WO2003016512A1 - Method for improving methods of immunotherapy - Google Patents

Abstract

The invention relates to a method for improving methods of immunotherapy. The areas of application of the invention include the field of medicine and molecular biology. The aim of the invention is to provide a simple method for influencing ligand exchange of MHC molecules for the purpose of developing therapeutical methods. The invention is put into practice according to the claims. It is based on the surprising finding that ligand exchange of MHC molecules (MHC class II molecules and MHC class I molecules) is influenced by H bridge donors and acceptors. It was further surprisingly found that hydrogen bridge donors or acceptors also influence the exchange of larger ligands (preferably > 3000 daltons, also >10000 daltons), such as for example proteins or complex protein mixtures, on MHC molecules (MHC class II molecules and MHC class I molecules). Preferably ligand exchange is influenced by low-molecular H bridge donors or acceptors.

Description

Methods for improving immunotherapeutic methods

The invention relates to a method for improving immunotherapeutic methods. Areas of application of the invention are medicine and molecular biology.

Immunotherapy methods have become increasingly important in the fight against various diseases in the past decade. Proteins of the MHC gene complex (Major Histocompartibility Complex) play a significant role in these processes. The most important molecules are the MHC class I and MHC class II molecules. They are receptor molecules whose task is to present short protein fragments on the cell surface. Among other things, also presented peptide antigens, which in conjunction with the MHC molecules form the target structure of the T cells and, after binding of the T cell receptor, initiates the antigen-specific activation of the T cells.

The MHC-associated peptide antigens are therefore causally responsible for triggering cellular immune responses. Depending on the origin of these peptide antigens, they can be divided into different groups. Pathogen-specific peptide antigens occur, for example, in the course of infections. They originate directly from pathogens (e.g. virus proteins) and trigger the pathogen-specific immune defense accordingly. Tumor-associated peptide antigens can appear after cell transformation. They are specific for the transformed cell and can trigger immune responses that are directed against tumors. A third group of peptide antigens form autoantigens. Autoantigens are peptide antigens that are derived from the body's own self-proteins and can trigger autoimmune reactions. These can e.g. B. manifest in diseases such as multiple sclerosis (MS), diabetes or rheumatoid arthritis (RA).

In addition to the antigens that are presented on the cell surface by the MHC molecules, the type and maturation of the antigen-presenting factors also play a role in the extent and direction of the immune response Cell matter. Antigens, presented in "mature" (matured) dendritic cells, induce productive immune responses, such as those required for fighting infections or eliminating tumor cells. 'Immature' (unripe) dendritic cells tend to trigger suppressive immune responses, which leads to the suppression of existing or emerging productive immune responses. The latter is of importance for the treatment of autoimmune diseases (Hackstein et al., 2001). At the present time, most antigen-specific immunotherapies are still in the experimental stage and cannot yet be used routinely. One of the reasons for this is that in order to trigger the specific immune response, the antigen-presenting cells suitable for the respective purpose must be loaded with sufficient amounts of antigen.

The invention has for its object to find a simple way to influence the ligand exchange of MHC molecules, in order to build up therapeutic methods. In order to expand the spectrum of the improvement of the immunotherapies, it is desirable to also use larger molecules, e.g. whole proteins to be used as antigens.

The invention is implemented according to the claims. It is based on the surprising finding that the ligand exchange on MHC molecules (MHC class II molecules and MHC class I molecules) is influenced by H-bridge donors or acceptors. The ligand exchange is preferably influenced by low molecular weight H-bridge donors or acceptors.

It was also surprising that the exchange of larger ligands (preferably> 3,000 Daltons, also> 10,000 Daltons), such as proteins or complex protein mixtures, on MHC molecules, also by H-bridge donors or - acceptors, preferably by low-molecular H-bridge donors or - acceptors (MHC class II molecules and MHC class I molecules) is influenced. The invention relates to a method for improving immunotherapeutic methods by influencing the ligand exchange of MHC molecules by H-bridge donors or acceptors. The invention also relates to a method for targeted antigen loading of cells by ligand exchange on MHC molecules, which is characterized in that H-bond donors or acceptors are used to influence the exchange of ligands, which also include large ligands, large ones being used Ligands preferably act as whole proteins, protein mixtures and / or complex protein mixtures. The invention also relates to the antigen-loaded (antigen-presenting) cells, possibly MHC molecules, and their use for the treatment of diseases, especially cancer (such as colon carcinomas, melanomas, kidney carcinomas, lymphomas) or infectious diseases (such as Influenza, malaria), as well as autoimmune diseases (such as multiple sclerosis, rheumatoid arthritis, diabetes) or to weaken aggressive immune reactions.

This influence can lead to an increase or a decrease in the antigen loading of MHC molecules, of which examples are given in the practical part. In order to trigger tumor-specific, pathogen-specific or autoreactive immune responses, an increase in the loading of antigen is desirable. To trigger the immune responses, antigen-representing cells such as dendritic cells (matured and unmatured), B cells, macrophages and others are loaded.

In order to weaken aggressive immune reactions, the load with low-affinity antigens must be lowered.

The method according to the invention brings about an enormous improvement in immunotherapeutic methods by the targeted increase or decrease in the load.

Low molecular weight H-bridge donors in the sense of the invention are lower aliphatic alcohols, preferably ethanol, propanol or butanol. Phenols, anilines or their derivatives, preferably parachlorophenol, are equally suitable. In a further preferred embodiment of the invention, well-known pharmaceuticals such as diprivan (2,6-diisopropylphenol) are also used as donors. The latter is particularly advantageous because these pharmaceuticals do not have to be approved separately. According to the invention, it is also possible to use combinations of the compounds mentioned as H-bridge donors.

