Selective agonists and antagonists of il-2
The invention relates to a polypeptide (I), a mutant protein of IL-2 people with numbering according to IL-2 wild-type human IL-2 is substituted by at least one position, 20, 88 or 126, allowing the specified mutant protein activates T-cells, preferably before natural killer (NK) cells; pharmaceutical composition having immunostimulatory activity, comprising the polypeptide of I; polynucleotide, representing a DNA sequence encoding a mutant protein of IL-2 persons; vector pBC1IL 2SA for expression of mutant protein IL-2 persons; line ovary cells of Chinese hamsters; cell line of African green monkeys, the E. coli strain, cell line Spodoptera fugiperda, transformirovannykh vector pBC1IL 2SA that produce mutant protein IL-2; the method of treating mammals suffering from oncological diseases, as well as to method of selection of mutant proteins IL-2 assessment in studies using IL-2Rin comparison with IL-2Rwhere the activity of the mutant protein IL-2 is increased in relation to IL-2 wild-type one is resistome level of technology
1. The scope of the invention
The invention in General relates to the fields of pharmacology and immunology. More precisely, the invention is directed to new compounds for the selective activation of T cells (PHA-blasts (T-cells activated by phytohemagglutinin)) and reduced activation of natural killer (“NK”) cells. New connectivity options include compounds of the family of cytokines, in particular human interleukin-2 (“IL-2”).
2. Description field of technology
Interleukin 2 (IL-2) is an effective stimulator of the immune system, activating different immune cells, including T cells, b cells and monocytes. IL-2 is also the potential and critical growth factor for T cells. Thanks to these activities of IL-2 was investigated for possible application in the treatment of cancer. Human IL-2 is approved by the FDA (food and drug administration (USA)) medication for the treatment of metastatic renal carcinoma and metastatic melanoma. The use of IL-2 in those patients is limited due to the high toxicity associated with treatment with Interleukin-2; according to estimates, at best 20% of patients tolerate the treatment. The toxicity associated with the treatment of IL-2, includes selfaction performance (objective response rate ~17%).
Despite the fact that there were extensive structural and functional analysis of murine IL-2 (Zurawski, S. M. and Zurawski, G. (1989) Embo J 8: 2583-90; Zurawski, S. M, et al., (1990) Embo J 9: 3899-905; Zurawski, G. (1991) Trends Biotechnol 9: 250-7; Zurawski, S. M. and Zurawski, G. (1992) Embo J 11: 3905-10; Zurawski, et al., EMBO J, 12: 5113-5119 (1993)), the analysis of human IL-2 was limited. Most studies with mutant proteins IL-2 was performed on murine cells; however, a limited number of studies have been conducted using human PHA blasts (T-cells treated with phytohemagglutinin) expressing high affinity IL-2 receptor IL-2R. Studies using PHA-blasts confirmed the importance of residues Asp-20 and D-helix of human IL-2. It was shown that the provisions Asp 20 and Gln-126 are key residues responsible for interaction withand-subunits of the receptor for IL-2, respectively (described in Theze, et al., Immunol. Today, 17,481-486 (1996)). Although it has been shown that residues in the C-helix murine IL-2 are involved in the interaction with murine IL-2R ((Zurawski, et al., EMBO J, 12 5113-5119 (1993)), has not been shown that the equivalent residues in human IL-2 have the same svoistva human and murine IL-2 (activity of human IL-2 in murine cells reduced ~ 100 times) it is difficult to predict whether similar interactions within a species. Nothing is known about the studies using cells expressing only the human receptor IL-2Rwith an average affinity.
We studied the activity of some mutant proteins IL-2 on human PHA-blasts (Xu, et al., Eur. Cytokine Netw, 6, 237-244 (1995)). It was shown that the mutant proteins containing the replacement of Asp-20 to leucine (D20L), and arginine, asparagine and lysine, very defective in their ability to induce the proliferation of PHA-blasts. Thus, it follows that the replacement of Asp-20 will result in the formation of mutant protein with reduced activity. In addition, in the work of Xu et al. it is argued that by 1995 was not identified mutant proteins IL-2, useful for clinical or research applications.
Mutant protein human IL-2 Q126D received Buchli and Ciardelli, Arch. Biochem. Biophys, 307(2): 411-415, (1993), has significantly reduced activity as their primary property, and agonize; in the experiment with T-cells of the person he has been active in 1000 times smaller than IL-2, and behaved as a partial agonist. In experiments with murine T-cells mutant protein was almost inactive. La shows the ability Q126D to be an antagonist of IL-2-mediated activity, although in the experiment with human T cells only partially.
Zhi-yong, W., et al., Acta Biochimica et Biophysica Sinica 25(5): 558-560 (September 1993) conducted experiments on replacement in IL-2 at positions 62, 69, 99 and 126, demonstrating 20 - and 30-fold lower activity 62-Leu-IL-2 and 126-Asp-IL-2, respectively, compared to IL-2 wild-type experiment with murine T-cells (CTLL-2). However, not made a conclusion or assumption that substitutions at position 126 can determine the activity selective for T-cells compared to NK cells, or to show whether such changes have a similar effect in human T cells.
In the work of Collins, L., et al., PNAS USA 85:7709-7713 (1988) reported that substitution of Asp at position 20 on Asn (D20N) or Lys (D20K) leads to loss of binding with high receptor (IL-2Ridentified in the work of Collins et. al. the P55/R) and (intermediate) average affinity (IL-2Rmarked R) 100-1000 times compared to human IL-2. Binding to IL-2Rwere unaltered for both mutant proteins. From this work it follows that the violation of binding to IL-2-receptor intermediate (average) affinity (IL-2R), it is assumed that separate binding or activation between IL-2Ror IL-2Runattainable by substitution of Asp at position 20.
Brendt, W. G., et al., Biochemistry 33 (21):6571-6577 (1994) used a combinatorial cassette mutagenesis for simultaneous replacement provisions 17-21 in natural IL-2, it is assumed that these provisions interact with the IL-2 receptor intermediate (average) affinity. From 2610 analyzed clones only 42 were active. It was found that the positions 20 and 21 are of paramount importance for biological activity. Not made assumptions or conclusions about individual substitutions, except L21V.
In U.S. patent No. 5229109 (Grimm et al.) approved low toxicity analogues of IL-2 when used in immunotherapy and cancer treatment. Properties of two analogues of IL-2 with substitutions in positions Arg38 (alanine) and Phe42 (lysine) were studied and compared with the properties of natural IL-2. It was found that the analogues are able to maintain the ability to bind with intermediate receptor of IL-2 with minimal binding to so-called “Vysocany what about from P75 (IL-2R), and the receptor with high affinity consists only of the P55 receptor complex+R75 (IL-2R). Also analogues was supported by the ability to stimulate mononuclear cells peripheral blood to the production of lymphokine-activated killer cells (LAK). It should be noted that the secretion of IL-1and TNFin response to analogues was significantly reduced compared to the natural molecule IL-2, this patent describes the amino acid residues that specifically interact with the IL-2R(P55), with the exception of the interaction with the IL-2Rwill lead to lower activity in cells bearing high-affinity IL-2 receptor, and will not affect the activity of cells bearing IL-2 receptor with an intermediate activity. Thus, the products of LAK cells (assuming they are proizvodnje NK-cells) will be supported. Mutations in the proteins described here is aimed at amino acid position 20, 88 and 126; it is considered that these provisions specifically interact with the IL-2R(R75; position 20 and 88) and IL-2R(at the time of filing of the patent Grimm et al. unknown; position 126). the FL mutant proteins, described by Grimm et al., will be decreased interaction with high-affinity receptor IL-2, but will not affect the activity of the IL-2 receptor with an intermediate (medium) affinity; mutant proteins described in this work have the opposite characteristics, they are characterized by a significant defect in their ability to interact with the IL-2 receptor with an intermediate affinity IL-2Rand very weak or non-defective functional interactions with high-affinity IL-2 receptor, IL-2R.
In U.S. patent No. 5206344 (Gordon et al.) described mutant proteins IL-2, in which one of the amino acids of the Mature native sequence IL-2 is replaced by a cysteine residue, then these proteins are processed through the substituted cysteine residue conjugatively with polymer, chosen from a number of homopolymers of polyethylene glycol or polyoxyethylene polyols, where the homopolymers are not substituted or substituted with one end of the alkyl group. These mutant proteins produced by expression in a host of mutant genes, these genes were delivered instead of genes encoding proteins of the host, the method of site-napii 125 of the Mature IL-2, which is not neobhodimim for the biological activity of IL-2. Mutations causing reduced toxicity not described.
In U.S. patent No. 4959314 (Lin et al.) described mutants of biologically active proteins such as IFN-and IL-2 in which the cysteine residues that are not essential for biological activity, have been deleted or substituted with other amino acid residues to eliminate sites of intermolecular linking or education illegal disulfide bridges. Such mutant proteins produced by expression in bacteria mutant genes, these genes were synthesized from genes encoding proteins of the host, method of oligonucleotide-directed mutagenesis. Mutations causing reduced toxicity not described.
In U.S. patent No. 5116943 (Halenbeck et al.) described that biologically active standard therapeutic protein protects against oxidation by a process comprising replacing each methioninamide balance, prone to oxidation by chloramine T or peroxide, a conservative amino acid, and more resistant to oxidation methionine residues do not replace. Oxidation resistant mutant protein, thus obtained, is preferably mutant beee preferably alanine. Mutations causing reduced toxicity not described.
U.S. patent No. 4853332 (Mark et al.) describes mutant proteins IFN-and IL-2 in which the cysteine residues that are not essential for biological activity, have been deleted or replaced by other amino acids to eliminate sites of intermolecular linking or education illegal disulfide bridges. The patent States that the replacement of IL-2 cysteine at position 125 to the serine leads to the formation of mutant protein with activity comparable to native IL-2.
U.S. patent No. 5696234 (Zurawski et al.) describes mutant proteins cytokines mammals and how to search for agonists and antagonists of cytokines mammals. In particular it is shown that the double mutant human IL-2, P82A/Q126D, has an antagonistic activity on the murine Baf3 cells, cotransfectionandthe subunits of IL-2R person. Shows the presence of small agonistic activity. Also it is shown that mutants of murine IL-2 show partial agonistic and antagonistic activity in NT2 cells, in particular mutants Q141D, Q141K, Q141V and Q141L. Activity of mutant proteins, confirming or suggesting izbiratelnogo IL-2, which is active in the cells, expressionwhich IL-2Rand inactive in cells expressing IL-2R(Zurawski.G., Trends Biotechnol. 9: 250-257 (1991); Zurawski, S. M. and Zurawski. G. Embo. J. 11:3905-3910 (1992)). Mutant mouse proteins IL-2, having such properties are replacing Asp-34 on Ser or Thr, and Gin-141 Lys. Asp-34 and Gln-141 of murine IL-2 is equivalent to Asp-20 and Gln-141 of the human IL-2, respectively. Although these references refer to mutant proteins IL-2 “selective agonists”, mainly described the potential of these proteins as antagonists of endogenous IL-2, but not the potential of their lower toxicity.
