DE19853066A1 - Use of hyaluronic acid fragments in vaccine production, especially for cancer treatment - Google Patents

Abstract

Use of optionally modified low molecular weight hyaluronic acid fragments for the production of a vaccine is new. Low molecular weight and optionally modified hyaluronic acid (HA) fragments are used for the production of a vaccine. Independent claims are also included for: (i) a method for the enrichment of dendritic cells, comprising: (a) separating mononuclear cells from blood; (b) concentrating cells having the CD14 surface marker; (c) cultivating the cells obtained in (b) in a medium containing GM-CSF and IL-4 cytokines; and (d) cultivating the cells obtained in (c) with the HA fractions; (ii) the use of the enriched dendritic cells for the production of a vaccine; (iii) a vaccine containing an antigen or peptide, an HA fragment (optionally chemically modified) and optionally a carrier system; (iv) a system containing an HA fragment coupled with an antigen or peptide; and (v) a vaccine containing a system (iv).

Description

The invention relates to the use of low molecular weight Fragments of hyaluronic acid in immunotherapy, in particular for the preparation of compositions used in immunotherapy can be used. Such compositions, which in the Immunotherapy can be used as part of this Application commonly referred to as "vaccines".

The provision of dendritic cells (DC) at the therapy of various diseases an ever increasing growing importance. With the help of dendritic cells it is possible to provide highly active immunomodulators that work with various antigens, especially tumor antigens, Load virus peptides or allergenic compounds can be. These cells are then adopted in the Immunotherapy in patients with tumors, viral diseases or Allergies used. It is therefore adoptive Immunotherapy a significant need for dendritic cells, these dendritic cells preferably over standardized, reproducible and inexpensive processes  should be provided. These procedures must be under GMP / GLP conditions can be carried out.

Various methods are known from the prior art Generation of dendritic cells from stem cells is known.

From the bone marrow or peripheral blood from with Granulocyte colony stimulating factor (G-CSF) treated Chemotherapy patients can use hematopoietic stem cells be isolated, which have the surface marker CD34. These stem cells are made with the addition of various cytokines cultivated and the dendritic cells are after a get relatively long cultivation time.

There are now procedures that use large dendritic cells Number in vitro from progenitor cells of peripheral blood generate (J. Immunol. Med. 1996, 196: 137-51; Exp. Hematology 1997, 25: 232-36; Ann. Surgery 1997, 226: 6-16).

A study in melanoma patients using no shows mature dendritic cells of the prior art Response rates in 5 out of 16 patients (Nature Medicine 1998, 4: 328-32), but all patients were able to use T cells Specificity for that coupled to dendritic cells Melanoma antigens are induced. However, there is the problem that if the amount of peptide is too low or with regard to their Binding strength modified peptides a tolerance to that injected peptide can be induced (J. Immunol. 1998, 160: 4449-56). With regard to tumor therapy, the situation would be fatal, instead of immunizing the patient, this would Situation a tolerance towards the induced Cause tumor antigens. Investigations by the inventors showed that the effect is not even by injecting a larger one Amount of dendritic cells to bypass. Rather is for a promising use of dendritic cells  Degree of their activation or maturation ("Maturation") crucial.

A method of maturing under dendritic cells Use of Monocyte Conditioned Medium (MCM), tumor necrosis factor alpha (TNFα) or CD40 ligation is in the state of the Technique known (J. Immunol. Methods 1996, 196: 121-135; J. Exp. Med. 1997, 185: 341-349; Blood 1998, 91: 4652-4661). At this procedure isolates monocytes from peripheral blood, which are cultivated with GM-CSF and IL-4. For complete Maturation of the cells to dendritic cells must end additional cytokines are added to the differentiation phase.

However, the methods known from the prior art have various disadvantages. The relatively long Cultivation time is not for use in the clinic suitable. In addition, the high cost of the cytokines and the batch dependency of the cytokines used to be disadvantageous.

With conventional vaccinations with antigens or peptides there is also a problem in that the Immune response is too weak or that, as mentioned above, the production of mature dendritic cells, the one could cause improved immune response, too expensive, too is expensive and not reproducible enough. In the Vaccination of tumor patients has also been shown that with a local antigen injection immune responses let trigger, but largely on the injected Extremity or even just the nearest lymph node station remain limited. There is therefore a need for a vaccination strategy, especially for tumor patients, where the antigen can also be given systemically to to induce a generalized immune response. The The most obvious solution to this problem, namely the Administration of intravenous administration of peptide and one  suitable adjuvant, has the disadvantage that by the Dilution effect in peripheral blood within the adjuvant becomes ineffective in the shortest possible time.

These problems can be due to the surprising finding be solved that low molecular weight hyaluronic acid fragments advantageous in different ways in the Immunotherapy, especially for vaccination of Tumor patients can be used.

The present invention therefore provides general use of low molecular weight hyaluronic acid fragments that can optionally be suitably chemically modified for Production of a vaccine, especially for vaccination by subcutaneous (s.c.), intracutaneous (i.c.) and intravenous (IV) vaccination, especially for the treatment of Tumor patients available.

In a first embodiment, the present invention provides a method for enriching mature dendritic cells, which comprises the following steps:

• a) monvuclear cells are obtained from blood,
• b) Cells that have the surface marker CD14 enriched,
• c) the cells which have the surface marker CD14 cultivated in a medium containing the cytokines GM-CSF and IL-4 includes, and
• d) the cells obtained in step c) are hyaluronic acid acid fragments, which are modified as appropriate can be cultured to mature the cells to induce dendritic cells.