The new procedure opens up a wide range of options for improving immunotherapy. A special variant "ex vivo" is to be specifically listed below.

For the treatment of diseases, especially cancer (such as colon carcinomas, melanomas, lymphomas) or infectious diseases (such as influenza, malaria), the body's own dendritic cells are loaded with tumor- or pathogen-specific antigens by H-bridge donors or acceptors, after which the Cells are then reinjected to trigger the immune response to the patient.

Furthermore, for the treatment of autoimmune diseases (such as multiple sclerosis, rheumatoid arthritis, diabetes), the body's own immature dendritic cells can be loaded with tissue-specific self-antigens by means of H-bridge donors or acceptors, after which the cells are then reinjected to trigger a suppressive immune response to the patient. Alternatively, mature dendritic cells can also be used if necessary.

An advantage of the new method is that both antigen components and whole proteins, protein mixtures and even relatively complex protein mixtures, such as myelin-containing spinal cord homogenate, can be loaded onto preferably MHC class II molecules. It is possible that, for. B. to trigger a tumor response, the antigen-representing cells ex vivo with tumor-associated tumor cell lysates can be loaded and reinforce this loading according to the invention by the small molecules (H-bridge donors or acceptors).

The invention will be explained in more detail below by means of exemplary embodiments and illustrations

Example 1:

Ethanol is one of the simplest small molecules with the ability to form an H bond (Peoples et al., 1996). In order to examine the influence on the peptide loading, soluble HLA-DR1 molecules were incubated with a high-affinity peptide ligand (HA306-318) in the absence or presence of ethanol. After 8 h the complex formation was analyzed by SDS-PAGE. As shown in Fig. 1, the presence of ethanol has significantly increased the amount of peptide complex. Visible by silver staining, the band representing the SDS-stable HA306-318 / HLA-DR1 complex was clearly visible in the ethanol-containing sample (Fig. 1, lane 4), while the corresponding band was far paler if no ethanol was included (lane 3). Quantification of the loading efficiency by ELISA showed that the amount of complex formed in the presence of 5% ethanol was about 5 times higher than in the absence (data not given).

In the next series of experiments, the influence of ethanol on the release of the .Invariant Chain'-CLIP peptide was examined (Fig. 2). The 'invariant chain' molecule binds to MHC class II proteins at early biosynthesis stages and directs them to the endosomal MIIC part, where the peptide loading mediated by HLA-DM takes place (Busch et al., 2000). After proteolytic degradation of the "invariant chain" protein, the CLIP peptide (IC106-120) usually remains at the binding site until it is replaced by ligands of higher affinity. The tests with ethanol have shown that for HLA-DR2 a dose-dependent release of CLIP was observed in the presence of 10 μg / ml free MBP86-100 peptide. In the absence of free high affinity peptide, ethanol did not release CLIP, which shows that ethanol did catalyze the exchange reaction of CLIP. In contrast to HLA-DR2 was found to have no effect on HLA-DR1. Even when a 10-fold higher affinity ligand (HA306-318) was present than in the case of HLA-DR2, ethanol was unable to catalyze the exchange. Apparently, the higher affinity of CLIP for HLA-DR1 prevents the release from this MHC class II molecule by ethanol.

The previous experiment showed similarities to the exchange reaction catalyzed by HLA-DM. For a further assessment of the ethanol-mediated ligand exchange, the release of the CLIP peptide by ethanol was compared directly with the CLIP release, which was triggered by soluble HLA-DM. For both HLA-DM (Fig. 3, middle row) and ethanol (bottom row), the release reaction was in the presence of titrated amounts of free peptides with high affinity (MBP86-100), with medium affinity (IC 106-120) and with low affinity (HA306-318) for the HLA-DR2 molecule and compared with the CLIP release of the uncatalyzed exchange reaction (top row). Since HLA-DM needs an endosomal pH to show optimal catalytic activity, the first part of the experiment was carried out at a pH of 5.0 (Fig. 3A). According to previous reports (Sloan et al., 1995), the influence of HLA-DM was only evident in the presence of a high-affinity binding peptide. An almost complete release of the biotinylated CLIP peptide was found at a concentration of only 10 μg / ml MBP peptide, whereas essentially no difference to the control reaction in the presence of CLIP 106-120 or HA306-318 peptide was observed. Exactly the same result was achieved with ethanol. In the presence of 5% ethanol, CLIP was displaced from the HLA-DR2 binding site by 10 μg / ml MBP86-100, but not by IC 106-120 or HA306-318. Ethanol thus mediates an exchange of the peptides on the HLA-DR2 molecule according to their affinity. In the second part of this experiment, the effectiveness of HLA-DM and ethanol in ligand release under physiological conditions was tested (Fig. 3B). HLA-DM molecules are known to have little or no catalytic activity at pH 7.0 (Busch et al., 1998) and according to these reports (Jensen et al., 1999) was at a pH Value of 7.3 almost no release of biotinylated CLIP peptide from HLA To observe DR2 molecules. In the presence of HLA-DM, approximately 80% of the CLIP / HLA-DR2 complex was therefore still detectable after the incubation. In contrast to HLA-DM, the ligand exchange capacity of ethanol was only slightly reduced at neutral pH. Less than 12% of the HLA-DR2 molecules were still loaded with CLIP after incubation with ethanol. This effect is probably due to the unchanged dipole moment of the hydroxyl group of ethanol, which, in contrast to the HLA-DM activity, is not influenced by the increased pH.

