WO2002077208A1 - Antigen-dependent reduction of specific immune reactions by influencing the co-stimulation - Google Patents

Abstract

The invention relates to a reduction in specific immune reactions. According to the method, antigen-producing cells are stimulated to present defined antigens with simultaneous production of a PD-1 binding molecule.

Description

Antigen-dependent reduction of specific immune reactions by influencing the co-stimulation

The present invention relates to antigen-presenting cells (APC), processes for producing antigen-presenting cells, medicaments containing antigen-presenting cells and use of the antigen-presenting cells.

description

For almost 10 years, gene therapy methods have been developed and used for various diseases and in animal models. So far, they have not become routine. Most of the time, this is the treatment of genetically determined, serious diseases for which other therapies are not available. Furthermore, gene therapies are used to treat severe and also non-treatable cancer.

Experimental medicine has so far paid little attention to the treatment of allergies and autoimmune diseases using genetic engineering methods.

General information about allergies

Allergies cause considerable healthcare costs in industrialized countries. After all, it is estimated that at least 20% of the population is allergic to any substance. Most suffer from allergic rhinitis, which includes hay fever in particular, or bronchial asthma: they sneeze or gasp for breath after inhaling certain pollen or other generally harmless substances. Many children and some adults are also allergic to food. Others experience rashes or even an allergic shock after taking medication such as penicillin. Call someone else Bee stings cause severe local swelling or severe systemic disorders that affect the entire organism. In extreme cases, allergic attacks can even lead to death. Immediate medical care for asthma patients in the United States consumed an estimated $ 3.6 billion in 1990, accounting for around one percent of all healthcare costs.

It is now known that some of the cellular and molecular interactions in allergic reactions are often similar, regardless of which substances respond to the individual and what symptoms he develops. Certain side effects of allergies usually only occur when the immune system is fighting parasites. The body responds to parasites as well as allergens with the massive production of molecules called immunoglobulin E antibodies (IgE). The production of these antibodies is induced by T helper cells, which in turn are activated by cells presenting antigen.

sensitization

Different allergens cause different symptoms, among other things, because they come into contact with the immune system in different parts of the body. In the upper airways, the misguided immune response causes sneezing and a stuffy nose. In contrast, the bronchi in the lower airways can narrow and become mucous, so that typical asthmatic symptoms appear. Accordingly, immune activities in the tissues of the gastrointestinal tract cause nausea, abdominal cramps, diarrhea or vomiting.

After all, an allergen that gets into the blood in some way can trigger anaphylaxis: an allergic reaction in regions of the body that are far from its point of entry. Severe anaphylactic shocks can upset all normal bodily functions and can be fatal.

Even if the allergic reactions manifest themselves differently, they are always started by the same mechanism: the sensitization. One-time contact with an allergen, typically a protein, are sufficient. In the airways or other tissues, the allergenic substance meets so-called phagocytes or macrophages. These gobble up the foreign substance, dismember it and present the fragments on the cell surface with MHC II molecules. In the further course, some T-helper lymphocytes recognize the fragments presented and bind to them. The T helper lymphocytes are activated by the macrophages and then in turn activate some B lymphocytes, which also recognize the allergen. The B cells then mature into plasma cells that produce antibodies. First of all, these are antibodies of the so-called IgM type; from a certain point in time, however, the plasma cells switch to IgE antibodies.

It can take days or weeks for the antibodies to be made, and the allergen that started their production may have long since disappeared. Not the IgE molecules. With their Fc region, they attach themselves to IgE receptors of two different classes of immune system cells. One is mast cells, which are usually found in the body tissues near blood vessels and epithelial cells. There is contact with the outside world via the epithelium (this also includes the epithelium of the respiratory tract and gastrointestinal tract). IgE antibodies also bind to basophilic granulocytes (basophils). However, these cells circulate in the bloodstream.

Once production of the IgEs begins, it apparently lasts for months, sometimes even years. As a result, they constantly occupy IgE receptors on mast cells and basophils - ready to take immediate action the next time they come into contact with allergens.

Acute symptoms

So while the first encounter with an allergen does not cause symptoms even in people who later turn out to be allergy sufferers, the second exposure initiates a stage of the hypersensitivity reaction, which also appears externally. The allergy-causing substance binds to the IgEs of the mast cells within seconds of contact with human tissue. If he attaches himself to two or more IgE molecules at the same time, he bridges between them. Such cross-links bring the affected IgE receptors closer together, and this activates the cell so that it releases highly effective substances that directly produce allergic symptoms. (The release can also be caused in other ways; allergic reactions are only spoken if IgEs are involved). The most important of these substances is histamine. It can stimulate the formation of mucus in the epithelia and thus contribute to the blockage of the air passages, as well as the smooth muscles that wrap around the bronchial tubes and intestines like an elastic band. It is also able to dilate the fine blood vessels and make them more permeable so that fluid can seep into the tissue. The result is redness and swelling. If these vascular changes affect large parts of the body, they can cause fatal circulatory failure: With such a shock, the blood pressure suddenly drops so much that the oxygen supply to the heart and brain is no longer guaranteed.

The second group of mediators consists mainly of prostaglandins and leukotrienes. They are only released after the allergen molecules have attached to the IgEs on the cells. Like histamine, they narrow the bronchi and dilate the blood vessels. However, their effects last longer.

In addition, stimulated mast cells emit a variety of potentially toxic enzymes. They also appear to release cytokines that regulate the activities of other immune cells.

Treatment: 1. Allergic rhinitis

Antihistamines are usually effective and still serve as standard therapy. The latest variants can no longer easily cross the blood-brain barrier and no longer make the patient tired. If antihistamines are ineffective in severe inflammation, inhalable corticosteroids, which are usually prescribed to relieve chronic inflammation in asthma, often help. In severe cases, immunotherapy or hyposensitization (also known as allergy shots or desensitization), which was introduced in 1911, can provide relief in the long term. With this treatment, doctors inject patients with increasing doses of the allergen to which they are sensitive. The dose is decisive in all cases: too little allergen does not confer tolerance. In addition, protection is rarely complete.