In EP 0267795 A2 (Zurawski et al.) describes the number of mutant proteins of murine IL-2 sequence, including mutants containing deletions and/or substitutions within the first third of the biologically important N-terminal amino acid residues but not included, will not be discussed and are not available amino acid substitutions proposed in this paper for the equivalent residues of murine IL-2. There is a need for improved molecule of IL-2 with reduced toxicity and greater overall tolerability.
U.S. patent No. 4738927 (Taniguchi et al.) describes a recombinant DNA comprising rayalaseema growth cell line cytotoxic T-lymphocytes, and vector DNA can replicate in prokaryotic and eukaryotic cells; the coding sequence of the specified gene is located in a downward direction from the promoter sequence, and the specified polypeptide in General represents from 132 to 134 amino acids in the amino acid sequence of the polypeptide. There is also described a gene, recombinant DNA vectors, host cells and recombinant methods of obtaining native IL-2. Taniguchi et al. do not describe variants of mutant proteins and do not indicate the position in the protein that is responsible for signaling and binding activity.
It is clear that the mutant proteins IL-2 with no DLT (dose limiting toxicity previously received recombinant protein of IL-2 required for use of therapeutic benefits of this cytokine.
Summary of invention
The invention describes a polypeptide comprising a mutant protein of human IL-2, numbered according to IL-2 wild-type, where the specified human IL-2 is substituted by at least one of positions 20, 88 or 126, whereby the indicated mutant protein activates T-cells, preferably before NK-cells. This invention is less toxic mutant protein of IL-2, DNIe proteins IL-2, containing a single mutation of the Aspartate 20, Asparagine 88 and Glutamine 126. Specific mutant proteins indicated D20X, N88X and Q126X, where X is a specific amino acid, which, when substituted in the human IL-2, causes selective activity of cells expressing IL-2Rreceptor (e.g. T cells), preferably cells expressing IL-2Rthe receptor (for example, NK-cells). Mutant proteins with more than 1000-fold selectivity include D20H, D20I, N88G, N88I, N88R and Q126L. In particular, these proteins also show significant activity of IL-2 wild-type T-cells. Also identified other mutations, providing less than 1000 but more than 10-fold selectivity. The invention also includes polynucleotide encoding included in the invention of the mutant proteins, vectors containing polynucleotide, transformed host cells, pharmaceutical compositions comprising the mutant proteins, and therapeutic methods of treatment using these proteins.
The invention also includes the method of selection of mutant proteins IL-2 test results SMD src="https://img.russianpatents.com/chr/946.gif">where the activity of the mutant protein of IL-2 in one analysis compared with other increases in relation to IL-2 wild type. IL-2Rand IL-2Rrepresent individual receptor subunit ectodomain in the appropriate configuration and are used for direct measurement of the binding of mutant proteins IL-2 with each receptor complex. Analysis using IL-2consider the response from IL-2-bearing cells, and analysis using IL-2response from IL-2-bearing cells. IL-2-bearing cells are PHA-blasts, and IL-2-bearing - NK-cells. In the analysis investigated the proliferation and IL-2-, and IL-2-chosen to replace the finding of the mutant protein, vector driving the expression of the mutant protein human IL-2 having the ability to activate PHA-blasts and a reduced ability to activate NK-cells, the vector is capable of transfection body of the target and subsequent expression in vivo mutant protein human IL-2, encoded by the specified polynucleotide.
The invention relates also to a method of treatment of patients affected IL-2-curable disease, by assigning therapeutically effective amounts of the mutant protein human IL-2, numbered in accordance with IL-2 wild type, with the ability to activate PHA-blasts and a reduced ability to activate NK cells. This method is applicable for conditions amenable to treatment with IL-2, such as HIV, cancer, autoimmune disease, infectious disease; as adjuvant in anticancer vaccine and conventional vaccine therapy for immune stimulation in old age or when immunity is weakened in other ways, as well as in patients with SCI (deep combined immunodeficiency) and for other therapeutic purposes, requiring the stimulation of the immune system.
Brief description of drawings
Fig.1-7 show curves from doses for calove Q126L (Fig.7). A: Individual response, the dose of IL-2 (filled circles) and mutant protein (unfilled circles) in the study of proliferation of primary T cells (PHA-blasts). In: Individual response, the dose of IL-2 (filled circles) and mutant protein (unfilled circles) in the study of proliferation of primary NK cells.
Fig.8A-8D. Toxicity Proleukin PROLEUKINthe chimpanzee was evaluated on two animals (H-159, triangles, and X-124, squares) compared to the filler as a control (X-126, diamonds). Toxicity was assessed by renal parameters (urea nitrogen, blood (BUN), AND creatinine) and liver function (total bilirubin, C; alanine aminotransferase (ALT), D).
Fig.9. A graph of the percentage change of body weight within 30 days. The body weight of the animals who were given IL-2/N88R (triangles), Proleukin (squares) or filler (diamonds), were measured on the indicated days.
Fig.10A-10D. The number of lymphocytes (A), total leukocyte count (), neutrophils (C) and platelets (D) in animals that were given IL-2/N88R (triangles), Proleukin (squares) or filler (diamonds), were measured on the indicated days.
Fig.11A-11D. Indicators of nitrogen of urea of blood (A, BUN), creatinine (In), phosphorus (C) and time of activated prothrombin (D) in animals, cfig.12A-12D. The levels of total bilirubin (A), ALT (b), fibrinogen (S) and time of activated prothrombin (D) in animals that were given IL-2/N88R (triangles), Proleukin (squares) or filler (diamonds), were measured on the indicated days.
Fig.13A-13D. The levels of sodium (A), chloride ions (In), calcium (C) potassium (D) in the blood of animals who were given IL-2/N88R (triangles), Proleukin (squares) or filler (diamonds), were measured on the indicated days.
Fig.14A-14C. Levels of albumin (A), hematocrit () and hemoglobin (in the blood of the animals who were given IL-2/N88R (triangles), Proleukin (squares) or filler (diamonds), were measured on the indicated days.
Fig.15A-15C. The effects of IL-2/N88R (squares), Proleukin (diamonds) and filler (triangles) are shown for the total population of T cells (A, CD3+ cells), the population of CD4+ T cells (C) and CD8+ T cells (C).
Fig.16A-16C. The effects of IL-2/N88R (squares), Proleukin (diamonds) and filler (triangles) are shown for the total population of T cells (CD3+ cells), the population of CD4+ T cells (C) and CD8+ T cells (C).
Fig.17. The effects of IL-2/N88R and Proleukin on the activation of T-cells in relation to the value of the fluorescence intensity of CD25-positive cells. The effects of IL-2/N88R (squares), Proleukin (diamonds) and filler (triangles) are shown for populations of CD3+CD4+ T cells. Received IL-2/N88R (squares), Proleukin (diamonds) and filler (triangles) are shown for populations of CD4+ T cells (A, CD3+/CD4+ cells, a population of CD8+ T cells, CD3+/CD8+ cells), the total population of T cells (CD3+ cells) and populations of NK-cells (D, CD3-/CD16+ cells).
Fig.19. Trend of the number of lung metastases in relation to dose in mice who were given Proleukin (empty circles) or IL-2/N88R (filled circles). Lung metastases were counted at the end of the study.
Fig.20. Plasmid map of IL-2/NK-vector pBC1IL2SA.
Description of the preferred embodiments
A. Prior art
The effects of IL-2 on T cells is mediated by the binding of IL-2-receptor proteins. Certain proteins on the surface of T-cells bind IL-2. The first was identified a single protein called IL-2Raverage of 55 kDa (p55), he appears in the activation of T cells and was originally named TAS-antigen (from T activation). IL-2Rassociated with IL-2 with constant Kdapproximately 10-8M, it is also known as “low-affinity” receptor. Binding of IL-2 with cells expressing only IL-2Rthat does not lead to any discernible biological response.
The Second IL-2-src="https://img.russianpatents.com/chr/947.gif">(with) chain, as it is found in a number of cytokine receptors. IL-2Rb is from 70 to 75 kD (called either R or P75) and is a member of the family, receptors of cytokines of type I, characterized by the presence of two sites of cysteine/WSXWS. IL-2Ris expressed in a coordinated fashion withS.The binding affinity of IL-2 to IL-2Rchigher than IL-2Rthe constant Kdapproximately 10-9M; IL-2Rcalso known as the IL-2 receptor with “intermediate affinity”. IL-2 causes the growth of cells expressing IL-2Rwithand premaxilla stimulation of growth was observed at a concentration of IL-2, leading to premaxillae binding (i.e., 110-9M). IL-2Rwithis the same signal receptor complex that can bind to IL-15.
The third known IL-2-receptor complex is the complex of IL-2Rwithable to bind IL-2 significantly stronger with constant Kdapproximately 10-11M, thus IL-2Rwithalso known as “high-affinity complex. Stimulation of the growth of such cells, respectively, at low concentrations of IL-2. Binding of IL-2 and growth stimulation can be blocked by antibodies to IL-2RIL-2Rorwithand more effectively by a combination of antibodies to several subunits of the receptor. These observations suggest that IL-2Rforms a complex with IL-2Rwithincreasing the affinity of the signaling receptor for IL-2, and enable the delivery of the signal growth with significantly lower concentrations of IL-2. I believe that the first IL-2 rapidly binds to IL-2Rand this contributes to the Association with IL-2RS.Resting T cells Express IL-2Rwithand only a small amount of IL-2Rthus decreases the concentration of IL-2 needed to stimulate growth. Binding of IL-2 to IL-2Rwithcauses the signal on the signal path Jac/STAT.
The authors have discovered mutant proteins human IL-2, which activate T cells (PHA-blasts; cells expressing the high-affinity IL-2 receptor IL-2R) preferably before estestvennimi killer (NK) cells (cells expressing IL-2 receptor with an intermediate affinity IL-2R). Mutant proteins in which Asp-20 replaced by histidine (D20H) or isoleucine (D20I), Asn-88 is replaced by arginine (N88R), glycine (N88G) or isoleucine (N88I) or Gln-126 is replaced by leucine (Q126L) or glutamate (Q126E), show unexpectedly full IL-2 activity in PHA-blasts, and low activity (or lack thereof) on NK cells. Previous studies of mutant proteins human IL-2 was based on murine cell systems analysis and studies on the tile is (such as NK-cells). In addition, studies using human PHA blasts showed that replacement of Asp-20 (Leu, Arg, Asp, or Lys; Xu, et al., Eur. Cytokine Netw, 6, 237-244 (1995)) or Gln-126 (Asp; Buchli and Ciardelli, Arch. Biochem. Biophys, 307(2): 411-415, (1993)) result in mutant proteins human IL-2 at a significantly reduced activity. In previous studies using murine IL-2 were identified mutant proteins IL-2 mouse with different activities (Zurawski, et al., EMBO J, 12, 5113-5119 (1993)), but none of the mutations leading to these results did not carry mutations identified in the proposed work. Mutant proteins human IL-2 containing identical substitutions in positions that are synonymous with the provisions of mutant proteins of murine IL-2, do not exhibit similar activity, thus, it can be assumed that the examples in mice can't predict functionality in the human system. Not known to the authors of the study of mutant proteins, IL-2, comparing the relative activity in the cells expressing IL-2 receptor IL-2R(for example, PHA-blasts) and in cells expressing IL-2 receptor IL-2R(therapeutic index in comparison with the human IL-2 wild-type due to reduced toxicity and provide therapeutically useful compounds for the treatment of diseases, requiring stimulation of the immune system.