Example 2:

In order to identify even more effective compounds, a number of small organic molecules with different functional groups were tested (a manuscript is created). The most effective proved to be hydrophobic compounds that had hydroxyl groups. For aliphatic alcohols it was found that the catalytic activity increases with the chain length (Fig. 4A). Methanol (Ci) has practically no effects, ethanol triggered a 50% release of the CLIP peptide at a concentration of 600 mM, n-propanol at 140 mM and n-butanol (C ₄ ) at a concentration of 40 mM. Replacing the hydroxyl group with (positively charged) amino groups led to a complete loss of catalytic activity. Therefore no release was observed with n-propylamine (Fig. 4B, top picture). Aromatic compounds tended to be more effective ligand exchangers than aliphatic molecules. The activity of 2 aromatic compounds is shown in the lower image of Fig. 4B. Phenol triggered a 50% release at a concentration of about 30 mM. A similar effect was also found with aniline, which has an uncharged amino group due to its mesomer structure (Fig. 4B, bottom picture). No release was found with ethanolamine or amino acids such as tyrosine. Apparently, the presence of charged substituents prevents the catalytic activity even if they are removed from the H bond donor group (no data given). However, the most important result of these studies was that the compound was found to have a functional group that can act as an H bond donor. As shown in Fig. 4C, the removal of the hydroxyl group or the blocking of the H donor function by substitution of the hydrogen completely abolished the catalytic effect.

For all subsequent experiments, the more effective small molecules n-propanol and phenol were used instead of ethanol. Their influence on the binding of high affinity ligands to HLA-DR / CLIP complexes is shown in Fig. 5. In the absence of these alcohols, only relatively low binding rates were measured. Therefore, after a 3 hour incubation less than 10% of HLA-DR1 was loaded with HA306-318 and about 38% of the HLA-DR2 molecules were loaded with MBP86-100. In contrast, the presence of 75 mM phenol reduced the time to reach half the maximum load (tι _{/ 2} ) to a few minutes. With HA306-318 and HLA-DR1 tι _{/ 2 was determined} to be about 15 minutes, while with MBP86-100 and HLA-DR2 t-ι _{/ 2 was} only 5 minutes. Such a rapid loading indicates the formation of peptide-receptive forms of MHC class II molecules (Rabinowitz and others, 1998). Compared to phenol, n-propanol showed only a slightly reduced effect for HLA-DR2, however a considerable decrease in the loading efficiency was observed for HLA-DR1. The receptive forms have been reported to occur after the release of the peptide ligands (Natarajan et al., 1999, Rabinowitz and et al., 1998). The difference in the loading capacity of phenol and n-propanol could therefore be due to the higher stability of the HLA-DR1 / CLIP complexes, which is less sensitive to the CLIP release due to the weaker n-propanol (see Fig.

2). Because small molecular H-bond donors maintain activity at a neutral pH, they could be tested under living cell physiological conditions. In contrast to the stronger phenol, n-propanol did not affect cell viability. A number of experiments were carried out in which the influence of n-propanol on the rate of ligand exchange on the cell surface was determined. First, the release of natural ligands of MHC class II molecules on the cell surface was tested by measuring the amount of "empty" MHC molecules after the action of n-propanol on dentritic cells. The analysis was performed using the conformation-specific KL304 antibody (LaPan et al., 1992). It was found that this antibody only binds to empty MHC class II molecules (Santambrogio et al., 1999a, Santambrogio et al., 1999b). A FACS analysis with dendritic cells (developed from mouse bone marrow cells) showed that the amount of empty MHC class II molecules was actually significantly higher after a 4-hour incubation with 2% n-propanol (Fig. 6A). The median fluorescence of the KL304 staining was 81, while in the absence of n-propanol a value of only 38 was measured. In order to ensure that the incubation with n-propanol produces peptide-receptive MHC class II molecules, the action on dendritic cells was repeated in the presence of high-affinity peptides (Fig. 6B). For these experiments, dendritic cells were incubated with encephalitogenic PLP139-151 peptide in the absence or presence of 2% n-propanol. The KL304 staining showed that the amount of empty MHC molecules was only slightly reduced in the absence of n-propanol. Only at the highest peptide concentration used (10 μg / ml) was there a 26% decrease in KL304 staining from 38 to 28. In contrast, in the presence of 2% n-propanol, the amount of detectable empty MHC class II molecules decreased by almost 85%. The median fluorescence of the KL304 staining of dendritic cells decreased with n-propanol exposure from 81 (found in the absence of the peptide) to 12 at a concentration of 10 μg / ml peptide. Thus, n-propanol is able to generate functional, empty MHC class II molecules on the surface of antigen-presenting cells, which can be effectively loaded with high-affinity peptide ligands. The peptide loading was further analyzed with biotinylated peptides. Staining with streptavidin labeled with phycoerythrin allowed the amount of peptide bound to the cell surface to be determined directly by FACS analysis. For these experiments, fibroblast cells expressing either HLA-DR1 or HLA-DR2 were incubated with titrated amounts of biotin HA306-318 or biotin MBP86-100 peptide (Fig. 7A). In the presence of n-propanol, the staining of HA306-318 on fibroblast cells expressing HLA-DR1 was approximately 10-fold, while for MBP86-100 and HLA-DR2 the amplification was even 20-fold. With no surface staining was observed with either peptide with non-transfected fibroblast cells that do not express MHC class II molecules, which confirms that MHC class II molecules were actually loaded with the peptides.

Finally, T-cell experiments were carried out to check whether the increase in loading efficiency was also translated into improved T-cell responses (Fig. 7B-C). Fibroblast cells expressing HLA-DR were loaded with peptide antigens in the absence or presence of n-propanol, and the immune response was charged with CH7CH17 T cells, a Jurkat cell line that expresses a T cell receptor specific for HLA-DR1 / HA306-318 (Wedderburn and et al ., 1995), or the mouse T cell hybridoma 08073 (Fig. 7B) specific for HLA-DR2 / MBP86-100. According to the peptide loading, the HLA-DR1 restricted T cell response was triggered at about 10 times lower peptide concentrations if 2% n-propanol was present during the incubation of the antigen presenting cell with the HA306-318 peptide. The HLA-DR2 restricted T cells even required less than 1/20 of the peptide concentration to be stimulated when loading MBP86-100 in the presence of n-propanol.

