The notch ligand


(57) Abstract:

The invention relates to the use of therapeutic compounds in the modification of T-cell interactions, antigen-presenting cells with T-cells and interactions between pathogens and the immune cells of the host. In particular, it relates to the use of these compounds in the modification of interactions between Notch proteins and their ligands, and the use of such compounds in the production of pharmaceuticals for the treatment of conditions such as graft rejection, autoimmune processes, allergies and asthma, and infectious diseases. The advantage of the invention is to develop tools that inhibits T-cell interactions, leading to infectious tolerance and which can be used in the production of medical drugs for immunotherapy. 3 S. and 9 C.p. f-crystals, 2 tab., 9 Il.

The invention relates to the use of therapeutic compounds in the modification of activated T cells. In particular, it relates to their use for the modulation of the interaction between proteins a family of Notch and its ligands and the use of such compounds in the treatment of such conditions as: the reaction of rejection Tran the action between T-cells and between antigen-presenting cells (AP-cells) and T cells is vital for the functioning of the human immune system. However, under certain pathological conditions, it may be useful to modify, positively or negatively, such interaction. For example, under such conditions as autoimmune reaction, Allergy or reaction, transplant rejection, it is desirable to induce regulation by type of negative feedback of the immune response by stimulating the negative T-cells or the interaction of T cells with AP-cells. Model "infectious tolerance" and "linked suppression" suggest that tolerance can be induced in a small number of T-cells, and these T cells then transmit that tolerance of other T-cells, thus preventing effective immunological attack. In other pathological conditions such as tumor induced immunosuppression, parasitic viral or bacterial infection, immunosuppression is a common phenomenon. In these circumstances it would be therefore desirable to inhibit T-cell interactions, leading to infectious tolerance.

However, to date, the mechanisms that support such interaction between T-cells and T-cells with AP-cells have not been studied.

In the application WO 92/19734 obnaruzhennymi proteins. It is shown that the family of Notch genes plays an essential role for the proper development of the differentiation of embryonic insect cells such as Drosophila.

Proteins belonging to the family of Notch are transmembrane receptors that contain several conservative peptide motifs. Each protein from this family is characterized by repetitions like extracellular epidermal growth factor (EGF) and colonelblinky motive Lin-12/Notch. In addition, each protein has a 6-8 anchirinah recurring motifs in the cytoplasmic tail together with the PEST sequence. The Notch ligands have diagnostic DSL domain (D, Delta, S, Serrate, L,Lag2), including 20-22 amino acids on aminocore protein and 3 to 8 EGF-like repeats in the extracellular region. Proteins have a short cytoplasmic tail with unsaved functional domains.

The obtained data indicate that Notch promotes differentsirovanogo commiteeman immature T cells in the thymus, directing the thymocytes towards differentiation into cells CD8+, not dependent on the MTL (E. Robey with the researcher. Cell, 1996, 87:483-492). In the process of maturation in the thymus T cells acquire the ability to recognize their own antigens and to distinguish them from others is AlertNet also exist on the periphery, and in many respects their value is under study. There are many experimental models of reaction, transplant rejection, autoimmune diseases and specific responses to allergens, which clearly illustrate the ability to induce a state of specific areactively (tolerance or anergy) in the recipient upon immunization with a specific antigen. The result can draw two important conclusions. First, immunization with a peptide fragment of the antigen in special conditions can suppress specific answers not only to himself but also to other areas of the same molecule in the case of the intact protein is used for provocative immunization (linked suppression; Hoyne GF. with TCS. J. Exp Med. 1993; 178:183 and Metzler Century, Wraith DC. Int. Immunol. 1993; 5: 1159). Secondly, best shown in experimental models of transplantation in the phenomenon of "infectious tolerance", which postulated that immune cells, being immune to a specific antigen, is able to suppress cell-mediated response in other cells and, moreover, this second population of cells becomes regulatory and tolerant (Qin SX. with TCS. Science, 1993; 258:974). Immunological mechanisms underlying this receptor family of Notch and its ligands Delta and Serrate expressed on the surface of normal Mature cells of the peripheral immune system.

Therefore, under the conditions according to the invention, Notch-ligands can be used in the production of medical drugs for immunotherapy.