The invention also relates to vaccines which contain appropriately mature dendritic cells, and the use of the matured with hyaluronic acid fragments dendritic cells used to make such vaccines.

In a further embodiment, the invention relates Vaccines, the low molecular weight hyaluronic acid fragments, the can optionally be chemically modified, as an adjuvant contain.

In a further embodiment, the invention relates Systems that contain an antigen or peptide, optionally with a Carrier system and coupled to it a low-molecular Hyalu ronic acid fragment, which may be suitably modified may contain. In a further embodiment, the Invention vaccines containing one with a hyaluronic acid contain acid fragment modified antigen or peptide.

The vaccines according to the invention are particularly preferred for subcutaneous (s.c.), intracutaneous (i.c.) or intravenous (i.v.) Vaccination used especially by tumor patients.

First, the first embodiment of the Invention, namely the method for enriching mature dendritic cells with hyaluronic acid fragments, which may be suitably modified, described, as well as the corresponding vaccines that these contain mature dendritic cells.

Mononuclear cells can be extracted from blood using a Density gradients are obtained, with more preferred Embodiment a leukocyte concentrate via a Ficoll Density gradient is separated.

In the second step, the Enriched cells with surface inhomogeneous CD14,  preferably with the help of at least one against the Surface marker CD14 directed antibody. For use Magnet-activated cell sorting (MACS) can come here or fluorescence-activated cell sorting (FACS). A the simpler, but not so efficient, method Enrichment via plastic adhesion.

Then the surface marker CD14 Cells cultivated in a medium that GM-CSF in a Concentration from 5000 to 10000 U / ml and IL-4 in one Has a concentration of 100 to 1000 U / ml. Possibly other suitable cytokines can be added.

Finally, those obtained in the cultivation step Cells cultured with hyaluronic acid fragments, these Have fragments 1 to 50 basic building blocks of hyaluronic acid. A basic building block is an aminodisaccharide from D- Glucuronic acid and N-acetyl-D-glucosamine in β1-3-glycosidic Binding. Hyaluronic acid fragments are preferably used with 1 to 10 basic modules each.

The cultivation of the surface inarker CD14 Cells take place in a medium that contains GM-CSF and IL-4, preferably for a period of at least 72 h to 7 days. This is followed by the cultivation in step d) for at least 48 hours with hyaluronic acid fragments. The Hyaluronic acid fragments are preferably in a concentration from 1 to 50 µg / ml and particularly preferably in one Concentration of 10 to 30 µg / ml.

The present invention thus relates to a Embodiment the use of low molecular weight Hyaluronic acid fragments for the maturation of dendritic Cells.  

Dendritic cells can be produced or enriched by the method according to the invention. The dendritic cells are antigen presenting cells that specialize in the initiation of the primary immune response. They perform different functions in the various stages of their development. In the immature state, the dendritic cells are very effective in processing native protein antigens for the MHC class II route. Mature dendritic cells, on the other hand, are less suitable for the uptake of new proteins for presentation, but stimulate the resting CD4 ⁺ and CD8 ⁺ T cells much better in terms of growth and differentiation.

The maturation of the dendritic cells then takes place in vivo, when the immature dendritic cells from the sites of the Antigen uptake to the T cell areas of the lymphoid organs hike. In vitro, the ripening in cultures of fresh isolated dendritic cells can be observed. The maturation The dendritic cells also translate into changes in the Morphology and phenotype. The characterization and Differentiation of the dendritic cells is usually done on the detection of various surface markers.

Because of their natural functions, they are dendritic Cells particularly suitable as a natural adjuvant in the Vaccination and immunotherapy, especially in tumor therapy, to be used. It is aimed at therapeutic use crucial, mature double rooms too do not use that after reinfusion in the patient differentiate back to macrophage-like preliminary stages can.

According to the current state of knowledge, it is assumed that the dendritic cells in the immature state the antigen (for example, a tumor antigen) and then process the antigen. The dendritic cells mature  and migrate to the T cell-rich areas of the secondary lymphoid organs. Mature dendritic cells express in high levels of MHC, co-stimulating molecules and adhesion molecules on their surface, creating an interaction between the dendritic cells and the T cells (T-cell clustering). Co-stimulatory signals are transmitted via B7-CD28 and CD40-CD40 ligand interactions transmitted and through the Production of cytokines such as IFN-α, IFN-γ and IL-12 which known to promote cellular mediated immunity, reinforced.

Based on current knowledge, it seems to be essential that specialized in the induction of primary T cell response Antigen presenting cells are present as the Presentation of antigens on T cells in the absence of a second signal (co-stimulation) either the death of the T- Cells or an antigen-specific tolerance can cause.

The advantages of the method according to the invention are therefore especially seen in the fact that the dendritic cells can be provided relatively easily. You also have to only relatively low concentrations of expensive Cytokines are used. The method according to the invention enables the deployment of a cell population that is quite consists predominantly of dendritic cells and which are located in a relatively uniform (synchronous) Ripening state. If the dendritic cells for the Presentation of antigens can be used appropriate antigens at the appropriate time dendritic cells are added and the antigens can then from the dendritic cells over the course of the Ripening process. The antigens can either as proteins, in the form of killed cells or Cell preparations or in the form of genetic engineering modified cells are added. If the  dendritic cells are in the desired state, can be used for therapy.