Example 3:

The improvement in n-propanol in the T cell reaction was particularly evident in a kinetic experiment in which 0.1 μg / ml MBP86-100 peptide was used to stimulate the 08073 T cells (Fig. 7C). After loading for 8 hours in the absence of n-propanol, the IL-2 release of the T cell hybridoma only reached a value of 0.4 U / ml. The peptide loading in the presence of 2% n-propanol, on the other hand, led to a much faster and stronger T cell reaction. After loading the APC for 2 hours, an antigen density was reached, the release of 1.2 U / ml was triggered and the loading time of 8 h increased the IL-2 release to 2.1 U / ml. The hyperbolic shape of the reaction curve suggests two-phase kinetics. After the originally rapid binding of the peptide to the existing MHC molecules on the cell surface, the Speed back and the delivery of recirculated or newly formed MHC class II complexes from the endosomal way to speed limiting.

In summary, the T cell experiments show that the presence of n-propanol significantly enhances the T cell response to foreign antigens such as the virus-derived HA306-318 peptide or self-epitopes such as the encephalitogenic MBP86-100 peptide , Small molecular H-bond donors can effectively reduce the threshold concentration of the (auto) antigens in order to trigger antigen-specific T cell reactions.

Example 4:

In the further embodiment of the invention, even more effective ligand exchange catalysts have been identified. p-Chlorophenol (pCP) is one of the compounds that work at 200 times lower concentrations than n-propanol. Fig. 8A shows a direct comparison of these two substances. When loading HLA-DR2 expressing fibroblast cells with biotinylated MBP86-100 peptide, a fluorescence of 10 is already achieved at a pCP concentration of 1mM, whereas with n-propanol a concentration of 200mM is necessary to achieve this value. Similar drastic results can also be achieved with mouse spleen cells expressing HLA-DR1. The FACS analysis in Fig. 8B shows the loading of these cells with biotinylated HA306-318 peptide. A clear shift in the cell population can be seen in the cells which were incubated with the peptides in the presence of 2mM pCP (bottom right). In contrast, for cells in which the loading took place in the absence of pCP, there is almost no shift at all (bottom left). In addition to streptavidin staining (determination of the peptide loading), the HLA-DR molecules were also stained with anti-HLA-DR-FITC antibodies in this experiment. The specificity of the pCP-mediated loading with biotinylated peptides can be seen very clearly from the fact that only the HLA-DR positive cells are stained with streptavidin-PE. Example 5:

Improved binding of MBP to soluble HLA-DR2 by parachlorophenol (pCP)

The influence of parachlorophenol (pCP) on the binding of MBP to HLA-DR2 was first tested in an ELISA with soluble MHC class II molecules (Fig. 1). HLA-DR2 molecules were loaded with biotinylated CLIP peptide (BiotinCLIP) in order to determine the binding of unbiotinylated ligands in a pCP-mediated exchange reaction (7). In this experiment, the binding of the peptides MBP 86-100 and HA306-318 and the purified MBP protein was indicated by the amount of BiotinCLIP that was released after incubation of the HLA-DR2 / biotinCLIP complex with these ligands.

MBP 86-100 (Fig. 1, left panel) binds to HLA-DR2 with high affinity, but the peptide alone only released about 15% of the CLIP peptide. In the presence of 10 mM pCP, however, more than 98% of the CLIP peptide dissociated from the HLA-DR2 complex (pCP alone had no effect). HA 306-318 has only a low affinity for the HLA-DR2 molecule. No significant CLIP release was observed despite the presence of pCP (Fig. 1, middle panel), since H-bridge donor compounds only catalyze a peptide exchange if the free ligand has a high affinity for the MHC molecule (7). An increase in CLIP release depending on the pCP amount was also observed when free MBP protein was used (Fig. 1, right panel). 35% of the CLIP release was observed in the absence of pCP, and nearly 99% of the CLIP peptide dissociated when the complex was incubated with both the purified MBP and pCP. So pCP mediates not only the binding of short peptides to soluble HLA-DR2 but also the binding of large proteins such as MBP.

Example 6: pCP-mediated binding of MBP to MHC class II molecules on the cell surface

pCP can be used for the catalysis of peptide exchange on soluble HLA-DR molecules as well as on MHC class II molecules on the cell surface (7). In order to test whether pCP mediates the loading of antigen-presenting cells with MBP, EBV-transformed B cells were incubated with biotinylated MBP protein and then stained with streptavidin-PE. The amount of MBP bound to the cell surface was then determined by a FACS analysis (Fig. 2). Three different EBV-transformed B-cells were used: HLA-DR1 ~ expressing 721.221 (Fig. 2, left panel), HLA-DR2 expressing MGAR (middle panel) and 721.174 (right panel), which - due to a larger deletion in the MHC gene region - do not express class II MHC molecules (22). The effect of pCP on the binding of biotinylated MBP (lower panel) was compared to the binding of biotinylated MBP 86-100 (upper panel) and biotinylated HA 306-318 (middle panel).

MBP86-100 binds to HLA-DR2 with high affinity, but only with low affinity to HLA-DR1. Therefore, no pCP-mediated increase in binding at 721.221 or 721.174 was observed (a pCP-independent signal of ~ 16, which was discovered at 721.221, is probably due to the binding of MBP 86-100 to HLA-DP or - Attributed to DQ). However, a pCP-mediated improvement in binding was observed when HLA-DR2-expressing MGAR cells were incubated with the peptide. In the absence of pCP, the geometric mean of fluorescence was determined to be 4, while in the presence of pCP the fluorescence increased from 8 (0.5 mM pCP), 13 (1, 0 mM pCP) to 19 (2.0 mM pCP) ) increased.