2. Asthma

Bronchodilators are the most widely used drugs for asthma. They relieve the symptoms caused by histamine and other bronchoconstrictors very quickly, but are unlikely to affect the underlying inflammation. In addition, their excessive use can cause a back reaction of the body, so that after their effects have subsided, the respiratory flow is more restricted than before. In addition, the methods listed under 1. are used.

3. Anaphylactic reactions

Insect bites cause anaphylaxis in some people. In severe cases these lead to death e.g. Circulatory failure or suffocation. Any severe anaphylaxis, whether it occurs for the first or fifteenth time, is an emergency, and one must first try to control the most threatening symptoms. This is usually done by injecting adrenaline, which inhibits the release of the mediators, opens the airways and counteracts the dilation of the blood vessels. One can preventively by immunization z. B. act with the poison of a health-threatening insect.

New approaches

Animal-based immunization with an allergen (Der p 5) of the mite Dermatophagoides pteronyssl- nus is currently being tested in an animal model. This immunization results in IgG production, but not IgE, and results in a 90% reduction in the amounts of specific IgE, which were caused by classic sensitization with Der p 5 and alum as adjuvant or allergen-induced rhinitis.

Another new strategy is the use of humanized monoclonal anti-IgE antibodies against the FcεRI binding region for IgE. This prevents the binding of IgE to the IgE receptor, so that no mediators of the allergic reaction from mast cells or basophils can be released. This strategy has shown in clinical studies in patients with allergic rhinitis and allergic asthma that these antibodies are well tolerated and reduce the allergic reactions.

(see also LM Lichtenstein: "Allergy and immune system"; in the spectrum of science special: The immune system; 1994; 74-83; SK Huang, KY Chua and KH Hsieh: allergen gene transfer. Current Opinion in Immunology 1997; 800-804; C. Heusser and P. Jardieu: Therapeutic potential of anti-IgE antibodies. Current Opinion in Immunology 1997; 805-814).

General information about transplants

Tissue transplantation to replace diseased organs is an important medical therapy today. In most cases, a response from the adaptive immune system to the graft poses the greatest threat to successful treatment. Responses from the adaptable immune system are induced by antigen presenting cells by activating T helper lymphocytes. When transfusing blood, which is the first and most commonly used graft, the ABO and Rh blood group antigens must be matched to avoid the rapid destruction of inappropriate erythrocytes. In the case of other tissues, the very polymorphic main histocompatibility complexes (MHC) have to be matched to one another, since these almost always trigger the immune response. Unfortunately, the perfect matching of the MHCs is almost impossible, except for relatives.

Although the immune response makes organ transplantation difficult, there are few alternatives to organ failure. The use of potent im- Organ-suppressive agents, especially cyclosporin A and FK-506, which prevent the activation of T cells, make organ transplants successful. Nevertheless, some problems arise here, since the suffering that has destroyed one's own organ often also destroys the foreign organ. The suppression of the immune system also increases the risk of developing cancer or infections. The procedure is also very expensive (Janeway and Travers 1997).

Co-stimulatory molecules

The product of the mouse PD-1 gene (ACCESSION NM_021893), a member of the IgG superfamily, is a receptor that is common in many tissues. Analyzes carried out by flow cytometry and immunoprecipitation with the monoclonal antibody J43mAk showed that the PD-1 gene product is a 50 to 55 kDa membrane protein. The PD-1 protein appears to be highly glycosylated since the calculated molecular weight of the amino acid sequence is 29310 Da. Normal mouse lymphoid tissue such as thymus, spleen, lymph nodes and bone marrow contain only a small number of PD-1 positive cells. Nevertheless, a significant PD-1 positive population appears in thymocytes as well as on T cells in spleen and lymph nodes due to the in vivo treatment with anti-CD3 monoclonal antibodies. Furthermore, PD-1 antigen expression was strongly induced by in vitro stimulation in various subgroups of thymocytes and spleen T cells, either with anti-CD3 mAb or Concavalin A, which can cause T cells to activate as well as to die , PD-1 expression on spleen B cells was similarly stimulated with anti-IgM Ab. In contrast, PD-1 was not significantly expressed after treatments such as growth factor deprivation, dexamethasone or lipopolysaccharide. These results suggest that the expression of PD-1 is strictly regulated and induced by signal transduction via the antigen receptor and does not exclude the possibility that the PD-1 antigen plays a role in the clonal selection of lymphocytes, although for the normal course of apoptosis PD-1 expression is not required (Agata et al 1996; Ishida et al 1992; Nishimura et al 1996). The PD-1 receptor induces the decrease in immune responses and its loss leads to the breakdown of tolerance in peripheral tissues. PD-1 deficient mice develop lupus-like proliferative arthritis and glomerulonephritis with predominant IgG3 deposition during aging. PD-1 is structurally similar to CTL-associated antigen 4 (CTLA-4), which binds B7-1 and B7-2 and plays a crucial role in the maintenance of T cell homeostasis (for reviews, see (Sperling & Bluestone 1996; Thompson & Allison 1997)). PD-1 does not contain the MYPPPY motif, which is critical for B7-1 and B7-2 binding. The extracellular region of PD-1 and CTLA-4 each consist of an IgV domain with 23% identity to each other. A ligand (PD-Ll) exists for PD-1. The binding of PD-1 by PD-Ll leads to the inhibition of TCR-mediated T cell proliferation and cytokine secretion (Freeman et al 2000).