The method according to the invention is for the provision of autologous dendritic cells. The term "autolog" means that the output cells of a donor (patient) be removed. These cells are then grown in vitro treated according to the invention in order to obtain dendritic cells. The enriched dendritic cells are then if necessary after contact with the antigen from the same donor, from which they originally came from.

As an alternative to this, the method can also be used for Provision of "allogeneic" dendritic cells used become. Here, the cells from a donor or from preserved blood obtained from blood donation services can, manufactured and then returned to other recipients. This enables standardized dendritic cells are provided, for example, optimally with a Tumor antigen are loaded. Especially with viral infections (HIV) can effectively increase cellular immunity become.

According to the invention, the dendritic cells are matured Fragments of hyaluronic acid (HA), if appropriate can be modified, used. The hyaluronic acid is a Macromolecular polysaccharide that acts as the body's own substance primarily for water storage in the dermis. Hyaluronic acid is mainly from keratinocytes in the basal Layers of the epidermis and fibroblasts of the Subcutaneous connective tissue is produced. Hyaluronic acid is found also in the vitreous of eyes, the synovial fluid of the Joints and is part of the connective tissue. At low Concentrations, hyaluronic acid forms a highly viscous aqueous Solution. Hyaluronic acid is a high molecular compound with a molecular weight between 50,000 daltons and several  Million daltons. The basic building block of hyaluronic acid is a Aminodisaccharide consisting of D-glucuronic acid and N-acetyl-D- Glucosamine exists in a β1-3 glycosidic bond. This The basic building block is β1-4-glycosidic with the next unit connected. This unbranched chain of hyaluronic acid exists from about 2000 to 10,000 such basic units. By Hyaluronidases are hydrolyzed β-glucosidic bonds and the hyaluronic acid becomes smaller fragments reduced. According to the invention, hyaluronidase is preferred from bull testicles or from Streptococcus hyaluronicus isolated hyaluronidase used. The invention Hyaluronic acid fragments used are preferred initially mechanically crushed by shear force and / or ultrasound and then the polysaccharide is further degraded with the help of a suitable hyaluronidase. The desired Fragments with preferably 1 to 50 basic units are then isolated by suitable separation processes.

The hyaluronic acid fragments can be chemically modified appropriately before or after isolation. Modification after isolation is preferred. Examples of modifications which can be mentioned are esterification, salt formation, amidation, reduction of an acid group to aldehydes or alcohol or elimination of an acid group. Salts and esters of hyaluronic acid fragments, for example alkali and alkaline earth metal salts and C ₁ -C ₁₀ -, preferably C ₁ -C ₄ -alkyl esters are preferred. Other chemical modifications are also possible and can easily be carried out by a person skilled in the art on the basis of his specialist knowledge.

The present invention is further illustrated by the examples described below. When carrying out the examples, particular attention was paid to contamination of the reagents with lipopolysaccharides (LPS). Lipopolysaccharides are components from the cell wall of grams of negative bacteria. Even the smallest contaminations with lipopolysaccharides are sufficient to irreversibly activate monocytes or CD14 ⁺ stem cells from peripheral blood. Therefore, in the experiments carried out according to the invention, care was taken to fall below a threshold value of 0.01 ng / ml lipopolysaccharide, which experience has shown to have no effect on monocytes, CD14 ⁺ stem cells or dendritic cells.

example 1 Cell isolation a) Obtaining mononuclear cells (PBMC)

A buffy coat from a healthy, human donor was over a density gradient with Ficoll Hyperpaque plus (Pharmacia, Uppsala, Sweden) by Centrifugation separated. The mononuclear cells (PBMC) are deposited in the interphase of the gradient, red Blood cells (erythrocytes) as well as neutrophil granulocytes are below the gradient and have been discarded. Ficoll-Hyperpaque plus is a mixture of macromolar sugars cellular origin, but is from the manufacturer on the LPS content tested (endotoxin content <0.001 ng / ml). This value could be in own tests are confirmed.

b) Labeling of the CD14 positive cells

To further purify the monocytes, the PBMC were kept for 45 min. incubated with anti-CD 14 mAb. The mixture was then washed with PBS and the cell pellet was resuspended in 2 ml of MACS buffer. MACS® (Miltenyi, Biotech, Bergisch Gladbach) = Magnetic activated Cell sorting; A method for purifying cells using antibodies bound to metal beads; Marked cells get stuck in the magnetic column matrix. [PBS w / o Ca ²⁺ / Mg ²⁺ (Gibco) with 5 mM ethylenedinitrilotetraacetic acid (EDTA) and bovine serum albumin (BSA); pH 7.2 adjusted by adding HCl]. The addition of EDTA prevents the cells from clumping, which is important for the purification in the column. The cells were then incubated with 50 μl of goat anti-mouse IgG microbeads under the same conditions, then washed with MACS buffer, centrifuged and resuspended in 5 ml of MACS buffer.

c) Enrichment of the CD14 positive cells via MACS

Enrichment took place at 4 ° C. The VS + ® column was inserted into the MACS magnet (both Miltenyi) and first washed with 5 ml MACS buffer. To obtain a single cell suspension, the cells were passed through a cell sieve (30 μm mesh size; Miltenyi) and applied to the column. The negative fraction from unlabeled cells that were not magnetically retained in the column matrix passed through and were discarded. The column was washed twice with 5 ml of MACS buffer in order to obtain the purest possible positive fraction. The column was then removed from the magnets and the cells, which were specifically retained in the matrix by means of microbeads, were rinsed under pressure with 5 ml of buffer into a sterile tube. The cells obtained were counted in the Neubau Chamber. In general, 3-5 × 10 ⁷ CD14-positive cells (monocytes) could be obtained from a buffy-coat using this method, with a purity of <90%. These cells can be completely converted into DC by adding cytokine.

