Mutein human il-4, a polynucleotide molecule, vector, line, sf9, transformed by the vector, the selection method muteena, the pharmaceutical composition for activating t-cells, a method of treating a patient suffering from a disease susceptible to treatment with il-4
The invention relates to biotechnology and can be used to obtain Malinov human IL-4 with an activating T-cell activity and reduced activates endothelial cell activity. Mutein IL-4, causing the proliferation of T-cells and reduced the secretion of IL-6 ACPWC compared with IL-4 wild type, obtained by mutation of surface residues of the D-helix of IL-4 persons, numbered in accordance with IL-4 wild type. Mutiny IL-4 contain single, double and triple mutations, represented by the symbols R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W; UA, Y124Q, Y124S, UT; Y124A/S125A, T13D/R121E and R121/122F/Y124Q, when numbering in accordance with IL-4 wild-type (His=1). Polynucleotide encoding mutiny according to the invention clone in the vector, and transform them cell owners with subsequent cultivation. Pharmaceutical compositions containing mutiny, used to treat patients suffering from a condition susceptible to treatment with IL-4. The invention allows to obtain a less toxic mutein IL-4, which makes possible a broader therapeutic applications of interleukin. 7 N. and 36 C.p. f-crystals, 23 ill., 1 PL.
The invention relates mainly to bertele activating T-cells and with reduced activating effect on endothelial cells or fibroblasts. New songs include variants of the family of cytokines, and in particular, human interleukin-4 (IL-4).
Interleukin-4 (IL-4) is a pleiotropic cytokine that activates immune cells, endothelial cells and fibroblastic type. Describes the effects detected after administration of IL-4 in vitro, include the proliferation of b-cells, switching of immunoglobulin classes in b cells. As for T cells, IL-4 stimulates proliferation of T cells after pre-activation with mitogens and exerts a downward regulating effect on the production of IFN-. In monocytes, IL-4 induces the expression of MHC molecules, class II, secretion lipopolysaccharideinduced tPA and the expression of CD23. In endothelial cells (EC) IL-4 induces the expression of VCAM-1 and secretion of IL-6 and reduces the expression of ICAM-1 (Maher, DW, et al., Human Interleukin-4: An Immunomodulator with Potential Therapeutic Applications, Progress in Growth Factor Research, 3:43-56 (1991)).
Because of its ability to stimulate proliferation of T cells activated by exposure to IL-2, was persecuted therapeutic application of IL-4. For example, IL-4 showed antitumor activity in animal models with malignant tumors of the kidneys and caused regression of tumors in mice (Bosco, M., et al., (1990)). However, its toxicity limits the size of dosage in humans (Margolin, K., et al., Phase II Studies of Recombinant Human Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J. Immunotherapy, 15:147-153(1994)).
Due to the immunoregulatory activity of IL-4 for it proposed a number of clinical applications. Among these clinical applications are diseases caused by an imbalance in the immune system, particularly diseases caused by imbalance in the reactions of T-helper (Th) cells to an antigen. These diseases include certain autoimmune diseases, rheumatic diseases, skin diseases and infectious diseases. A large amount of experimental work has allowed to establish that Th cells are divided into two broad classes, referred to as Th1 and Th2 (Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L., Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins, J. Immunol, 136:2348-2357 (1986); Mosmann, T. R., Cytokines, differentiation and functions of subsets of CD4 and CD8 T cells, Behring Inst. Mitt., 1-6 (1995)). These classes of T cells are defined by the cytokines they Express: Th1 cells produce IL-2, IFN-and TNF-whereas Th2 dysbalance-cells produce IL-4 and IL-5. Th1-and Th2 dysbalance cells are formed from not subjected to CD4+ T cells. �="https://img.russianpatents.com/chr/947.gif">and IL-12 directs the process of differentiation not exposed to the cells towards the Th1 phenotype, whereas IL-4 directs the process of differentiation towards the Th2 phenotype. Despite the fact that Th1-and Th2 dysbalance-subgroups among the many phenotypes of Th cells can be represented extreme cases (for example, have been described Th0 cells, which Express low levels of expression of both IFN-and IL-4), however, this classification is dominant in the field of immunology to describe the nature of the immune response.
Observations were made that certain organspecific autoimmune diseases associated with T-cell response to autoantigen carried out predominantly Th1 (Liblau RS; Singer SM; McDevitt HO, Th1 and Th2 CD4+T cells in the pathogenesis of organ-specific autoimmune diseases, Immunol. Today, 16:34-38 (1995)). One such autoimmune disease is insulin-dependent diabetes mellitus (IDDM), a disease characterized by mediated T-cell destruction-cells of the pancreas. Some data suggest that for destruction-cells of the pancreas mainly responsible cells Th1-type (overview Tisch, R. et al., Review: Insulin-dependent Diabet the respective impact on the population of Th1 and significantly slows down the development of diabetes (Rapoport, et al., IL-4 Reverses T cell Proliferation Unresponsiveness and Prevents the Onset of Diabetes in NOD Mice, J. Exp.Med., 178:87-99 (1993)). Other autoimmune disease is multiple sclerosis (PC), a disease characterized by an autoimmune attack on the myelin sheath surrounding nerve cells. Studies conducted on people with PC, demonstrated that the aggravation of the PC associated with the presence autoantigenic Th1 and Th0 cells, and that remission is associated with the presence autoantigenic Th2 and Th0 cells (Correale, J. et al., Patterns of cytokine secretion by autoreactive proteolipid protein-specific T cell clones during the course of multiple sclerosis, J. Immunol., 154:2959-2968 (1995)). In mice with experimental autoimmune encephalomyelitis (EAE), an animal model of PC is also evident polarization of Th1 cells (Cua DJ, Hinton, DR, and Stohlman, S.A., J. Immunol., 155:4052-4059 (1995)). Indirect data obtained in the study of models of EAE, lead to the assumption that IL-4 plays a key role in reducing the severity of the disease resulting from the treatment tolerogenic peptide (Brocke, S. et al., Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein, Nature, 379:343-346 (1996)).
Other autoimmune diseases such as rheumatoid arthritis (RA), are also targets for therapy on the basis of IL-4. In animal models of RA was shown imbalance show the level of expression of TNF-anti-TNF-antibodies reduced the severity of disease, suggesting that treatments based on IL-4, resulting in decreasing the regulation of populations of Th1-cells, may also have anti-TNF-action (see Feldmann, M., et al., Review: Rheumatoid Arthritis, Cell, 85:307-310 (1996)).
Psoriasis is a chronic skin disease characterized by infiltration of the affected skin monocytes and T-cells. Several reports indicate that T cells and forestry of the psoriatic skin lesion have a predominantly Th1 phenotype (Uyemura K, Yamamura M; Fivenson DF, Modlin RL; NickolofT BJ, The cytokine network in lesional and lesion-free psoriatic skin in characterized by a T-helper type 1 cell-mediated response, J. Invest. Dermatol, 101:701-705 (1993); Schlaak JF; Buslau M; Jochum W; Hermann E; Girndt M; Gallati H, Meyer zum Buschenfelde KH; Fleischer b, T cells involved in psoriasis vulgaris belong to the Th1 subset, J. Invest. Dermatol, 102:145-149 (1994)). Moreover, it was shown that monomethylfumarate, drug, reported that it brings to the clinical improvement of patients suffering from psoriasis, selectively stimulates the secretion of cytokines MCPC Th2 (de Jong R; Bezemer AC; Zomerdijk TP; van de Pouw-Kraan T; Ottenhoff TH; Nibbering PH, Selective stimulation of T helper 2 cytokine responces by anti-psoriasis agent monomethylfumarate, Eur. J. Immunol., 26:2067-2074 (1996)). Thus, expect the s infectious diseases associated with polarized Th cell responses to infectious agent. Th2 dysbalance-mediated reactions in some cases have been associated with immunity to infectious agent. An example is Borrelia burgdorferi, the causative agent of Lyme disease. People infected with C. burgdorferi, is observed predominantly Th1-like profile of cytokines (Oksi J; Savolainen J; Turnips J; Bousquet J; Laipalla P; Viljanen MK, Decreased interleukin-4 and increased gamma interferon production by peripheral blood mononuclear cells of patients with Lyme borreliosis, Infect. Immun., 64:3620-3623 (1996)). In the model Century burgdorferi-induced arthritis in mice resistance to disease associated with the production of IL-4, whereas susceptibility is associated with the production of IFN-(Matyniak JE; Reiner SL, T helper phenotype and genetic susceptibility in experimental Lyme disease, J. Exp.Med., 181(3):1251-1254 (1995); Keane-Myers A; Nickell SP, Role of IL-4 and IFN-gamma in modulation of immunity Borrelia burgdorferi in mice, J. Immunol., 155:2020-2028 (1995)). The treatment of infected Century burgdorferi mice IL-4 increases resistance to infection (Keane-Myers A; Maliszewski CR; Finkelman FD; Nickell SP, Recombinant IL-4 treatment augments resistance to Borrelia burgdorferi infections in both normal susceptible and antibody-deficient susceptible mice, J. Immunol., 156:2488-2494 (1996)).
It was reported that IL-4 has a direct inhibitory effect on the development of lymphomas and leukemias (Akashi, K., The role of interleukin-4 in the negative regulation of leukemia cell growth, Leuk. Lymphoma, 9:205-9 (1993)). For example, it was reported that IL-4 induces apoptosis in cells of patients suffering from acute lymphoblastic leukemia is to patients, suffering from non-Hodgkin b-cell lymphoma (Defrance, T., et al., Antiproliferative effects of interleukin-4 on freshly isolated non-Hodgkin malignant B-lymphoma cells, Blood, 79:990-6 (1992)).