HA306-318 binds to HLA-DR1 with high affinity and to HLA-DR2 with medium affinity. Binding experiments with biotinylated HA306-318 showed pCP-mediated improvement in binding to both MGAR and 721.221 cells, but was more pronounced in the case of 721.221 Cells. For 721,221 the fluorescence signal increased from 3 (no pCP), 8 (0.5 mM pCP), 13 (1, 0 mM pCP) to 20 (2.0 mM), whereas in MGAR cells an increase of 2 ( no pCP), 3 (0.05 mM pCP), 5 (1, 0 mM pCP) except 7 (2.0 mM). No signal was confirmed on 721.174 cells confirming the specificity of binding to HLA-DR molecules.

A pCP-dependent increase in binding was also observed for purified MBP protein (lower panel). It is known that MBP can bind HLA-DR1 and HLA-DR2 to some degree. In HLA-DR2-expressing MGAR cells, the fluorescence, which was determined in the absence of pCP, was 10 and increased in the presence of pCP from 17 (05, mM pCP) to 22 (1, 0 mM pCP) to 29 ( 2.0 mM). In contrast to the MBP 86-100 peptide, a pCP-dependent increase was also observed in 721,221 cells (14 (no pCP), 24 (0.5 mM pCP), 32 (1, 0 pCP) and 39 (2 mM pCP)). The HLA-DR1 restricted epitope is MBP 151-170 while the HLA-DR2 restricted epitope is MBP 86-100. This is probably why different areas of the MBP protein bind to HLA-DR1 and HLA-DR2. In both HLA-DR1 and HLA-DR2, however, the binding is clearly improved by the presence of pCP. No or a very small signal (~ 3) was detected on 721.174 cells, which confirms that MBP protein specifically binds MHC class II molecules to the cell surface.

Example 7:

Improved MBP-specific T cell response through pCP

The binding experiments with EBV-transformed B cells showed that pCP was able to improve the binding of MBP protein to HLA-DR on the cell surface. To test whether the improved binding also leads to improved T cell responses, / nv / fro experiments were carried out. MGAR cells were incubated with the antigen in the absence or presence of pCP and used in an assay to stimulate MBP 86-100 specific T cells. Fig. 3A shows a comparison of the T cell response after the addition of the MPB86-100 peptide (left panel) or the MBP protein (right field) was measured. Without pCP, a concentration of 1 μM MBP86-100 peptide was not sufficient to trigger a detectable T cell reaction. Even after 8 hours, there was almost no IL-2 release. However, a clear response was measured when the binding was in the presence of pCP. After 8 hours of incubation, the IL-2 release gradually increased from 0.6 U / ml (0.5 mM pCP) to 3.3 U / ml (1 mM pCP) and up to 9.8 U / ml ( 2 mM pCP). A similar improvement dependent on pCP was also observed with purified MBP protein. After 8 hours of incubation with 0.5 μM MBP protein, the amount of IL-2 increased from 2.5 U / ml (no pCP) to 5.9 U / ml (0.5 mM pCP), 8.6 U / ml (1 mM pCP) to 11.6 U / ml (2 mM pCP). The hyperbolic shape of the curve when loaded in the presence of 2 mM pCP shows biphasic kinetics. This is even more pronounced at higher antigen concentrations (data not given) and suggests that, after the initial rapid binding, additional ligands for the binding become speed-limiting for the supply of newly synthesized or recycled HLA-DR molecules.

Purified MBP is free of lipids and also partially denatured. To ensure that the improvement in binding by pCP also takes place in the natural lipid-bound form, the experiment was repeated using a myelin-containing tissue homogenate, which was produced from the spinal cord of mice (Fig. 3B). MGAR cells were incubated with 500 μg / ml of this homogenate in the absence or presence of pCP, as described above, and in a T cell experiment with the MBP-specific T cell (left field) and with a control T cell, which does not react to MBP, tested (right field). With the MBP-specific T cell, an IL-2 release of only 0.1 U / ml was found in the absence of pCP. In the presence of pCP, however, this amount increased from 0.2 U / ml (0.5 mM pCP) to 0.4 U / ml (1 mM pCP) to 0.7 U / ml (2 mM pCP). No pCP-dependent increase was detected with the control T cell, which indicates that the reaction is actually MBP-specific.

Both the purified (partially denatured) MBP and the natural lipid-bound form can bind directly to the HLA-DR. During MHC If associated associated MBP is recognized directly by T cells, the lipid-bound form must first be taken up and processed endosomally. MHC class II molecules therefore not only have an immediate function in antigen presentation, they are also receptors that take up proteins from the extracellular space, mediate their internalization and transport the processed peptide antigens back onto the membrane. In addition to MBP, other proteins are known, such as chicken egg lysozyme and influenza hemagglutinin, which are known to follow this path. Empty MHCKIasse II molecules, which can naturally mediate the uptake of these proteins, can be found on immature DC and on microglia. The MHC-mediated uptake of protein therefore appears to be part of the general antigen presentation pathway.

This new study has now shown that the efficiency of antigen uptake can be influenced by small-molecule compounds. The fact that proteins can also be transferred to MHC class II molecules by pCP also significantly extends the range of potential therapeutic use for H donor molecules. For example, the E-v / Vo loading of DC with tumor-associated proteins or even tumor cell lysates now seems to be feasible.

In the context of immunotherapy, they could therefore be valuable means for the controlled loading of antigen-presenting cells with defined protein antigens.