Interleukin 2 (IL-2) is currently used in clinical practice for the treatment of metastatic kidney cancer. However, its strong toxicity limits its use by a group of the most healthy patients, and DLT (dose limiting toxicity reduces its overall effectiveness. Suggest that the acute toxicity of IL-2 is due to the activation of NK-cells, whereas the efficiency is due to direct activation of T cells (Jacobson, et al., Proc Natl Acad Sci USA (United States), Sep. 17, 1996, 93(19) p. 10405-10; Smith KA, Blood 1993, 81(6), R. 1414-23; Kaplan, et al., Biotechnology, 10 (2), p. 157-62). T-cells and NK-cells Express various receptors for IL-2 (T cells: IL-2R, high-affinity IL-2 receptor; NK-cells: IL-2RIL-2-receptor with intermediate activity). Described in this paper mutant proteins human IL-2 activate the IL-2 receptor of T cells and does not activate the IL-2-receptor NK-cells.
In order to avoid activation of NK-cells with some success was applied therapy with low doses of IL-2 (Jacobson, et. El. (1996). Proc Natl Acad Sci USA 93: 10405-10). This strategy was based on the concept that at low doses of IL-2 will be activerow the Orme IL-2-receptor with intermediate activity, IL-2Rexpressed on NK-cells, while T cells Express high-affinity form of the IL-2R.
However, the authors approached the problem of toxicity with other positions. An assumption was made that the violation of the interaction of IL-2 to IL-2Rand/or IL-2Rby certain modifications of the specific binding residues on the binding surface IL-2 prevents efficient binding (and thus activation) with cells expressing only IL-2R. However, cells expressing IL-2Rinitially binds IL-2Rthus, binding to the cell will still occur and therapy with low doses proposed by Jacobs et al., can give toxic side effects. Due to the binding of IL-2on the cell surface can occur effective use of IL-2Rand IL-2R. despite the weak interaction to modetsa capable of signaling IL-2/IL-2R. We believe that the variant IL-2 may selectively activate high-affinity IL-2 receptors on T-cells, preferably in relation to IL-2 receptors with intermediate activity on NK-cells will have an increased therapeutic index in comparison with IL-2 wild type due to a reduced toxicity profile. Variant of IL-2 with a high therapeutic index will have a much wider application in the treatment of cancer (direct and/or adjuvant therapy), and immunodeficiency (e.g. HIV and tuberculosis). Other possible applications of IL-2 due to its immunostimulating activity and include, in addition, the direct treatment of cancer, immunodeficiency in patients with HIV or CT man; infectious illnesses like tuberculosis; application of adjuvant methods “cancer vaccine” and when shown to stimulate the immune system, such as improving the standard procedures of vaccination or treatment for the elderly.
The corresponding changes in the provisions of the Asp-20, Asn-88 or Gln-126 reduced binding interaction or for IL-2R(Asp-20 and Asn-88), or IL-2R(Gln-126).the ne and had a reduced activity of NK-cells.
Since it was not possible to predict the result of this replacement to research on T - and NK-cells, were made replacement at all possible natural amino acids (except Cys) at positions Asp-20, Asn-88 and Gln-126 and a limited but diverse set of mutations at position Asp-84 to test their interaction with the IL-2R. If observed visible interaction with the IL-2Rwould have made an additional mutation at position Asp-84. Data on 46 individual substitutions at these positions are shown in table. 1.
Mutations were introduced using site-directed mutagenesis of cDNA of IL-2 wild type. The desired clones were subcloned in the expression vector suitable for expression in a heterologous system (e.g., E. coli, baculovirus, yeast cells or mammalian cells, such as ovarian cells of the Chinese hamster (Cho). Purified proteins were examined in studies of proliferation of T cells (PHA-blasts) and NK-cells. Different answers, which gave individual proteins between these studies, i.e. EU50point mutations affecting these activities. Specifically, mutant proteins, stimulating a relatively stronger response in the study of PHA-blasts (T-logo type) correspond to substitutions, providing cell specificity, based on the ability of specific mutants to bind and activate IL-2Rexpressed on these cells, but the lack of ability to communicate and thus to activate cells expressing only IL-2R. Mutant proteins IL-2, having this property, in vivo will have immunostimulating properties of IL-2 with reduced toxicity, manifested in vascular permeability and hypotension associated with treatment with IL-2.
Cell line Cho used to produce recombinant protein described herein, were laid in ATS (American Type Culture Collection, 12301 Paraklawn Drive, Rockville, MD, 20852, USA, on 5 may 1999, Deposit No. MOUTH-8. Because this strain belongs to the strains registered under the terms of the Budapest agreement, he will be available to a patent office of a state signatory to the Budapest Treaty.
In this work, “IL-2 wild-type” refers IL-2, natural or recombinant containing the sequence 133 normally occurring amino acids corresponding to the natural IL-2 person (minus sknote described in Fujita, et al., Pnas USA, 80, 7437-7441 (1983), with or without additional N-terminal methionine, which necessarily contains, when the protein is expressed as an intracellular fraction of E. coli.
In this work, “mutant protein IL-2” means a polypeptide in which a specific substitutions relative to the Mature protein of interleukin-2. In table. 1 described 46 obtained mutant proteins with individual replacements and relevant data on their relative activity. The preferred products are mutant proteins with at least 100-fold relative activity. Most preferred are the following proteins with more than 1000-fold relative activity: the residue (Asp) aspartate (D) in regulation 20 (“D20”), with numbering according to IL-2 wild-type, replaced by isoleucine (“D20I”), or histidine (“D20H”); residue asparagine (Asn) at position 88 (N88) substituted for isoleucine (N88I) or arginine (N88R) and the residue glutamine (Gln) at position 126 (Q126) replaced by leucine (Q126L), glutamate (Q126E) or aspartate (Q126D). For the rest, not replaced, the provisions of the preferred mutant proteins IL-2 have an amino acid sequence identical to IL-2 wild type. However, the mutant proteins IL-2, described in this paper, mahuika sites on these or other residues of the polypeptide chain of the native IL-2. In accordance with this invention any such insertions, deletions, substitutions and modifications can result in mutant proteins IL-2, preserving the PHA-blast-selective activity with reduced ability to activate NK cells, and constitute the essence of this invention.
The combination of the preferred or particularly preferred substitutions described above, in a single recombinant molecule of IL-2 can lead to combinations of mutants with activity similar to that described for the single mutants. For example, it can be expected that the molecule of IL-2 containing a combination of two or more mutations selected from table. 1, may lead to IL-2-agonist, selective with respect to T cells, with a relative activity, similar relative activity of molecules with single substitutions described herein. Raman mutants are the scope and essence of the present invention.
The authors prefer conservative modifications and substitutions by other provisions of the IL-2 (i.e., those that have minimal influence on the secondary or tertiary structure of the mutant protein). Such conservative substitutions include substitutions 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, DVD;
is lys, arg, his;
is phe, tyr, trp, his;
The authors also prefer modification or replacement that doesn't make sites for additional intermolecular linking or education illegal disulfide bonds. For example, it is known that IL-2 contains three cysteine residue in the wild type, this position 58,105 and 125 of the Mature sequence.
Under “numbering according to IL-2 wild-type” authors understand the identification of the selected amino acids in accordance with the position in which this amino acid normally found in the Mature sequence of IL-2 wild type. If the insertions or deletions, specialists in the art will understand that Asp, normally located in position 20 may be shifted according to the position in the mutant protein. However, the situation out of Asp, you can easily determine the collation and comparison of the flanking amino acids with amino acids flanking Asp IL-2 wild-type.
The term “types of cells bearing IL-2Rthe receptor” refers to cells, the presence of this type of receptor which is known, i.e., T-cells, activated T-cells, b-cells, activated monocytes and activated NK cells. The term “pile types of receptors have known, i.e., b cells, resting monocytes and resting NK cells.
Mutant proteins IL-2, presented in this invention can be obtained by any suitable method, izvestnim in the art. Such methods include constructing a DNA sequence that encodes a mutant protein of IL-2, and the expression of these sequences in the correspondingly transformed host. This method can be used to obtain mutant proteins presented in this invention. However, these mutant proteins can also be obtained, although less preferably, by chemical synthesis or by a combination of chemical synthesis and recombinant DNA. Methods of mass production or perfusion of production in General correspond to a given field of technology. Cm. Freshey, R. I. (ed), “Animal Cell Culture: A Practical Approach”, 2nd ed., 1992, IRL Press, Oxford, England; Mather, J. P. “Laboratory Scaleup of Cell Cultures (0.5 to 50 liters)”, Methods Cell Biology 57: 219-527 (1998); Hu, W. S., and Aunins, J. G., “Large-scale Mammalian Cell Culture, Curr Opin Biotechnol 8: 148-153 (1997); Konstantinov, K. B., Tsai, Y., Moles, D., Matanguihan, R., “Control of long-term perfusion Chinese hamster ovary cell culture by glucose auxostat”, Biotechnol Prog 12:100-109 (1996).
In one embodiment, the implementation of the recombinant method of producing a mutant protein described in the invention, the DNA sequence design by highlighting or izina (I) site-specific mutagenesis. This method is well known. See, for example. Mark et al., “Site-specific Mutagenesis Of The Human Fibroblast Interferon Gene”, Proc. Natl. Acad. Sci. USA 81, p. 5662-66 (1984); and U.S. patent No. 4588585 included in this description by reference.
Another method of constructing a DNA sequence that encodes a mutant protein of IL-2, described in this invention, is a chemical synthesis. It includes, for example, direct chemical synthesis of a protein sequence that encodes a mutant protein of IL-2, having the properties described in this invention. This way you can include both natural and unnatural amino acids in positions that affect the interaction of IL-2 to IL-2Ror IL-2R. Alternate the gene encoding the desired mutant protein of IL-2, can be synthesized chemically using oligonucleotide synthesizer. Such oligonucleotides designed based on the amino acid sequence of the desired mutant protein of IL-2 and selecting those codons that are preferred host cells that received recombinant mutant protein. In this respect take account of the fact that the genetic code is degenerated, one amino acid may be encoded by more than one codon. N is(W) is encoded by a single codon, TGG. Accordingly, you should note that for a given sequence of DNA that encodes a specific mutant protein of IL-2, there are several degenerate DNA sequences encoding this protein. Consider, for example, that in addition to the DNA sequence to protein D20I shown in SEQ ID NO:1, there are several degenerate sequences encoding this protein. Such degenerate DNA are considered in the scope of this invention. Thus, the term “degenerate variants” in the context of this invention means all DNA sequences coding and allowing the expression of specific mutant protein.