It was also reported that IL-4 is active, the presence of which suggests that he will be able to cause clinical improvement in osteoarthritis. Osteoarthritis is a disease in which the main pathological process is the destruction of the cartilage (Sack, KE, Osteoarthritis, A continuing challenge, West J. Med., 163:579-86 (1995); Oddis CV, New perspectives on osteoarthritis, Am. J. Med., 100:10S-15S (1996)). IL-4 inhibits the production of TNF-and IL-1 by monocytes and synoviocytes patients suffering from osteoarthritis (Bendrups, A, Hilton, A, Meager, A and Hamilton, JA, Reduction of tumor necrosis factor alpha and interleukin-1 beta levels in human synovial tissue by interleukin-4 and glucocorticoid, Rheumatol. Int., 12:217-20 (1993); Seitz, M., et al., Production of interleukin-1 receptor antagonist, inflammatory chemotactic proteins, and prostaglandin E by rheumatoid and osteoarthritic synoviocytes-regulation by IFN-gamma and IL-4, J. Immunol., 152:2060-5 (1994)). It was also reported that IL-4 directly inhibits the breakdown of cartilage in cartilage explants ex vivo (Yeh, LA, Augustine, AJ, Lee, P, Riviere, LR and Sheldon, A, Interleukin-4, an inhibitor of cartilage breakdown in bovine articular cartilage explants, J. Rheumatol., 22:1740-6 (1995)). Activity data suggest that IL-4 to induce clinical improvement in osteoarthritis.
However, the clinical use of IL-4 was limited p., et al., Phase II Studies of Recombinant Human Interleukin-4 in Advanced Renal Cancer and Malignant Melanoma, J. Immunotherapy, 15, 147-153 (1994)). In the literature there are no data that describe the mechanism of acute toxic effect of IL-4, any data that can be described analogs or mutants of IL-4, which saved immunoregulatory activity, but which have low acute toxicity.
Known mutant proteins ("mutiny") IL-4. Mutein IL-4 IL-4/Y124D is a T-cell antagonist (Kruse N, Tony HP, Sebald W, Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement, Embo J., 11:3237-44 (1992)).
Therapeutic use of IL-4, described in patents or patent applications include the following: the use of IL-4 for potentiating antitumor effects of chemotherapeutic agents particularly Hodgkin's disease and non-Hodgkin's lymphoma (see international patent application WO 9607422); the use of antigenic fragments of IL-4 for producing antibodies for the treatment of diseases associated with IL-4, by suppressing or mimic the binding activity of IL-4 (see international patent application WO 9524481) and to determine measurement and immunological purification of IL-4 (see international patent application WO 9317106); for induction of differentiation of precursor b cells veterans with impaired functioning of the immune system (see international patent application WO 9404658); when applied in combination with IL-10 as a treatment for leukemia, lymphoma, inflammatory bowel and delayed-type hypersensitivity (e.g., ulcerative colitis and Crohn's disease) (see international patent application WO 9404180); treatment of HIV infection through injection of IL-4 to inhibit virus replication in monocytes and macrophages and to enhance their cytotoxicity against some tumor cells (see WO 9404179); to stimulate the proliferation of skin fibroblasts for the treatment of wounds in patients diabetes and immune disorders (see international patent application WO 9211861); to strengthen the primary immune response when introduced bacterial, toxoids and viral vaccines, especially vaccines tetanus toxoid (see international patent application WO 9211030); to suppress IL-2-induced proliferation in b-cell malignancies especially in chronic lymphocytic leukemia, non-Hodgkin malignant lymphoma (see international patent application WO 9210201); the use of IL-4 for the treatment of malignant melanoma, malignant renal tumors and basal cell carcinoma (see international patent application WO 9204044).
In the patent literature describes proteins and some Mut is t "691"), issued to Lee et al., directed by produced by mammalian proteins and mutiny human IL-4 that detect activity of growth factors In cells and the activity of growth factors T cells. It describes nucleic acids encoding polypeptides exhibiting activity of IL-4, are polypeptides and methods for their preparation. Described mutiny IL-4 wild-type amino acid positions that retain their in vitro ability to stimulate the proliferation of both B-and T-cells. However, in U.S. patent 5017691 offered nothing about any Malinov IL-4 selectively acting on T cells, the expected activation of the ER or increase the permeability of endothelial cells, which is accompanied by the introduction of IL-4. Thus, it is IL-4 cannot be used as therapeutic effects due to the limiting dose toxicity.
In U.S. patent No. 5013824 described peptide derived hIL-4, containing from 6 to 40 amino acids of native hIL-4. Also described immunogen containing conjugates of peptides and media. Carriers include red blood cells, bacteriophages, proteins, synthetic particles, or any substance capable of causing the production of antibodies against the conjugated peptide. Malinov IL-4 is not described by the quadratic agonists hIL-2 and hIL-13. Data regarding IL-4, not shown. In the international patent application WO 95/27052 described splicing mutants of IL-2 and 1L-4, containing exons 1, 2 and 4.
There is a need for an improved molecule IL-4, which has reduced toxicity and generally more tolerant.
The invention is directed to mutiny human IL-4, numbered in accordance with IL-4 wild type, with an activating T-cells activity, but with lower activity of activated endothelial cells. In particular, mutiny human IL-4, where the exposed surface on the remains of the D-helix of IL-4 wild-type motivovany, resulting in mutein causes the proliferation of T-cells and causes reduced secretion of IL-6 ACPWC compared with IL-4 wild type. This invention is less toxic mutein IL-4, which makes possible a wider therapeutic application of this interleukin.
In addition, the invention is directed to mutiny IL-4 containing single, double and triple mutations, represented by the symbols R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W; Y124A, Y124Q, Y124S, Y124T, Y124A/S125A, T13D/R121E and R121T/E122F/Y124Q, when numbering in accordance with IL-4 wild-type (His=1). The invention also includes the new cells-owners, pharmaceutical compositions containing mutiny, and therapeutic methods of treatment.
The invention is also directed to a vector containing polynucleotide encoding mutein of this invention, a vector that directs the expression of mutein human IL-4, possessing activity of activated T-cells, but with low activity of activated endothelial cells, and the vector enables transfection of the body-target and subsequent expression in vivo specified mutein human IL-4, encoded by the specified polynucleotide. The invention is also directed to a method of selecting mutein human IL-4, numbered in accordance with IL-4 wild type, with the activity of activated T-cells, but with low activity of activated endothelial cells, including mutation of surface residues of the D-helix of IL-4 wild type, whereby the received mutein causes the proliferation of T-cells and causes reduced secretion of IL-6 ACPWC compared to wild type.
The invention is also directed to a method of treating a patient suffering from a condition susceptible to treatment with IL-4, by introducing a therapeutically effective amount mutein human IL-4, about the low activity of activated endothelial cells. This method is applicable when the condition susceptible to treatment with IL-4, is an autoimmune disease, in particular multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes mellitus, systemic lupus erythematosus, infectious disease, particularly Lyme disease, Th1-polarized disease, in particular psoriasis, cancer, particularly acute lymphoblastic leukemia and nahodkinskuju lymphoma, a disease of the cartilage, in particular osteoarthritis.
A brief description of the drawings. In Fig.1 shows the amino acid sequence (TH. ID. no:1) Mature human IL-4 wild type, used in this study. Spirals are underlined and consistently marked a, b, C and D. the Provisions that are being motivovany allowed to get agonists of IL-4, selectively acting on the cells, indicated irnm font.
Fig.2 is a graphical representation of the concept of agonist, selectively acting on T cells.
In Fig.3 presents cumulative curve "dose-response" Malinov-selective agonists in the study of the secretion of IL-6 ACPWC. Panel A: Oh, IL-4 wild type;, R121E;, R121P;, R121T/E122F/Y124Q. Panel V: ILp>In Fig.4 presents the individual curves "dose-response" Malinov-selective agonists in the study of the secretion of IL-6 ACPWC. Panel A: Oh, IL-4 wild type;, R121E. Panel V: IL-4 wild type;, R121P. Panel: IL-4 wild type;Y124Q. Panel D: Oh, IL-4 wild type;, Y124R. Panel E: Oh, IL-4 wild type;, Y124A/S125A. Panel F: IL-4 wild type;, R121T/E122F/Y124Q.
In Fig.5 presents the total curve "dose-response" Malinov-selective agonists for the biological response of Malinov IL-4 in the analysis of proliferation 1T-cells. Panel A: O IL-4 wild type;, R121E;, R121P;, R121T/E122F/Y124Q. Panel: O IL-4 wild type;, Y124Q;, Y124R;, R124A/S125A.
In Fig.6 presents individual curves dose-response studies of proliferation 1T-cells. Panel A: Oh, IL-4 wild type;, R121E. Panel V: IL-4 wild type;, R121P. Panel: O IL-4 wild type;, Y124Q. Panel D: IL-4 is IL-4 wild type;, R121T/E122F/Y124Q.
In Fig.7 presents the individual curves dose-response, showing the antagonism of IL-4-induced secretion of IL-6 ACPWC and Malinov-agonists IL-4 selectively acting on T cells, R121E (and Y124Q (2). Curve dose-response of antagonist IL-4 R121D/Y124D (O) is included as a control.
In Fig.8A, 8B presents the individual curves dose-response: the Panel In the biological response to mutein R121D in the analysis of proliferation 1T cells (O=IL-4,=R121D); Panel a shows the inability R121D to induce the secretion of IL-6 ACPWC (O=IL-4,=R121D).
In Fig.9A presents the individual curves dose-response for IL-4 (O) and for Malinov agonist, selectively acting on T cells, R121E (A) and T13D/R121Ein the analysis of proliferation 1T-cells.
In Fig.9B presents the individual curves dose-response, showing the antagonism of IL-4-induced secretion of IL-6 ACPWC and Malinov-agonists IL-4 selectively acting on T cells, R121E (a) and T13D/R121E.