The expression pattern of the receptors of the Notch family and their ligands in the peripheral immune system healthy adult still has not been described, but the present invention is shown that T cells constitutively Express Notch 1 mRNA, whereas the expression of Delta is limited to only a subpopulation of T-cells in peripheral lymphoid tissues. The expression of Serrate limited to the subpopulation of antigen-presenting cells (AP) cells at the periphery (figure 1). Therefore, the family of receptor ligands may continue to regulate the fate of cells in the immune system, as has been shown in other tissues, after embryonic development (Ellisen LW. with TCS. Cell 1991; 66: 649). The Notch signaling may play a Central role in the induction of peripheral reaction areactively (tolerance or anergy) and may provide a physical explanation linked suppression and infectious tolerance.

The present invention is, furthermore, connected with the use of Notch ligands to influence the reaction, mediated by T-cells. Thus, it was observed that outstay antigen or allergen Notch ligands can cause tolerance, T-cell population to a specific antigen or allergen. Moreover, this population of T cells with further exposure with the second population of native T cells, are also capable of transmitting the second population tolerance to the specified antigen or allergen.

Thus, the invention also relates to use of Notch ligands to influence the associated tolerance and/or transferred tolerance (also called infectious tolerance).

Another aspect of the invention is associated with the use of Notch ligands to modulate the expression of functional protein Notch or signalizovania Notch.

In the above aspects, according to the invention, the Notch ligand may be exposed to T cells by incubation mix of T cells with AP-cells or the like, which Express or redundantly Express Notch ligands in the presence of an allergen or antigen. Overexpression of the gene of Notch ligand can be called up by the cells during transfection their genome is capable of causing the expression of the Notch ligand, or AP-cells exposed to an agent capable of increasing expression of an endogenous gene (genes) ligand Notch.

Appropriate agents influencing the expression of Notch ligands, include agents that affect lcov bones, Wilson, P. A. and Hemmati-Brivanlou, A., 1997, Neuron 18: 699-710, Hemmati-Brivanlou A. and Melton D. Cell 88:13-17) to their receptors leads to reverse the transcriptional regulation of the Delta by inhibiting the expression of the transcription factor complex Achaete/Scute. There is reason to believe that this complex is directly involved in the regulation of expression of Delta. Thus, any agent which inhibits the binding of BMPs to their receptors, can cause increased expression of Delta and/or Serrate. Such agents include Noggin, Chordin, Follistatin, Xnr3, FGFs, Fringe, as well as their derivatives and other variants (see links on the noggin-Smith W. C. and rland, R. M Cell 70:829-840, chordin-Sasai, Y. et al., 1994 Cell 79: 779-790). Thus, communication Noggin and Chordin BMPs prevents activation of their signaling cascade, which leads to suppression of transcription Delta.

Certain medical conditions can make the immune system is weakened and you may need to suppress the expression of Delta and/or Serrate to overcome immunosuppression. In such circumstances can be used by agents capable of inhibiting the expression of Delta and/or Serrate. Examples of agents that can suppress the expression of Delta and/or Serrate, can serve as protein Toll (Medzhitov R. al., 1997 Nature 388: 394-397) BMPs and other agents that can reduce or prepjatstvovanie type of negative feedback to reduce the protein expression of Delta or Serrate, in the manufacture of medical products for use in the treatment of repeated bacterial infection or immunosuppression caused by the tumor.

As discussed above, the invention also relates to modification of the expression of the Notch protein, or presentation on the cell membrane, or signaling pathways. It was shown that all this is involved in mediated T-cell responses, which take part in the emergence of tolerance (linked and/or infectious). Agents that enhance the presentation of a fully functional protein Notch on the cell surface include matrix metalloproteinases, such as the gene product of Drosophila Kuzbanian (Dkuz, Pan, D and Rubin, G. M. 1997 Cell 90: 2.71-280) and other genes of the ADAMALYSIN family. Agents that reduce or inhibit its presentation as a fully functioning protein in the cell membrane can serve as inhibitors of matrix metalloproteinases (MMP), such as inhibitors, based on hydroximate.

The term "antigen-presenting cell or equivalent" here means not only the AP cells. Expert it is clear what is meant any filler that can provide the desired Notch ligand populations of T-cells; to refer to all such stucley also dendritic cells, L-cells, hybridoma, lymphoma, macrophages, b-cells or synthetic AP-cells, such as lipid membranes.

