA was extracted using Trizol reagent according to the manufacturer's instructions.

[0046] Degenerate primers were prepared on an Applied Biosystems DNA Synthesizer with the primer sequences (d) and (e) as set out below:

(d)5′-GAX ACY GAX GGY TTX TGG AA-3′(e)3′-GCY GTX TTZ TCZ AAC CAC AT-5′

[0047] wherein X is C or T, Y is A, C, G or T, and Z is G or A.

[0048] Single stranded cDNA was prepared using total RNA and reverse transcri...

example 2

Experimental Procedures

[0058] Cell culture—The cell lines, HEL, K562, KG-1, THP-1, U937, Mann and Jurkat were obtained from the American Type Culture Collection (Rockville, Md.). L428 cells were provided by V. Diehl (Klinik for Innere Medizin, Cologne, Germany). HDLM2 and KMH2 cells were obtained from the German Collection of Micro-organisms and Cell Culture (Braunscfweig, Germany). Mono Mac 6 cells (Bufler et al (1995) Eur. J. Immunol. 25, 604-610) were provided by H. Engelmann (Institute for Immunology, Munchen, Germany). All cell lines were maintained in RPMI 1640, 10% fetal calf serum 100 U / ml penicillin, 100 ug / ml streptomycin except that HDLM2 cells were with 20% fetal calf serum.

[0059] Isolation of leukocytes-Leukocyte populations were isolated using standard laboratory procedures.

[0060] Isolation of cDNA encoding for human DEC-205—A set of degenerate oligonucleotide primers were designed based on the published amino acid sequence of mouse DEC-205 (Jiang et al (1995), abov...

example 3

Production of Anti-DEC-205 Antibodies

[0099] A BALB / c mouse was immunized ip / sc with L428 cells and boosted SC with two peptides derived from the DEC-205 cDNA sequence. DEC-205 peptide 1 ATTQDEVHTKC (aa1267-aa1277) and DEC-205-peptide 2 TEKEVKPVDSVKC (aa1227-aa1239) were synthesized by Chiron Mimotopes Pty Ltd (Clayton, Victoria, Australia). After a third immunization with the two DEC-205 peptides sc / ip / IV the mouse was sacrificed and a spleen cell suspension prepared. The spleen cells were fused with the NS-1 myeloma cell line using standard techniques (Hock et al, Immunology 1994;83:573). A hybridoma was subsequently isolated, 2F5, which produced monoclonal antibody binding to the DEC-205-peptide 1 but not the DEC-205-peptide 2 or a third control DEC-205-peptide 3 (KCLGLDITKSVNELR) (aa82-aa96). This is shown by FIG. 9.

E. Constructs

[0100] The invention also provides constructs. The constructs will generally include antigens against which an immune response is desired but can als...

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Abstract

The invention provides isolated human DEC-205, its extracellular domain and functionally equivalent fragments thereof. Also provided are polynucleotides encoding same and vectors which include such polynucleotides. Further provided are methods of recombinantly producing human DEC-205, an extracellular domain thereof or a functionally equivalent fragment, and ligands that bind to human DEC-205 or a fragment thereof. Also provided are constructs for use in prophylaxis or therapy comprising such a ligand, human DEC-205 or an extracellular domain thereof coupled to a toxin or to an antigen capable of inducing a protective immune response in a patient.

Description

FIELD OF THE INVENTION [0001] This invention relates to dendritic cell receptors. In particular, it relates to human DEC-205, to the production and use thereof, and to ligands which bind to it. Human DEC-205 and its ligands are useful in prophylaxis and therapy. BACKGROUND OF THE INVENTION [0002] Dendritic cells perform important immunoregulatory functions by presenting antigens in the form of peptides bound to cell-surface major histocompatibility complex (MHC) molecules to T cells. Identification of the mechanism by which this antigen presentation function is achieved therefore has important implications in manipulating immune response in prophylaxis and therapy, particularly in humans. [0003] Jiang et al, Nature 375: 151-155 (1995) disclose a murine dendritic cell receptor having a molecular weight of 205 kDa (murine DEC-205). However, they do not disclose a receptor on human dendritic cells. [0004] The applicant has now identified a receptor on human dendritic cells. It is broad...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/385C07K14/705G01N33/569C12N15/09
CPCA61K39/385A61K2039/6031G01N33/56972C07K2319/00C07K14/705A61P37/02
Inventor HART, DEREK
Owner THE OF THE TRUSTEES OF THE SISTERS OF MERCY & QUEENSLAND
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