The DNA sequence encoding a mutant protein of IL-2, described in this invention, obtained by site-directed mutagenesis, chemical synthesis or by other methods can include or not to include DNA encoding the signal sequence. This signal sequence, if present, must be recognized by the cell selected for expression of mutant protein of IL-2. The sequence can be prokaryotic, eukaryotic, or a combination of these two types. It could also be a signal sequence prirodnogo protein of IL-2 from recombinant cells in which it was received. If selected prokaryotic cells, it is generally preferable that the DNA sequence does not encode a signal sequence. If selected eukaryotic cells, it is generally preferable that was coded signal sequence, and it is most preferable to use a signal sequence of IL-2 wild-type.
For the synthesis of the gene encoding the mutant protein of IL-2, corresponding to this invention, it is possible to apply standard methods. For example, you can use the full amino acid sequence for constructing back translated gene. It is possible to synthesize a DNA oligomer containing a nucleotide sequence encoding a mutant protein of IL-2. For example, it is possible to synthesize several small oligonucleotides coding for part of the desired polypeptide, and then ligitamate them. Individual oligonucleotides usually contain overlapping 5’ or 3’ sites for complementary Assembly.
Being assembled (by synthesis, site-directed mutagenesis or another method), the DNA sequence encoding a mutant protein of IL-2, must be entered in the vector for expression and is associated with controlling the expression of polnost Assembly can be confirmed by nucleotide sequencing, restriction mapping and expression of biologically active polypeptide in a suitable host. In the art it is known that to obtain high levels of expression transfitsirovannykh gene in host gene to be associated with the control of transcriptional and translational expression sequences, functional in the selected host.
The choice of controlling the sequence and expression vectors for expression depends on the choice of the owner. May be involved in a wide range of combinations of host/vector for expression. The vectors used for expression in eukaryotic hosts include, for example, vectors containing regulatory expression sequence from SV40, human papilloma virus of cattle, adenovirus or cytomegalovirus. The vectors used for expression in bacterial hosts include known bacterial plasmids, such as plasmids from E. coli including col E1, pCR1, pER32z, pMB9 and their derivatives, plasmids with a wide possess narrow specificity, such as RP4, phage DNA, such as the numerous derivatives of phage lambda, e.g NM989, and other DNA phages, such as M13 and filamentous phages with single-stranded DNA. The vectors used for axpw insect cells, include pVL 941. We prefer pFastBac1 (GibcoBRL, Gaithersburg, MD). Gate et al., “Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells”, Cell, 45, p.685-98 (1986).
In addition, these vectors can be used any of a wide set of controlling the expression of sequences. Used to control the expression sequences include sequences associated with the structural genes of the above-mentioned vectors for expression. The examples used in controlling the expression of the sequences are, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the areas of the main operator and promoter of phage lambda, e.g. PL, control plots fd envelope protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, such as PhoA, the promoters of the yeast system-mating, polyhedral promoter of Baculovirus and other known sequences controlling the expression of genes of prokaryotic or eukaryotic cells or viruses, and various combinations of these sequences.
To obtain mutant proteins IL-2, described in annulosa yeast), plants, insects, mammals, or other suitable cells or cell lines of animals; as well as transgenic animals or plants. In more detail, these hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (Sf9), animal cells such as ovarian cells of the Chinese hamster 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 and plant cells in cell culture. For expression in animal cells, we prefer cell culture Cho and COS 7, and especially the line Cho cells Cho (DHFR-) or LCI.
Note that not all vectors and controlling the expression of sequences will function equally well when the expression of these DNA sequences. And also not all hosts are equally suitable for one expression system. However, the specialist in the art may make a selection among these vectors, which controls the expression sequences and hosts without undue experimentation. For example, the choice of the vector should be considered the owner, SQL chopinot and the expression of any other proteins, encoded by this vector, such as markers of antibiotics. For example, the vectors preferred for carrying out this invention include those that allow encodes a mutant protein DNA to amplificates with a certain number of copies. Such amplificatoare vectors are well known in the art. They include, for example, vectors that can accumulate DHFR amplification (see, for example, Kaufman, United States Patent 4470461, Kaufman and Sharp, “Construction Of A Modular Dihydrafolate Reductase cDNA Gene: Analysis Of Signals Utilized For Efficient Expression”, Mol. Cell. Biol, 2, p.1304-19 (1982)) or by amplification with glutamine synthetase (GS) (see, for example, U.S. patent 5122464 and published European application 338841).
When choosing a sequence controlling the expression, you should consider a variety of factors. These factors include, for example, the relative strength of the sequence, its controllability, and compatibility with the DNA sequence that encodes a mutant protein of IL-2, especially the possible secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded described in the invention, the DNA sequences of the characteristics of their secretion, their ability to properly kladiva what sledovatelnot DNA.
Given these parameters, a specialist in the art may select various combinations of the vector/sequence control expression/owner that will Express the desired DNA sequence in fermentation or large-scale use of animal culture, for example, when using cells Cho or COS 7.
Obtained according to this invention the mutant proteins IL-2 can be glycosylated or deglycosylated depending on the host organism used to produce protein. If the owner of the selected bacteria, mutant proteins IL-2 will be deglycosylated. On the other hand, eukaryotic cells will glycosylate mutant proteins IL-2, although perhaps not quite as glycosylated natural IL-2. Mutant protein produced by the transformed host can be cleaned by any suitable method. There are various methods for the purification of IL-2. See, for example, Current Protocols in Protein Science, v. 2. Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc.). The authors prefer pereosazhdeniya from inclusion bodies produced in E. coli, or from conditioned medium of cell cultures of yeast or mammals, producing danfei. See below examples 1 (E. coli) and 10 (perfusion Cho cells).
Biological activity described in the invention of mutant proteins IL-2 can be monitored by any suitable method known in the art. Such studies include the proliferation of PHA-blasts and NK-cells. Mutant proteins with the appropriate activity, i.e. fully active against IL-2Rwith low activity in cells bearing IL-2Rdetermine the results of the two analyses. “The relative activity of the mutant protein is measured with respect to IL-2 wild-type and as described in the examples below represents the ratio of the activity of proliferation of PHA-blasts to activity proliferation of NK-cells.
Described in the invention of the mutant protein IL-2 should be prescribed at a dose approximately corresponding to or greater than the dose used in the treatment of natural or recombinant IL-2 wild type. It is preferable to administer an effective amount of a mutant protein of IL-2. “Effective amount” means an amount capable of preventing or reducing the severity or spread of the condition or testimony, in which the spas protein IL-2-dependent disease, dose, the schedule of reception of mutant protein of IL-2 that is assigned if the mutant protein of IL-2 alone or in combination with other drugs, from time-life composition in serum and General health of the patient.
The mutant protein of IL-2 is preferably introduced into the composition, comprising a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means a carrier that does not have adverse effects on patients, in whom it is prescribed. Such pharmaceutically acceptable carriers well known in the art. We prefer 2% HSA/PBS at pH 7.0.
Mutant proteins IL-2 can be included in the pharmaceutical compositions is well-known methods. For example, in Remington''s Pharmaceutical Science by E. W. Martin, included in the description of the invention by reference, describes suitable compositions. Pharmaceutical composition with a mutant protein of IL-2 can be prepared in various forms, including liquid, gel, liofilizirovannoe substance or other suitable shape. The preferred form depends on the specific indications for use and will be obvious to a person skilled in the art.
Pharmaceutical composition with a mutant protein of IL-2 can naznachatb the integral way. The preferred method of administration depends on the specific indications for use and will be obvious to a person skilled in the art. A pharmaceutical composition comprising a mutant protein of IL-2 may be administered together with other drugs. These tools can be introduced into the composition of a given pharmaceutical composition or applied separately from the mutant protein of IL-2, either simultaneously or according to another schedule of admission. In addition, the pharmaceutical composition with a mutant protein of IL-2 can be used as an adjunct with other therapies.
Thus, this invention provides compositions and methods for the treatment of HIV, cancer, autoimmune diseases, infectious diseases, for use as adjuvant cancer vaccines and conventional vaccine therapy, to stimulate the immune system in the elderly or other patients with a weakened immune system, as well as in patients with SCI, or for other therapeutic methods, requiring General stimulation of the immune system of animals, preferably mammals, most preferably humans. As mentioned previously, IL-2 has different activities. These include stimulation of PHA-blasts, rest is completely on the cells, expressing only the high-affinity IL-2 receptor, such as resting T cells, but not those that will expressyour receptor with intermediate activity, such as NK cells or monocytes.
It also assumes the use of DNA sequences encoding mutant proteins IL-2 gene therapy. Application in gene therapy involves the treatment of those diseases in which it is expected that IL-2 will provide a therapeutic effect due to the activation of T cells, for example, HIV, cancer, autoimmune disease, infectious diseases, for use as adjuvant cancer vaccines and conventional vaccine therapy, to stimulate the immune system in the elderly or other patients with a weakened immune system, as well as in patients with SCI, and other diseases or infectious agents that are susceptible to IL-2 mediated immune response.
Local delivery of mutant proteins IL-2 when using gene therapy may provide a hit of the drug in the desired area. Techniques for gene therapy both in vitro and in vivo. There are several methods of delivery of potentially therapeutic genes to specific cell populations. See, for example, Mulligun, “The BasGene transfer Into Mouse Muscle In Vivo”, Science, 247:1465-68 (1990);
2) delivery of DNA via 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. Biophvs. Res. Comm., 179:280-85 (1991);
3) DNA transfer via 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); Anderson, “Human Gene Therapy”, Science, 256:808-13 (1992);
4) the transfer of DNA by DNA-containing viruses. Such DNA viruses include adenoviruses (preferred vectors based on the Ad-2 or Ad 5), herpes viruses (preferred vectors based on herpes simplex virus and parvovirus (preferred vectors on the basis of defective or non-Autonomous parvoviruses, preferred vectors based adenoassociated viruses, the most preferred on the basis of AAV-2). See, for example, li et al., “The Use Of DNA Viruses As Vectors For Gene Therapy”, Gene Therapy, 1:367-84 (1994); U.S. patent 4797368 included in the description by reference, and U.S. Patent 5139941 included in the description by reference.
The choice of a particular vector system to transfer the desired gene depends on various factors. One of the important factors is the nature of a population of target cells. the, not suitable for infection of non-dividing cells. In addition, retroviruses have oncogenic potential.
Advantages of adenoviruses consist in the fact that they have a wider possess narrow specificity can infect silent and finally differentiated cells such as neurons, hepatocytes, and essentially neocolony. See, for example, Ali et al., “The Use Of DNA Viruses As Vectors For Gene Therapy”, Gene Therapy, 1:367 (1994). Adenoviruses, apparently, does not integrate into the host genome. Because they exist extrachromosomal, significantly reduces the risk of insertional mutagenesis. li et al., “The Use Of DNA Viruses As Vectors For Gene Therapy”, Gene Therapy, 1:373 (1994).