Description of the preferred embodiments of the invention
It was shown that IL-4 uporabe on endothelial cells (Maher DW, Davis I, Boyd AW, Morstyn G: Human interleukin-4: an immunomodulator with potential therapeutic applications. Prog. Growth Factor Res. 3:43-56, 1991; Powrie F, Coffman RL: Cutokine regulation of T cell function: potential for therapeutic intervention. Immunol. To day 14:270-4, 1993). In particular, the increasing regulation of the adhesion molecule vascular cell-1 (VCAM-1; (Swerlick RA, Lee KH, Li LJ, Sepp NT, Caughman SW, Lawley TJ: Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells. J. Immunol. 149:698-705, 1992)) and the induction of IL-6 (Colotta F, Sironi M, Borre A, Luini W, Maddalena F, Mantovani A: Interleukin 4 amplifies monocyte chemotactic protein and interleukin 6 production by endothelial cells. Cytokine 4:24-8, 1992) and macrophage chemoattractant protein-1 (MCP-1; Colotta F, Sironi M, Borre A, Luini W, Maddalena F, Mantovani A: Interleukin 4 amplifies monocyte chemotactic protein and interleukin 6 production by endothelial cells. Cytokine 4:24-8, 1992; Rollins BJ, Pober JS: Interleukin-4 dosage the synthesis and secretion of MCP-1/JE by human endothelial cells. Am.J.Pathol. 138:1315-9, 1991)) are direct effects of IL-4 in cultured endothelial cells; increasing regulation of VCAM-1 correlated with enhanced adhesion of lymphocytes both in vitro (Carlos TM, Schwartz BR, Kovach NL, Yee E, Rosa M, Osborn L, Chi-Rosso G, Newman B, Lobb R, Rosso M, et al.: Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine - activated cultured human endothelial cells. Blood 76:965-70, 1990; Thornhill MH, Wellicome SM, Mahiouz DL, Lanchbury JS, Kyan-Aung U, Haskard DO: Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-1-dependent and-independent binding mechanisms. J. Immunol. 146:592-8, 1991) and in vivo (Briscoe DM, Cotran RS, Pober JS: Effects of t IL-4 IL-4/Y124D (substitution of tyrosine to aspartic acid at position 124) is a T-cell antagonist (Kruse N, Tony HP, Sebald W: Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement. Embo J. 11:3237-44, 1992). The in vivo experiments conducted by the authors of the present invention have demonstrated that IL-4/Y124D shows acute toxicity, similar to that of IL-4 wild type in monkeys that were not previously described observation. The cellular processes associated with toxicity of IL-4 wild-type and IL-4/Y124D-mediated toxicity include increasing regulation of VCAM-1, increasing regulation of MCP-1 in serum, increased the content of circulating monocytes together with a concomitant decrease in the content of circulating lymphocytes and increased hematocrit. Similar cellular processes observed in clinical trials using IL-4 in humans (Wong HL, Lotze MT, Wahl LM, Wahl SM: Administration of recombinant IL-4 to humans regulates gene expression, phenotype, and function in circulating monocytes. J. Immunol. 148:2118-25, 1992). Because it is an antagonist of T-cells, these results suggest that the toxic effects exhibited by IL-4/Y124D, due to agonistic activity and through mediated by cells other than T cells. The observed in vivo toxic effects of using IL-4/Y124D and known effects of IL-4 on endothelial cells are consistent with a mechanism, consisting in the fact that oxychlordane the authors of the present invention installed, what on endothelial cells (EC) can be a new IL-4 receptor. This possibility has led to the attempts of synthesis of Malinov IL-4, which are selectively activated would T cells, but not EC. Whereas T cells Express the IL-4 receptor, consisting of subunits of IL-4Rand IL-2Rthe authors of the present invention found that the endothelial cells of the umbilical vein of a person (ACPWC) Express IL-4Rbut do not Express IL-2R. Studies using cross-linking showed that the cell surface ACPWC expressed two receptor chains: the molecular weight of one coincides with the IL-4Rand the second circuit with lower molecular weight. These results suggest that ACPWC is expressed component of the new IL-4 receptor, similar in function to IL-2Rbut differing from it in sequence. Differences in specific molecular structures between these two receptors were used to create variants of IL-4, which is selective with respect to one receptor over another (for example, selectively acting on T cells agonist the L-4, containing subunit of IL-4R/IL-2Rand the IL-4 receptor of endothelial cells containing IL-4R/-like receptor subunit. Despite the fact that two of the IL-4 receptor pictured here together for the sole purpose of illustration, they are expressed on different cell types. T-cell receptor consists of IL-4Rand IL-2R; the binding of IL-4 induces the formation of a heterodimeric receptor, which leads to the transmission of cellular signals. Induced IL-4 education heterodimeric receptor similarly happens on the screen except that the IL-4 receptor consists of IL-4Rand-like receptor component,-like receptor component is distinct from IL-2R. Selective effect on T-cell agonists of IL-4 are variants of IL-4, which retain their ability to interact with T-cell receptor IL-4R/IL-2Rbut not able to induce heterodimerization and, thus, signaling IL-4R; their ability to distinguish between IL-2Rand-like subunit provides their properties to selectively activate the cells.
Two components of T-cell receptor, IL-4Rand IL-2Rinteract with different parts of the molecule IL-4 and, thus, the authors of the present invention have focused on the modification of a small plot of IL-4. Assuming that the new receptor subunit to interact with the same site of IL-4 and IL-2Rthe authors of the present invention has made a number of substitutions in the D-helix, in particular residues 121, 124 and 125.
D-helix is involved in the interaction with IL-2Rand with the proposed new receptor on ACPWC (characteristically, mutein IL-4 R121D/Y124D is an antagonist ACPWC). Mutiny containing modifications of the D-helix of IL-4 (residues from 110 to 126; His=1) were subjected to screening for their ability to either stimulate the proliferation of T-cells or secretion of IL-6 by endothelial cells of the umbilical vein of a person (ACPWC). Mutiny that induced the response of T-cells, non-response ACPWC, further characterized further using mutagenesis.
The source is s to this, were held scanner replacement of alanine in the AB-loop, since it is assumed that this fragment involved in the interaction of the cytokine ligand and receptor subunits that interact with the D-helix. In particular, the exposed surface residues Glu-110, Asn-111, Glu-114, Arg-115, Lys-117, Thr-118, Arg-121, Glu-122, Tight-124, Ser-125 and Lys-126 were selected by the research purpose and are the preferred targets for mutational analysis. More preferred are the sites 118-126, and sites 121-125 are the most preferred. Comparison between IL-2, IL-4, IL-7 and IL-15 in this fragment also reveal differences between IL-4 and IL-2, IL-7 and IL-15, which might imply the existence of specific residues responsible for the interaction with the receptor ACPWC. Specific replacement, taken from alignment performed between IL-2 and IL-4 was made in IL-4. They included: Arg 115 on Phe; Lys-117 for Asn; Glu-122 on Phe; Lys-126 on Il; and three simultaneous replacement of Arg-121 on Thr, Glu-122 on Phe and Tight-124 on Gln.
Mutations were made using siteprovides mutagenesis on cDNA of human IL-4 wild type. Suitable clones were subcloned into expressing a vector suitable for expression in a heterologous system (for example, E. Li, baculovirus or cells SNO). Purified proteins of testireba what toynami, obtained in these analyses, either in EC50or maximum response (plateau), point mutations that affect the activity data. Specifically, mutiny that stimulate a relatively stronger response in T-cell analysis (anti-IL-4 wild-type) compared to the response ACPWC (anti-IL-4 wild-type) may suggest the presence of provisions which are more important for the interaction of IL-4 with IL-2Rthan for the interaction of IL-4 with the new IL-4 receptor ACPWC. Further analysis and mutagenesis (for example, combinatorial replacement, replacement of all amino acids) certain provisions will give the opportunity to get mutein IL-4, having the properties of selective agonist with respect to T-cell receptor IL-4. This protein will also be a selective antagonist of IL-4-induced reactions ACPWC.
Here we describe new mutiny and the mechanism for obtaining new Malinov IL-4, having the properties of selectively acting on T cells agonists and reduced toxicity. A similar strategy can be applied to identify selectively acting on T cells antagonists.
The term "IL-4 wild-type" means here IL-4, native or recombinant containing boneprone, in Fig.1.
The term "mutein IL-4" here means a polypeptide resulting from the fact that in human Mature protein of the Il-4 produced a special replacement.
In particular, as described here, the residue is arginine (R) at position 121 ("Arg-121"), numbered in accordance with IL-4 wild type, replaced by alanine (A), aspartate (D), glutamate (E), phenylalanine (F), histidine (H), isoleucine (I), lysine (K), asparagine (N), Proline (P), threonine (T) or tryptophan (W); or a glutamate residue (E) in position 122 replaced by phenylalanine (F); or the tyrosine residue at position 124 is substituted by an alanine (A), glutamine (Q), arginine (R), serine (S) or threonine (T) or serine residue (S) at position 125 is substituted with alanine (A). The most preferred by the authors of the present invention mutiny IL-4 have an amino acid sequence identical to IL-4 wild-type on the other, unsubstituted residues. However mutiny IL-4 of this invention can also be characterized by the inserts, divisions, substitutions and modifications of amino acids at one or more sites in other residues of polypeptide chain of the native IL-4. In accordance with this invention any such insertions, deletions, substitutions and modifications lead to the formation of mutein IL-4, bone to activate endothelial cells.
The authors of the present invention prefer conservative modifications and substitutions in other positions of IL-4 (i.e., those that have minimal impact on the secondary and tertiary structure mutein). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative substitutions:
- ala, pro, gly, gln, asn, ser, thr;
is cys, ser, tyr, thr;
- val, ile, leu, met, ala, phe;
is lys, arg, his;
is phe, tyr, trp, his, and
- asp, glu.
The authors present invention also prefer modifications or substitutions that do not contribute additional sites of intermolecular crosslinking or incorrect formation of disulfide bonds. For example, it is known that IL-4 contains six cys residues in positions 3, 24, 46, 65, 99 and 127 protein wild-type.
The term "numbered in accordance with IL-4 wild type," the authors of the present invention involve the identification of the selected amino acids in relation to the position in which this amino acid is usually in IL-4 wild type. If mateine IL-4 produced insertions or deletions, for a specialist in this area will be valuable to the fact that ser (S), normally located in isace shifted ser (S) can be easily determined by inspection and correlation of flanking amino acids such flanking ser IL-4 wild-type.
Mutiny IL-4 of the present invention can be obtained using any suitable method known in this field. Such methods include constructing a DNA sequence that encodes mutiny IL-4 of the present invention, and expressiona data sequences in a suitable transformed host. This method will allow you to obtain recombinant mutiny according to this invention. However mutiny according to this invention can also be obtained, though less preferably, by chemical synthesis or recombinant DNA technology.