Example 2 Cell culture

The isolated CD14 positive cells were in 12 ml c-RPMI [(cRPMI: "RPMI 1640" (Gibco, Paisley, Scotland) with 10% heat-inactivated fetal calf serum, 1% L-glutamine and 1% penicillin-streptomycin), all components contain less than 0.025 international endotoxin units / ml (recommended value for aqua ad injectabilia)]. In order to mature them into dendritic cells (DCs), they were given human GM-CSF for clinical use (Leukomax 400®, Sandoz AG, Nuremberg) in a concentration of 8000 units / ml medium and IL-4 (Genzyme, Rüsselsheim, Batch tested for LPS content: <0.001 ng / ml) added in a concentration of 500 units / ml medium. Then the cell suspension was pipetted out in a six-well plate to 2 ml each and cultivated in an incubator at 37 ° C. and 5% CO ₂ . On the fourth day, 2 ml / well c-RPMI with GM-CSF and IL-4 were added.

Example 3 Hyaluronic acid preparations a) Fractionation of Hvaluronic acid and separation of the Fragments

Purified hyaluronic acid (HA) from cockscombs (Healon®; Pharmacia, intended for clinical use, endotoxin Content <0.001 ng / mg) was first with an ultrasound device (Branson Sonifier) for 2 min. split into larger fragments. In order to further crush these fragments, they were Hyaluronidase (type I from bovine testicles; Sigma) added. The Reaction solution was adjusted to pH 5 with sodium acetate, Incubated at 37 ° C for 12 hours and then by heating inactivated to 90 ° C. The fragments obtained were now used separated by their size. For this they were in a 1.5 m long Glass column on a polyacrylamide gel (Bio-Gel P-10, Bio-Rad, Munich) layered. Aqua served as the rinsing liquid bidestillata, under the column took a fraction collector (Pharmacia) every 20 min. the factions on. The tubes were closed, stored cool and protected from light.  

b) detection of the size of the hyaluronic acid fragments 1. Larger fragments after ultrasonic comminution

Sonified HA was measured in a 5% agarose gel Gel electrophoresis separated. The polysaccharide bands were in the gel by staining with stains-all (3,3'-diethyl-9-methyl- 4,5,4 ', 5'-dibenzothiacarbocyanine; Sigma) made visible. The fragments thus obtained have a size of 10,000 to 50,000 kDa.

2. Small fragments after additional hyaluronidase digestion ANTS mark

First, the individual samples with the fluorophore ANTS be marked [ANTS solution: 0.15 M 8-amino-naphthalene-1,3,6- Trisulfonic acid disodium salt in acetic acid / water (3/17, v / v), dissolved by slowly heating to 60 ° C]. ANTS marks each Sugar molecule at the chain end. The little fragments were made also by gel electrophoresis using a 30% Acrylamide gel separated. The gel could now be exposed to UV light (ANTS dye) made visible and photographic be documented.

c) Quantitative determination of the hyaluronic acid concentration of fragmentation and separation

0.1 ml of each sample was removed and placed in an ice bath 0.6 ml staining solution (5.2 g di-sodium tetraborate in 1 l concentrated sulfuric acid) mixed. Now were each 0.02 ml 0.1% carbazole in ethanol added, mixed and again 10 min. cooked long. After cooling down The HA concentration could be measured photometrically at room temperature 520 nm can be determined. Aqua dest. Served as the blank Standard 0.2 µM HA / ml in aqua dest.  

Example 4 Cell stimulation

The cells were stimulated on the fourth day of stimulation Cultivation of different HA fragments in one Concentration of 0.025 mg / ml (25 µg / ml) medium was added. As Lipopolysaccharides (LPS) from served as positive controls Escherichia coli serotype 0177 (Sigma).

Results:

• 1. Separation of the fragments obtained using Gel electrophoresis and ANTES staining. The invention fragments used range in size from 2 to 12 fold up sugar. The concentration measurement gave values of about 1 mg / ml split HA. Another split this up Fraction into individual sugars is principally by means of an HPLC (High Pressure Liquid Chromatography) possible. The results allow the conclusion that especially the small fragments for are responsible for the stimulation.
• 2. Influence of DC stimulation by means of HA fragments on the Expression of surface markers.

The surface density can be determined using the FACS images different receptors detected using antibodies become. If you integrate the area under the curves, you get the so-called MFI (Mean fluorescence intensity). These values are listed in Table 1 for various receptors.  

Table 1

The meaning of the individual listed in Table 1 Surface marker:

ICAM-1 is an almost ubiquitous InterCellular Adhesion molecule. The results show an easy one Upregulation, as in various types of Cell activation is observed. CD1a is a marker for dendritic and Langerhans cells of the skin; during monocytes express only CD14, it happens in the course of Maturation more and more to the loss of CD14 and expression of CD1a. LPS, HA fragments and MCM medium push this process back slightly, functional consequences of this Regulation is not known.  

Table 2

Table 2 shows the stimulation of other surface markers (CD44s, CD83 and CD115). Another sign of double maturation are a down regulation of CD115 (the G-CSF receptor) as well as an upregulation of CD83, a functional relevance these changes are not yet known. In Table 2 it can be clearly seen that the criteria for the maturation of the double room fully satisfied by stimulation with small HA fragments become.