When AP-cells transliterowany gene capable of expression of the Notch ligand, transfection can be carried out by a virus such as retrovirus or adenovirus, or other medium or method capable of delivering a gene of the cell. The effectiveness of any such media or methods shown in gene therapy, and these include retroviruses, liposomes, electroporation method, as well as other viruses, such as adenoviruses, adeno-associated virus, herpes virus, vaccinia virus, DNA, precipitiously by calcium phosphate, transfection with DEAE-dextran, microinjection, polyethylene glycol, protein-DNA complexes.

The use of such media or methods separately or in combination with each other makes it possible to carry out sitepreview the transfer of a gene in a specific cell population, making possible the implementation of the method of the present invention in vivo. For example, a virus may be used in combination with liposomes to increase the efficiency of absorption of DNA. Sitespecifically delivery can be achieved by the inclusion of specific proteins the liposomes.

Preferably the use of the structures by which gene expression (for example, Serrate) would be associated with the expression of the antigen. AP-cells, excessively expressing Serrate, would have, in addition, the high expression level of the bound antigen, and preferably reacted with T-cells of the appropriate specificity.

A further embodiment of the present invention is associated with a molecule containing a fragment of Notch ligand, operatively associated with T-cell fragment of an allergen or antigen, so that when the exposure with T-cells both fragments are able to communicate their respective sites. This molecule is able to turn the antigen/allergen-specific T-cell tolerance to an allergen or antigen containing this fragment, because the specificity required of a fragment of the Notch ligand, due to the close contact portion of the allergen or antigen.

A fragment of the antigen or allergen can be, for example, synthetic MHC-peptide complex. This fragment of the MHC molecules carrying antigenic peptidase plot bearing element of the antigen. Such complexes have been described in Altman JD with TCS. Science 1996 274:94-6.

In the following passengeronly Fcpreferably IgGFcor IgMFc.

In each of the above aspects of the present invention it is preferable that the Notch ligand proteins were family Serrate or proteins of the Delta family, their derivatives, fragments or analogs.

In the list of diseases or infections that can be characterized as mediated by T-cells, can include one or more of the following: asthma, Allergy, graft rejection, autoimmune manifestations, tumor-induced aberrations in the system T-cells, as well as infectious diseases such as caused by species of Plasmodium, microfilaria, worms, mycobacteria, HIV, cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, Haemophilus influenzae type b agents measles, hepatitis C or Toxicara. Thus, when using a suitable allergen or antigen, the Notch ligand may be used according to the invention for the treatment of specified diseases or infections.

The invention also provides a method of detecting the immunosuppression induced by damaging the body. Such organisms are able to create a soluble form of the members of the families Serrate, Notch and/or Delta, or from their derivatives, or proteins, geistliche analysis for the presence of in vivo membranosvyazannyh Serrate, Delta, Notch, or derivatives thereof, and preferably is associated with antibodies against Serrate, Delta or Notch, or derivatives thereof.

Methods of screening assays for the registration of reduced or enhanced expression and/or processing of Notch, Delta/Serrate include:

1. The expression of the Delta/Serrate, Notch and Fringe installed after exposure of isolated cells with the test compounds in culture, for example,

a) on the protein level by specific staining antibodies using methods of immunohistochemistry or flow cytometry,

b) at the level of RNA by quantitative polymerase chain reaction with reverse transcriptase RT-PCR. RT-PCR can be implemented using a control plasmid with the set standards for the measurement of endogenous gene expression using specific for Notch 1 and Notch 2, Serrate 1 and Serrate 2, Delta 1 and Delta 2, Delta 3 and Fringe primers. This design can be modified once you have identified new members of the ligand group.

c) on the functional level through analysis of cell adhesion.

Increased expression of Delta/Serrate or Notch should lead to increased adhesion between cells expressing Notch, and the League is active or fluorescent-labeled target cells (transfetsirovannyh protein Notch/Delta/Serrate). The mixture of cells are incubated at 37oC for 2 hours non-stick cells are washed away, and the degree of adhesion is determined by the level of radioactivity/immunofluorescence assay on the surface die.

The use of such methods allows to identify compounds or ligands Notch violating the expression or processing of the protein Notch or Notch ligand. The invention also relates to compounds or Notch ligands, which can be identified such analytical methods.

The scope of this invention also includes the method of analysis, comprising contacting (a) Notch protein and a ligand capable of contact with the Notch protein (b); and determining whether the compound with the binding of the ligand with the protein Notch, if Notch protein associated with T-cell.

