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Carbohydrate specific cellular immunity inducing microorganisms and fractions thereof

A technology of carbohydrates and cellular immunity, applied in the direction of microorganisms, microorganism-based methods, biological tests, etc., can solve the problems of non-CH-specificity, ineffective immune response, lack of specificity, etc.

Inactive Publication Date: 2009-12-09
GLYCOTOPE GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 4 Few reports have shown that complex carbohydrates are not removed by antigen-presenting cells during glycoprotein processing and can be presented together with peptides to major histocompatibility complex II restricted T cells
MUC1 with short sialylated carbohydrates induces dendritic cell activation and maturation phenotypically similar to that induced by bacterial LPS and thus lacks specificity, but these DCs cannot be induced after co-culture with allogeneic CD4+ T cells Th1 type immune response or cytotoxic CD8 response (Carlos et al. (2005) J Immunol 175:1628-1635)
[0006] 6 MUC1-derived peptides O-glycosylated with a Tn epitope induce cellular immunogenicity in mice, but immunization with glycopeptide-loaded dendritic cells shows no immunotherapeutic, nonselective cleavage of human MUC1 The expressing murine cell line and induced CTLs showed cross-reactivity between glycosylated and non-glycosylated forms of the same peptide, so the immune response was not efficient and not CH-specific (Stepensky et al. (2005) Clin Exp Immunol 143:139-149)
In contrast, in the case of MUC1, desialylated MUC1 (presenting short sugar moieties such as Tn or TF) was unable to induce T cell cytokine production and thus no T cell activation, or in the case of sialylated sugars , the induction of secretion of cytokines such as TNFα and IL-6 associated with tumor metastasis, tumor development, and poor prognosis occurs, thus failing to activate the required TH1 response, and even thought to be involved in tumor escape from the immune response (Carlos et al., J. Immunol, )
[0011] 11 The prior art teaches that natural molecules including human carbohydrate tumor antigens do not induce potent cellular Th1 and cytotoxic immunity against carbohydrate epitopes, and that polysaccharides from bacteria, especially capsular polysaccharides, do not induce antigen-specific immunity answer

Method used

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  • Carbohydrate specific cellular immunity inducing microorganisms and fractions thereof
  • Carbohydrate specific cellular immunity inducing microorganisms and fractions thereof
  • Carbohydrate specific cellular immunity inducing microorganisms and fractions thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0789] Example 1 Anaerobic culture technology and culture medium

[0790] 568 The anaerobic technology for bacterial culture is based on the previously described method summarized by Breznak and Costilow. Disperse the medium prepared with cysteine ​​hydrochloride (cysteine·HCl) as a reducing agent in an anaerobic culture tube (Ochs, Bovenden, Germany) or a glass serum bottle, leaving about half to one third of the total container volume as The gas headspace is sealed with a butyl rubber plug. The solution prepared without using a reducing agent (such as PBS-a) is boiled before dispersion. Before autoclaving, use N 2 / CO 2 (80 / 20, v / v) instead of gas phase. In order to achieve this, a needle was pierced through a butyl rubber stoppered bottle, and the bottle was evacuated by means of a vacuum pump (Vacuubrand, Wertheim, Germany). After evacuating, use N to shake the bottle repeatedly throughout the process 2 / CO 2 (80 / 20, v / v) inflation. The evacuation and inflation steps are perfo...

Embodiment 2

[0793] Example 2 Affinity enrichment of core-1 positive microorganisms

[0794] 571 2.1 TF1 and TF2 coated Preparation

[0795] The respective volume of 100μl (M-450 Rat Anti-Mouse IgM, Dynal Biotech ASA, Oslo, Norway) placed in a 2ml Safe-Lock Eppendorf tube (Eppendorf, Hamburg, Germany), using Dyna Magnetic Particle -S( -S, Dynal Biotech, Oslo, Norway), with 2ml of phosphate buffered saline a (PBS-a: 8.1g l -1 NaCl, 0.16g l -1 NaH 2 PO 4 ·H 2 O, 0.98g l -1 Na 2 HPO 4 ·2H 2 O, 1g l -1 BSA, pH 7.4) washed twice, and suspended in 25μl PBS-a. The lyophilized TF1 or TF2 cell culture supernatant was dissolved in 1 ml milli-Q synthetic grade water (Millipore, Billerica, MA, USA). Add the dissolved TF1 or TF2 cell culture supernatant (1ml) to the And incubate on a test tube rotator (model 34528, Snijders Scientific, Netherlands) at 4C for 30 min. Tube placed -S, and let it stand for 3 minutes before removing the liquid with a pipette. Make Resuspend in 2ml PBS-a and place -S, an...

Embodiment 4

[0825] Example 4 Growth and immobilization of bacteria based on ELISA screening

[0826]590 well-separated clones were randomly selected from selective and non-selective agar plates, and re-streaked three times on non-selective medium. Pick a single clone and depending on which one gives the best growth, inoculate it into ST (agar omitted), WC or MRS broth, and grow overnight at 37°C. These cultures were inoculated (1%) into 300 ml of fresh ST, WC or MRS broth and grown overnight at 37°C. The cells were pelleted (8000×g, 15min, 4C) and resuspended in 10ml PBS-c (8g l -1 NaCl, 0.2g l -1 KCl, 1.44g l -1 Na 2 HPO 4 , 0.24g l -1 KH 2 PO 4 )(12). The suspension was fixed at 4C for 3 to 4 hours by adding 30 ml of 4% paraformaldehyde (PFA) solution in PBS-c (prepared according to (8)). Next, the sample was washed with 40ml PBS-c (8000×g, 15min, 4C), and the pellet was suspended in 15ml PBS-c, and then an equal volume of 96% ice-cold ethanol was added. The samples are stored at -20C until...

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Abstract

The present invention relates to the field of prevention and treatment of disorders associated with the occurrence of certain carbohydrate epitopes. More particularly, the present invention relates to the prevention and treatment of carbohydrate epitope positive tumors. It relates to formulations and methods for the induction of an effective carbohydrate specific cellular immune response.

Description

Technical field [0001] 1 The present invention relates to the field of prevention and treatment of diseases characterized by the appearance of specific carbohydrate epitopes. The present invention provides nutraceuticals and pharmaceutical compositions, which include microorganisms and fragments thereof that are positive for carbohydrate epitopes, and the microorganisms and fragments thereof induce effective carbohydrate-specific cells against carbohydrate-positive cells and diseases Immune response. In addition, it provides a method for selecting, isolating and identifying carbohydrate-positive microorganisms, which are suitable for use as an effective part of a health food or pharmaceutical composition. It provides methods for generating carbohydrate-specific dendritic cells and cell lines, as well as carbohydrate-specific T cells, T cell clones, and T cell lines. The present invention also provides preparations and methods for preventing and treating diseases related to specifi...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61K39/40G01N33/53A61P35/00A23L1/30A23L33/135C12N1/20C12R1/19G01N33/50G01N33/569
CPCY02A50/30
Inventor S·高尔兹P·尤瑟米尔A·劳弗勒
Owner GLYCOTOPE GMBH
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