CD44 is an adhesion molecule that is a described function the binding of HA.  

Table 3

Table 3 shows the expression of further surface markers.

In addition to the maturation markers, factors are shown here that for the function of the antigen presentation, d. H. of stimulation of T lymphocytes play a crucial role. For this include the MHC class II molecule HLA-DR and the two costimulatory factors B7-1 and B7-2. Without that Upgrading these factors is also an application of the DZ in the applications described above does not make sense. According to the invention, a clear upregulation of all Factors found after treatment with HA fragments, comparable to the maximum stimulation after LPS administration.

Example 5

In order to functionally represent the increased expression of surface molecules, the DCs were pre-stimulated and incubated for 5 days with purified naive allogeneic T cells, which were also obtained from leukocyte concentrate (buffy coats) (so-called mixed leukocyte reaction, MLR). Depending on the stimulatory capacity of the DCs, there is more or less pronounced T cell proliferation, which was determined by incorporating radioactive ³ H-thymidine. The radioactive counts per minute (Cpm) of the individual samples were shown, which correlate directly with the T cell proliferation that took place. It can be seen that DCs treated with small HA fragments are significantly more potent APCs, comparable to the value obtained for LPS stimulation (shown in Table 4 B).

This example also shows that according to the invention with small HA fragments between 2-12 UDP sugar molecules a distinct one Maturation of DC is obtained, which is quite comparable with published data from other methods. A strict one Dependence on a single sugar appears unlikely, but larger molecules (20-30 UDP- Sugar) obviously no influence, since the invention used fractions no differences in effect show, even if they contain larger fragments. Sounded HA also has no effect.

Example 6

Interestingly, the effect according to the invention obviously specific for double rooms, because with interferon-gamma (IFNγ) treated monocytes that mature into macrophages no increase in their stimulatory capacity after Show treatment with HA fragments (see Table 4 A).  

Table 4

Ask 45: No evidence of small in gel electrophoresis HA fragments, in the uronic acid assay less than 0.001 mg HA Negative fraction to rule out an effect of Column components.

Ask 17: The largest are in gel electrophoresis detectable bands with approx. 16-fold sugars, strongly represented are sugar up to a size of 6 times sugar. Uronic acid assay: 1 mg / ml.

Ask 14: Gel electrophoresis: up to approx. 22 times sugar, strong Bands up to 10-fold sugar, uronic acid assay: 1 mg / ml.

Example 7

This example shows that, surprisingly, by mature hyaluronic acid fragments dendritic cells conventional dendritic cells in the  Induction of a peptide-mediated hapten specific Immunity are superior.

Mice (C57 / BL6, female, 6-12 weeks, 60 pieces, 5 mice each experimental group, 20 g per mouse) were dendritic Cells previously injected with a synthetically produced Protein with a defined amino acid sequence, a peptide (SIINFEK * L, SIIK * FEKL; * = TNP-lysine). The Peptide is chosen so that it is directly linked to the Antigen binding site of MHC molecules on the cell surface can react from antigen presenting cells; this means, it is immediately after the addition of dendritic cells presented on their surface. At this peptide is in turn coupled to the hapten trinitrophenyl (TNP), which is the antigen Represents determinant. That is, a T cell that over that from the dendritic cell presented at trinitrophenyl coupled peptide is activated, becomes reactive against Trinitrophenyl or chemical structural homologue of Trinitrophenyl, e.g. B. that during the below trinitro- Chlorobenzene (TNCB). The strength of the trinitrophenyl-specific In this system, the T cell reaction is directed accordingly according to the degree of dendritic cell activation; or the Effectiveness with the peptide coupled to trinitrophenyl from the dendritic cells to the T cells. The The method represents a recognized model for in vivo investigation of mechanisms that a. during T cell activation Role-play.

Corresponding methods are for example in the J. Invest. Dermatol. 1998, 110: 441-48; Eur. J. Immunol. 1995, 25: 92-101 and J. Immunol. 1993, 151: 678-87 described in more detail.

The injected dendritic cells dissolve within one Week a T-cell mediated immune response. That means, you  migrate into the lymphatic organs and after injection trigger the T cell activation described above. The Efficiency of the immunization can take place after this time based on an ear swelling reaction caused by direct Application (ear brushing with a 1% TNCB solution) one Trinitrophenyl analogs, in our case Trinitro-Chlorobenzen (TNCB) to be quantified on the ear. This treatment triggers an inflammatory reaction within 24 hours, the strength of which on the number of memory T cells in the skin depends. The ear swelling reaction is called an increase in density of the ear measured in µm. The results are as follows Table 5 summarized.

Table 5

In this system, a significant increase in Ear swelling reaction after pretreatment of the dendritic Cells with low molecular weight hyaluronic acid fragments be detected. The injection of untreated  dendritic cells (group 6) releases a barely detectable Ear swelling reaction, as well as the sole one Pretreatment with high molecular hyaluronic acid (group 4) or low molecular weight hyaluronic acid fragments (group 2). Become the dendritic cells pretreated with hapten peptide (group 5), there is a clear ear swelling reaction of approx. 0.15 mm. The T cell response can be increased by additional Pretreatment of dendritic cells with low molecular weight Hyaluronic acid fragments significantly improve and shows a Ear swelling response of approximately 0.24 mm (group 1). A Pretreatment with high molecular hyaluronic acid and hapten In contrast, peptide (group 3) shows no significant improvement compared to the untreated control group 5.