The human and mouse PD-Ll molecule (EMBL / GenBank / DDBS under accession nos. AF233516 and AF233517, (Freeman et al 2000)) are members of the B7 gene family and have a similar structural organization, which consists of an IgV and an IgC domain in the extracellular region (Boussiotis et al 1996), a hydrophobic transmembrane domain, followed by a short, charged intracellular region. Human PD-Ll is identical to B7-H1 at 290 amino acids, which is reported to activate T cell stimulation (Dong et al 1999). The mouse PD-Ll cDNA encodes a polypeptide with 70% amino acid identity to the human PD-Ll. PD-Ll has amino acid identities of 21, 20, and 23% to B7-1, B7-2, and ICOS (ligand of inducible co-stimulator) (Boussiotis et al 1996; Ling et al 2000; Swallow et al 1999; Yoshinaga et al 1999). The expression patterns of B7-1, B7-2, and PD-Ll are different. B7-2, one of the ligands of CD28 and CTLA-4, is constitutively expressed on monocytes. However, the constitutive expression of B7-1 and B7-2 cannot be observed in any organ. B7-1 and B7-2 expression can be induced in dendritic cells, macrophages and B cells (Boussiotis et al 1996), as well as some types of fibroblasts and epithelial cells. In contrast, PD-Ll is constitutively expressed by non-lymphoid, parenchymal organs such as the heart, placenta, skeletal muscles and lungs, but not the small intestine (Dong et al 1999). PD-Ll is also expressed in some cancers. These tumors may be using PD-Ll to inhibit anti-tumor immune responses. Since PD-1 is expressed on activated T and B cells (2), the expression of PD-Ll in non-lymphoid tissues and on dendritic cells could regulate po- enable potentially autoreactive lymphocytes. This could be an important mechanism to limit T and B cell activity in the heart, lungs, kidneys and placenta where PD-Ll is highly expressed (Freeman et al 2000). PD-1 Ligand 2 (PD-L2) is a second ligand for PD-1. Binding of PD-1 to PD-L2 dramatically inhibits T cell receptor (TCR) mediated proliferation and cytokine production by CD4 T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. At high antigen concentrations, they reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle residue in G ₀ / Gx (cell cycle phases) but do not increase the number of dead cells. PD-L2 expression on APCs is increased by treatment with interferon γ and could also be detected in some other tissues and tumor cell lines. PD-Ll and PD-L2 thus have overlapping functions (Latchman et al 2001). mPD ~ L2 (previous name: Protein AF142780) codes for a polypeptide with 38% amino acid identity to mPD-Ll. Murines and human PD-L2 have 70% amino acid identity. The five members of the B7 family - B7-1, B7-2, ICOS-L, PD-Ll and PD-L2 - have 21-27% amino acid identity and a structural organization consisting of a signal sequence, an IgV-like one IgC-like and a transmembrane domain and a short cytoplasmic tail. The cytoplasmic tail of PD-Ll is conserved between mice and humans, which is in contrast to the poor conservation of the cytoplasmic tail of PD-L2 of humans and mice (Latchman et al 2001).

The PD-L2 tissue distribution is similar to that of PD-Ll. There is expression in the placenta, heart, pancreas, lungs and liver, low expression in the spleen, lymph nodes and thymus, no expression in unstimulated monocytes, but this could be induced by interferon γ. However, the kinetics of this induction is slower than that of PD-Ll.

General information on autoimmune diseases

Autoimmune diseases are chronic diseases. These include rheumatism in various clinical forms, diabetes, multiple sclerosis, certain forms of cardiac muscle inflammation and Thyroid disease. The series of autoimmune diseases could be continued as desired, although approximately 90% epidemiologically only make up a small percentage.

The clinical course of autoimmune diseases, even with one and the same diagnosis, can be very different. Relapsing courses of the disease are sometimes observed. Accordingly, the treatments are individually designed by the doctor.

Some of the autoimmune diseases are antibody-mediated. In immunology, this means that the organism produces antibodies for mostly unknown reasons, which are directed against the body's own cellular structures (autoantigens). Once these antibodies have been formed, their interaction and binding to the respective organ or cell-specific structures trigger a cell and tissue-destroying reaction of the immune system. Ultimately, this leads to the clinical picture of the disease (Steinman 1994).

An example of this is the thyroid disease Graves' disease, but also a form of cardiac muscle inflammation that leads to dilated cardiomyopathy. In some cases, both the targets against which the autoantibodies are directed and the autoantibodies themselves are precisely characterized.

PCT / EP 00/03984 discloses a method for reducing specific immune reactions by suppressing the B7 expression of cells presenting antigen while simultaneously expressing predetermined (in particular pathological) antigens.

WO-A-01/14557 relates to PD-1 as a receptor for B7-4. The document discloses that B7-4 can inhibit immune cell activation due to binding to an inhibitory receptor or to an immune cell. Active substances are described for the modulation of PD-1, B7-4 and their interaction in order to modulate co-stimulating or inhibiting signals in an immune cell, which results in a modulation of the immune response. A problem on which the invention is based is, inter alia, to provide genetic engineering, therapeutically usable products for the reduction of specific immune reactions in which targets (antigens, autoantigens) and antibodies, autoantibodies are known.

The problem is solved by the antigen-presenting cell according to the invention, which predominantly presents certain antigens beforehand (monoantigenic antigen-presenting cell) and is characterized in that molecules which bind PD-1 are preferably produced in the monoantigenic antigen-presenting cell and if appropriate, CTLA4-binding molecules are produced and, if appropriate, one of the functions of stimulatory receptors, such as a B7 and / or CD40 receptor, is suppressed.

1 shows a diagram of the function of gene manipulation at the cellular level:

a) Normal mechanism of T helper cell activation via monocytes

b) Mechanism of monocytes after genetic engineering. Absence of T helper cell activation after gene manipulation of the monocytes.

2 shows a “normal” immune reaction for antibody formation, antigen-presenting monocytes as inducers of antibody production. Monocytes take up molecules recognized as foreign, disassemble them and bring the fragments of MHC II molecules onto the cell surface (antigen presentation). At the same time, a surface molecule (B7) is expressed that can bind to both CD28 and CTLA4. The antigen presented is recognized by T helper lymphocytes, which in turn stimulate B cells to produce antibodies. The scheme is very simplified. CTLA4 (CD152) is an analogue of CD28, also binds B7, but induces apoptosis of the T cell. CTLA4 occurs mainly in intracellular vesicles and is only released after activation of the cells. PD-1 is a CD28 and CTLA4 analogue and develops CTLA4-like effects. Fig. 3 shows schematically the function of gene manipulation at the molecular level: PD-1 is bound by a ligand (PD-Ll), which is located on the plasma membrane of the monocyte (antigen-presenting cell), and thus inactivates the T cell.