Adeno-associated viruses (AAV) have the same advantages as vectors based on adenoviruses. However, AAV demonstrate the ability to site-specific integration into human chromosome 19. Ali et al., “The Use Of DNA Viruses As Vectors For Gene Therapy”, Gene Therapy, 1:377 (1994).
In a preferred variant of the invention, the DNA encoding a mutant protein of IL-2 used in gene therapy for immunodeficiency diseases such as HIV; infectious diseases, such as tuberculosis; and cancers such as carcinoma of the kidney.
According to this variant implementation of gene therapy using Diallele or immediately after diagnosis.
This approach has the advantage of selective activity of mutant proteins IL-2 and prevents toxicity and adverse effects. Specialist in the art will understand that according to the method of the invention can use any suitable vector for gene therapy comprising DNA encoding a mutant protein of IL-2. Methods for constructing such vectors are known. See, for example, Anderson, W. F., Human Gene Therapy, Nature, 392 25-30 (1998); Verma, I. M. and Somia, N., Gene Therapy - Promises, Problems, and Prospects, Nature, 389 239-242 (1998). Introduction of a vector containing DNA encoding a mutant protein of IL-2 in the desired site can be accomplished using known techniques.
For a better understanding of the invention examples. These examples are only illustrative and are not considered as limiting the scope and essence of the invention in any way.
Example 1. Obtaining mutant proteins in E. coli. Mutant proteins were obtained from site-directed mutagenesis using primers containing codons related to the desired mutation, as described Kunkel TA, Roberts JD, and Zokour RA, “Rapid and efficient site-specificmutagenesis without phenotypic selection” (1987), Methods Enzymol 154:367-382. Briefly, the human cDNA of IL-2 containing sites Resta sites. cDNA of IL-2 wild type was obtained using polymerase chain reaction (PCR) with a pool of cDNA generated from mRNA isolated from peripheral blood lymphocytes from human induced for 24 hours 12-myristate-13-phorbol acetate (10 ng/ml). In PCR primers were used: 5’ end of the open reading frame of IL-2:
5’-CCT CAA CTC CTG AAT TCA TGT ACA GGA TGC-3’ (SEQ ID NO: 3);
and for the 3’ end of the open reading frame of IL-2:
5’-GGA AGC GGA TCC TTA TCA AGT CAG TGT TGA G-3’ (SEQ ID NO: 4).
In each oligonucleotide were included sites of enzymes Eco RI (5’ end) and Barn HI (3’ end), these are shown in italics. PCR was performed under the following conditions: 1 minute at 94C, 1 minute at 58,7C and 1 minute at 72C for 25 cycles. The accuracy obtained in this way IL-cdnc was confirmed by sequencing using the Sequenase kit(Amersham Life Sciences, Arlington Heights, IL) according to the Protocol provided by the manufacturer. Containing the single-stranded uracil DNA (U-DNA) was obtained by transformation of strain CJ236 E. coli (Bio-Rad Laboratories, Hercules, CA) vector M13 mp19 containing U-cdnc. For site-directed mutagenesis was used primers containing 15 nucleotides homologous matrix U-DNA stow, homologous matrix U-DNA with a 3’ end from the last modified nucleotide. Original site-directed mutagenesis was used to introduce restriction site NcoI at the beginning of the Mature sequence of human IL-2. Introduction this site comprises the N-terminal methionine residue, which directs expression in the cytoplasmic space of E. coli when using, for example, the vector for the expression of pET3d. The primer used for this purpose consisted of:
5’-GCA CTT GTC ACA AAC ACC ATG GCA CCT ACT TCA AGT-3’ (SEQ ID NO: 5)
Specific primers used to introduce mutations at position D20, N88 and Q126, consisted of:
D20X: 5’-GGA GCA TTT ACT GCT GNN NTT ACA GAT G-3’ (SEQ ID NO:6)
N88X: 5’-GGG ACT TAA TCA GCN NNA TCA ACG TAA TAG-3’ (SEQ ID NO:7)
Q126X 5’-GGA TTA CCT TTT GTN NNA GCA TCA TCT C-3’ (SEQ ID NO:8)
where NNN is replaced with the appropriate codons histidine (CAC) or isoleucine (ATC) (position D20), arginine (CGT), glycine (GGT), or isoleucine (ATC) (position N88), or leucine (CTG) (position Q126). Other mutations were made using similar strategies and the corresponding codon for this mutation. Primers were fosforilirovanii T4 polynucleotide kinase (New England Biolabs, Beverly, MA) according to the Protocol provided by the manufacturer. After annealing of primer on the matrix U-DNA and �r/8482.gif">(GibcoBRL, Gaithersburg, MD) were transformed with 5 μl of the reaction mixture and were sown on LB medium containing 0.7% agar. After incubation at 37With the material of the colonies was multiplied by selection of individual colonies and transfer them into 2 ml of LB medium, followed by incubation at 37With during the night. Single-stranded DNA was obtained using the dial to highlight M13 (Quiagen, Inc., Chatsworth, CA) according to the Protocol provided by the manufacturer, and clones containing the desired mutation were identified by sequencing single-stranded DNA using a kit Sequenase sequencing(Amersham Life Sciences, Arlington Heights, IL) according to the Protocol provided by the manufacturer. cDNA mutant protein of IL-2 from the replicative form DNA corresponding to the colonies containing the correctly mutated sequence was isolated using Nco I and b I and was subcloned into the plasmid vector rate (Stratagene, San Diego, CA) (Strat). The strain E. coli BL21 transformed containing mutant protein vector rate, raised to ABS280(optical density at 280 nm) of 0.60 to 1.0, and at this point was added to 0.4 mm IPTG (isopropylthioxanthone) for the induction of production of mutant protein of IL-2.
Example 2. Extraction and purification p://img.russianpatents.com/chr/215.gif">g. For renaturation and purification of recombinant mutant proteins IL-2 cells were first dispersible in 10-fold volume (volume/wet weight) buffer sucrose/Tris/EDTA (0,375 M sucrose, 10 mm Tris Hcl pH 8.0, 1 mm EDTA). Dispersed cells were voiced by ultrasound at 300 W with 30-second intervals in an ice bath on the device Missonix model XL2020, equipped with 1-inch standard electrode. Then voiced the material was centrifuged at 17,000g for 20 minutes at 4C. the Precipitate, which at this stage should be white, washed with resuspending and centrifugation once in the buffer sucrose/Tris/EDTA, twice in buffer Tris/EDTA (50 mm Tris Hcl, pH 8,0,1 mm EDTA), then resuspendable in 10-fold volume of buffer 0.1 M Tris Hcl, pH 8.0 (at this stage, we selected a sample for analysis in the gel) and centrifuged for 20 minutes at 17,000g.
The precipitate was dissolved by adding 3-fold volume of 8 M guanidine chloride in 0.1 M Tris Hcl (pH 8.0) and 0.1% (vol/vol) 2-mercaptoethanol. After incubation for 2 hours at room temperature the material was centrifuged for 20 minutes 17,000g. The resulting solution were dialyzed for was brilliantdigital at 17,000g for 20 minutes, brought triperoxonane acid up to a concentration of 0.1% and filtered through a 0.22 μm filter. The solution is immediately transferred into siliconized vial and loaded on a C8 column (Vydac 208TP54). Mutant proteins IL-2 was purified using 20-minute linear gradient using 45-85% acetonitrile in 0.1% TFU. The concentration lirovannomu protein was determined by measure A and amino acid analysis. Then the protein was divided into aliquots (100 ml) in siliconized test tubes and kept at -20C. Thus, the purified mutant protein was a single band in SDS-polyacrylamide gel (SDS-page) (SDS - sodium dodecyl sulphate) (silver staining) and was estimated by amino acid analysis (typical accuracy of >90%).
Example 3. Study the proliferation of T-cells
Menagerie cells peripheral blood (RVMS) was isolated from approximately 100 ml of normal human blood (Irwin Memorial Blood Bank, San Francisco, CA), diluted in the ratio 1:2 cold Dulbecco phosphate buffer (free of CA2+and Mg2+, DPBS). The material was centrifuged on a substrate Ficoll-Paque (Pharmacia) to separate cells RUMS, which are then washed with large quantities of cold DPBS. PHA-blasts (activated T-chattaroy was added to 1% (weight/volume) of each of the components: L-glutamine; non-essential amino acids; sodium pyruvate; and antibiotic - protivolakoznoe substance (RPMI medium) at a density of 1106cells/ml was Added phytohemagglutinin (PHA-P; Sigma) to a final concentration of 10 μg/ml, and incubated the cells at 37C, 5% CO2within 3 days. The cells were collected and washed twice in DPBS, resuspendable in RPMI medium and were sown in 96-well tablets with a flat bottom with a density of 1106cells/well in 200 μl of various concentrations of IL-2 or mutant protein in RPMI medium. The plates were incubated for 48 hours at 37With, and treated with 1 mccu3H-thymidine (DuPont NEN, Boston, MA)/well in 6 hours, collected and measured radioactivity after collecting the cells on the filters fiberglass.
Example 4. The study of the proliferation of NK-cells
Menagerie cells peripheral blood (RVMS) was isolated from approximately 100 ml of normal human blood (Irwin Memorial Blood Bank, San Francisco, CA), diluted in the ratio 1:2 cold Dulbecco phosphate buffer (free of CA2+and Mg2+, DPBS). The material was centrifuged on a substrate Ficoll-Paque (Pharmacia) to separate cells RUMS, which are then washed out ballymaloe NK cells Miltenyi Biotec (Bergisch Gladbach, Germany; Cat# 465-01). The set consists of two reagents, columns for separation and very powerful magnetic tripod for the column. The first reagent is a mixture of conjugated with a hapten monoclonal antibodies CD3, CD4, CD19, CD33 isotype IgG1 mouse. This reagent is intended for separation of cells RUMS from T cells, b cells and myeloid cells. Apparently, you can use any suitable set of antibodies that recognize these types of cells. The second reagent consists of colloidal superparamagnetic particles microbasin MAC conjugated with antiJapanese antibody. Cells resuspendable in PBS containing 0.5% bovine serum albumin and 2 mm EDTA (PBS/EDTA). The volume of the suspension depends on the number of cells and is shown in the graph Miltenyi Biotec. Usually 2-5108cells RVMS resuspendable 800 ál of buffer and then used 200 μl of each reagent. After incubation with reagents cells (resuspendable in 2 ml buffer) was applied on the column. All cells are NK cells, are associated with the magnet, a NK-cells are separated and collected at the outlet. Cells were washed, resuspendable in RPMI medium (contains: RPMI 1640, which added up to 1% of each of the components: L-glutamine; non-essential amino acids; sodium pyruvate; and antibiotic - and 96-well tablets with a flat bottom with a density of 1105cells/well in 200 μl. The cells were collected and washed twice in DPBS, resuspendable in RPMI medium and were sown in 96-well tablets with a flat bottom with a density of 1x105cells/well in 200 μl of various concentrations of IL-2 or mutant protein in RPMI medium. The plates were incubated for 48 hours at 37With, and treated with 1 mccu3H-thymidine (DuPont NEN, Boston, MA)/well in 6 hours, collected and measured radioactivity after collecting the cells on the filters fiberglass.