The Notch ligands according to the invention are preferably proteins are members of the families of Delta or Serrate or polypeptides, or derivatives thereof. Preferably, they received standard recombinant methods well known to specialists in this field. The corresponding sequences of the genes for the formation of such compounds according to the invention can be obtained from publications such as WO 97/01571, WO 96/CAE. Even more preferably, the members of the families of Notch, Delta or Serrate, proteins or polypeptides, or derivatives thereof were fragments of the extracellular domains of Notch, Delta or Serrate or members of their families, or derivatives of such fragments. Here the term "ligand Notch" further includes any ligand or a member of a family of ligands that interact with a member of a family of proteins Notch and includes a group of proteins belonging to both the "toponimicheskii proteins, i.e. proteins - products of genes Delta, Serrate, Deltex and Enhancer of split (enhancer of sublocus), and to the other members of this family of genes identified by the ability of their gene sequences to gibridizatsiya with or homology protein Notch, Delta and Serrate, or the ability of their genes to show phenotypic interaction.

Notch, Delta or Serrate were first described in Drosophila, and, therefore, represent a protein-prototypes Notch receptor and, accordingly, family members of the Notch ligand. Multiple proteins and Notch ligands currently described in many species of vertebrates and invertebrates, but their nomenclature may differ from that of the flies. For example, Notch is a homologue of Lin-12 and Glp 1, Serrate/Delta are homologues Jagged, Apx1 and Lag-2.

FA is the specialists in this field principles. Thus, the nature of the filler and the level of activity will depend on the compounds according to the invention, which should be included in the composition.

Preferably, the pharmaceutical compositions were presented in the form of a single dosage.

The dosage of the compounds of the present invention, assigned to the patient in the form of a pharmaceutical composition, can be determined by the treating physician.

The preferred method of appointment of the compositions according to the invention is one of the usual ways, including intramuscular and intraperitoneal, intravenous injection, intranasal inhalation, pulmonary inhalation, introduction subcutaneously, intradermally, intra-articular, vnutriobektovyh, local methods of administration, and route of administration through the digestive tract (for example, through Peyer's patches).

The term "derivative" is used here to refer to proteins or polypeptides of the present invention, may include any substitution of, variation of, modification, deletion, replacement or Supplement one or more amino acid residue in the sequence, provided that the resulting protein or polypeptide has the ability to modulate peptido of the present invention, may include any substitution of, variation of, modification of, replacement of, deletion of or Supplement one or more amino acid residue in the sequence, provided that the resulting protein or polypeptide has the ability to modulate the interaction of ligands of Notch-Notch.

The term "analog" is used here to refer to proteins or polypeptides of the present invention, includes any peptidomimetic, which is a chemical compound with the ability to modulate the interaction of ligands of Notch-Notch similar to the parent protein or polypeptide. These include compounds that can positively or negatively influence the expression or activity of the protein Notch or Notch ligand.

The connection can be considered as modulating the interaction of ligands of Notch-Notch, if it is able to either inhibit or enhance the interaction of Notch with its ligands, preferably sufficient to provide therapeutic effectiveness degree.

Under the expression "ligand Notch-Notch" here refers to the interaction between family member Notch and ligand, capable of binding one or more of such members.

The concept of therapy used is t in use in veterinary and human.

Here the concepts of protein and polypeptide can be taken as synonyms, the term protein is usually more often used to refer to relatively long amino acid sequence than that of the polypeptide.

Hereinafter the present invention will be described non-limiting examples with reference to the relevant drawings, in which:

the figure 1 shows the results of in situ hybridization performed as described in example 1;

the figure 2 shows the results of the experiment described in example 4;

the figure 3 shows the results of the experiment described in example 5;

the figure 4 shows the results of the experiment described in example 6;

the figure 5 shows the results of the experiment described in example 7;

the figure 6 shows the results of the experiment described in example 8;

the figure 7 shows the results of the experiment described in example 9;

in figures 8A and 8b shows the results of the experiment described in example 10;

the figure 9 shows the results of the experiment described in example 11.

Example 1

Notch, Delta and Serrate is expressed in the peripheral immune system.