The effect can be intensified if B7 is additionally suppressed in the antigen-presenting cell and, if appropriate, a CTLA4-binding molecule is also introduced.

Fig. 4 shows the function of gene manipulation at the molecular level: the autoantigen is synthesized by the PD-Ll producing antigen presenting cells.

5 and 6 show a comparison of the antigen presentation of "normal" and genetically modified monocytes. Figure 5 shows autoantigen levels presented on the MHC II. Normal monocytes rarely present the autoantigen and if so, only a few MHC II present it. 6 shows that the genetically manipulated monocytes almost exclusively present the autoantigen.

7a and 7b demonstrate the function of the genetically modified monocytes in the body. According to FIG. 7a, natural autoantigen-presenting monocytes induce the production of (pathological) autoantibodies if they are B7-positive. According to FIG. 7b, genetically manipulated monocytes compete with the natural autoantigen-presenting monocytes and reduce autoantibody production.

The monoantigenic antigen-presenting cell according to the invention is preferably a monocyte, a dendritic cell and / or a macrophage.

The APCs are the switching points of the adaptable immune system. Only they can trigger a T cell-mediated immune response. This is shown in the following figures using the example of monocytes and the antibody production triggered by T helper cells: Within the immune system, the antibodies normally play an important role as specific defense molecules ("humoral immune response"). They are produced by mature B lymphocytes. However, the induction and production of soluble antibodies is not independent of the remaining cells in the immune system; rather, this humoral immune response is controlled by other cells in the immune system.

Antibody production - and thus also the production of pathological autoantibodies - cannot be maintained without the help and mediation of monocytes and T-helper lymphocytes, which in turn activate the B-lymphocytes in an antigen-specific manner (Fig. 1).

The molecular mechanisms of the interaction - of cell-cell contact - of monocytes with the T helper lymphocytes is known in detail at the receptor level (FIG. 2).

If the antigen is e.g. a bacterial protein, the immune response is useful for the organism. However, if the antigen is an endogenous structure, one speaks of a (pathological) autoimmune reaction.

In addition to the antigen to be presented (against which the antibodies to be produced are directed), various receptors and auxiliary receptors on the cell surface are involved in cell-cell contact and cell activation. An essential molecule of the intercellular interaction in the antigen presentation (contact of monocyte with the T helper lymphocytes) is a costingulatory receptor with the designation B7 (FIG. 2).

The monoantigenic antigen-presenting cell according to the invention has an increased expression of an antigen, preferably by transfection of an antigen-presenting cell with nucleic acid-containing material, the antigen-presenting cell essentially presenting only predefined antigens.

The gene therapy method according to the invention is based on parallel genetic engineering interventions on the patient's own blood monocytes. The interventions are carried out using suitable probes and cause the production of PD 1 binding molecules, preferably PD-L1 and possibly CTLA4 binding molecules, preferably antibodies (not shown), and possibly the reduction of B7 molecules by hindering or preventing the formation of the molecule on the surface of the monocytes (not shown) (Fig. 3) and at the same time a strong presentation of autoantigen (Fig. 4 - 6).

Reduction of specific antibody production through gene therapy

The production of pathological autoantibodies is specifically ended by the manipulation of monocytes (or DCs) and the resulting shutdown of antigen-specific T cells. This takes advantage of the fact that T cells produce the receptor PD-1. When bound to PD-Ll, this ensures that T cells no longer proliferate. Binding of PD-1 can thus shut down T cell responses. Binding molecules other than PD-Ll can also be used for this.

In addition, it can be exploited that T cells produce two receptors that recognize B7 (1 or 2). One receptor is CD28, the other is CTLA4. CD28 has a stimulating effect, while CTLA4 does not. The contact of CTLA4 with B7 switches off the respective T cell. This then either goes into apoptosis via programmed cell death, is switched off, or is converted into a tolerance-generating T cell. In this case, the tolerance is generated against the specific antigen recognized by this T cell. (Gribben et al., 1994; Judge et al., 1999; Lee et al., 1998; Lin et al., 1998; Metzler et al., 1997; Olsson et al., 1999; Oosterwegel et al., 1999a; Oosterwegel et al., 1999b; Peach et al., 1994; Walunas et al., 1996; Wu et al., 1997; Wulfing and Davis, 1998).

For binding to CTLA4 instead of CD28, molecules such as antibodies are used which bind to CTLA4 but not to CD28. These can then induce the T cell response promoted by CTLA4. These CTLA4 binding molecules can preferably be antibodies. The co-receptor B7, without which the antigen presentation or the induction cascade for antibody production does not start, can also be suppressed in order to suppress preferential binding of CD28. The co-receptor is two different co-receptors called CD80 (B7-1) and CD86 (B7-2). Their structures are known.

After the antibody production is switched off, the autoantibodies circulating in the blood disappear due to the natural degradation and the lack of replenishment.

After the in vitro manipulation of the monocytes, the cells are returned to the patient's bloodstream. The genetically modified monocytes now switch off the pathological T helper lymphocytes in the organism. The genetically modified cells compete directly with the autoantigen-presenting monocytes already present in the organism (preferably the bloodstream and lymphatic system), which however carry B7 on the cell surface and normally activate the T helper lymphocytes and thus the antibody production (Fig 7a and 7b).

Antigen presentation in genetically modified monocytes

The cDNA of a protein that causes the production of pathological autoantibodies as an autoantigen is integrated into the monocyte genome. This genetic information then serves to overproduce the autoantigen. Peptides of this autoantigen are then preferably presented on MHC II and / or MHC I [see also FIGS. 5 and 6]. MHC II presents the peptides to the T helper cells and tries to find those that specifically recognize these presented peptides. If a PD-1 binding molecule activates PD-1 instead of CD28 and a CTLA4-binding molecule also activates CTLA4 instead of CD28 and the co-receptor B7 does not appear on the cell surface at the same time, the T helper cells are shut down and may experience premature cell death.

All genetically modified monocytes present the autoantigen on most of their MHC II complexes, while very few "normal" in vivo

Monocytes present the autoantigen and then only at a few MHC II complexes. The aim of the treatment is to displace the "normal" monocytes which present the autoantigen and activate the T helper cells in vivo by the genetically modified monocytes which have been programmed to switch off the antibody production or T cell response.