Example 8

This example was about a mouse model with specificity for the Lympho-Choriomeningitis Virus (LCMV) vaccinations with dendritic cells with low molecular weight Hyaluronic acid fragments had been treated for induction tested for antiviral immunity. The used The test method is in principle from J. Virol. 1998, 72: 3812-18.

In the same way as in Example 7, this was done dendritic cells treated with GP-33, a peptide which for the antigenic determinant of a surface antigen of the Coded LCM virus, and injected intravenously into the mouse. This causes, as described above, depending on the degree of activation or Pretreatment of the dendritic cells more or less effective immunization of the animal against LCMV. The The effectiveness of the immunization was then assessed after a week determined by vaccinating the mouse with the complete LCMV. A successful immunization against the virus can be in the form a clonal expansion of GP-33 specific T cells in the Blood and the lymphoid organs of the treated animals  read off. In another assay, the determination of the virus Load (number of viruses) in peripheral blood determined.

In a second embodiment, described below the invention provides a vaccine Available that contains at least one antigen or peptide and a low molecular weight hyaluronic acid fragment, which if necessary can be modified as an immune response enhancer (adjuvant). It was surprisingly found that low molecular weight Hyaluronic acid fragments, which may be modified can, advantageously as an adjuvant in direct vaccination can be used with antigens or peptides.

This method uses a vaccine (vaccine) that contains the antigen contains, the patient subcutaneously (s.c.), intracutaneously (i.c.) or also injected intravenously (IV). The injected antigen will of the body's own antigen presenting cells (APZ) e.g. B. the dendritic cells and recorded in regional Lymph node presents what is an antigen-specific Triggers immune response. It was surprisingly shown that the simultaneous injection of low molecular weight Hyaluronic acid fragments, optionally chemically can be modified by the immune response Recruitment, activation and maturation of APZ at the Injection site reinforced.

The low molecular weight hyaluronic acid fragments or their chemical modifications are already at the first Embodiment of the invention has been described in detail. The in this second embodiment of the invention usable low molecular hyaluronic acid fragments or their chemical modifications are the same as for the first embodiment described. The production takes place also as described above.  

The vaccine according to the second In principle, any antigen or peptide that is said to cause an immune response. The Hyaluronic acid fragments or the chemical modifications thereof are preferably contained in the same solution in which the antigen or peptide is included. It is also possible to separate the antigen or peptide from the low molecular weight hyaluronic acid fragments or their chemical To administer modifications. A vaccine that does both Antigen or peptide as well as the low molecular weight Hyaluronic acid fragment, which may be chemically modified contains, contains the antigen or peptide in one Concentration of approx. 1 to 1000 µg / ml, preferably in one Concentration from 10 to 100 µg / ml and the low molecular weight Hyaluronic acid fragment or its chemical modifications in a concentration of 10 µg / ml to 1000 µg / ml, preferably in a concentration of 10 µg / ml to 100 µg / ml, e.g. B. 30 µg / ml. A suitable daily dose of antigen or peptide is z. B. 1 to 1000 ug / day, preferably 10 to 100 ug / day and the daily dosage on the low molecular weight Hyaluronic acid fragments and their chemical modifications can e.g. B. 10 ug / day to 1000 ug / day, preferably 10 ug / day to 100 µg / day, e.g. B. 30 µg / day.

The compositions of the invention can also Antigen or the peptides and the low molecular weight Hyaluronic acid fragments, optionally chemically can be modified, still others in the prior art contain known auxiliaries, e.g. B. Freund's adjuvant.

According to the invention, the administration and use of the low molecular weight hyaluronic acid fragments or their chemical Modifications take place as is customary in the field and is familiar to the expert.  

The following example explains this second embodiment the invention.

Example 9

To test the in vivo effectiveness of low molecular weight hyaluronic acid fragments, healthy test subjects (N = 3) were given 100 µl of a 1 mg / ml preparation of low molecular weight hyaluronic acid fragments (one basic unit and fragments with 2 to 20 basic units) and 100 µl isotonic saline solution as a control (0, 9% NaCl) injected subcutaneously for two consecutive days. On day three, punch biopsies were taken and the preparations were processed immunohistologically. Already after 24 hours after subcutaneous injection of low molecular weight hyaluronic acid fragments, there was a mild inflammatory reaction in the form of a discreet reddening around the injection site, as well as a clear infiltrate, which mainly consists of activated "mature" (mature) dendritic cells. The results of the immunohistological preparations are summarized in Table 6. The cells infiltrating into the dermis were counted under the microscope under 40x magnification using a raster eyepiece, 3 × 12 fields were counted per preparation and the number of infiltrating cells per mm ^{2 was} calculated from this. Table 6 shows the significant increase in infiltrating cells as early as 24 h after the injection of low molecular weight hyaluronic acid fragments; in contrast, the control injection carried out in parallel shows no such effect. The exact immunophenotyping of the infiltrate shows a clear predominance of CD1a and HLA-DR positive cells at the sites treated with low-molecular hyaluronic acid fragments, ie the cells show the phenotype of mature dendritic cells. A significant increase in lymphocytes or individual subclasses (CD19 = B cell marker, CD3 = T cell marker, CD4 = T helper cell marker, CD8 = marked cytotoxic T cells) was not observed at least in the observed period (Table 6). In clear contrast to this finding, the infiltrate after control injection of isotonic saline shows a much more homogeneous distribution, comparable to the distribution of the individual subclasses as found in an unspecific inflammatory reaction (Table 6).