In one embodiment, the implementation of the recombinant method of obtaining mutein of the present invention the DNA sequence design by selection or synthesis of DNA sequences encoding IL-4 wild type, followed by the replacement of the codon encoding arg121, the codon encoding alanine (A), aspartate (D), glutamate (E), phenylalanine (F), histidine (H), isoleucine (I), lysine (K), asparagine (N), Proline (P), threonine (T) or tryptophan (W) using sitespecific mutagenesis. This technique is well known. See, e.g., Mark et al., "Site-specific Mutagenesis of the Human Fibroblast Interferon Gene", Proc. Natl. Acad. Sci. USA 81, pp.5662-66 (1984); patent Saadiq, coding mutiny IL-4 according to this invention, is a chemical synthesis. For example, the gene that encodes the desired mutein IL-4, can be synthesized chemically synthesized oligonucleotides. Such oligonucleotides are indicated on the basis of the amino acid sequence of the desired mutein IL-4 and preferably selecting those codons that are preferred in the cell host, which will be produced recombinant mutein. It is widely known that the genetic code is degenerate - that the amino acid may be encoded by multiple codons. For example, phe (F) is encoded by two codons, TTC or TTT, tyr (Y) is encoded by TAC or TAT, his a (N) is encoded by CAC or CAT. Trp (W) is encoded by a single codon TGG. Consequently valuable is that for a given sequence of DNA that encodes a specific mutein IL-4, will be many degenerate DNA sequences that will encode this mutein IL-4. For example, valuable is that in addition to the preferred DNA sequence that encodes mutein R121E shown in PEFC, ID. no:3, there will be plenty of degenerate DNA sequences that encode shown mutein IL-4. D. the "its degenerate variants" in the context of this invention means all DNA sequences which encode specific mutein.
The DNA sequence encoding mutein IL-4 according to this invention, obtained using sitespecific mutagenesis, synthesis or other methods may also include or not to include DNA sequences that encode a signal sequence. This signal sequence if there should be a sequence recognized by the cell selected for expression mutein IL-4. It may be prokaryotic, eukaryotic, or a combination of them. It can also be a signal sequence of the native IL-4. The inclusion of a signal sequence depends on whether the desired secretion mutein IL-4 recombinant cells in which it is produced. If the selected cells are prokaryotic, as a rule, it is preferable that the DNA sequence does not encode a signal sequence. If the selected cells are eukaryotic, as a rule, it is preferable that the signal sequence is encoded, and it is most preferable to use a signal sequence of IL-4 wild-type.
For measures to construct back-translated gene may be applied full amino acid sequence. Can be synthesized DNA oligomer containing a nucleotide sequence encoding mutein IL-4. For example, several small oligonucleotides coding for part of the desired polypeptide may be synthesized and then legirovanyh. Individual oligonucleotides typically contain 5’- or 3’-end redundancy for complementary Assembly.
Collected (using synthesis, sitespecific mutagenesis or another method) the DNA sequence encoding mutein IL-4 according to this invention, will be inserted in expressing vector and operatively sewn to controlling the expression of a sequence suitable for expression mutein IL-4 in the desired transformed host. The correctness of the Assembly can be confirmed using sequencing of nucleic acids, restriction mapping and expression of biologically active polypeptide in a suitable host. As is well known in this field, in order to achieve high levels of expression made by the method of gene transfection into the host gene must be efficiently cross-linked to transcriptional and translational what that is,
The choice of controlling the expression of the sequence and expressing the vector will depend on the choice of the owner. Can be applied to a wide range of combinations of host/vector. Applicable expressing vectors for eukaryotic hosts include, for example, vectors containing regulatory expression sequence from SV40, human papilloma virus of cattle, adenovirus and cytomegalovirus. Applicable expressing vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli including col El, pCRl, pER32z, pMB9 and their derivatives, plasmids from a wider range of hosts, such as RP4, phage DNA, such as the numerous derivatives of phage lambda, e.g NM989, and other DNA-containing phages, such as M13 and filamentous phages containing single-stranded DNA. Applicable expressing vectors for yeast cells include plasmid 2. and its derivatives. Applicable vectors for insect cells include pVL941. The authors of the present invention prefer pFastBacTM1 (GibcoBRL, Gaithersburg, MD). Cate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance and Expression of the Human Gene in Animal Cells", Cell, 45, pp.685-98 (1986).
In addition, in D. the values. Such applicable regulatory expression sequence include controlling the expression of the sequence associated with the structural genes of the above expressing vectors. Examples of applicable controls the expression sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, major operator and promoter sites of phage lambda, e.g. PL, regular parts of the protein shell fd, the promoter for 3-phosphoglycerate or other glycolytic enzymes, the promoters of acid phosphatase, such as PhoA, the promoters of the system-mating of yeast, poliedro the promoter of the baculovirus and the other sequences controlling the expression of genes of prokaryotic and eukaryotic cells or their viruses, and various combinations thereof.
For the production of Malinov IL-4 according to this invention can be applied to any suitable host, including bacteria, fungi (including yeast), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. More specifically, these hosts may include well from the, is rozga, insects such as Spodoptera frugiperda (Sf9), animal cells such as oocytes Chinese hamster (Cho and mouse cells such as NS/O cells of the African green monkey, such as COS 1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells, as well as plant cells in culture cell tissue. For expression in animal cells, the authors of the present invention prefer to use cells Cho cells and COS 7 cultures and in particular the SNO cell line Cho (D HFR-).
Of course, it should be understood that not all vectors and controlling the expression of sequences will function equally effectively in expression of the DNA sequences described herein. Also, not all hosts will function equally well with the same expressing system. However, the person skilled in the art is able to make a choice between these vectors, which controls the expression of sequences and hosts without redundant experimental work. For example, the choice of the vector must be taken into account factor host, because the vector will be replicated. You should also take into account the factors of the number of copies of the vector, the ability to control a given number of preferred vectors for use in this invention include such which allow DNA coding mutiny IL-4, to be amplified in the number of copies. Such mnogoopytnyi vectors are well known in this field. They include, for example, vectors capable of amplificates by DHFR amplification (see, for example, Kaufman, patent of the United States 4470461, Kaufman and Sharp, "Construction of a Modular Dihydrofolate Reductase cDNA Gene: Analysis of Signals Utilized for Efficient Expression", Mol. Cell. Biol., 2, pp.1304-19 (1982)) or glutamylcysteine ("GS") amplification (see, for example, a patent United States 5122464 and European published application 338841).
When choosing which controls the expression of the sequence should also take into account many factors. They include, for example, the relative length of the sequence, its controllability, and its compatibility with the authentic DNA sequence coding mutiny IL-4 according to this invention, particularly as regards potential secondary structures. Hosts should be selected taking into account their compatibility with the chosen vector, the toxicity of the product encoded by the DNA sequences of this invention, their characteristics secretion, their ability to collapse the polypeptides, the requirements for their fermentation or cultivation and ease of cleaning product is combinatii vector/controlling the expression of the sequence/owner, which will Express the desired DNA sequence in the fermentation process or during large-scale cultivation of animal cells, for example, using cells Cho or COS cells 7.
Mutiny IL-4, obtained in accordance with the present invention may be glycosylated or deglycosylation depending on the host body, used for the production of mutein. If the owner selected bacteria produced mutein IL-4 is deglycosylated. On the other hand, eukaryotic cells will glycosylate mutiny IL-4, although perhaps not in the same way as glycosylases native IL-4.
Mutein IL-4 produced by the transformed host may be purified according to any suitable technique. For the purification of IL-4 are known various methods. See, for example, the patents of the United States 5013824; 5017691; and international patent application WO 9604306-A2. The authors of the present invention prefer to apply immunoaffinity cleaning. See, for example, Okamura et al., "Human Fibroblastoid Interferon: Immunosorbent Column Chromatography and N-Terminal Amino Acid Sequence", Biochem., 19, pp.3831-35 (1980).
The biological activity of Malinov IL-4 according to this invention can be analyzed using any suitable methods is Vesti, induction proteinkinases, oligoadenylate-2,5-And-synthetases or phosphodiesterase activities, as described in European patent application EP-B1-41313. Such analyses also include immune-based assays (see, for example, a patent United States 4753795), analyses of inhibiting the growth, proliferation of T cells, induction of IL-6 (MCP-1 or VCAM-1), EK and determination of binding to cells that Express the receptor of interleukin-4. See also Spits N., Yssel H., Takebe Y., et al., Recombinant Interleukin-4 Promotes the Growth of Human T Cells, J. Immunol. 139:1142-47 (1987).
Mutein IL-4 of the present invention will be administered in dosages corresponding to or exceeding those used in the treatment of native wild-type IL-4 or recombinant IL-4. Preferably administered an effective amount mutein IL-4. The term "effective amount" means the amount that can prevent or reduce the severity or development of the condition or symptom being treated. For the person skilled in the art it will be obvious that the effective number mutein IL-4 will depend on, among other things, from disease, dosing schemes mutein IL-4, enter whether mutein IL-4 alone or in combination with other drugs, from time-life to lucaus in themselves pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means a carrier that does not cause any adverse effects in patients, which it is introduced. Such pharmaceutically acceptable carriers well known in this field. The authors present invention I prefer to use 2% HSA/FBI at pH 7.0.
Mutiny IL-4 according to the present invention can be incorporated into pharmaceutical compositions in accordance with well known techniques. See, for example, suitable compositions are described in Remington''s Pharmaceutical Science by E. W. Martin, included here as a reference. The pharmaceutical composition mutein IL-4 may be executed in multiple forms, including liquid, gel, lyophilized, or any other suitable shape. The preferred form will depend on the specific symptom is treated, and will be obvious to a person skilled in this field.
The pharmaceutical composition mutein IL-4 may be administered orally in the form of aerosol, intravenously, intramuscularly, intraperitoneally, intradermally, or subcutaneously, or in any other suitable manner. The preferred route of administration will depend on the specific symptom is treated, and will be obvious to a person skilled in diversified funds. These funds can be included as part of the same pharmaceutical composition or may be introduced separately from mutein IL-4, either simultaneously or in accordance with any other appropriate treatment. In addition, the pharmaceutical composition mutein IL-4 can be used as a Supplement to other courses of treatment.