The monoantigenic antigen-presenting cell according to the invention can in particular show an increased number of homing receptors, such as CD44. Overexpression of homing receptors will show the monoantigenic antigen-presenting cell the way into the lymph nodes, as a result of which the genetically modified APCs increase and accumulate faster in lymph nodes. The lymph nodes are where most of the responses of the adaptive immune system are triggered. There, the genetically modified APCs have a much greater effect than outside the lymph nodes.

In a preferred embodiment of the monoantigenic antigen-presenting cell according to the invention, transfection causes an increase in the number of homing receptors and or an increase in the number of PD-1 binding molecules and / or a suppression of functions of the B7, CD40 receptors and / or an increase in the number of CTLA4 binding molecules.

The PD-1 binding molecules can preferably be PD-Ll, PD-L2, antibodies, monoclonal antibodies. These can be such that they remain in the plasma membrane of the cell.

The CTLA4 binding molecules can preferably be antibodies, monoclonal antibodies. These can be such that they remain in the plasma membrane of the cell.

In particular with antisense nucleic acids, the B7 and / or CD40 receptor expression in the monoantigenic antigen-presenting cell according to the invention can be prevented or reduced. Alternatively, the expression of the B7 and / or CD40 receptors can be suppressed by nucleic acids by co-suppression in the monoantigenic antigen-presenting cell according to the invention.

The monoantigenic antigen-presenting cell according to the invention can contain or be transfected with nucleic acids which bring about an expression of proteins or peptides which have structures affine with B7 and / or CD40 receptors. In this way, proteins are formed which, by forming complexes with B7 and / or CD40 receptors, practically neutralize these receptors. In particular, CTLA4, CD28, antibodies, F (ab) ₂ , scFv and / or F _ab fragments can be considered as proteins.

In a further preferred embodiment, the monoantigenic antigen-presenting cell according to the invention contains nucleic acids which code for a signal sequence or expression products of a signal sequence which determine whether the expression products remain in the endoplasmic reticulum, the Golgi apparatus, the Trans-Golgi network or intracellular vesicles causes.

The monoantigenic antigen-presenting cell according to the invention is transfected for the expression of antigens with nucleic acids which enable the transport of the expressed antigens in MHC II compartments of the cells.

All genetically modified monocytes present the autoantigen on most of their MHC complexes, while very few "normal" monocytes present the autoantigen at all and then only on a few MHC complexes. The aim of the treatment is to displace the "normal" monocytes that present the autoantigen and activate the T helper cells by means of the genetically manipulated monocytes programmed to switch off the antibody products. It is very important to also administer the antigen (s) in a form that allows them to be transported into the MHC II endosomes. This is the only way to reliably achieve a presentation at MHC II. In the case of a genetic engineering intervention, this is usually done by manipulating the open reading frame, so that the reading frame is preceded by a signal sequence for this compartment. Genetic manipulation has the additional advantage that it has a much longer half-life than, for example, oligo- nucleotides or peptides. A stable integration of genes even leads to a permanent change in the properties of the target cells.

The corresponding nucleic acids can be DNA, RNA, oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNA).

The DNA preferably has regulatory elements such as enhancers, promoters, polyA-coding 3 "ends for transcribing the DNA into RNA, the RNA regulatory elements for translating the RNA into protein. The regulatory elements ensure efficient expression of the genes.

The monoantigenic antigen-presenting cell according to the invention is produced, for example, by ex vivo or in vivo methods. An antigen-presenting cell is preferably ex vivo or in vivo by treatment with viruses, viral vectors, bacterial vectors, plasmids by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a monoantigenic antigen-presenting cell transfected.

In another embodiment, an antigen-presenting cell or a monoantigenic antigen-presenting cell can be obtained by treatment with viruses, viral vectors, bacterial vectors, plasmids, by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with an increased amount of PD-1 binding molecules and optionally with an increased amount of CTLA4 binding molecules and possibly suppressed function of co-stimulatory receptors or the expression of co-stimulatory receptors by preventing their expression or the co-stimulatory receptors are reacted with affine structures prevented from stimulating T cells bound to the monoantigenic antigen presenting cell.

Different strategies can be used to suppress co-stimulation, which ultimately means suppressing the production of one or more proteins or hindering the protein or proteins: 1. Antisense approach

2. Co-suppression

3. Binding of the co-stimulatory molecule.

Re 1.: In this case, antisense nucleic acids must come into contact with the mRNA of the co-stimulatory molecule. You will then likely bind the molecule and prevent translation. A wide range of molecules, such as RNAs, come as nucleic acids

DNAs, PNAs, ribozymes, in question. Here, too, a genetic engineering intervention would ensure the longest half-life of the effect.

Re 2.: It has now been found that the production of a gene product can also be achieved by integrating a homologous one

Sense gene sequence can achieve. The mechanism is still completely unknown.

Re 3.: The binding of the co-stimulatory molecule e.g. by specific antibodies prevents this molecule from contacting the notified

Receptor takes up on a T cell and thus prevents activation of the T cell. The external addition of such binding molecules has the disadvantage that they act on all antigen-presenting cells and thus prevent any immune reaction. In order to specifically hinder immune reactions, we have designed a model in which the co-stimulatory molecules are bound in the cell, in an intracellular compartment. An extension of this model is intended to ensure that the binding molecule is retained in the intracellular compartment so that the co-stimulatory receptor does not reach the plasma membrane in the first place. Signal sequences are known for this, which must be added to the open reading frame of the binding molecule. This intracellular approach can be carried out well on isolated cells. Here, too, genetic engineering intervention is preferable. The desired effects can also be achieved if only one of the two goals is carried out with a genetic engineering intervention. In this case, z. B. the following other molecules are used:

1. Impairment of co-stimulation by

Nucleic acids (mostly complementary to the target sequence), which are e.g. B. can be oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNAs),

Antibodies or other molecules that bind the co-stimulatory molecules.

2. Antigen contacting through

Proteins, peptides, peptidomimetics.