Example 5. Mutant proteins that activate T-cells, preferably before NK-cells
Mutant proteins were obtained using site-directed mutagenesis (Kunkel et al. (1987), Methods Emzymol 154: 367-382) in provisions Asp-20, Asn-88 and Gln-126, and expressed in E. coli using the system for the expression of pet-3A according to the description provided by the manufacturer (Stratagene). Mutant proteins were purified by recovery of the bodies included in the guanidine-HCl, perioadele and subjected to HPLC as described previously. The obtained protein was characterized by a purity >95% according to the analysis in painted silver SDS-page; the concentration and purity of the protein was determined by amino acid analysis (AAA) (accuracy AAA usually >90%). The sequence of the mutant be subjected to the above studies on T - and NK-cells. The relative activity of mutant proteins IL-2 in these studies are shown in table. 1.
Activity of mutant proteins described as the relative concentration of the mutant protein, required for obtaining 50% of maximum response (EC50), in comparison with EC50for IL-2 wild type in the same study; in the case of multiple analyses of a single mutant protein is the geometric mean of the values obtained. Values EU50for IL-2 wild-type lie in the range between -10 and 150 PM in the research on T-cells, and -50 and -200 RM in research on NK-cells. This ratio is calculated for each mutant protein with regard to activity, as defined in the research on T - and NK-cells obtained from a single donor.
Activity of mutant proteins can be divided into 6 broad categories: 1) 1000-fold selectivity relative to T cells; 2) 100- <1000-fold T-cell selectivity; 3) 10- <a 100-fold selectivity relative to T-cells; 4) activity against T-cells, increased IL-2; 5) selectivity with respect to NK cells; 6) 10-fold selectivity or T, or NK-cells.
Class 1: D20H, I, and Y; N88G, I and R.
TO THE Q126A, N.
Class 6: D20A, T and V; N88H, K, L, W, and Y; Q126K, P, T, W and y
Inside classes 1-3 are preferred mutant proteins with T-cell activity, which is close to or greater than the activity of IL-2 wild type. In class 1 are proteins D20H and I; N88G, I and R; in class 2 - N88M and T, Q126D, E, G, L and V; grade 3 - N88A, Q126F and G. Predicted that the mutant proteins of Class 4 have more than IL-2 wild-type, potential activity in vivo.
According to the table. 1 you will notice that no single mutation did not lead to inactivation of the mutant protein of IL-2 in both studies. However, it can be concluded that the combination of mutations at specific positions, which led to a significant decrease in activity could potentially lead to antagonistic activity of IL-2 on T cells or other cells bearing high-affinity IL-2 receptor. The receipt of such antagonist is predicted on the basis of these data, as mutations were made only to change the interaction with the IL-2Rand IL-2R. One such example can be a double mutant D20R/Q126T. It is assumed that the combination of mild mutations in a single molecule will have combinatory effect, i.e. the activity D20R on T-cells 0,00018 activity Q126T is 0.0001, and R According to the information provided can be other combinations of mutations.
Example 6. The biological activity of IL-2 wild-type and mutant proteins IL-2
In Fig.1-7 shows the curves of the dose for IL-2 wild-type (IL-2) and D20H (Fig.1), IL-2 and D20I (Fig.2), IL-2 and N88G (Fig.3), IL-2 and N88I (Fig.4). IL-2 and N88R (Fig.5), IL-2 and Q126E (Fig.6) and IL-2 and Q126L (Fig.7). A: Individual response, the dose of IL-2 (filled circles) and mutant protein (unfilled circles) in the study of proliferation of primary T cells (PHA-blasts) person. In: Individual response, the dose of IL-2 (filled triangles) and mutant protein (unfilled triangles) in the study of proliferation of primary NK cells.
In particular, according to the schedule shown in Fig.1 experiment the dose of IL-2 wild-type, giving 50% of maximal proliferation (EC50), ~1.510-10M in the study on T-cells (a) and ~110-10M - NK-cells). The EC50for D20H was ~210-10M in the study on T-cells and assessed >110-5M in NK-cells. Thus, in this study, the final excess activity D20H on T-cells on the activity of NK cells is >50,000 times. Similar results were obtained when using the CSO type, giving 50% of maximal proliferation (EC50), is -1,510-10M in the study on T-cells (a) and ~310-10M - NK-cells). The EC50for D20I was also -1,510-10M in the study on T-cells and was estimated ~510-6M in NK-cells. Thus, in this study, the final excess activity D20I on T-cells on the activity of NK cells is ~16,000 times.
Similar results were obtained using blood donors (data not shown).
According to the schedule shown in Fig.3 experiment the dose of IL-2 wild-type, giving 50% of maximal proliferation (EC50), is ~410-11M in the study on T-cells (a) and ~210-10M - NK-cells). The EC50for N88G was ~510-12M in the study on T-cells and was estimated ~310-8M in NK-cells. Thus, in this study, the final excess activity N88G on T-cells on the activity of NK cells is ~1,200 times. Similar results were obtained when using the CSO type, giving 50% of maximal proliferation (EC50), ~1.510-10M in the study on T-cells (a) and ~110-10M - NK-cells). The value of EC50for N88I was ~410-10M in the study on T-cells and was estimated ~510-6M in NK-cells. Thus, in this study, the final excess activity N88I on T-cells on the activity of NK cells is ~18,000 times. Similar results were obtained using blood donors (data not shown).
According to the schedule shown in Fig.5 experiment the dose of IL-2 wild-type, giving 50% of maximal proliferation (EC50), ~1.510-10M in the study on T-cells (a) and ~110-10M - NK-cells). The value of EC50for N88R was ~910-11M in the study on T-cells and was estimated ~310-7M in NK-cells. Thus, in this study, the final excess activity N88R on T-cells on the activity of NK cells is ~5,000 times. Similar results were obtained using blood trauma 50% of maximal proliferation (EC50), is ~810-12M in the study on T-cells (a) and ~510-11M - NK-cells). The EC50for Q126E was ~810-13M in the study on T-cells and was estimated ~210-9M in NK-cells. Thus, in this study, the final excess activity Q126E on T-cells on the activity of NK cells is ~400 times. Similar results were obtained using blood donors (data not shown).
According to the schedule shown in Fig.7 experiment dose IL-2 wild-type, giving 50% of maximal proliferation (EC50), is ~510-11M in the study on T-cells (a) and ~810-11M - NK-cells). The value of EC50for Q126L was ~210-11M in the study on T-cells and was estimated ~210-8M in NK-cells. Thus, in this study, the final excess activity Q126L on T-cells on the activity of NK cells is ~625 times. Similar results were obtained using blood donors (data not shown).
Example 7 is about a pronounced selective agonistic activity against T-cells in research on primary T - and NK-cells. Compared with IL-2 wild type it in -6,000 times more active on T-cells than NK cells, as compared to T cells has activity essentially equal to the activity of IL-2 wild type. IL-2/N88R received in the cells of Cho, purified and evaluated the activity and toxicity in models of chimpanzees. On this model, in vivo the protein detected activity comparable to the activity of commercially available recombinant variants of IL-2 (PROLEUKIN, Chiron Corporation, Emeryville, CA), in contrast to the mutant protein which has only mild side effects (both objective and clinical parameters).
A. Experimental work
1. Materials and methods
Part of the research, including work with animals was conducted at the research center New Iberia Research Center, New Iberia, LA, Sponsor Bayer Corporation, Berkeley, CA) and consisted of two phases of research and 11 male chimpanzees from young to adult age, with body weight ranging from 45 to 70 kg
Phase I was the stage of determining the dose and included a subcutaneous injection of doses of filler or subcutaneous administration of doses Proleukin, constituting 1.2 mg/m2twice a day (BID) for 5 days. Phase II was to compare the actions of filler, Proleukin and IL-2/N88R, introduced subcutaneously, every nom, based on the pharmacokinetic analysis. Blood samples were collected for analysis of chemical composition of blood, SHS, Hematology/coagulation, and for FSK analysis (FGC - fluorescent sorting cells) populations of T - and NK-cells, as described in detail in sections 5 and 6.
2. Procedure dose
In the days when the blood was collected before the introduction of the analyte or filler animals were given General anesthesia using ketamine IM in the amount of approximately 10 mg/kg on days when sampling blood was not required, before the introduction of the analyte or filler animal was physically immobilized in the coop. The dose was administered subcutaneously every 12 hours for 5 days, the coat in the area of injection sheared on the first day of the study. Place and time of injection was recorded at each dose.
3. Clinical observations
(a) Daily observations and food consumption. Each animal was observed twice a day, and any deviations reported to the head of research. The animals looked sick, was taken under the supervision of a study leader, vet project and a representative of the sponsor. Food consumption was confirmed and recorded is the introduction of dose on the first day and each time the euthanasia of the animal blood collection.
(c) Observation of the injection site. The injection sites were examined daily. Any abnormalities such as redness or swelling, were recorded.
4. Working with samples
(a) Chemical analysis of the serum. From each animal in the above mentioned deadlines were collected approximately 2 ml of blood in tubes without anticoagulant. Documented the sampling time and left blood at room temperature for coagulation. Then the samples were centrifuged, collected serum and send it to the laboratory NIRC Clinical Pathology Laboratory. Standard panel study of serum laboratory NIRC presented in table. 4.
(b) Hematology. From each animal in the above mentioned deadlines were collected approximately 2 ml of blood in tubes with EDTA and sent to the laboratory NIRC Clinical Pathology Laboratory. Conducted standard for NIRC hematological study of all samples, including complete blood count, differential and platelet-derived formula.
(c) Research sponsor. In the above mentioned deadlines were collected approximately 6 ml of blood in tubes with EDTA as anticoagulant. Documented the time of blood collection. The samples were centrifuged, plasma was separated and carried aliquo the AI research was sent to Bayer Corporation, Berkeley, CA.
5. The study FGC
(a) Method. Approximately 5 ml of blood was collected in sodium heparin anticoagulant within the time specified for FSK analysis.
Full blood sample was obtained in EDTA or ACD, or heparin. The number of cells brought up to a level 2-20 thousand in mm3.
To conduct research on antibodies marked accordingly 1275 glass or plastic tubes. To each tube was added antibody or mixture of antibodies in the amount suggested by the manufacturer.
To each tube was added 100 μl of well-mixed blood sample and the mixture incubated for 30 minutes at room temperature protected from light.