Accordingly, the invention provides compositions and methods for treatment of immune diseases, cancers or tumors, abnormal cell growth or immunomodulation in any suitable animal, preferably a mammal, most preferably a human. As stated above in the "Background" section, IL-4 has many effects. Some of them represent the stimulation of proliferation of T cells, the differentiation of T-helper cells, induction of activation and proliferation of human b cells and impotenceprevention switching classes of immunoglobulin. The effects exerted on the lymphoid system include an increase in the level of expression of the antigen MHC class II (Noelle, R., et al., Increased Expression of la Antigens on Resting In Cells: a New Role for Cell Growth Factor, PNAS USA, 81:6149-53 (1984)) and CD23 on b cells (Kikutani, H., et al., Molecular Structure of Human Lymphocyte Receptor for Immunoglobulin, Cell 47:657-61 (1986)). T-helper the progression Th1. Thus, any of a Th1-mediated disease treatable with IL-4 or its equivalent.
Also discusses the use of DNA sequences encoding mutiny IL-4 according to this invention, in gene therapy applications. Consider gene therapy applications include the treatment of such diseases in which it is expected that IL-4 will provide effective treatment due to its immunomodulatory activity, for example, multiple sclerosis (PC), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), uveitis, orchitis, primary biliary cirrhosis, malaria, leprosy, Lyme disease, contact dermatitis, psoriasis, b-cell lymphoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, malignant tumors, osteoarthritis and diseases somehow sensitive to IL-4, or infectious agents that are sensitive to IL-4-mediated immune response.
Local delivery Malinov IL-4 using gene therapy may provide the availability of medicines in the area of the target. Techniques of gene therapy both in vitro and in vivo. There are several ways of transferring potentially medicinal Kenobi include:
1) Direct gene transfer. See, for example, Wolff et al., "Direct Gene Transfer into Mouse Muscle In Vivo", Science, 247:1465-68 (1990).
2) DNA Transfer using liposomes. See, for example, Caplen et al., "Liposome-mediated CFTR Gene Transfer to the Nasal Epithelium of Patients with Cystic Fibrosis", Nature Med., 3:39-46 (1995); Crystal, "The Gene As a Drug", Nature Med., 1:15-17 (1995); Gao and Huang, "A Novel Cationic Liposome Reagent for Efficient Transfection of Mammalian Cells", Biochem. Biophys. Res. Comm., 179:280-85 (1991).
3) DNA Transfer using retroviruses. See, for example, KAU et al., "In Vivo Gene Therapy of Hemophilia B: Sustained Partial Correction in Factor IX-Deficient Dogs", Science, 262:117-19 (1993); Andersen, "Human Gene Therapy", Science, 256:808-13 (1992).
4) the Transfer of DNA using DNA-containing viruses. Such DNA-containing viruses include adenoviruses (preferably the vectors on the basis of Ad-2 or Ad 5), herpes viruses (preferably the vectors based on herpes simplex virus and parvovirus (preferably based vectors are not able to play without a virus-assistant or non-Autonomous parvovirus, more preferably vectors based on adeno-associated virus, most preferably vectors based on AAV-2). See, for example, li et al., "The Use of DNA Viruses As Vectors for Gene Therapy", Gene Therapy, 1:367-84 (1994); United States patents 4797368, 5139941.
The choice of a particular vector system for transferring the gene of interest will depend on many factors the nutrient studied and applied in many gene therapy applications, these vectors are generally not applicable to infect non-dividing cells. In addition, retroviruses have the potential oncogenicity.
Adenoviruses have the advantage that they possess a wide spectrum of hosts, can infect resting or completely differentiated cells, such neurons or hepatocytes, and are essentially non-carcinogenic. See, for example, Ali et al., supra, p.367. Adenoviruses do not integrate into the host genome. Because they are outside the chromosomes, the risk of insertional mutagenesis is sharply reduced. Ali et al., supra, p.373.
Adeno-associated viruses are advantages similar to those vectors based on adenoviruses. However, AAV are sitespecific integration into human chromosome 19. Ali et al., supra, p.377.
In the preferred implementation of the DNA encoding mutiny IL-4, in the present invention are used in gene therapy of autoimmune diseases, such as PC, IDDM and RA, infectious diseases such as Lyme disease and leprosy, malignant tumors, such as non-Hodgkin's lymphoma and ALL diseases of the cartilage such as osteoarthritis, and psoriatic conditions such as psoriasis.
Gene therapy DNA coding mutiny IL-4 on this izobreteny is this approach uses selective activity of Malinov IL-4 according to this invention to prevent unwanted autoimmune stimulation. For the specialist in this field is valuable that any suitable gene therapy vector containing DNA encoding mutein IL-4, can be applied in accordance with this implementation. Known methods of constructing such a vector. See, e.g., Ohno et al., supra, p.784; Chang et al., supra, p.522. Introduction of a vector containing DNA encoding mutein IL-4, site-target can be carried out using known methods, for example, as described in Ohno et al., supra, p.784.
The following examples are set forth in order to better understand the present invention. These examples are intended for purposes of illustration and should not be construed as limiting in any way the scope of the invention.
Below shows the amino acid sequence of Mature human IL-4 used in this study. Amino acids, the substitutions which received agonists, selective effect on T-cells, are shown in bold:
Mutiny Express in baculovirus system, purified to homogeneity, and research in biological analyses, which reflect different uses of the IL-4 receptor. To test for the activity of selectively dei 1T cells to the subject of positive activity of IL-4. Compounds that have the ability to induce proliferation 1T cells, while low ability to induce the secretion of IL-6, are agonists of IL-4 selectively acting on T cells, and are within the scope of this invention. More specifically, to evaluate the activity through an alternative receptor IL-4 (IL-4/-like receptor component) using endothelial cells from umbilical veins person (ACPWC).
Example 1. Products Malinov
Mutiny get through sitespecific mutagenesis using primers containing the codons corresponding to the desired mutations, essentially as described by Kunkel TA, Roberts JD, and Zakour RA, "Rapid and efficient site-specific mutagenesis without phenotypic selection" (1987), Methods Enzymol. 154:367-382. Briefly, cDNA of human IL-4, containing the restriction sites Bam HI and b I subcloning in the vector M13 Mr of M13 phage (New England Biolabs, Beverly, MA), using the same sites. cDNA of IL-4 wild type was obtained using polymerase chain reaction (PCR, "PCR") with a pool of cDNA obtained from mRNA isolated from peripheral blood lymphocytes of human induced within 24 hours of phorbol-12-MIA IL-4
5’-CGC GGA TCC ATG GGT CTC ACC TCC -3’ (TH. ID. no:22);
and for the 3’ end of the open reading frame of IL-4
5’-CGC TCT AGA HUNDRED GCT CGA ACA CTT TGA AT-3’ (TH. ID. no:23).
The restriction sites Bam HI (5’ end) and b I (3’ end) include in each oligonucleotide and denote italics. The applied PCR conditions are 1 minute at 94C, 1 minute at 58,7C and 1 minute at 72C for 25 cycles. The correctness of the thus obtained cDNA sequence of IL-4 confirmed using sequencing using kit Sequenase sequencing(Amersham Life Sciences, Arlington Heights, IL) as described by the manufacturer. Brazilsugarexp single-stranded DNA (U-DNA) obtained by transformation of E. coli strain CJ236 (Bio-Rad Laboratories, Hercules, CA) M13 mpl9 containing the cDNA of IL-4. In sitespecific mutagenesis mainly used primers containing 15 nucleotides homologous matrix U-DNA, Noah is at the 5’end of the codon(s), subjected to mutagenesis, the nucleotides that comprise the desired replacement, and an additional 10 nucleotides homologous matrix U-DNA, located on the 3’-end from the last replacement nucleotide. Applied specific Prime time is mutated nucleotides. Primers phosphorylate using polynucleotides T4 (New England Biolabs, Beverly, MA), using the Protocol of the manufacturer. After hybridization of the primer with U-DNA matrix and chain elongation by DNA polymerase T7 (Bio-Rad Laboratories, Hercules, CA) cells of E. coli strain DH5TM(GibcoBRL, Gaithersburg, MD) transform 5 ál of the reaction mixture and plated on the tablet with LB medium containing 0.7% agar. After incubation at 37With plaques propagated by taking one of the plaques and transfer 2 ml of LB medium and cultured overnight at 37C. single-Stranded DNA allocate, using the cleaning kit M13 (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's Protocol, and the clones containing the desired mutation identified by sequencing single-stranded DNA, using the kit Sequenase sequencing(Amersham Life Sciences, Arlington Heights, IL) according to the manufacturer's Protocol. cDNA mutein IL-4 with replicative form DNA, corresponding to plaques containing appropriately mutated sequence, allocate, using Bam HI and b I, and subcloning in plasmid vector pFastBacTM1 (GibcoBRL, Gaithersburg, MD). After sublimirovanny receive recombinant BA the DNA muteena, strain DHl0BacTME. coli (GibcoBRL, Gaithersburg, MD) as described by the manufacturer. Mutiny Express in Spodoptera frugiperda (Sf) 9 using baculovirus expressing system Bac-to-Bac (GibcoBRL, Gaithersburg, MD). All incubation of insect cells is carried out at 28C. In short, the volumes of the cultures of Sf9 cells in 2 ml transferout 5 μl of recombinant Bacmid using CellFECTIN (GibcoBRL, Gaithersburg, MD). After 60 hours after transfection, the supernatant collected and used for infection of 100-200 ml of culture 1106the Sf9 cells/ml in the medium of Grace (GibcoBRL, Gaithersburg, MD). In accordance with the Protocol of the manufacturer after 48-60 h after infection supernatant harvested by centrifugation at 5000 rpm for 10 minutes in a Sorvall centrifugeRC-5B. using the GSA rotor (Dupont Instrument Co., Willmington, DE), and analyze the titer of virus (typically receive a plaque-forming number 1108/ml). For production of the protein 2-3106the Sf9 cells/ml in 500 ml of medium SF900 II (GibcoBRL, Gaithersburg, MD) infect with a multiplicity of infection of 4 to 10 and after 60-72 h after infection, the supernatant is collected by centrifugation at 5000 rpm for 10 minutes in a Sorvall centrifugeRC-5B, using a GSA rotor (Dupont Monoclonal antibodies S400.1 and S400.17 against human IL-4 develop in mice using standard protocols using as immunogen recombinant human IL-4 (Genzyme Diagnostics, Cambridge, MA), produced as part ascitic fluid, purified and attached to CNBr-activated sepharose (Pharmacia, Uppsala, Sweden) according to the manufacturer's Protocol. Supernatant Sf9 cells obtained by infection of Sf9 cells with recombinant baculovirus containing the corresponding mutein IL-4, applied to a 1 ml column affinity to IL-4 media, washed with 100 mm NaHCO3, 500 mm NaCl, pH 8,3, to remove the salts washed with water and elute with 8 column volumes of 100 mm glycine, pH 3.0. Fractions are collected in siliconized vessels containing 0.1 volume of 1 M Tris, pH 8.0. Protein-mutein further purified according to the method of reversed-phase chromatography, using a column of C18 Dynamax-300(Rainin Instrument Co., Woburn, MA) with a gradient from Buffer a to B 0-100% Buffer A, water; Buffer B, acetonitrile, 0.1% of triperoxonane acid). Fractions analyzed by SDS-PAGE and mainstreami faction lyophilizer for storage and for analyses resuspended in sterile phosphate-buffered solution. Thus purified mutein in the study, usually appears as a single band on SDS-PAGE (colour silver), and its estimate using amino acid analysis (usually with fine which Vezha blood of healthy donors and purified by centrifugation using Ficoll-PaquePlus (Pharmacia, Uppsala, Sweden) essentially as described in Kruse, N., Tony, H. P. and Sebald, W. "Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement", Embo J. 11:3237-44 (1992). Purified mononuclear cells peripheral blood incubated for 7 days with 10 μg/ml of phytohemagglutinin (Sigma Chemical Co., St. Louis, MO), collected by centrifugation and washed in medium RPMI 1640 (GibcoBRL, Gaithersburg, MD). 5104activated T-cells/well (PHA-blasts) incubated with varying amounts of IL-4 or mutein in medium RPMI 1640 containing 10 amniotic bovine serum, 10 mm HEPES, pH 7.5, 2 mm L-glutamine, 100 units/ml penicillin G and 100 μg/ml streptomycin sulfate, in 96-well tablets within 72 h at 37With, mark 1 µci3H-thymidine (DuPont NEN, Boston, MA)/well for 6 hours, harvested and radioactivity measured by scintillation counter TopCounTM(Packard Instrument Co., Meriden, CT).