In particular, molecules such as antibodies, proteins, peptides, peptidomimetics, PD-L1, PD-L2, CTLA4, CD28, CD40L and / or constituents and / or combinations of these molecules, which e.g. B7-1, B7-2, CD40 bind, which prevents co-stimulation of the T cell taking place in the presence of an antigen presentation, brought into contact with the monoantigenic antigen presenting cell or the antigen presenting cell.

It can be advantageous to use molecules such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, in particular biodegradable nanocapsules or particles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or other techniques to transfer molecules into the monoantigenic antigen-presenting cell or the antigen-presenting cell.

Nucleic acids can in particular by viruses, viral vectors, bacterial vectors, plasmids by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for the introduction of molecules are transferred into the monoantigenic antigen-presenting cell or the antigen-presenting cell.

A medicament containing at least one monoantigenic antigen-presenting cell according to the invention is further claimed. The medicament according to the invention is preferably formulated as an infusion solution for intravenous or intraperitoneal administration. The formulation is chosen such that when the medicament is administered there is no significant impairment of the effectiveness of the monoantigenic antigen-presenting cell according to the invention.

For example, physiological saline is preferred as the infusion solution. In principle, other solutions with a pH of 5.5 to 8.5 are also suitable. Serum, for example human serum, autologous serum or serum of other species, solutions with plasma substitutes, such as polyvinylpyrrolidone, are also suitable. Typically 0.5 ml to 500 ml should be applied. These quantities and pH values are of course not absolute, but can be varied and adapted to the specific needs of a patient by a specialist, depending on the conditions and requirements.

The monoantigenic antigen-presenting cell according to the invention can be used in particular for the production of a medicament for the treatment of unwanted immune reactions such as autoimmune diseases and allergies or deliberately induced immune reactions such as in immunizations.

Furthermore, the monoantigenic antigen-presenting cell according to the invention can be used for the production of a medicament for the treatment of immune reactions against allologic and / or xenologic tissue characteristics.

In particular, uses are claimed according to the invention in which the immune reactions to be treated are related to antigens or their gene sequences and / or parts thereof and are selected from the group consisting of Enzymes, their gene sequences and / or partial sequences, in particular glutamic acid decarboxylase (GAD), receptor type protein tyrosine phosphatase IA-2Beta, antigen: H ⁺ K ⁺ ATPase, U1RNP, transglutaminase, argininosuccinate lyase (ASL), tyrosinase related protein-2, thyroid peroxidase, factor VIII, factor IX;

Receptors, their gene sequences and / or partial sequences, in particular acetylcholine receptor of the nicotine type, β1-adrenergic receptor, αl-adrenergic receptor, angiotensin-2-ATl receptor, glutamate receptor, thyrotropin-stimulating hormone (TSH) receptor, LFA receptor 1,

HLA-B27, Epididymal Protein DE, Zona Pellucida (ZP) -3 Glycoprotein, Zona Pellucida (ZP) -4 Glycoprotein, Follicle-Stimulating Hormone (FSH) receptor, Sperm Immunogen SP-10 or Sperm Protein SP-10;

Hormones or messenger substances, their gene sequences and / or partial sequences, in particular insulin, thyroglobulin, follicle-stimulating hormones (FSH), prostaglandin F2 alpha, gonadotropin-releasing hormones (GnRH), oestradiol-17beta, estrogen, luteinizing hormone (LH) receptor, inhibin , Testosterone, androgen, chorionic gonodrophin (CG), interleukins, interferons, cytokines, chemokines, bone morphogenetic factors, ß-interferon, estradiol;

Structural proteins, their gene sequences and / or partial sequences, in particular myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), α-fodrin, non-erythroid α-

Spectrin, beta-amyloid precursor protein (beta-APP), type 2 collagen, sperm plasma membrane protein PH-20;

Antigens, their gene sequences and / or partial sequences, in particular CENP-A autoantigens, Beta2GP-I, ribosomal P protein, Ro / SSA,

La / SSB, Sm / RNP, Sm, Scl-70, Jo-1, BCOADC-E2, albumin, glucagon, islet cell antigens, Retinal S Ag;

Allergens that trigger an IgE response, their gene sequences and / or partial sequences, in particular Der f 1, Der f 2, Der f 3, Der p

1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 8, Eur m 1, Lep d 2, Fei d 1, Can f 1, Can f 2, Mus m 1, Rat n 1, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Per a 1, Bee venom Phospholipase A ₂ (PLA ₂ ), Group V major allergen Phl p 5b by Timothy Grass Pollen, Hom s 1.

Furthermore, the invention claims a use in which the immune reactions to be treated are associated with allologic and / or xenological tissue characteristics, their gene sequences and / or partial sequences, in particular MHC I, MHC II, rhesus factor.

Development of a gene therapy

The method presented here for reducing immune responses, e.g. B. for the treatment of autoimmune diseases with a clearly defined autoantibody profile, is based on the genetic manipulation of certain blood cells, preferably outside the body, which are then subsequently returned to the organism. The aim of this treatment is, among other things, the specific switching off of a chronic, immune system-driven production of autoantibodies that trigger the disease.

Areas of application of gene therapy

The procedure for the specific deactivation of immune reactions always makes sense where one or more molecularly defined targets (antigens, autoantigens) are known. These can be associated with an autoimmune disease or allergy, for example.

For example, these are diseases with autoantibody-mediated autoimmune reactions, i.e. diseases in which the "binding structures" of these autoantibodies (autoantigens, targets) are known and have pathogenetic significance.

Examples of diseases with known molecular structures (epitopes) that bind autoantibodies are:

• myasthenia gravis (autoantibodies against the acetylcholine receptor) • Graves' disease (autoantibody against the TSH receptor of the thyroid gland)

Dilated cardiomyopathy (DCM, autoantibodies against the β1-adrenergic receptor of the heart muscle cells)

• certain forms of insulin-dependent diabetes (autoantibodies against insulin)

• certain forms of malignant hypertension (autoantibodies against the angiotensin and / or αl adrenergic receptor).