Antisense sample RNA specific for Notch 1, Del buffer, heated to 70oC for 5-10 min, and added to the covered TESPA microscopic preparations containing 10 mm environment of the spleen and thymus, which previously were fixed in 4% p-d paraformaldehyde + PBS. Microscopic preparations were hybridized overnight at 65oC. the next day, these drugs are washed twice at 65oC and twice at room temperature (RT) 1SS/50%formamide/0.1%tween-20. Microscopic preparations were washed twice (IATA)-buffer 0.1 M maleic acid /0.15 M NaCl/0.1% tween-20, pH 7.5, at RT, and then blocked for 2 h IATA + 20% goat serum + 296-blocking reagent Behringer (BBR). The specimens were incubated overnight at RT with antidigoxigeninAB-fragments. After 4 washes IATA, the specimens were washed 2 more times in alkaline substrate buffer. For related samples antisense RNA was determined by incubating the specimens in the dark in substrate buffer containing NBT + BCIP. The specimens were stained with hematoxylin and mounted in the middle fixer Depx.


The results of hybridization is shown in figure 1, where it is shown that in the spleen three-month mouse Delta and Serrate expressibility isolated is este cells, again in periarteriolar vagina.

Example 2

Getting fused protein Delta-Fc< / BR>
The expression vector pIG-1 [D. Simmons, "Cloning of cell surface molecules by transient surface expression in mammalian cells", pp. 93-128, Cellular Interactions in Development, Ed. D. Hartley, pub. Ox. Uni. Press (1993)] allows to obtain a fused protein containing the extracellular part of the Delta 1 associated with IgGl-Fcdomain person. A fragment of the restriction enzyme containing only the extracellular domain of the protein Delta 1, cloned in the vector pIG-1, the resulting plasmid was transformed into MC 1061 E. coli and grown in SOB containing 10 μg/ml tetracycline/ampicillin. The purified vector was used for transfection of COS cells in vitro. The COS cells were grown to 50-75% of confluently and were transliterowany 10 μg of plasmid DNA per Cup method, DEAE-dextran. 24 h after transfection the culture medium was replaced with medium containing 1% FCS, cells were cultured for another 3-6 days in vitro. Cells were centrifuged for 5 min at 5000 rpm before the deposition of cellular debris and formation of cellular precipitate, the supernatant was removed and retained for subsequent experiments. The merger of Delta-Fcwas purified from culture supernatants by adding 2 ml of a 50% mixture of protein-sepharose (Pharmacia) and was stirred for overnight at 4oC. Grannational column. Fused design Delta-Fcwas suirable 2 ml lucynova buffer, pH 4.0. The eluate optionally neutralized by adding 1M Tris.

Example 3

Examples of models in which you can explore signalizovania ligand Notch

Peripheral tolerance to self-antigen can be investigated in the T-cell receptor (TCR) transgenic mice, in which the TCR ligand expressed as native antigen only on the periphery. Peripheral tolerance to transplantation antigens can be induced in several ways, including pereobrabotka recipient antibodies against T cells or blockade of costimulation. Through this it becomes possible to demonstrate how linked suppression and infectious tolerance. Peripheral tolerance to allergens can be induced by intranasal introduction isolated from allergen proteins. The expression of Notch ligands-Notch was measured on cells taken from the pneumatic paths and/or lymphoid tissues adjacent to areas in the inhalation of the allergen, and identified changes in tolerance. Moreover, in experimental models of infectious agents, the expression of Notch ligands-Notch can be measured in the body (patat to suppress responses premirovany antigen lymphocytes

Mice were immunized with a synthetic peptide containing the immunodominant epitope of the allergen house dust mite (NDM), Der p1 (p110-131), or ovalbumin (OVA) protein chicken eggs). A week later the cells of the lymph nodes (cells LU) allocated and received cell suspension. Lymph nodes of animals immunized with other antigens, kept separately. These cells were named premirovanii cells LOU.

T-cell hybridoma also tranditional full-sized Delta or a control plasmid, such that Delta expressives as a membrane protein. After two days of cultivation hybridoma irradiated to prevent their proliferation or cytokine production. Thus, the only response that was measured in the analysis proceeded only from cells of the lymph nodes.

Irradiated hybridoma added in a larger amount in the culture containing bromirovannye cell LU. Appropriate antigen (e.g., R-131 or OVA) was added to the cell culture for 24 hours. Supernatant then collected and measured the concentrations of IL-2 (the main growth factor for T-cells). The proliferative response of cells of the lymph nodes was measured after 72 hours.