After incubation were added to 2 ml lyse solution (Becton Dickinson FACS brand lysing solution, BD# 92-0002), gently mixed and left for 10 minutes at room temperature. Then the tubes were centrifuged at room temperature for 5 minutes at 300g. Decantation of supernatant, removing excess liquid, and to each cell precipitate was added 1 ml of PBS buffer (Gibco 14190-144). After careful mixing, the tubes were centrifuged at room temperature for 5 minutes at 300xiaoshahe solution (0.5% solution of formaldehyde, obtained by dilution of 10% formaldehyde (Polyscience, Inc. #0418) PBS buffer in the ratio 1:20), and gently resuspendable mixing. Then the samples were analyzed cytometer Coulter EPICS SL Flow Cytometer.
The study used the following antibodies: isotype control MigGl/MigG1 (Becton Dickinson, Cat. # 349526), CD45-PerCP (Becton Dickinson, Cat. # 347464), CD8-FITC (Becton Dickinson, Cat. # 347313), CD25-PE (Becton Dickinson, Cat. # 30795X), CD4-FITC (Becton Dickinson, Cat. # 340133), CD16-PE (Becton Dickinson, Cat. # 347617), CD3-PerCP (Becton Dickinson, Cat. # 347344).
6. The coagulation profile
The terms stated above were taken approximately 2 ml of full blood in tubes with sodium citrate as anticoagulant. The coagulation profile consisted of prothrombin time (PT), time activated partial thromboplastin (ART) and fibrinogen.
C. Results and discussion
1. Dose determination Proleukin
The primary goal of this phase was to determine the optimal dose Proleukin for comparison with IL-2/N88R. This dose Proleukin should be portable (smaller maximum tolerated dose) and ideally should cause clinically significant, but moderate and reversible toxic effect. Dose Proleukin, component 1.2 mg/m2twice a day, was defined as the start on the basis of cross-extrapolation of the requirements for doses prolactin specified. Throughout the course animals showed a significant decrease of activity, concern, and dehydration. Both animals on 3 or 4 day developed severe gastrointestinal symptoms, including decreased appetite, diarrhoea, vomiting. The introduction of the drug to one of the animals (room H-159) was discontinued after 3 days due to severe kidney (Fig.8A and C) and moderate hepatic (Fig.8C and D) dysfunction, reflected in the chemical levels in the blood. These indicators included increased levels of urea nitrogen, blood (BUN), creatinine and total bilirubin, ALT (alanine aminotransferase) (SGPT (serum glucopyranoside transaminase levels). Animal X-159 on the 3rd and 4th day, and the animal X-124 only on day 4 was injected intravenously solution of lactate ringer as a reducing agent and to prevent further dehydration. On the 8th day animal X-159 were excluded from the study due to renal dysfunction and a possible blood clot in his right leg, which was indicated very weak pulse (thigh) and the fact that all the eggs were cold and enlarged. Although significant toxicity was observed in one animal, the other was less severe symptoms, and the profile of adverse effects in both animals was reversible. Based on Alanta (exposure) dose IL-2/N88R, every 12 hours.
2. Comparison of IL-2/N88R and Proleukin
Clinical observations. In table. 5 shows the clinical observations made during the study. Values measured on a scale from 0 to 5, where 5 means “heavy”. All animals, which were injected Proleukin, were ill; were observed one death associated with the introduction of Proleukin. Values given after 6 days, for a group Proleukin reflect the data received from the remaining two animals. The most obvious side effect of IL-2/N88R was mild gastrointestinal upset (vomiting), although the introduction of drug induced conditional toxicity, the estimated parameters are given in table. 4. Body weight of animals of the group, which was introduced Proleukin, decreased by 6% on day 6 and 10% on day 10, then slowly recovered (Fig.9). In contrast, in animals from groups IL-2/N88R and Filler maximum observed decrease in weight was 1-3%.
(a) Hematology. IL-2/N88R caused cellular effects, consistent with activation of Proleukin, in particular lymphocytosis (Fig.10A), also observed a marked increase in the number of leukocytes (Fig.10B) and neutrophils (Fig.10C). For studies of IL-2/N88R caused neznicitelna 6 and 8 days, the BUN levels were significantly higher in animals which was introduced Proleukin (Fig.11A). The BUN levels were more than 130 mg/DL in two of these animals; also two animals raised creatinine levels at 6 and 8 days (Fig.11B), indicating that complete cessation of work of kidneys. In addition, in animals that were injected Proleukin, has greatly increased the deficit as phosphorus serum and anions at 6 and 8 days. In contrast, all animals that were injected IL-2/N88R, these settings work of the kidneys mainly remained normal throughout the study (Fig.11C and D).
(C) liver Function. Animals of the group, which was introduced Proleukin, total bilirubin more than tripled on day 6 and remained high up to 10 days (Fig.12A). In contrast, only one animal of the group IL-2/N88R was observed temporary slight increase of this parameter on the 3rd and 6th day. The level of SGPT serum dramatically increased and reached more than 100 IU/l in all animal groups, which were introduced Proleukin, on day 6 (Fig.12V). The SGPT level the animal A reached 651 u/l, and the level of SGOT (serum glutaredoxin transaminase levels) - 2789 u/l (Lab Log: NIR#8754-9852-Phase II, table 1. Individual and group average chemical indicators, page 17 of 21 table. ), which indicates severe liver failure.
(d) Coagulation. Euroweg maximum at b day (Fig.12C). The increase in animal groups, which were taken Proleukin, occurred earlier than in animals of group IL-2/N88R. This is reflected by the indicators 51% vs. 5% on day 3. Changes in the level of fibrinogen are more likely the result of acute protein response than defects of coagulation, as in this period there were no significant changes in the levels of ART or RT (Fig.12D). However, since 10 days, the animals of the group, which was introduced Proleukin, observed a clear trend towards increasing the level of ART, although the absolute values remained within normal limits. The same trend, albeit to a lesser extent, was observed in animals of group IL-2/N88R. Interesting was the fact that in the corresponding period of time, fibrinogen level decreased in both groups.
(e) Homeostasis. Two of the three animal groups, which were introduced Proleukin, the level of sodium in the serum decreased to values less than 135 mEq/l on day 8, but remained within the normal range in the remaining animals (Fig.13A). The level of chloride in animals group, which was introduced Proleukin, also decreased to values less than 95 mEq/l on day 3 and remained low up to 15 days (Fig.13B). The level of calcium was lower in two of the three animal groups, which were introduced Proleukin, 6 and 8 days and reached values of 4.9 and 3.1 mg/DL the th was introduced Proleukin, in addition to animal And 199, whose potassium level actually decreased to the level of toxicity 7.2 mEq/l before its exclusion from the study (Fig.13D).
(f) Signs of vascular permeability. The level of serum albumin was decreased in the group, which was administered (37%), and IL-2/N88R (19%) (Fig.14A). Increased levels of hematocrit was observed in the group which was administered Proleukin, 3 and 6 days (Fig.14V). On the contrary, as in the group treated filler, and IL-2/N88R the haematocrit level was decreased in the same period and remained low, indicating secondary anemia, which was expected due to repeated sampling of blood (Fig.14B and C). Increasing the level of hematocrit in the group, which was introduced Proleukin, together with a decrease in albumin is consistent with the development of the syndrome capillary permeability.
3. Activation of cells
Efficiency Proleukin and IL-2/N88R tracked by the difference in the percentage of CD25-positive lymphocytes (movement + proliferation) and on the value of the fluorescence intensity of CD25 or the number SW-antigens expressed on the surface of this T-cells (CD25=low-affinity IL-2R). The expression of CD25 tracked in the General population of T cells (CD3+ cells), as well as on the populations of CD3+CD4+ and CD3+CD8+.
The activity of IL-2 on NK-cells is Yali by multiplying the percentage of cells in the lymphocyte count in mm, the data received during the hematological analysis.
(a) SR-regulation of expression on the surface of T cells. According to the level of CD25-positive cells, the percentage of activated T-cells was regulated increase on day 6 of the study, mainly on CD3+CD4+ population of T cells. It turned out that Proleukin induces the expression of CD25 in a higher percentage of the total population of CD3+, CD3+CD4+ and CD3+CD8+ subpopulations of T cells on day 6. However, on day 8 the percentage of cells expressing CD25 antigen was identical in lymphocytes chimpanzees, which was introduced Proleukin, and chimpanzees, which was administered IL-2/N88R (Fig.15A, b and C). Neither the chimpanzee, which was introduced Proleukin, nor those who were injected IL-2/N88R, was not observed staining of CD25 on the surface of a population of NK-cells. It is assumed that this result is due to the lack of expression of IL-2R(antigen, which directed the staining of CD25 on the cell surface) on the surface of selected populations of NK cells (CD3-/CD16+).
The absolute number of CD3+CD25+ T cells was determined as the percentage of CD3+CD25+ T cells, and it turned out that Proleukin more active than IL-2/N88R (Fig.16A). Found that IL-2/N88R has the potential activation of T cells, the gathering is+ lymphocytes, caused by IL-2/N88R, identical to the same effect, caused by Proleukin (Fig.16). Discovered that Proleukin increases the number of CD3+CD8+CD25+ T cells to a greater extent than IL-2/N88R (Fig.16C).
With the introduction of as Proleukin and IL-2/N88R was observed identical kinetics of the number of molecules CD25 (fluorescence value), expressed on CD3+CD4+ subpopulation of T cells (Fig.17).
(b) the Movement of lymphocytes: the impact of the introduction Proleukin and IL-2/N88R
Activity Proleukin and IL-2/N88R on populations of T - and NK-cells was determined by analysis of differences in the absolute number of circulating lymphocytes before, during and after injection (Fig.18). Found that IL-2/N88R has more than Proleukin, activity increase in the absolute number of circulating CD3+CD4+ lymphocytes (Fig.18A) and slightly reduced in comparison with Proleukin activity increase in the absolute number of circulating CD3+CD8+ lymphocytes (Fig.18V). Both compounds have comparable, although a moderate effect on the increase in the total number of CD3+ lymphocytes (Fig.18C). No Proleukin, neither IL-2/N88R not affect the movement of NK-cells (Fig.18D).
IL-2/N88R was selected by analysis of mutant proteins IL-2 in studies on primary T - and NK-cells. It has 6,000 times the lo postulated, IL-2/N88R will cause only mild side effects at the dosage that will cause significant activation of T cells. Experiments on chimpanzees, comparing Proleukin IL-2/N88R, confirmed that IL-2/N88R has a much better safety profile than Proleukin while maintaining comparable ability to induce activation of T cells.
Example 8. The effectiveness of IL-2-selective agonist N88R in a murine model of metastatic lungs of tumor CT-26.
Methods: Mice (Balb/c, female, age 6-8 weeks) at day 0 were injected intravenously (IV) in the lateral tail vein 1105cells CT-26 (carcinoma of the colon of a mouse) in 0.2 ml PBS. The treatment was performed Proleukin or IL-2/N88R in different doses (solvent: 5% solution of dextrose in water (D5W) or D5W, administered intravenously once daily for 8 days (QD8), starting 1 day after implantation. IL-2/N88R was prepared at room temperature by dilution in D5W, using siliconized vials and tuberculin syringes, and introduced animals within 2 hours after cooking. Proleukin were prepared by adding each ampoule 0.7 ml of sterile water for injection (SWFI) (to a final concentration of 1.86 mg/ml). Cultivation was prepared as described in d ' in the solution Bowen (Bouin). After 24 hours, the tissue was transferred into a 10% formalin. The number of metastatic colonies in the lungs were counted under the microscope for dissection.