Example 4. Analysis of the secretion of IL-6 ACPWC
Endothelial cells of the umbilical vein of a person (ACPWC) obtained from Clonetics®Corp. (San Diego, CA) and support in accordance with the protocols of the supplier. Cells (3-6 passage) are harvested by incubation with trypsin/EDTA, washed and seeded with sublapsarianism plotstyle brain (EBM; CloneticsCorp., San Diego, CA). Upon reaching the continuity (3-4 days at 37C) the medium removed and replaced with EGMwithout ABM. After 24 hours the cells in fresh EGMwithout ABM add varying concentrations of IL-4 or mutein and allowed to incubated for a further 24 hours Supernatant collected and the concentration of IL-6 analyzed using ELISA human IL-6. Conditions are identical except for analyses antagonists, varying concentrations mutein added to a constant concentration of IL-4 in 100 gr. Briefly, 96-well tablets Immunolon2 (Dynatech Laboratories, Inc., Chantilly, VA) cover 5 μg/ml of MABS against human IL-6 Cat#1618-01 (Genzyme Diagnostics, Cambridge, MA) overnight at 4C. Standard human IL-6 (Genzyme Diagnostics, Cambridge, MA) or samples titrated in duplicate and incubated with coated tablet; after leaching add secondary rabbit PAT against human IL-6 (Caltag Laboratories, South San Francisco, CA, Cat#PS-37) in a dilution of 1:1000. The existence of linked rabbit anti-IL-6 PAT determined by applying an ass PAT against rabbit Ig, stitched with alkaline phosphatase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, Cat#711-055-152) at a dilution of 1:2000, and are, prameet reader Vmax(Molecular Devices Corp., Menio Park, CA).
Example 5. Activity Malinov.
The table below summarizes the results for muteena in the two analyses described above. EC50, PM represents the effective concentration that provides 50% reaction of the maximum measured concentration picomole/liter. Activity is a function of both strength (EC50) and maximum response (Rmax). Selectively acting on the cells mutiny show differential activity in relative decline in Rmaxand/or relative reduction in force (lower EC50in the analysis ACPWC compared with T-cell analysis. "Rmax, wt.%" represents the maximum response measured relative to IL-4 wild type. By definition, IL-4 wild type causes 100% reaction. All mutiny are active in the analysis of the proliferation of T-cells. Mutiny R121D, R121E, R121P and R121T/E122F/Y124Q in this analysis have a stronger effect than IL-4 wild type, although mutein R121T/E122F/Y124Q causes reduced maximum response. Mutiny Y124Q, Y124R and Y124A/S125A show values EC50exceeding 2-3 times those of IL-4 wild type, as well as reduced the maximum response. However, they retain a significant part of the activity of IL-4 on one, testifies to their selectivity with respect to T-cells and, thus, the selectivity with respect to the IL-4 receptor expressed on T-cells (IL-4R/IL-2R). Data mutiny are antagonists of IL-4 in relation to endothelial cells, because despite the fact that they normally interact with IL-4Rthey do not activate the IL-4R/-like particles. Mutiny R121P and Y124R are active in the analysis ACPWC, but their values EC50increased 50-150 times and cause reduced maximum response relative to their ability to stimulate T cells. Although these two proteins was not entirely selectively acting on T cells, they activate the IL-4 receptor T cells preferably before the IL-4 receptor ACPWC.
Example 6. Biological response to mutiny IL-4 in the analyses ACPWC
In Fig.2A and B presents a number of graphs "dose-response" secretion of IL-6 ACPWC under the action of selectively acting on T cells agonists compared with IL-4 wild type. Each tablet used for activity analysis Malinov, IL-4 include as NR is the Oia with IL-4 wild type. Similarly to Fig.4B-E presents curves dose-response for R121P, Y124Q, Y124R, Y124A/S125A and R121T/E122F/Y124Q, respectively, compared to IL-4 wild type. Activity values lead relative to control values response to IL-4. Mutiny R121E, Y124Q and R121T/E122F/Y124Q do not show activity in this assay. Mutiny R121P and Y124R, though, and show partial agonistic activity in this analysis are relatively less strong in comparison with IL-4 wild type than in 1T-cell analysis. Thus, despite their activity, they still show the preferred activation of the IL-4 receptor on T-cells.
Example 7. Biological response to mutiny IL-4 in 1T-cell assays
In Fig.4A and B shows the curves "dose-response" Malinov agonist, selectively acting on T cells, characterized by the analysis using cells of a healthy donor. Each tablet used for activity analysis Malinov, IL-4 include as an internal control; the characteristic curve. In Fig.6A presents curve dose-response for R121E compared with IL-4 wild type. Similarly to Fig.6B-E presents curves dose-response for R121P, Y124Q, Y124R, Y124A/S125A and R121T/E122F/Y124Q respectively compared to the 24Q and R121T/E122F/Y124Q have a stronger effect, than IL-4 wild type despite the fact that R121T/E122F/Y124Q is only a partial agonist in this analysis. Although mutiny Y124Q, Y124R and Y124A/S125A have in this analysis is not such a strong impact, as IL-4 wild type, they are still effective partial agonists (values of Rmax60-70% from those of IL-4 wild type). In Fig.6 activity corresponds to the response to IL-4, observed on the same tablet on each mutein. Particularly noteworthy mutiny R121E and Y124Q that show significant activity in T-cell analysis, showing no visible activity in the analysis ACPWC. Each of these Malinov is an agonist, selectively acting on T cells.
Example 8. Antagonistic effect of Malinov, selectively acting on T cells, IL-4-induced secretion of IL-6 ACPWC
Mutiny IL-4, selectively acting on T cells, R121E (and Y124Q (and the antagonist of IL-4 R121D/Y124D (O) (Fig. 7) was titrated against a constant concentration of 100 PM IL-4. Under these conditions, the antagonist of IL-4 R121D/Y124D has an antagonistic effect with respect to IL-4 with KI~1.5 nm. ACPWC not Express IL-2Rbut Express-podisi on the interaction of IL-4 with IL-2R(interoperability) and with-like subunit (interaction is absent or dysfunctional interaction), but do not affect the ability of these Malinov contact with IL-4R. Thus, agonists R121E and Y124Q, selectively acting on T cells, due to its ability to selectively interact with the IL-2Ron T-cells without stimulation activation-like receptor subunit in endothelial cells, is able to inhibit IL-4-induced reactions on data endothelial cells (in these conditions, KI~ 0,8-1 nm). This antagonism of Malinov IL-4 selectively acting on T cells, can inhibit the effects of endogenously produced IL-4 on endothelial cells in the process aimed at the T-cell therapeutic effects specified mottainai.
Example 9. Biological response to mutein IL-4 R121D
Mutein IL-4 R121D (Arg-121 replaced by Asp) receive, as described and analyzed in the analysis 1T cells and ACPWC. In Fig.8A and 8B shows IL-4 (O) and R121D (in tests such as T cells (Fig.8A), and ACPWC (Fig.8B). In T-cell analysis R121D aktivnym (EC50=; Rmax=O). These results show that only the replacement of Arg-121 human IL-4 Asp allows you to get the protein that has a selective activity against T-cells and does not have any visible activity toward endothelial cells. Although mutein R121D is a partial agonist in T-cell analysis, it is far more potent than IL-4 wild type. However, as mutein R121E, R121D is inactive in the analysis ACPWC, which suggests that it is an agonist, selectively acting on T cells.
Example 10. Biological response to mutein IL-4 T13D/R121E
Mutein IL-4 T13D/R121E (Thr-13 replaced by Asp, and Arg-121 replaced by Glu) receive, as described and analyzed in the analysis 1T cells and ACPWC, Specifically regarding figures 9A-B, T-cell analysis (Panel A) T13D/R121Eexhibits EC50~100 PM, about 2-3 times higher than IL-4 (O) or R121E (), and the value of Rmax100% from that of IL-4. In the analysis antagonists ACPWC (Panel B) T13D/R121Emanifests itself in ~27 times more potent antagonist activity of IL-4 (O), than R121E (Example 11. Evaluation of selective agonist of IL-4 in the model pathology
For these studies use adult males of Manakov-Griboedov (Masasa fascicularis, Charles River Primate Imports, Boston, Mass.), weighing approximately 4 to 6 kg. Animals are placed individually in rooms with a controlled internal environment in the open honeycomb cells and provide food twice a day and water ad libitum. All animals are deprived of food for approximately 12 h before the first day of the studies.