Results: the cell is in, continued to proliferate, as shown in figure 2, and secretively IL-2 when stimulated in culture with the appropriate antigen. Their ability to answer were maintained in a 1:1 ratio of cells LU:hybridoma. In contrast, the proliferative response and IL-2 production by cells of the lymph nodes decreased by 88% during the cultivation in the presence of hybridomas expressing the full-sized Delta (in the ratio 1: 1), with the corresponding antigen. Hybridoma was transfusional control virus (empty circles), Delta virus (empty squares). The figure 2 shows the data account of radioactivity per minute (CPM) labels3H-Tdr incorporated within 72 hours from start of culture growth. Account/min for cells of lymph nodes, grown in the presence of hybridomas expressing Delta or control design. The total number of cells per well = 4105(i.e., the number of lymph node cells varies, depending on the relationship to hybrid cells LU, T. O., CPM will vary). Cells LU p110-131 are cells, premirovanii Der p1 (p110-131), OVA-cells LOU are cells, premirovanii OVA. 2BB11 and VSS are two different Der p1-hybridomas.

These data show that inhibition of responses expressing Delta T-cells can be accroche Der P1, demonstrated the ability to inhibit the response premirovany OVA T cells, this apparent loss of specificity, apparently due to the close proximity of the cells that are reinforced in the culture system. Indeed, the data presented in figures 8A and 8b show that the animal hybridoma expressing Delta, must be antigenic specificity with immunogen, which is a modulation effect on the immune response to the immunogen. In this case, it is likely that T cells expressing Delta, can be in close proximity with only responding T-cells, if they recognize the same antigen on the same AP-cage.

Example 5

Dendritic cells expressing Serrate, prevent premirovanii antigen T-cell

Dendritic cells (DC) are the main antigen-presenting cells in the immune system and is essential for the stimulation of T-cell response. DC were obtained from spleen and transfusional or retrovirus, resulting in the expression of full-Serrate protein or a control retrovirus. Also DK for 3 hours continuously with which protein HDM p110-131 at a temperature of 37oIn vit 7 days facing cells LOUP were collected and re-stimulated in culture with peptide 4105cells/well. Because mice were immunized only dendritic cells treated with protein when shaken, it gave us the opportunity to assess the ability of these cells to premirovanii immune response.

Results: figure 3 shows the data account of radioactivity per minute in cells LU allocated after 72 hours of culture; these cells were obtained from animals immunized with control transfitsirovannykh (empty circles) or transfitsirovannykh Serrate (empty squares) dendritic cells (DC).

Immunization of mice Serrate expressing dendritic cells resulted in 10-fold fewer cells obtained from lymph nodes, compared to immunization with DC control. In addition, we found that cells LU mice immunized with DC+Serrate, did not show proliferative activity (93% reduction relative to control values, figure 3) and not secretively IL-2 compared with mice immunized with DC control.

Example 6

Expressing Delta T-cell hybridoma able to inhibit the development of immunity to the Per p1 antigen in animals

T-cell hybridoma (reactive with Der p1) was transfusional a retrovirus, sammysam was injected intraperitoneally 10 million irradiated hybrid and were immunized subcutaneously with 50 µg of Der P1, emulsified in complete Freund's adjuvant (CFA). After 7 days were collected emerging from lymph node cells and grew them in the amount of 4105cells/well in the presence of Der P1 (10 μg/ml), peptide 110-131 Der P1 (10 μg/ml) or peptide 81-102 Der p1 (10 µg/ml). Cultures were incubated for 72 hours at 37oWith and 8 hours before the end of the incubation were added tritium-labeled thymidine. Results cell proliferation in animals injected control transfitsirovannykh (puro) or transfitsirovannykh Delta (Delta-FL), shown in figure 4.

Results: cells LU animals injected control virus-transfitsirovannykh the hybridomas were produced high levels of IL-2 and proliferated in culture in the presence of Der p1, peptide 110-131 or peptide 81-102. Conversely, cells from animals injected with hybridomas expressing Delta, did not answer a single one of Der P1 antigens (Fig.4).

Example 7

Expressing Delta T-cells can block the response of normal T cells

Reactive to influenza a clone of T cells (HA1.7) people were transfusional mouse Delta using retroviral design to protein Delta expressively glue on the ability of these normal HA1.7 with peptide N-318 and antigen-presenting cells. HA1.7 in the number of 5105mixed with 1106irradiated DRB1*0101 mononuclear cells of peripheral blood (RVMS) + 1 μg NA-318 and cultivated at 37oC. After 6 hours was added 5% infoculture (a medium containing IL-2) to a total volume of 1 ml 24 hours after initiation of culture was added to Delta or control retrovirus, or have not added anything. 7 days after the beginning of cultivation, the cells were collected, washed, and transfetsirovannyh cells were irradiated. Transfetsirovannyh cells were mixed in the ratio 2:1 with untreated HA1.7 and were cultured for 2 days. Then mixed culture were harvested, washed and transferred to die in the number 2104viable cells per well together with:

a) 2,5104DRB1*0101 PBMCs (Wednesday)

b) 2,5104DRB1*0101 PBMCs + peptide (BP+AP-cells)

c) 5% infoculture (IL-2)

Cells were collected after 68 hours, last 8 hours, the cells were cultured in the presence of tritium-labeled thymidine.