Figure legends

Fig. 1 Influence of ethanol on the loading efficiency of

HA306-318 on soluble HLA-DR1

Soluble HLA-DR1 molecules were incubated with HA306-318 peptide in the absence (lane 3) or in the presence of 5% ethanol (lane 4). HLA-DR1 incubated without peptide were used as controls (lane 2). After an 8-hour incubation at 37 ° C. (pH 7.3), the reaction mixtures were separated by SDS-PAGE and the bands were visualized by silver staining. Loaded MHC class II molecules are visible as 60kD bands (SDS-stable HLA-DR1 / peptide complex), unloaded, empty HLA-DR1 molecules dissociate and are isolated as individual chains with Mw _ap of about 25kD (α chain) and 20kD ( ß chain) detected.

Fig. 2: Effect of ethanol on the exchange of CLIP peptide from

HLA-DR1 and HLA-DR2

The release of biotinylated CLIP from soluble HLA-DR1 (upper part) and HLA-DR2 molecules (lower part) was determined after incubation with titrated amounts of ethanol. Preformed CLIP complexes of soluble HLA-DR1 and HLA-DR2 molecules were generated by loading the MHC class II molecules with biotinylated CLIP peptide. These complexes were incubated with titrated amounts of ethanol in the absence or presence of the high affinity peptide ligands HA306-318 or MBP86-100. After 3 hours of incubation, the undissociated amount of biotin-CLIP / HLA-DR complex was determined in a sandwich ELISA using an α-HLA-DR, Capture 'antibody (L243) and avidin-peroxidase to detect the CLIP peptide , The experiment was carried out at pH 7.3.

Fig. 3: Comparison of the CLIP release by HLA-DM and ethanol

The release of biot. CLIP from soluble biot. CLIP / HLA-DR2 complexes were either without catalyst (upper part), with 0.1 mg / ml soluble HLA- DM (middle part) or tested with 5% ethanol (lower part). The release was compared at pH 5.0 and pH 7.3. A. Ligand exchange at pH 5.0. The release of CLIP took place for 3 hours at 37 ° C in the presence of titrated amounts of peptides of different affinity for the HLA-DR2 molecule: high affinity MBP86-100 (left part), medium affinity IC106-120 (middle part) or low affinity HA306-318 (left Part). B. Exchange at pH 7.3. The effects of HLA-DM and ethanol were compared at pH 7.3 in the presence of the high affinity MBP86-100 peptide. The values plotted on the ordinate were determined by ELISA as described in Fig. 2 and represent the relative amount of biotin-CLIP / HLA-DR2 after the release reaction.

Fig. 4: Structural requirements of small molecules for the release of CLIP

The release of biotin-CLIP from soluble biotin-CLIP / HLA-DR2 has been tested with various aliphatic and aromatic compounds. A. Influence of the chain length of aliphatic alcohols. Biotin-CLIP / HLA-DR2 was titrated with methanol (CH ₃ -OH), ethanol (CH ₃ -CH ₂ -OH), n-propanol (CH ₃ -CH ₂ -CH ₂ -OH) and n-butanol (CH ₃ -CH ₂ -CH ₂ -CH ₂ -OH) incubated. The data shown in the figure were obtained in the presence of 20 μg / ml MBP86-100. In the absence of the peptide, little or no release was observed (data are not reported). B. Effect of hydroxyl or amino groups which are linked to alkyl or aryl radicals. The peptide release by functional hydroxyl (left side) and amino groups (right side) in combination with aliphatic n-propyl (upper part) or aromatic benzyl residues (lower part) is shown. The experiments were carried out in the absence (open symbols) or in the presence of 20 μg / ml MBP86-100 (closed symbols). C. Influence of the H donor functional group. The effects of benzene, phenol and anisole on CLIP / HLA-DR2 were compared. The release occurred at a concentration of 60mM of the small molecule in the absence (open crossbars) or presence (filled crossbars) of 20 μg / ml MBP86-100. In all experiments, the release reaction of biotin-CLIP / HLA-DR2 complexes was at pH 7.3 for three hours and the amount of CLIP-HLA-DR2 remaining after incubation was determined by an ELISA as shown in Fig. 2 described, determined.

Fig. 5 Binding of peptides to MHC molecules in the presence of

Phenol or n-propanol

The kinetics of the binding of biotinylated HA306-318 and biotinylated MBP86-100 to soluble HLA-DR1- (left side) and soluble HLA-DR2-

Molecules (right side) was analyzed. The binding was with HLA-DR-

Tested molecules pre-loaded with CLIP peptides by incubation for 48 hours. The binding took place in the presence of 75 mM phenol

(filled square), 300 mM n-propanol (filled triangle) or without an added H donor molecule (open circle). The amount of peptide / HLA-DR

Complexes formed with biotinylated peptides were determined at the indicated times by ELISA using Eu ³⁺ -labeled streptavidin. The amount of peptide complex formed was reported as cpm.

Fig. 6 Determination of the empty MHC class II molecules after the

Effect of n-propanol on dendritic cells

Dendritic cells derived from bone marrow were incubated with a 2% n-propanol. After incubation, the amount of empty MHC molecules was determined by FACS analysis using the conformation-specific KL304 antibody. The dendritic cells were isolated from SJL / -J mice (H2 ^S ). A. Generation of empty MHC class II molecules on the cell surface. The figure shows a FACS histogram that was generated after 4 hours of incubation of dendritic cells at 37 ° C in the absence (thin solid line) or presence of 2% n-propanol (thick solid line). An isotype control is shown as a dashed line. B. Loading the surface MHC class II molecules with high affinity peptide. The same incubation took place in Presence of the indicated amounts of lA ^s -restricted peptide PLP139-151. The bars show the fluorescence intensity (median) of the KL304 staining after incubation in the absence (open bars) or presence of 2% n-propanol (filled cross bars).