Treatment with genetically manipulated patient's own blood cells: principle of the proposed gene therapy

Within the immune system, the antibodies play an important role as defense molecules ("humoral immune response"). They are produced by mature B lymphocytes. However, the induction and production of the antibodies is not detached from the remaining cells of the immune system; rather, this humoral immune response is controlled by other cells in the immune system. The immune response cannot be maintained without the help and mediation of antigen presenting cells and T helper lymphocytes. The molecular mechanisms of the interaction - of cell-cell contact - of antigen-presenting cells with the T-helper lymphocytes is known in detail at the receptor level.

In addition to the antigen to be presented, various receptors and auxiliary receptors are involved in cell-cell contact and cell activation on the cell surface. An essential molecule of the intercellular interaction in antigen presentation (contact of antigen-presenting cells with the T-helper lymphocytes) are costimulatory receptors with the names PD-Ll, PD-L2, B7h (LICOS), B7-1 and B7-2 , CD40 also plays a role in this signal transduction. The functions of ICOS, PD-1, CD28, CTLA4, B7 and CD40 are the contents of numerous publications (Daikh et al., 1997; Greenfield et al., 1998; Johnson-Leger et al., 1998; McAdam et al., 1998 ; Vyth-Dreese et al., 1995, (Freeman et al 2000; Hutloff et al 1999; Kopf et al 2000; Tamatani et al 2000)) and (apart from ICOS, PDl) are also extensively dealt with in textbooks [see eg (Janeway and Travers, 1997)].

The designed procedure is based on at least two parallel interventions on the patient's own antigen-presenting cells. The interventions are carried out using suitable probes and have an effect

The production of a PD-1 binding molecule, e.g. PD-Ll, which via the action of PD-1, which occurs in T cells, prevents the T cells from activation and proliferation

• At the same time, a strong presentation of autoantigen on the antigen-presenting cells

And optionally the production of a CTLA4 binding molecule, e.g. Antibodies that prevent the T cells from activating through the action of CTLA4, which occurs in T cells

• and, if necessary, the shutdown of B7 and thus the prevention of the formation of the molecule on the surface of the antigen-presenting cells and / or the shutdown of CD40.

Should the manipulation of the cells z. B. are monocytes, ex vivo, the cells are returned to the donor's bloodstream. The genetically manipulated monocytes now switch off the corresponding T helper lymphocytes in the organism. The genetically manipulated cells compete directly with the autoantigen-presenting monocytes already present in the organism (preferably the bloodstream and lymphatic system), which however carry B7 and no additional PD-1 binding molecule, CTLA4 binding molecule on the cell surface and usually the T-helper - Activate lymphocytes and, among other things, antibody production. Treatment of autoimmune diseases, allergies and transplants: state of the art

Causal therapies for the treatment of autoimmune diseases and allergies do not exist. With a few exceptions (extracorporeal elimination of antibodies from the patient's blood or plasmapheresis), autoimmune diseases and allergies are treated with medication by inhibiting immune system reactions.

Drug treatment of autoimmune diseases, allergies and transplants with immunosuppressive preparations such as cortisone and its derivatives, as well as cyclosporin, beta interferon or cytostatics (methotrexate) is still the most common. Furthermore, antibodies, antagonists and oligonucleotides against various signal components of the immune system are tested with the purpose of preventing the activation of T cells. Such drugs are at best selective, but never specifically directed against the wrongly programmed autoimmune reactions. Immunosuppressants therefore have a negative effect on important, necessary and useful parts of the immune system; for this reason and their side effects, their use is limited.

The concept of gene therapy for the reduction of specific immune reactions differs fundamentally from immunosuppressive therapies. A specific shutdown of misdirected immunological reactions by genetically reprogrammed patient's own blood cells is carried out.

The methodology shown is intended as a generally applicable concept for reducing immune responses. With this basic concept it should be possible to stop any immune reaction of the adaptable immune system. In addition, such immune reactions can be triggered at will and then switched off again. literature