The results are shown in figure 5.


The described culture as such or after transfection with control virus HA1.7; raw HA1.7 give a good response on protein + antigen-presenting cells. Incubation in the presence Delt However, these cells give the same good response to IL-2, as untreated or incubated with control virus HA1 cells.7, which shows that they are simply not capable of proliferation (Fig.5).

Example 8

The expression of Serrate antigen-presenting cells inhibits T-cell responses

Clone H1.7 was mixed with peptide N-318 (1.0 microgram/ml) in the presence of L-cells expressing HLA-DRB1*0101 (as antigen-presenting cells), using 2104cells of each type. L-cells were transfusional control (puro) or Serrate (Serrate L-cells)- a retrovirus expressing. The proliferative response was measured after 72 hours, last 8 hours cultivation was carried out in the presence of tritium-labeled thymidine. The results are given in figure 6 for crops HA1.7:

a) without additives

(b) HL-2

c) + peptide + DR1*0101-L-cells

d) + peptide + DR1*0101-L-cells, transfetsirovannyh control virus

e) + peptide + DR1*0101-L-cells, transfetsirovannyh virus Serrate

Results: H1.7-stimulated L-cells expressing Serrate, showed a weak response to the antigen compared with those stimulated control transfitsirovannykh L-cells (Fig.6).

Example 9

Expressing Serrate antigen-presenting cells induce reg is presenting cells) in the presence of 2% IL-2. L-cells were transfusional or control, or Serrate - expressing retro-virus. After 7 days of culturing cells HA1.7 was collected, washed and irradiated before mix with fresh HA1.7 (using 2104cells of each population). Then cells were cultured for another 2 days before stimulation with peptide (10 μg/ml) + normal antigen-presenting cells (DRB1*0101 PBMCs). The proliferative response was assessed after 72 hours after the start of cultivation, and 8 hours before the end of the cultivation was introduced 3H-thymidine. The results are shown in Fig. 7 for vegeculture cell H1.7:

a) without additives

b) + IL-2

c) + control virus-induced HA1.7, then the peptide + DRB1*1010 PBMC

d) + Serrate - virus-induced HA1.7, then the peptide + DRB1*0101 PBMC

Results: H1.7, induced Serrate-expressing L-cells (Serrate-induced HA1.7), has lost the ability of normal cells HA1.7 respond to normal antigenic stimulus (Fig.7). This indicates the ability of the cells became tolerant under the action Serrate, to overcome their tolerance, turning into a native population of cells (infectious/migrated tolerance).

Example 10

Regulation induced Delta-Express the ohms, transductional Delta or a control retrovirus. This hybridoma WS has specificity to protein 110-131 Der P1. At the same time, the animals received either Der P1 or ovalbumin, emulsified in complete Freund's adjuvant. After 7 days the cells of the lymph nodes were removed, and 4105cells were cultured in the presence of Der P1 or ovalbumin (i.e. used the same antigen, as they have already been immunized). The IL-2 production was measured 24 hours after the beginning of cultivation. Indexes stimulate the production of IL-2 are shown in figure 8A (responses in animals immunized with Der P1) and figure 8b (responses in animals immunized with OVA) in animals that were injected with hybridoma, transfetsirovannyh control (puro) or Delta (delta)-retroviruses.

Results: the response to Der P1 in animals, which were introduced hybridoma delta expressing US, was almost completely suppressed, whereas the response to ovalbumin was unaffected. This is shown in Fig.8A and 8b, where each point represents the production of IL-2 from each individual mouse to the antigen as the stimulation index (IP) control (no added antigen).