At each study animal anaesthetize using intramuscular injection of ketamine hydrochloride (Ketaset, 10 mg/kg). The upper part of the back of each animal clip out and cleaned with 70% alcohol-Betadine. Using a 1 ml tuberculin syringe animals in the back intradermally in a volume of 0.1 ml is injected IL-4, a selective agonist of IL-4 or the media (with 0.2% human serum albumin, HSA). The injection sites shares, at least 10 cm, and are marked with an indelible marker. Biopsy samples of tissues receive, using 6 mm tool to puncture the BBC after the injection.
System response to IL-4, a selective agonist of IL-4 or the media evaluated in the following way. The test substance is injected subcutaneously twice per day (with an interval of approximately 10-12 h) during four consecutive days in a volume of 0.1 ml/kg at doses of 0, 2, 5, 25 or 250 mg/kg total daily dose of 0, 5, 50 and 500 μg/kg, respectively. A sample of peripheral blood is obtained from each animal prior to the first injection of the carrier or IL-4 and at the beginning of each day of the research and spend a CBC and differential count of aliquot and analysis of surface markers of mononuclear cells in peripheral blood by the method of flow cytometry. The remainder of the blood sample centrifuged and aliquots of plasma stored at -70For further analysis of the levels of chemokines.
Prepare frozen sections and allow them to reach room temperature, air-dried and fixed in acetone at 4C for 5 minutes. VCAM-1 localize with S.3, a monoclonal antibody against human VCAM-1. As a negative control used izotopicheskii similar immunoglobulin with a different specificity in a suitable concentration. Endogenous Biotin blocks the ow temperature in a wet chamber with these antisera, diluted FBI 0,1% BSA and 1 normal rabbit serum. After three washes, the FBI slices stained with a set of Vector ABC Elite in accordance with the manufacturer's instructions and conjugate or antibody-based test is determined by incubating sections in 3-amino-9-ethylcarbazole/hydrogen peroxide (set of substrates AEC, Vector). The slices are washed thoroughly with 0.1 M acetate buffer, washed with distilled water and fixed in Lerner AQUA-MOUNT (Lerner Laboratories, Pittsburgh, PA).
Samples evaluated by two independent observers blinded fashion using scales from 0 to 3+ for assessing intensity, as well as distribution of color. The system of evaluation of the expression of VCAM-1 is: 0 absent or pale coloration random vessel; 1+ pale staining of several vessels; 2+ staining of moderate intensity most of the vessels; 3+ intense staining of the majority of the vessels. Blood vessels is determined on serial microslides, painted on the factor a background of Villebranda (polyclonal rabbit antibodies against FPV; Dakoplatts, Carpinteria, CA).
The counting of red blood cells, determination of hematocrit, blood count and platelets count is performed on heparinized blood samples using Serono 9000 Blood Analy the complexity of two hundred cells and record the percentage of each cell type.
Analysis of surface markers of mononuclear cells in peripheral blood (MCPC) carried out as follows. a 4 ml sample of heparinized blood diluted in balanced salt solution Hanks (USR, without Mg++ and CA++) and layered on 4 ml Percoll (density 1,070 g/ml). The tubes centrifuged at 1800 rpm (Beckman GS-6R) for 20 minutes at 24C. Lymphocytapheresis layer is sucked off and centrifuged at 1100 rpm for 10 minutes. The obtained cellular precipitate resuspended in 6 ml of a phosphate buffer solution (FBI), containing 0.1 azide and 5 goat serum. 1 ml of the aliquot used for analysis of cell surface markers, as described below.
For analysis by the method of flow cytometry using antibodies against CD2, CD4, CD8, CD1lb, CD16, CD25, CD49 and HLA-DR (R&D Systems, Minneapolis, MN). Aliquots twenty ál of marker antibodies incubated with 1 ml aliquot of cell suspension in the dark for 60 minutes at 4And the samples centrifuged (1000 rpm, 10 min at 4C). The precipitate washed three times with 1 ml of the FBI, containing 0.1% azide and 5 goat serum, followed by analysis using FACS. Plasma samples obtained during each study, analyze on the subject of level contains the given antibodies against MCP-1 for 16 h at 4C and then washed FBI, pH 7.5, 0.05 to Tween-20 (wash buffer). The nonspecific binding sites blocked with 2% BSA in FBI (200 μl) and tablets incubated for 90 minutes at 37C. the Tablets washed three times with wash buffer and add diluted (undiluted, 1:5 and 1:10) sample (50 μl) in duplicate, followed by incubation for 1 h at 37C. the Tablets four times wash add streptavidin-peroxidase conjugate (100 μg/ml) (Dakoplatts, Carpinteria, CA) and tablets incubated for 30 minutes at C. Tablets washed three times and add 100 μl of chromogenic substrate (0,67 mg/ml orthophenylphenate (Dakoplatts, Carpinteria, CA). Tablets incubated at 25C for 6 minutes and the reaction stopped with 50 μl/well of 3 M solution of H2SO4in wash buffer with addition of 2% of ACS. Tablets read at 490 nm in the ELISA reader. The standards represent a 0.5 log cultivation of recombinant MCP-1 from 100 ng/ml to 1 PG/ml (50 μl/well). ELISA allows to accurately determine the concentration of MCP-1>50 PG/ml
Example 12. The treatment of multiple sclerosis selective agonist of IL-4
The use of animal models as a way of predicting frownie selective agonist of IL-4 in multiple sclerosis (PC) is carried out in model igranka, using recombinant blogspiration agonist of the human IL-4. These studies are conducted in order to study the effect of prophylactic and therapeutic treatment on the occurrence and severity of acute symptoms and chronic relapsing-remitting disease.
Experimental autoimmune encephalomyelitis (EAE) is an animal mediated CD4+ T-cells in autoimmune inflammatory disease of the Central nervous system. The emergence of EAE cause marmosets (C. jacchus), weighing 300 to 400 g, by immunization 200 mg posthumously frozen homogenate white matter of the human brain (GM), emulsified in complete Freund Freund (PAF) containing 3 mg/ml of killed Mycobacterium tuberculosis, as described Massacesi et al., Ann. NeuroL, 37:519 (1995). On the day of immunization and again after 2 days 1010inactivated microorganisms Bordetella pertussis diluted in 10 ml saline and administered intravenously.
AAA evaluated according to clinical and pathological criteria. To register the severity of clinical disease use a standardized evaluation system: 0 = normal neurological indicators; 1 = drowsiness, anorexia, weight loss; 2 = ataxia and either ParaPa the same; 3 = paraplegia or hemiplegia; 4 = quadriplegia.
It was shown that magnetic resonance imaging (MRI) is an applicable method of characterizing early as well as late immune-mediated damage in PC (Stewart et al., Brain, 114:1069 (1991)). MRI is used to assess the condition of animals after immunization to monitor disease progression over time. MRI data receive cryogenic ‘2000’ NMR system Picker International, operating at a field strength of 0.15 Tesla; for images used take-up spool aperture 15 see Used a multilayer pulse spin echo and treatment-relaxation. In spin-echo sequences are used, the time delay of the echo or 40 and 60 MS, or 40 and 80 MS. In the sequence of treatment-relaxation Merimbula delay is 400 MS.
Marmosets anaesthetize ketamine hydrochloride and placed in the scanner using a laser is used to align the patient so that the inner corners of the eye slits are aligned perpendicular to the direction of the constant magnetic field. Animals crawl before immunization and then daily from 9 days after immunization. To daily scan ibremen after immunization. CNS isolated and fixed in 10% formalin. Prepare paraffin sections of brain and spinal cord and stained with hematoxylin and eosin. Each coronal slice of the brain or horizontal slice of the spinal cord analyze on the subject of histopathological signs of inflammation or demyelination in accordance with the conditional scale: inflammation; 0 = no inflammation, + = rare perivascular accumulations of leukocytes/average whole slice; ++ = moderate amount of perivascular accumulations of cells/slice; there can be inflammation of the membranes of the brain; +++ = widespread perivascular accumulations of leukocytes and parenchymal infiltration by cells of inflammation. Assessment of demyelination: 0 = demyelination absent; + = rare foci of demyelination; ++ = moderate demyelination; +++ = extensive demyelination with large confluent lesions.
In studies of pre-treatment of acute diseases of the test drug is injected subcutaneously in the range of doses from 1 to 500 μg/kg with subsequent scheme introduction 1 introduction in the days before 1 introduction in the week before the onset of the symptoms of the disease. For therapeutic intervention with advanced disease testerone is from 1 injection per day to 1 injection per week for several months.
Example 13. Treatment of rheumatoid arthritis
Rheumatoid arthritis (RA) is a debilitating inflammatory disease in which chronic activation of resident and infiltrating synovial cells induces the degradation of cartilage and bone, leading to fibrosis and loss of function. It is believed that cytokines, secreted activated T-cells, play a role in maintaining the chronic inflammatory response.
RA induce in mice DBA/1, applying collagen type II, as described in Joosten et al., Arthritis & Rheumatism; 39:797 (1996). Kollageninducyruemuyu arthritis (KIA) cause by immunization of mice by intradermal injection at the base of the tail with 100 μl of emulsion containing 100 μg of collagen. At 21 days the animals are subjected to secondary immunization by intraperitoneal injection of collagen type II (100 μg) dissolved in a phosphate buffer solution (FBI).
Assessment KIA is carried out by visual examination of the mice on the subject of developing arthritis in peripheral joints and expose the severity of arthritis. Mice consider patients with arthritis when you notice significant changes of redness and/or swelling (0 = no changes, 0,5 = significant, 1,0 = moderate, 1,5 = expressed and 2.0 = heavy maximum swelling is I some animals score and get the tissues of the feet and joints for pathological and histopathological studies. Fabric is subjected to immunohistochemical staining (frozen sections) or register and sign in paraffin, make slices and stained H&E for analysis of cellular infiltration.