Table 6

The example shows that low molecular weight Hyaluronic acid fragments obviously next to the potent Activation of dendritic cells in vitro according to the first Embodiment of the invention also directed immigration of activated dendritic cells to the injection site in can cause vivo. These dendritic cells migrate then after inclusion of co-injected or low molecular weight Hyaluronic acid fragments coupled antigens / peptides to the regional lymph nodes where they have a specific T cell mediated immune response can induce.

The following is the third embodiment of the invention described, according to the vaccines provided that contain a system in which the low molecular weight Hyaluronic acid fragments or their chemical modifications an antigen or peptide are coupled. Through this  Embodiment of the invention particularly addresses the problem solved that with a local antigen injection Allow immune responses to be triggered, but largely to the injected extremity or even just the next one Lymph node station remain limited. With the Vaccines according to the invention according to the third embodiment it is possible to use the antigen systemically e.g. B. intravenously Administer without the adjuvant (the low molecular weight Hyaluronic acid fragments, which may be chemically modified due to the thinning effect in peripheral blood becomes ineffective within a short time.

The low molecular weight hyaluronic acid fragments or their chemical modifications are the same as for the first embodiment described and can be the same Way to be made.

As an antigen or peptide, optionally with a carrier system, can usually in vaccines, preferably in vaccines for tumor therapy, called antigens or peptides become.

The way of coupling between low molecular weight Hyaluronic acid fragment, which may be chemically modified can be and peptide or antigen, optionally with a Carrier system is not particularly limited. Prefers finds the coupling through chemical covalent bonding of Antigen or peptide with the adjuvant (low molecular weight Hyaluronic acid fragment or chemical modification thereof) instead, or by the joint inclusion of antigen or peptide and adjuvant in a microsphere, e.g. B. Cancer Res. 1998, 58: 3385-90.

It is particularly preferred to couple the basic building block UDP-β-DN-acetylglucosamine used in hyaluronic acid synthesis to the antigen or preferably to the peptide. This means that the hyaluronic acid fragment with a basic unit is particularly preferred in the third embodiment of the invention. As already explained above, this is an aminodisaccharide composed of D-gluconic acid and N-acetyl-D-glucosamine in a β ₁ -3-glycosidic bond. UDP-β-DN-acetylglucosamine is commercially available.

The vaccines according to the third embodiment of the invention can be produced by processes and with additives, as they are known in principle in the prior art. It is only essential that the new combinations of peptide or Antigen, optionally with a carrier system and low molecular weight hyaluronic acid fragment, which if necessary can be chemically modified. It understands it goes without saying that in the present case the chemical Modification of the hyaluronic acid fragment can also be chosen in this way may have a suitable chemical coupling to the antigen or peptide can be carried out advantageously. Suitable chemical Modifications and coupling methods are known to the person skilled in the art Known principle, and any method of the prior art Technology can be used.

A vaccine according to the invention according to the third Embodiment of the invention includes e.g. B. 1 µg / ml to 1000 µg / ml, preferably 10 µg / ml to 100 µg / ml of the antigen or Peptide, optionally with a carrier system. A suitable one Daily dose of antigen or peptide, optionally with a Carrier system is e.g. B. 1 ug / day to 1000 ug / day, preferably 10 µg / day to 100 µg / day. Is the coupling between antigen or Peptide and low molecular weight hyaluronic acid fragment or chemical modification thereof takes place by covalent bonding, is the concentration or daily dose of the low molecular weight Hyaluronic acid fragments or their chemical modification necessarily equal to the corresponding concentration or Daily dose of the antigen or peptide, optionally with a Carrier system. If the low-molecular hyaluronic acid fragment is  which may be chemically modified, and Antigen or peptide not chemically bound before, but e.g. B. by common inclusion in a microsphere, that is preferred concentration of low molecular weight Hyaluronic acid fragments or its chemical modification preferably also equal to the concentration of the antigen or Peptides.

Possible additives to the vaccine are common Additives, e.g. B. Freund's adjuvant. The production of the In principle, vaccine takes place in the prior art known way.

The example explains the third embodiment of FIG Invention.

Example 8

In order to achieve the greatest possible proximity of vaccine peptide and adjuvant, the low-molecular hyaluronic acid fragment or the basic building block used in hyaluronic acid synthesis UDP-β-DN-acetylglucosamine (fragment with a basic unit) was coupled directly to the peptide. UDP-β-DN-acetylglucosamine is available as a pure substance from Oxford Glucosciences and shows a similar effect on dendritic cells as the hyaluronic acid cleavage products, in terms of both the required concentration of the substance and the degree of activation. The generally known conversion of aldehydes and amino groups to Schiff bases is used for coupling. A certain percentage of the sugar molecule is in its open-chain aldehyde form and, in aqueous solution, undergoes the above-mentioned reaction with the protein.

To stabilize the product, the compound is reduced by adding sodium borohydrite (NaBH ₃ ), which forms a covalent bond between the carbon atom of the aldehyde group and the nitrogen atom of the amino group on the peptide with elimination of water.

To check the effectiveness, the so produced Compound either intra- or subcutaneously in 50-100 µl isotonic Saline can be applied. The peptide-HA compound binds to MHC molecules on the Surface of antigen presenting cells e.g. B. dendritic cells and is said to simultaneously activate the Effect cells. Administration can also be intravenous respectively.

Preferred examples of peptides against tumor and virus antigen, which are preferred in all embodiments according to the invention, are listed in Table 7:

Table 7

The vaccines according to the invention according to all embodiments can the antigen or peptide, optionally with a suitable carrier system. Such carrier systems are e.g. B. liposomes and microsomes, as in the prior art is known to be made from phospholipid compounds can.