Example 11

T cells Express Delta during induction of tolerance

HA1.7 cultiv development in H1.7 tolerance. After 0, 30, 120, 240, or 360 minutes cells (1,5106) was collected in capsules and froze. RNA was obtained from cellular precipitation by homogenization in a solution of guanidine thio-cyanate and then by centrifugation in CsCl density. 1 μg RNA was converted into cDNA using oligoprimers dT. From the obtained cDNA 1/20 was subjected to PCR (40 cycles) using specific delta homolog of human primers. The PCR samples were analyzed by gel electrophoresis (figure 9), where: lane 1 - marker, lane 2 - t=0 min, lane 3 - t=30 min, lane 4 - t=120 min, lane 5 - t=240 min, track 6 - t=360 min, lane 7 is the negative control.


Resting HA1.7 is not expressed Delta-mRNA, but the transcripts appeared after 2 hours after the beginning of the development scheme for the initiation of tolerance.

Example 12

Analyses of the expression of Notch 1, Serrate 1 and Delta 1 during the induction of tolerance immunohistological method and in situ hybridization

Mice S BL/6J was administered intranasally or 100 µg 110-131 protein Der p1 or the control solution (saline phosphate buffer, PBS) for 3 days consecutively. It is shown that intranasal introduction of antigen in this way leads to the development of immunity to this antigenaemia tail. Superficial lymph nodes and spleen of animals from this time was allocated through different periods of time (d0 is the first day of the introduction of antigen or re-immunization) and used in immunohistological studies or for in situ hybridization. For immunohistological studies, tissue was frozen and did fine 3 μm sections that were fixed in ice-cold acetone and were labeled polyclonal antibody, specific for Notch 1 or Serrate 1. Bound antibodies were determined using horseradish peroxidase conjugated goat anti-rabbit antibodies obtained in the presence of diaminobenzidine as substrate. Antibodies specific to Delta 1, the research did not apply. For in situ hybridization did sections of frozen tissues and fixed them in 4% paraformaldehyde. The sections were subjected to hybridization with digoxigenin-related samples antisense RNA specific to Notch I, Serrate 1 and Delta 1, at 65oC. the Associated sample was determined using alkaline phosphatase conjugated goat antidigoxigenin antibodies obtained using NBT and BCIP as substrate. The data shown in tables 1 and 2 refer to immunohistological studies and hybridization only one peptide (primary PBS/p110-131) or intranasal and subcutaneous injection of antigen (PBS/p110-131 re).

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The initial levels of Notch, Delta and Serrate expressionlist the control mice received only PBS. Mice treated with PBS intranasally and antigen subcutaneously, showed a moderate increase in expression of all three molecules within 8 days after the second immunization. Animals receiving either one intranasal peptide or intranasal peptide followed by re-antigen, showed the same picture of increased expression of Notch, Delta and Serrate, which developed more quickly and to higher values than the control mice.

1. The use of Notch ligand in the manufacture of medicinal products for the treatment mediated T-cell disease or infection.

2. Application under item 1, in which mediated T-cell disease or infection is the result of one or more conditions selected from allergies, autoimmune diseases, transplant rejection induced by tumor violations in the system T-cells, and infectious diseases, such as caused by species of Plasmodium, microfilaria, worms, mycobacteria, HIV, cytomegalovirus, Pseudomonas, Toxoplasma, Agin the Notch ligand selected from Serrate, Delta or their fragments, derivatives or analogues.

4. The way of development of tolerance of T cells to the allergen or antigen, comprising the incubation/exposure of these T cells with antigen-presenting cells expressing or excessively expressing the Notch ligand in the presence of the specified allergen or antigen.

5. The method according to p. 4, where the (over) expression of the Notch ligand is a consequence of transfection of antigen-presenting cells by the virus, are able to Express the indicated ligand.

6. The method according to p. 4, where the (over) expression of the Notch ligand is a consequence of the stimulation of antigen-presenting cells with an agent that increases the expression of a gene expressing a Notch ligand.

7. The method according to p. 6, where the agent is selected from Noggin, Chordin, Follistatin, Xnr3, growth factors, fibroblast, or their derivatives, or fragments.

8. The method according to p. 4, 5, 6 or 7, in which the antigen presenting cells are selected from dendritic cells, L-cells, hybridomas, lymphomas, macrophages, b-cells or artificial antigen-presenting cells, such as lipid membranes.

9. The method according to any of paragraphs. 4-9, in which the Notch ligand selected from Serrate, Delta or their fragments, derivatives sludge is segment of immunoglobulin.

11. Protein under item 10, where the extracellular domain of Notch ligand obtained Notch, Delta or Serrate.

12. Protein under item 10 or 11, where Fwithsegment is IgGFwithor IgMFwith.


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