In table. 7 shows the number of metastases counted in each mouse. With the introduction of Proleukin and IL-2/N88R was observed comparable efficiency. With the introduction of high doses of IL-2/N88R (group 8, 60 mg/kg) in all mice, except one, were observed 12 metastasis or less. This result contrasts with the result of applying higher doses Proleukin, in which all surviving mice were observed 12 metastasis or more.
Significant reduction in the number of metastases was observed in mice which were injected IL-2/N88R in doses of 10,30 and 60 mg/kg (groups 6, 7 and 8, respectively) and in mice which were injected Proleukin in a dose of 10 mg/kg (group 3). These results are displayed graphically In Fig.19. Graph built using the logarithm of the dose: for Proleukin doses were 3 and 10 mg/kg; for IL-2/N88R - 1,3, 10, 30 and 60 mg/kg According to the curve for the non-linear law (4 parameters), the value of the IC50amounted to 5.2 mg/kg for Proleukin and 10.9 mg/kg for IL-2/N88R.
According to this experiment were calculated value IC50in relation to the reduction in the number of metastat 95%).
Data on survival of mice are presented in table. 8. In the group of mice that were administered 10 mg/kg Proleukin, one (1) mouse died on day 7 and five (5) - on the 8th day. One (1) mouse fell on day 8 in groups, which was introduced 3 or 10 mg/kg IL-2/N88R, and there was no deaths in mice which were injected 1, 30, or 60 mg/kg IL-2/N88R. In addition, the majority of mice that were injected 3 or 10 mg/kg Proleukin were dying, among mice who were injected with IL-2/N88R, dying was not observed.
Animals of both groups, which was introduced Proleukin, were dying. Among the animals, which were injected IL-2/N88R, dying was not observed.
So, in these studies it was shown that IL-2/N88R as effective in reducing the severity of neoplastic disease, as Proleukin (according to the calculation of lung metastases model ST-26). In addition, it was shown that IL-2/N88R significantly less toxic than Proleukin.
Example 9. Obtaining a stable, high-yielding cell lines Cho expressing IL2N88R
Cell lines with stable products, secreting large amounts of mutant protein IL2N88R, were obtained by transfection of cells CHO(dhfr-) vector for expression shown in Fig.20 (ATSS Deposit No. RTA-8). The individual elements of the vector for the=polyadenylation signal from SV40; DHFR=cassette expression of dihydrofolate reductase.
The vector is constructed using standard techniques of recombinant DNA. General references: Sambrook et al., Molecular Cloning, 2d ed., 1989, Cold Spring Harbor Press; Short Protocols in Molecular Biology, 2d ed., 1992, John Wiley & Son; Methods in Enzymologv, v. 185, Ed. Goeddel et al. Academic Press, Inc., London, 1991. The expression vector contains a separate cassettes for gene expression IL2N88R and amplifierarava and selective gene DHFR (dihydrofolate reductase). About 1106cells Cho (ovaries of Chinese hamsters) were transfusional 10 µg PBC1IL2SA when using Lipofectin reagent (Life Technology Inc., Bethesda, Maryland) according to the manufacturer's instructions. Then the cells were selected in the presence of 50 nm methotrexate and raised in the environment of DME/F12 deficient in thymidine and gipoksantin, with the addition of 5% cialisbuynow fetal bovine serum. Cell populations were analyzed for products IL2N88R using a commercial ELISA kit (R&D Systems). Then vysokoplodorodnye populations were selected in medium containing increasing concentrations of methotrexate (from 100 to 400 nm methotrexate) and analyzed for the production IL2N88R. Then used the clone with limited dilution to obtain clones with high and stable productivity. Reflection is>the example 10. Getting IL2N88R without the use of serum in the perfusion bioreactor
Continuous production IL2N88R was organized using a continuous perfusion fermentation. In 19-liter fermenter Wheaton (Wheaton) downloaded the stable cells lines SNO from example 9 at a concentration of 2106cells/ml and incubated at level sharing environment 5 l/day. Used the environment based on the environment DME/F12 (Life Technologies, Inc., Rockville, MD) supplemented with recombinant human insulin (10 μg/ml) (HUMULIN, li Lilly, Inc., Indianapolis, IN) and FeSO4EDTA (50 μm). Cell density was maintained at a level of 4106cells/ml Average daily output of the fermenter was ~200 mg/day. Products IL2N88R were stably maintained within 30 days.
Example 11. Purification of IL-2/N88R obtained in cells SNO
Perfusion eluate described above, were processed as follows. Perfusion medium was collected and applied to a column of S-separate. The column was called 20 mm phosphate buffer with 5 mm NaCl at pH 7.0. The conductivity of the source material (“TCF”) brought up 4 MSM by addition of water and the pH brought up to the specified values phosphoric acid.
After downloading TCF column was washed with the same b is mm ethanolamine, pH of 10.5, with the formation of S-eluate.
Anion exchange was performed by passing the S-eluate through a column of QAE Fast Flow(Pharmacia), adjusted to 10 mm bicarbonate buffer at pH of 10.5. The speed was maintained at a level of 250 cm/h After washing to the line of Foundation of IL-2SA was suirable 20 mm phosphate pH 4.0.
Chromatography on hydroxyapatite (NAR) was performed by passing the diluted eluate QAE (1:1 WFI (water for injection)) through a column Packed with ceramic hydroxyapatite (Type P, BioRad, Hercules, CA), adjusted to 10 mm phosphate at pH 7.0. The speed was maintained at a level of 250 cm/h. After washing to the line of Foundation of IL-2SA was suirable 100 mm phosphate pH 7.0.
The eluate with hydroxyapatite were subjected to ultrafiltration to a volume of 300 ml using a device Millipore Pelicon-2 with three cartridges PES 5K (Millipore Corporation, Bedford, MA).
The eluate NAR after ultrafiltration was purified by passing through a gel filtration column S100HR (Pharmacia) at a speed of 35 cm/h Column equalised 10 mm phosphate and 150 mm NaCl pH 7.0.
Fraction after gel filtration was diluted with WFI to achieve a conductivity of 4.0 MS/cm and repeatedly inflicted on the S-sepharose in the above-described conditions. IL2SA was suirable 10 mm Fosfatnym buffer with 1M NaCl at pH 7.0.
The final fraction after for the purpose of 6 mg/ml The resulting solution was sterile filtered, divided into aliquots and frozen at -70C. the Total yield was 65%.
Specialist in the art may develop other embodiments of the invention. From the invention follows the method of obtaining mutant proteins, not necessarily specifically described in this paper, but with the ability to activate T cells, as evidenced by the proliferation of PHA-blasts and reduced proliferation of NK-cells, and, thus, these proteins make up the volume and nature of the present invention. The concept and the experimental approach described in this paper are applicable to other cytokines using heterologous multivariate receptor systems, in particular this applies to the cytokines IL-7, IL-9 and IL-15, IL10, interferonand interferon.
This application contains the following sequence:
SEQ ID NO: 1: amino acid sequence of interleukin-2 (hIL-2)
SEQ ID NO: 2: cDNA sequence of IL-2
SEQ ID NO: 3: 5’ Primer for PCR of IL-2
SEQ ID NO: 4: 3’ Primer for PCR of IL-2
SEQ ID NO: 5: Primer for mutagenesis for IL-2 expression vector
SEQ ID NO: 6: Primer for mutagenesis for yle="text-align:center; margin-top:2mm;">Claims
1. The polypeptide representing the mutant protein interleukin-2 people with numbering according to IL-2 wild-type human IL-2 is substituted by at least one position 20, 88 or 126, allowing the specified mutant protein activates T-cells, preferably before natural killer (NK) cells.
2. The polypeptide representing the mutant protein IL-2 man under item 1, in which position 20 substituted compared to IL-2 wild-type.
3. The polypeptide representing the mutant protein IL-2 man under item 2, in which position 20 substituted with isoleucine.
4. The polypeptide representing the mutant protein IL-2 man under item 2, in which position 20 substituted with histidine.
5. The polypeptide representing the mutant protein IL-2 man under item 1, in which position 88 is substituted compared to IL-2 wild-type.
6. The polypeptide representing the mutant protein IL-2 man under item 5, in which position 88 is substituted with arginine.
7. The polypeptide representing the mutant protein IL-2 man under item 5, in which position 88 is substituted with isoleucine.
8. The polypeptide representing the mutant protein IL-2 man under item 5, in which position 88 is substituted for glycine.
9. Polypeptid is anyone type.
10. The polypeptide representing the mutant protein IL-2 man under item 9, in which position 126 replaced by aspartate.
11. The polypeptide representing the mutant protein IL-2 man under item 9, in which position 126 replaced by glutamate.
12. The polypeptide representing the mutant protein IL-2 man under item 9, in which position 126 replaced by leucine.
13. Pharmaceutical composition having immunostimulatory activity, comprising the polypeptide under item 1 in combination with a pharmaceutically acceptable carrier.
14. Polynucleotide, representing a DNA sequence encoding a mutant protein of IL-2 man under item 1, and degenerate variants.
15. Vector pBC1IL2SA for expression of mutant protein IL-2 containing polynucleotide under item 14.
16. Line ovary cells of Chinese hamsters, transformed by the vector according to p. 15, which produces a mutant protein is IL-2.
17. Line of African green monkey cell transformed by the vector according to p. 15, which produces a mutant protein is IL-2.
18. The strain of E. Coli transformed with the vector under item 15, which produces a mutant protein is IL-2.
19. Cell line Spodoptera frugiperda transformed by the vector according to p. 15, which produces mutantninja treatment of IL-2, by introducing a therapeutically effective amount of the mutant protein IL-2 man under item 1.
21. The method according to p. 20, where the disease is treatable with IL-2, is a cancer, including renal carcinoma, carcinoma, colon cancer, and malignant melanoma.
22. The method of selection of mutant proteins IL-2 assessment in studies using IL-2Rin comparison with IL-2Rwhere the activity of the mutant protein IL-2 is increased in relation to IL-2 wild type in one study, preferably in front of others.
23. The method according to p. 22, IL-2Rand IL-2Rare separate receptor subunit ectodomain in the correct combination and are used to assess direct binding of mutant proteins IL-2 with each receptor complex.
24. The method according to p. 22, where the study using IL-2Rtake into account the response of IL-2R-bearing cells, and the="https://img.russianpatents.com/chr/946.gif">-bearing cells.
25. The method according to p. 24, where IL-2R-bearing cells are PHA-blasts, and IL-2R-bearing cells are NK cells.
26. The method according to p. 25, where it is investigated proliferation as IL-2R-bearing cells, and IL-2R-bearing cells.