Claims (33)

1. use of low molecular weight hyaluronic acid fragments, which may be modified as appropriate, for Production of a vaccine. 2. Use according to claim 1, wherein the vaccine for Treatment of cancer can be used. 3. Use according to claim 1 or 2, wherein the Hyaluronic acid fragments, which are modified as appropriate can consist of 1 to 50 basic units. 4. Use according to claim 3, wherein the Hyaluronic acid fragment UDP-β-D-N-acetylglucosamine (fragment from a basic unit). 5. Use according to one of claims 1 to 4, wherein the low molecular weight hyaluronic acid fragments, if appropriate can be suitably chemically modified for the enrichment of serve dendritic cells, which are then used as a vaccine become. 6. A method for enriching dendritic cells, comprising the following steps:

• a) mononuclear cells are obtained from blood,
• b) cells which have the surface marker CD14 are enriched,
• c) the cells having the surface marker CD14 are cultivated in a medium which contains the cytokines GM-CSF and IL-4, and
• d) the cells obtained in step c) are cultivated with hyaluronic acid fragments, which may optionally be modified in a suitable manner, in order to induce the irreversible maturation of the cells to dendritic cells.

7. The method according to claim 6, characterized in that the mononuclear cells from blood using a density gradient, especially a Ficoll density gradient from one Leukocyte concentrate can be obtained. 8. The method according to any one of claims 6 or 7, characterized characterized in that the surface marker CD14 Cells using at least one against the surface marker CD14-directed antibodies can be enriched. 9. The method according to any one of claims 6 to 8, characterized characterized in that the surface unarker CD14 Cells are cultivated in a medium that GM-CSF in a Concentration from 5000 to 10000 U / ml and IL-4 in one Has a concentration of 100 to 1000 U / ml. 10. The method according to any one of claims 6 to 9, characterized characterized in that the cells in step d) with Hyaluronic acid fragments are cultivated, the 1 to Have 50 basic building blocks of hyaluronic acid, the Basic building block an aminodisaccharide from D-glucuronic acid and N- Acetyl-D-glucosamine in βl-3-glycosidic bond. 11. The method according to claim 10, characterized in that the hyaluronic acid fragments each 1 to 10 aminodisaccharides exhibit.   12. The method according to any one of claims 6 to 11, characterized characterized in that the surface marker CD14 Cells between 72 hours and 7 days in a medium cultured that contains GM-CSF and IL-4. 13. The method according to any one of claims 6 to 12, characterized characterized in that the cells in step d) for at least Can be cultivated for 48 hours with hyaluronic acid fragments. 14. The method according to any one of claims 6 to 13, characterized characterized that chemically modified hyaluronic acid fragments are used. 15. Enriched dendritic cells, available after a Method according to one of claims 6 to 14. 16. Use of enriched dendritic cells after Claim 15 for the manufacture of a vaccine. 17. Use according to claim 16, wherein the vaccine for Treatment of cancer is used. 18. Use according to any one of claims 1 to 4, wherein the Vaccine low molecular weight hyaluronic acid fragments that can optionally be suitably chemically modified and an antigen or peptide, optionally with a carrier system, contains. 19. Vaccine containing an antigen or peptide, optionally with a carrier system and a low molecular weight Hyaluronic acid fragment, which may be suitable chemically can be modified. 20. Vaccine according to claim 19, wherein the vaccine for the subcutaneous, intracutaneous or intravenous administration is prepared.   21. A vaccine according to claim 19 or 20, wherein the vaccine consists of a formulation that contains the antigen or peptide, optionally with a carrier system and the low molecular weight Hyaluronic acid fragment, which is modified as appropriate can be contains. 22. A vaccine according to claim 19 or 20, wherein the vaccine comprises two separate formulations, one formulation the antigen or peptide, optionally with a carrier system, contains and the other formulation the low molecular weight Hyaluronic acid fragment, which is modified as appropriate can be contains. 23. Vaccine according to one of claims 19 to 22 Use in the treatment of cancer. 24. Vaccine according to one of claims 19 to 23, wherein the low molecular weight hyaluronic acid fragment, which if necessary can be suitably modified from 1 to 50 basic units consists. 25. Use according to any one of claims 1 to 4, wherein the Vaccine includes a system in which the low molecular weight Hyaluronic acid fragment, which is modified as appropriate may be coupled to a peptide or antigen. 26. System containing a low molecular weight Hyaluronic acid fragment that is modified as appropriate may be coupled to an antigen or peptide. 27. The system of claim 26, wherein the low molecular weight Hyaluronic acid fragment, which is modified as appropriate can be chemically bound to the antigen or peptide is present.   28. The system of claim 26, wherein the low molecular weight Hyaluronic acid fragment, which is modified as appropriate can be and the peptide or antigen as separate molecules in in a microsphere. 29. The system according to any one of claims 26 to 28, wherein the low molecular weight hyaluronic acid fragment, which if necessary can be suitably modified, comprises 1 to 50 basic building blocks. 30. The system of claim 29, wherein the low molecular weight Hyaluronic acid fragment, which is modified as appropriate UDP-β-D-N-acetylglucosamine (fragment from a Basic module). 31. The system according to any one of claims 26 to 30, wherein the Antigen or peptide with a carrier system is present. 32. Vaccine containing a system according to one of the claims 26 to 31. 33. Vaccine according to claim 32 for the treatment of Cancer.