Fig. 7 Improved antigen loading and increased sensitivity of the T cell response after treatment with n-propanol

The loading of the surface MHC class II molecules of the antigen presenting cells with peptide antigens and the T cell reaction triggered by these cells were determined. Antigen was loaded in the absence (circles) or presence (triangles) of 2% n-propanol. A. Loaded with antigen. Fibroblast cells transfected with HLA-DR1 or HLA-DR2 (solid symbols) were incubated for 4 hours with titrated amounts of biotinylated HA306-318 peptide (for HLA-DR1; top left image) or biotinylated MBP86-100 (HLA-DR2; bottom left) Image). The amount of peptide loading was determined by FACS analysis after staining the cells with streptavidin labeled with phycoerythrin. L cells that do not express HLA-DR (open symbols) were used to control specific peptide binding. B. T cell response. Fibroblast cells expressing HLA-DR1 or -DR2 were exposed to titrated amounts of HA306-318 (upper picture) or MBP86-100 (lower picture) in the absence (full circles) or presence (full triangles) of 2% n -Propanol incubated. After the incubation, the cells were washed and used for the T cell assay. The specific T cell response was determined using CH7C17, TCR-transfected Jurkat cells (HLA-DR1 / HA306-3 8), and T cell hybridoma 08073 (HLA-DR2 / MBP86-100). The response was measured by determining IL-2 release. C. Kinetics of Antigen Loading. Fibroblast cells expressing HLA-DR2 were in the specified time with 0.1 μg / ml MBP86-100 peptide in the absence (circles) or presence (triangles) of 2% n-propanol or alone with 2% n-propanol ( open squares). After the incubation, the cells were washed and used for the assay with the MBP86-100-specific T cell hybridoma 08073. Fig. 8 Loading of antigen-presenting cells with peptides in

Presence of parachlorophenol (pCP).

Fibroblast cells (A) and splenic lymphocytes from HLA-DR1 transgenic mice (B) expressing MHC class II were incubated with biotinylated peptide antigens for three hours and the loading subsequently determined with phycoerythrin-labeled streptavidin in the FACS. A) Comparison of n-propanol and parachlorophenol. Fibroblast cells expressing HLA-DR2 were loaded with 2.5 μg / ml biotinylated MPB86-100 in the presence of titrated amounts of n-propanol (open circles) or parachlorophenol (filled circles). From the curves, it can be seen that about 200 times lower concentrations were required with parachlorophenol than with n-propanol in order to achieve a corresponding loading. The streptavidin fluorescence was plotted on the ordinate of the medium as the measurement parameter for the loading. B. Specificity of loading. Spleen cell suspensions of HLA-DR1 transgenic mice were in the absence of parachlorophenol (left half of the figure) or in the presence of 2 μg / ml parachlorophenol (right half of the figure) with 10 μg / ml biotinylated HA306-318 (lower half of the figure. ) and as a control without peptide (upper half of the figure) for three hours. After the incubation, the cells were stained with FITC-labeled anti-HLA-DR antibody (ordinate) and phycoerythrin-labeled streptavidin (coordinate) and then measured in the FACS. Dotplot analysis shows a marked increase in streptavidin staining in cells in which the loading was carried out in the presence of parachlorophenol (bottom right), while in the spleen cells in which the loading was carried out in the absence of pCP, no or only a slight shift was measured was (bottom left). In addition, the parachlorophenol-mediated load was only measured in the HLA-DR positive (bottom right, upper quadrant), but not in the HLA-DR negative cells (bottom right, lower quadrant). This underlines that parachlorophenol catalyzes the specific loading of the MHC class II molecules. Fig. 9 Binding of MBP 86-100, HA306-318 and purified MBP to soluble HLA-DR2 molecules

Binding was determined in an exchange experiment with biotinylated CLIP peptide ligands. Biotinylated the CLIP was loaded onto soluble HLA-DR2 molecules and the HLA-DR2 / biotinCLIP complex in the presence or absence of 10 mM parachlorophenol (pCP) with 70 μM unbiotinylated MBP 86-100 (left panel), HA 306-318 ( middle field) or MBP protein (right field). The exchange reaction was carried out at pH 7.4 and the amount of biotinylated CLIP which was still bound to HLA-DR2 after the incubation was determined in an ELISA reaction. The bars represent the relative amount of the BiotinCLIP / HLA-DR2 complex, which after incubation without pCP or a free ligand (control), only with pCP, only with a free ligand (MBP or HA), or with a free ligand and pCP was measured.

Fig. 10 Loading of MHC class II molecules on the cell surface of antigen presenting cells

EBV-transformed B cells 721.221 (HLA-DR1; left fields), MGAR (HLA-DR2; middle fields) and 721.174 (no MHC class II molules; right fields) were treated with 0.5 μM biotinylated MBP 86-100 ( upper fields), HA 306-318 (middle fields) or purified MBP protein (lower fields) in the presence of the indicated amounts of pCP. The incubation was carried out for 4 hours in a DMEM / 5% FCS medium under cell culture conditions. The binding of peptide or protein to the antigen presenting cell was determined by FACS analysis after staining with streptavidin-PE. The values shown on the ordinate represent the geometric mean of streptavidin-PE fluorescence.

Fig. 11 T cell reaction against MGAR cells, which with purified

MBP or spinal cord homogenate were loaded

A. Kinetic experiment with purified MBP MGAR cells expressing HLA-DR2 were loaded with 1 μM MBP 86-100 peptide (left field) or 0.5 μM purified MBP (right field) in the indicated periods. The loading was carried out in the presence of 2 mM pCP (closed circle), 1 mM pCP (open circle), 0.5 mM pCP (closed triangle) or without pCP (open triangle) in a DMEM / 5% FCS medium under cell culture conditions , After loading, the MGAR cells were washed and used to stimulate the 08073-T cell hybridomas that respond specifically to the MBP 86-100 antigen. The values shown on the ordinate represent the IL-2 release of the T cells, which was measured after 24 hours of incubation with the MGAR cells.

