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Immunostimulatory nucleic acid molecules for activating dendritic cells

Inactive Publication Date: 2003-05-29
UNIV OF IOWA RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

0010] As described in co-pending parent patent application U.S. Ser. No. 08/960,774 the vertebrate immune system has the ability to recognize the presence of bacterial DNA based on the recognition of so-called CpG-motifs, unmethylated cytidine-guanosine dinucleotides within specific patterns of flanking bases. According to these disclosures CpG functions as an adjuvant and is as potent at

Problems solved by technology

Many potent adjuvants in mice or other animals, like the Freunds complete adjuvant, cannot be used in humans due to toxicity.

Method used

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  • Immunostimulatory nucleic acid molecules for activating dendritic cells
  • Immunostimulatory nucleic acid molecules for activating dendritic cells
  • Immunostimulatory nucleic acid molecules for activating dendritic cells

Examples

Experimental program
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example 1

[0132] Generation and Characterization of Dendritic Cells

[0133] Methods

[0134] Isolation of dendritic cells: Dendritic cells represent a small population of peripheral blood mononuclear cells (0.1 to 0.4%). They express substantial levels of CD4, but lack the T cell molecules CD3, CD8, and T cell receptor, and other lineage markers (CD19, CD14, CD16, CD56) (O'Doherty U, et al., "Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium", J Exp Med, 1993; 178: 1067-1076). Using these characteristics, dendritic cells can be separated by high gradient immunomagnetic cell sorting using the VARIOMACS technique (Miltenyi Biotec Inc., Auburn, Calif.). Peripheral blood mononuclear cells were obtained from buffy coats of healthy blood donors (Elmer L. DeGowin Blood Center, University of Iowa) by Ficoll-Paque density gradient centrifugation (Histopaque-1077, Sigma Chemical Co., St. Louis,...

example 2

[0139] CpG Substitutes for GMCSF for DC Survival

[0140] Methods

[0141] Oligodeoxynucleotides Unmodified (phosphodiester) and modified nuclease-resistant (phosphorothioate) oligodeoxyribonucleotide were purchased from Operon Technologies (Alameda, Calif.). The optimal motif recognized by human immune cells is different from the optimal mouse motif. Based on other studies in which we tested a large number of oligonucleotides for their ability to activate human B-cells and NK-cells, we selected particularly potent oligonucleotides as examples of a family of active CpG-containing oligonucleotides for the use in the present study. The CpG oligonucleotides used were: 2006 (24-mer), 5'-TCG TCG TTT TGT CGT TTT GTC GTT-3' (SEQ ID NO: 84), completely phosphorothioate-modified, and 2080 (20-mer), 5'-TCG TCG TTC CCC CCC CCC CC-3'(SEQ ID NO: 94), unmodified phosphodiester. The non-CpG control oligonucleotides used were: 2117 (24-mer), 5'-TQG TQG TTT TGT QGT TTT GTQ GTT-3'(SEQ ID NO: 95), Q=5 methy...

example 3

[0146] Increased Size and Granularity of DC Induced by CpG is Associated with Enhanced Expression of MHC II

[0147] Methods

[0148] Surface antigen staining At the indicated time points, cells were harvested and surface antigen staining was performed as previously described. Monoclonal antibodies to HLA-DR (Immu-357), CD80 (MAB104) and to CD83 (HB15A) were purchased from Immunotech, Marseille, France. All other antibodies were purchased from Pharmingen, San Diego, Calif.: mABs to CD1a (HI149), CD3 (UCHT1), CD14 (M5E2), CD19 (B43), CD40 (5C3), CD54 (HA58), CD86 (2331 (FUN-1)). FITC-labeled IgG.sub.1, .kappa.(MOPC-21) and PE-labeled IgG.sub.2b, .kappa.(27-35) were used to control for specific staining.

[0149] Results

[0150] Flow cytometric analysis suggested that differentiation of DC is enhanced by CpG and is associated with an increase of cell size (FSC) and granularity (SSC) (FIG. 1). The surface expression of MHC II is known to be positively correlated with differentiation of DC. DC iso...

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Abstract

The present invention relates generally to methods and products for activating dendritic cells. In particular, the invention relates to oligonucleotides which have a specific sequence including at least one unmethylated CpG dinucleotide which are useful for activating dendritic cells. The methods are useful for in vitro, ex-vivo, and in vivo methods such as cancer immunotherapeutics, treatment of infectious disease and treatment of allergic disease.

Description

[0001] This application is a divisional of co-pending U.S. Ser. No. 09 / 191,170, filed on Nov. 13, 1998, now allowed which is a continuation-in-part of U.S. Ser. No. 08 / 960,774, filed Oct. 30, 1997, now issued as U.S. Pat. No. 6,239,116B1 on May 29, 2001, which is a continuation-in-part of U.S. Ser. No. 08 / 738,652, filed Oct. 30, 1996 and which is now issued as U.S. Pat. No. 6,207,646B1 on Mar. 27, 2001, and which patent is a continuation-in-part of U.S. Ser. No. 08 / 386,063, filed Feb. 7, 1995 and which is now issued as U.S. Pat. No. 6,194,388B1 on Feb. 27, 2001, and which patent is a continuation-in-part of U.S. Ser. No. 08 / 276,358, filed Jul. 15, 1994 and which is abandoned, each of which are incorporated by reference.[0002] The present invention relates generally to methods and products for activating dendritic cells. In particular, the invention relates to oligonucleotides which have a specific sequence including at least one unmethylated CpG dinucleotide which are useful for act...

Claims

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Application Information

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IPC IPC(8): A61K31/00A61K31/4706A61K39/39A61P37/04C07H21/00C12N5/0784C12Q1/68
CPCA61K31/00A61K31/4706A61K31/7048A61K31/711A61K31/7125A61K39/39C12Q1/68A61K2039/5158A61K2039/55561C07H21/00C12N5/0639C12N2501/056A61K2039/5156A61P37/04A61K39/464A61K39/4615A61K39/4622
Inventor KRIEG, ARTHUR M.HARTMANN
Owner UNIV OF IOWA RES FOUND
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