Evaluation of murine analog of the selective agonist of IL-4 according to the present invention in model KIA spend using equivalent murine protein molecule. The person skilled in the art are able to compare the structure of the murine IL-4 human IL-4, to obtain a similar mutiny murine IL-4 and make any necessary adjustments based on the reactions in vitro using cell lines expressing either IL-4R/IL-2Ror IL-4R/-like subunit, similar to those used for Malinov human IL-4 with T-cells and ACPWC. Animals injected dose per day to re-immunizing injection of collagen and apply the scheme of introduction from daily to weekly throughout the study (40+ days). Animals injected dose in the range of concentrations of the selective agonist of IL-4 from 1 to 100 µg/kg
Example 14. The treatment of insulin-dependent diabetes mellitus (IDDM)
In the literature there n the RCTs of the effectiveness of murine equivalent of a selective agonist of IL-4 in the treatment of IDDM apply non-obese individuals diabetic (NOD) mice. The person skilled in the art are able to compare the structure of the murine IL-4 human IL-4, to obtain a similar mutiny murine IL-4 and make any necessary adjustments based on the reactions in vitro using cell lines expressing either IL-4R/IL-2Ror IL-4R/-like subunit, similar to those used for Malinov human IL-4 with T-cells and ACPWC. Prediabetics NOD mouse (aged approximately 7 weeks) are proliferative immunity in vitro after stimulation of T cells. The timing of this immunity is not associated with insulator, and it persists to develop diabetes, which occurs at the age of 24 weeks.
Evaluation of selective agonist of IL-4 in NOD mice performed similar to the studies described Rapoport et al., J. Exp.Med.; 178; p.87 (1993). The NOD mice administered the test substance at the age of about 3 weeks, followed by a scheme of administration in the form of daily injection or weekly injection for 12 weeks up until the mouse has not reached the age of 15 weeks. A control group of mice will be treated inert protein aquarii glycosuria in the next two weeks. After 52 weeks, the animals slaughtered for taking various organs and tissues for evaluation of pathological changes. The tissue of the pancreas, submandibular salivary glands and kidneys of each mouse fix and conclude in paraffin, make the cuts and paint. Staining with aldehyde fuchsin sections of the pancreas is used to determine the extent to which insulite infiltrates reduced the content of granulated-cells. The spleen leukocytes count by FACScan analysis using MAT ascitic fluid against Thy-1.2, CD4 and CD8, as described Zipris et al., J. Immunol. 146; R. 3763 (1991).
Other embodiments of the invention will be apparent to the person skilled in the art. This invention shows how to get mutiny, not particularly described here, but with activating T-cells capacity and low activating endothelial cells ability, and, thus, the data mutiny responsible entity and scope of the invention. The concept and the experimental approach described here will be applicable to other cytokines that involve heterologous multivariate receptor systems, in particular IL-2 and related cytokines (eg, IL-7, IL-9 and IL-15), IL-10,E. sequence:
PEFC. ID. No.:1: hIL-4 (amino acid)
PEFC. ID. no:2: hIL-4 (amino acid, cDNA)
PEFC. ID. no:3: R121A (amino acid, cDNA)
PEFC. ID. no:4: R121D (amino acid, cDNA)
PEFC. ID. no:5: R121E (amino acid, cDNA)
PEFC. ID. no:6: R121F (amino acid, cDNA)
PEFC. ID. no:7: R121H (amino acid, cDNA)
PEFC. ID. no:8: R121I (amino acid, cDNA)
PEFC. ID. no:9: R121K (amino acid, cDNA)
PEFC. ID. No.:10: R121N (amino acid, cDNA)
PEFC. ID. No.:11: R121P (amino acid, cDNA)
PEFC. ID. No.:12: R121T (amino acid, cDNA)
PEFC. ID. No.:13: R121W (amino acid, cDNA)
PEFC. ID. No.:14: Y124A (amino acid, cDNA)
PEFC. ID. No.:15: Y124Q (amino acid, cDNA)
PEFC. ID. No.:16: Y124R (amino acid, cDNA)
PEFC. ID. No.:17: Y121S (amino acid, cDNA)
PEFC. ID. No.:18: Y124T (amino acid, cDNA)
PEFC. ID. No.:19: Y124A/S125A (amino acid, cDNA)
PEFC. ID. No.:20: T13D/R121E (amino acid, cDNA)
PEFC. ID. No.:21: R121T/E122F/Y124Q (amino acid, cDNA)
PEFC. ID. No.:22: 5’- terminal primer PCR, IL-4
PEFC. ID. No.:23: 3’ end of the PCR primer, IL-4
PEFC. ID. No.:24: primer mutagenesis for R121A
PEFC. ID. No.:25: primer mutagenesis for R121D
PEFC. ID. No.:26: primer mutagenesis for R121E
PEFC. ID. No.:27: primer mutagenesis for R121F
PEFC. ID. No.:28: primer mutagenesis for R121
PEFC. ID. No.:29: primer mutagenesis for R121I
PEFC. ID. No.:30: ol>The donkey. ID. No.:33: primer mutagenesis for R121
PEFC. ID. No.:34: primer mutagenesis for R121W
PEFC. ID. No.:35: primer mutagenesis for Y124A
PEFC. ID. No.:36: primer mutagenesis for Y124Q
PEFC. ID. No.:37: primer mutagenesis for Y124R
PEFC. ID. No.:38: primer mutagenesis for Y124S
PEFC. ID. No.:39: primer mutagenesis for Y124T
PEFC. ID. No.:40: primer mutagenesis for Y124A/S125A
PEFC. ID. No.:41: primer mutagenesis for T13D
PEFC. ID. No.:42: primer mutagenesis for R121T/E122F/Y124Q
Note: for muteena 13D/R121 used the primers of TH. ID. nos:26 and 41.
1. Mutein human IL-4, numbered in accordance with IL-4 wild type, with an activating T-cells activity, but with a lower activating endothelial cells activity, where the surface remains of the D-helix specified IL-4 wild-type motivovany by conservative substitutions at one or more positions selected from positions 121-125, whereby obtained mutein causes the proliferation of T-cells and causes reduced secretion of IL-6 in endothelial cells of the umbilical vein of a person (ACPWC) compared with IL-4 wild type, or where the above mutein contains in addition at least one amino acid substitution in the binding pereception IL-4Rat least with greater affinity than native IL-4.
2. Mutein human IL-4 p. 1, where the position of the 121 substituted compared to IL-4 wild-type.
3. Mutein human IL-4 p. 2, where the regulations are replaced by alanine.
4. Mutein human IL-4 p. 2, where the specified position is substituted by aspartic acid.
5. Mutein human IL-4 p. 2, where the specified position is substituted by glutamic acid.
6. Mutein human IL-4 p. 2, where the regulations are replaced by phenylalanine.
7. Mutein human IL-4 p. 2, where the regulations are replaced by histidine.
8. Mutein human IL-4 p. 2, where the specified position is replaced with isoleucine.
9. Mutein human IL-4 p. 2, where the specified position is substituted by lysine.
10. Mutein human IL-4 p. 2, where the regulations are replaced by asparagine.
11. Mutein human IL-4 p. 2, where the regulations are replaced by Proline.
12. Mutein human IL-4 p. 2, where the specified position is replaced with threonine.
13. Mutein human IL-4 p. 2, where the regulations are replaced by tryptophan.
14. Mutein human IL-4 p. 1, where the position 124 is substituted by comparison with IL-4 wild-type.
15. Mutein human IL-4 p. 14, where the specified position C the human IL-4 p. 14, where the regulations are replaced by arginine.
18. Mutein human IL-4 p. 14, where the regulations are replaced by serine.
19. Mutein human IL-4 p. 14, where the specified position is replaced with threonine.
20. Mutein human IL-4 p. 1, where position 124 and 125 substituted compared to IL-4 wild-type.
21. Mutein human IL-4 p. 20, where both clauses 124 and 125 replaced by alanine.
22. Mutein human IL-4 p. 1, where position 121, 122, and 124 substituted compared to IL-4 wild-type.
23. Mutein human IL-4 p. 22, where the position of the 121 substituted by threonine, the position 122 replaced by phenylalanine, and position 124 is substituted by glutamine.
24. Mutein human IL-4 p. 1, where the position 13 is substituted by aspartic acid, and position 121 is substituted by glutamic acid.
25. Pharmaceutical composition for activation of T cells with low activating effect on endothelial cells, containing an effective amount mutein human IL-4 p. 1 in combination with a pharmaceutically acceptable carrier.
26. Polynucleotide molecule encoding mutein human IL-4 p. 1 having the nucleotide sequence corresponding to the amino acid sequence under item 1, or its virodene the nucleotide molecule on p. 26.
28. Cell line Spodoptera frugiperda 9 transformed by the vector according to p. 27, producing mutein human IL-4.
29. The selection method mutein human IL-4 p. 1, characterized in that analyze the proliferation of T-cells caused by muteena, then the analysis of the secretion of IL-6 ACPWC caused by muteena, and choose mutein human IL-4 with an activating T-cells activity, but with a lower activating endothelial cell activity compared with IL-4 wild-type.
30. A method of treating a patient suffering from a condition susceptible to treatment with IL-4, including the introduction of a therapeutically effective amount mutein human IL-4 p. 1.
31. The method according to p. 30, where the specified condition susceptible to treatment with IL-4, is an autoimmune disease.
32. The method according to p. 31, where the specified autoimmune disease represents multiple sclerosis.
33. The method according to p. 31, where the specified autoimmune disease is a rheumatoid arthritis.
34. The method according to p. 31, where the specified autoimmune disease is an insulin-dependent diabetes mellitus.
35. The method according to p. 31, where the specified autoimmune disease is a systemic lupus erythematosus.
36. The method according to p. 30, where the decree is shown an infectious disease is a disease Lyme.
38. The method according to p. 30, where the specified condition susceptible to treatment with IL-4, is a Th1-polarized disease.
39. The method according to p. 38, where the specified Th1-polarized disease is a psoriasis.
40. The method according to p. 30, where the specified condition susceptible to treatment with IL-4, is a malignant tumor.
41. The method according to p. 40, where specified malignant tumor selected from the group consisting of acute lymphoblastic leukemia and non-Hodgkin's lymphoma.
42. The method according to p. 30, where the specified condition susceptible to treatment with IL-4, is a disease of the cartilage.
43. The method according to p. 42, where the disease of cartilage represents osteoarthritis.