B. T cell reaction after incubation of the MGAR cells with spinal cord homogenate MGAR cells were incubated for 4 hours with 500 μg / ml spinal cord homogenate in the presence of the indicated amounts of pCP. After washing the MGAR cells, they were used to stimulate the MBP-specific 08073-T cell hybridomas (left panel) and a control T cell (right panel). The experiment was carried out as described above and the bars represent the amount of IL-2 released by the T cells after 24 hours incubation with the MGAR cells.

Claims

claims 1. A method for improving immunotherapeutic methods, characterized in that the ligand exchange on MHC molecules is influenced by H-bridge donors or acceptors. 2. The method according to claim 1, characterized in that the ligand exchange of MHC class II molecules is influenced by H-bridge donors or acceptors. 3. The method according to claim 1, characterized in that the ligand exchange of MHC class I molecules is influenced by H-bridge donors or acceptors. 4. The method according to claim 1-3, characterized in that the ligand exchange is influenced by low molecular weight H-bridge donors or - acceptors. 5. The method according to claim 1-4, characterized in that an increase in the loading of MHC molecules takes place. 6. The method according to claim 5, characterized in that to trigger tumor-specific, pathogen-specific or autoreactive immune responses there is an increase in the loading of antigen 7. The method according to claim 1-6, characterized in that for triggering the immune responses, a loading of antigen-presenting cells, such as dendritic cells (matured and unmatured), B cells, macrophages and others takes place. 8. The method according to claim 1, characterized in that there is a reduction in the loading of MHC molecules. 9. The method according to claim 1 and 8, characterized in that a decrease in the attenuation of aggressive immune reactions Antigens are loaded. 10. The method according to claim 1-9, characterized in that lower aliphatic alcohols, preferably ethanol, propanol or butanol, are used as low molecular weight H-bridge donors. 11. The method according to claim 1-9, characterized in that phenol, aniline or their derivatives, preferably parachlorophenol, are used as low molecular weight H-bridge donors. 12. The method according to claim 1-9, characterized in that already known as low molecular weight H-bridge donors such as pharmaceuticals Diprivan (2,6-diisopropylphenol) can be used. 13. The method according to claim 1-9, characterized in that combinations of H-bridge donors or acceptors are used as low molecular weight H-bridge donors. 14. A method for the treatment of diseases, especially cancer (such as colon carcinomas, melanomas, kidney carcinomas, lymphomas) or infectious diseases (such as influenza, malaria), characterized in that a loading of the body's own dendritic cells with tumor- or pathogen-specific antigens by H -Bridge donors or - acceptors, which are then reinjected to trigger / strengthen the immune response to the patient. 15. A method for the treatment of diseases, especially autoimmune diseases (such as multiple sclerosis, rheumatoid arthritis, diabetes), characterized in that a loading of the body's immature dendritic cells with tissue-specific self-antigens by H-bridge donors or acceptors, which then takes place for triggering / amplification a suppressive immune response Patients are reinjected. 16. The method according to claim 1, characterized in that H-bridge donors or acceptors are used to influence the exchange of large ligands for targeted antigen loading. 17. The method according to claim 16, characterized in that act as large ligands with a molecular weight> 3,000 Dalton whole proteins, protein mixtures and / or complex protein mixtures. 18. The method according to claim 16 or 17, characterized in that H-bridge donors or acceptors are used which, depending on the affinity of the ligand, cause an increase or decrease in the loading of MHC molecules, preferably low molecular weight H-bridge donors. 19. The method according to claim 16-18, characterized in that lower aliphatic alcohols, phenols, anilines or their derivatives or known per se as low molecular weight H-bridge donors Pharmaceuticals or combinations of H-bridge donors or acceptors can be used. 20. The method according to claim 19, characterized in that ethanol, propanol or butanol is used as the lower aliphatic alcohol. 21. The method according to claim 19, characterized in that parachlorophenol is used. 22. The method according to claim 19, characterized in that Diprivan (2,6-diisopropylphenol) is used as pharmaceutical. 23. The method according to claim 16-22, characterized in that the ligand exchange is influenced by MHC class II molecules. 24. The method according to claim 16-22, characterized in that the ligand exchange is influenced by MHC class I molecules. 25. Use of antigen-loaded cells, possibly of MHC molecules, produced by a process according to claims 1-24 for triggering tumor-specific, pathogen-specific or autoreactive Immune responses. 26. Use according to claim 25 for the treatment of diseases, especially cancer (such as colon carcinomas, melanomas, kidney carcinomas, lymphomas) or infectious diseases (such as influenza, malaria), wherein loading of the body's own dendritic cells with tumor- or pathogen-specific antigens, if necessary with whole proteins, protein mixtures and / or complex protein mixtures, which then reinjected to trigger / enhance the immune response to the patient. 27. Use according to claim 25 for the treatment of diseases, especially autoimmune diseases (such as multiple sclerosis, rheumatoid arthritis, Diabetes), whereby the body's own immature dendritic cells are loaded with tissue-specific self-antigens, possibly with whole proteins, protein mixtures and / or complex protein mixtures, which are then reinjected to trigger / reinforce a suppressive immune response to the patient. 28. Use of antigen-loaded cells, possibly of MHC molecules, produced by a method according to claims 1-24 for weakening aggressive immune reactions. 29. Antigen-loaded cells, produced by a method according to claims 1-24.