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Claims

claims 1. Antigen-presenting cell which predominantly presents certain antigens beforehand (monoantigenic antigen-presenting cell), characterized in that a PD-1 binding molecule is produced in the monoantigenic antigen-presenting cell and, if appropriate, a CTLA4-binding molecule is produced and, if appropriate one of the functions of co-stimulatory receptors, such as B7 and / or CD40 receptors, are suppressed. 2. Monoantigenic antigen-presenting cell according to claim 1, characterized in that the monoantigenic antigen-presenting cell is a monocyte, a dendritic cell and / or a macrophage. 3. Monoantigen antigen-presenting cell according to claim 1 and / or 2, characterized in that by transfection of an antigen-presenting cell with nucleic acid-containing material an increased expression of the predefined antigens takes place and the antigen-presenting cell presents essentially only these antigens. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 3, characterized in that the monoantigenic antigen-presenting cell has an increased number of homing receptors, such as CD44. 5. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 4, characterized in that a transfection an expression of PD-1 binding molecules and optionally of CTLA4 binding molecules and optionally a suppression of functions of the B7, CD40 receptors and possibly increasing the number of homing receptors. 6. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 5 containing nucleic acids that encode PD-1 binding molecules and optionally encode CTLA4 binding molecules and optionally antisense nucleic acids to prevent B7 and / or CD40 receptor expression. 7. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 6 containing nucleic acids which code for PD-1 binding molecules which remain in the plasma membrane of the cell. 8. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 6, in which the PD-1 binding molecules are preferably PD-L1, PD-L2, antibodies, monoclonal antibodies. 9. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 8 containing nucleic acids, which suppress the expression of the B7 and / or CD40 receptors by co-suppression. 10. Monoantigenic antigen-presenting cell according to at least one of claims 1 to 9 containing nucleic acids which cause expression of proteins or peptides having structures affine with B7 and / or CD40 receptors. 11. Monoantigenic antigen-presenting cell according to claim 10, wherein the proteins which have structures affine to B7 receptors are CTLA4, CD28, antibodies, F (ab) _2A scFv and / or F _ab fragments. 12. Monoantigenic antigen-presenting cell according to claim 10 and / or 11, wherein the nucleic acids code for a signal sequence or the expression products have a signal sequence which indicates the whereabouts of the expression products in the endoplasmic reticulum, the Golgi apparatus, the Trans-Golgi network or intracellular vesicles. 13. Monoantigenic antigen-presenting cell according to at least one of claims 3 to 12, characterized in that the monoantigenic antigen-presenting cell for the expression of antigens is transfected with nucleic acids, which in the transport of the expressed antigens MHC II compartments of the cells enabled. 14. Monoantigenic antigen-presenting cell according to at least one of claims 3 to 13, characterized in that the nucleic acids are DNA, RNA, oligonucleotides, polynucleotides, ribozymes, peptide nucleic acids (PNA). 15. Monoantigenic antigen-presenting cell according to claim 14, characterized in that the DNA regulatory elements such as enhancers, promoters, polyA-coding 3 'ends for the transcription of the DNA into RNA contains the RNA regulatory elements for translating the RNA into protein. 16. A method for producing the monoantigenic antigen-presenting cell according to at least one of claims 1 to 15 by ex vivo or in vivo procedures. 17. The method according to claim 16, wherein an antigen-presenting cell ex vivo or in vivo by treatment with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for Molecular introduction into a monoantigenic antigen-presenting cell is transfected. 18. The method according to claim 16 and / or 17, wherein an antigen-presenting cell or a monoantigenic antigen-presenting Cell by treatment with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with increased production of PD-1 binding molecules and possibly CTLA4 binding molecules and optionally transfected with suppressed function of co-stimulatory receptors, the expression of co-stimulatory receptors by preventing their expression or the co-stimulatory receptors by reacting with affine structures to stimulate T cells bound to the monoantigenic antigen presenting cell are prevented. 19. The method according to at least one of claims 16 to 18, wherein molecules such as antibodies, proteins, peptides, peptidomimetics, PD-Ll, PD-L2, PD-1 binding molecules, CTLA4 binding molecules, CTLA4, CD28, CD40L and / or constituents and / or combinations of these molecules, for example PD-1, CTLA4, B7-1, B7-2, CD40, which prevents co-stimulation of the T cell taking place in the presence of an antigen presentation, are brought into contact with the monoantigenic antigen-presenting cell or the antigen-presenting cell , 20. The method according to at least one of claims 16 to 19, wherein the molecules according to claim 17 by vehicles such as liposomes, hydrogels, cyclodextrins, nanocapsules, nanoparticles, bio-adhesive microspheres and / or by electroporation techniques, iontophoresis, ballistic methods and / or others Techniques for introducing molecules into the monoantigenic antigen-presenting cell or the antigen-presenting cell are transferred. 21. The method according to at least one of claims 16 to 20, wherein nucleic acids by viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for the introduction of molecules into the monoantigenic antigen presenting cell or the antigen presenting cell. 22. Medicament containing at least one monoantigenic antigen-presenting cell according to at least one of claims 1 to 15. 23. Medicament according to claim 22, wherein at least one monoantigenic antigen-presenting cell is formulated as an infusion solution for intravenous or intraperitoneal application. 24. Use of at least one monoantigenic antigen-presenting cell according to at least one of claims 1 to 15 for the manufacture of a medicament for the treatment of unwanted immune reactions, such as autoimmune diseases and allergies or deliberately induced immune reactions, such as immunizations. 25. Use of at least one monoantigenic antigen-presenting cell according to at least one of claims 1 to 15 for the manufacture of a medicament for the treatment of immune reactions against allologic and / or xenologic tissue characteristics. 26. Use according to claim 24, wherein the immune reactions to be treated are associated with antigens or their gene sequences and / or parts thereof are selected from the group consisting of Enzymes, their gene sequences and / or partial sequences, in particular glutamic acid decarboxylase (GAD), receptor type protein tyrosine phosphatase IA-2Beta, H + K + ATPase, U1RNP, transglutaminase, argininosuccinatease (ASL), tyrosinase-related protein -2, thyroid peroxidase, factor VIII, factor IX; Receptors, their gene sequences and / or partial sequences, in particular acetylcholine receptor of the nicotine type, myasthenia gravis, β1-adrenergic receptor, αl-adrenergic receptor, angiotensin-2-ATl receptor, GI Uta mat receptor, thyrotropin-stimulating hormone (TS ) - Receptor, LFA-1, HLA-B27, Epididymal Protein DE, Zona Pellucida (ZP) -3 Glycoprotein, Zona Pellucida (ZP) -4 Glycoprotein, Follicle-Stimulating Hormone (FSH) Receptor, Sperm Immunogen SP-10 or Sperm Protein SP-10; Hormones or messenger substances, their gene sequences and / or partial sequences, especially insulin, thyroglobulin, follicle-stimulating hormones (FSH), prostaglandin F2 alpha, gonadotropin-releasing Hormones (GnRH), oestradiol-17beta, estrogen, luteinizing hormone (LH) receptor, inhibin, testosterone, androgen, chorionic gonadotrophin (CG), interleukins, interferons, cytokines, chemokines, bone morphogenetic factors, ß-interferon, estradiol; Structural proteins, their gene sequences and / or partial sequences, in particular myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), α-fodrin, non-erythroid α-spectrin, beta-amyloid precursor protein (beta-APP ), Type 2 collagen, sperm plasma membrane protein PH- 20; Antigens, their gene sequences and / or partial sequences, in particular CENP-A autoantigen, Beta2GP-I, ribosomal P protein, Ro / SSA, La / SSB, Sm / RNP, Sm, Scl-70, Jo-1, BCOADC-E2, albumin , Glucagon, islet cell antigens, Retinal S Ag; Allergens that trigger an IgE response, their gene sequences and / or partial sequences, in particular Der f 1, Der f 2, Der f 3, Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 8, Eur m 1, Lep d 2, Fei d 1, Can f 1, Can f 2, Mus m 1, Rat n 1, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Per a 1 , Bee venom phospholipase A2 (PLA ₂ ), Group V major allergen Phl p 5b by Timothy Grass Pollen, Hom s 1. 27. Use according to claim 25, wherein the immune reactions to be treated are associated with allologic and / or xenologic tissue features, their gene sequences and / or partial sequences, in particular MHC I, MHC II, rhesus factor.