Polypeptides of directed action
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to cell biology, molecular biology, cancer biology and medicine and represents a pharmaceutical composition for targeting to the cells bearing BLyS receptor, containing an effective amount of fused protein consisting of BLyS polypeptide fused with cytotoxic polypeptide where said cytotoxic polypeptide is located on the N-end of fused protein, and BLyS polypeptide is located on the S-end of fused protein, and a pharmaceutically acceptable carrier.
EFFECT: invention provides treatment and prevention of B-cell proliferative disorder.
11 cl, 13 ex, 4 tbl, 20 dwg
The present invention claims the priority of provisional patent application U.S. serial number 60/649478, filed February 1, 2005, which is included in the present description by reference in full.
The technical FIELD TO WHICH the INVENTION RELATES
The present invention relates, at least, to the field of cell biology, molecular biology, cancer biology and medicine. More specifically the present invention relates to compositions containing a BLyS polypeptide conjugated with a cytotoxic agent, and to the use of such compositions in therapy.
BACKGROUND of INVENTION
B-lymphocyte stimulator is representative of the superfamily of tumor necrosis factor (TNF), which induces proliferation and differentiation of b-cellsin vivoandin vitro(Mooreet al., Science 285: 260-263 (1999)). BLyS is different from other b-cell factors proliferation and differentiation, such as IL-2, IL-4, IL-5, IL-6, IL-7, IL-13, IL-15, CD40L or CD27L (CD70), its gene, specific for monocytes and the expression pattern of the protein, as well as by specific receptor distribution and biological activity against b-lymphocytes. Expression of BLyS not found in natural cells natural killer (NK)T-cells or B-cells, and is limited by the myeloma cells of origin. Express what I BLyS on resting monocytes exposed to positive regulation of interferon-gamma (IFN-gamma). The gene encoding BLyS, was mapped on chromosome 13q34.
BLyS person is expressed in the form associated with the membrane of the polypeptide type II, consisting of 285 amino acids, and soluble polypeptide consisting of 152 amino acids (Mooreet al., 1999, supra). Associated with the membrane form of BLyS has a transmembrane domain between amino acid residues 47 and 73. NH2the end of the soluble form of BLyS starts from Ala134associated with the membrane form of BLyS. It was shown that soluble recombinant BLyS induced the proliferation of b-cells in the spleen of the mousein vitroand binds to a receptor on the cell surface in these cells (Mooreet al., 1999, supra). It was also shown that the introduction of soluble BLyS mice leads to increased CD45Rdull, Ly6Dbright(also known as ThB) B-cells and increased serum levels of IgM and IgA (Mooreet al., 1999 supra). Thus, BLyS demonstrates In-cell tropism as the distribution of its receptor, and biological activity.
Successful development of therapeutics aimed at the tumor, depends in part on the capabilities of the site-specific delivery of therapeutic agents, as well as from the biological activity of the delivered agent. Monoclonal antibodies are used to impart selectivity of cytotoxic agents, which otherwise do not distinguish the camping selectivity, such as toxins, radionuclides and growth factors (Williamset al., 1990; Rowlinson-Buszaet al., 1992; Wahl, 1994). One such molecule is gelonin, inactivating the ribosome plant toxin size of 29 kDa, with efficiency and mechanism of action similar to the a-chain of ricin (RTA), but has increased stability and reduced toxicity (Stirpeet al.,1992; Rosenblumet al., 1995). In the previous studies failed to identify and explore the biological properties of various chemical conjugates of plant toxin gelonin and different antibodies (Boyleet al., 1995; Xuet al., 1996; Rosenblumet al., 1999). In previous studies, the antibody ZME-018 was subjected to chemical binding with purified Galanina, and the specified immunoconjugate showed specific cytotoxicity against antigen-positive melanoma cells in tissue culture models with xenograft tumors of human (Rosenblumet al., 1991; Mujooet al., 1995).
Thus, there is a need for the development of improved therapeutic agents having specificity for an abnormally proliferating b-cells.
A BRIEF DESCRIPTION of the INVENTION
The present invention relates to methods for production and application of molecules that possess mistaway activity against, for example, cancer cells in Odesa part of the tumor, and cytotoxic activity. This molecule may include, for example, conjugated polypeptide. The present invention also relates to compositions, which is obtained on the basis of these kongugirovannah polypeptides. For example, blocksdelaware conjugates of the present invention relate to a connection that includes both the toxin and the BLyS polypeptide. In some embodiments, implementation of the present invention conjugated to polypeptides designed using recombinant methods of obtaining fused protein. Conjugated compounds can also be attached to each other, for example, through a linker.
In specific embodiments, the implementation of the present invention mishenina and cytotoxic activity given molecule at the expense of a separate fragment of the molecule, while the alternative one fragment may have partially or completely as mistaway and cytotoxic activity.
For specialists in this sphere, it is clear that the term BlyS can be used interchangeably with the terms polynucleotide or polypeptides BAFF, BLYS, TALL1, THANK, ZTNF4, TALL-1, TNFSF20 and Delta BAFF, although in alternative embodiments they may not be interchangeable. In addition, the specialist in this field know how to determine whether the specific molecules that could potentially b shall be interchangeable with BLyS, so appropriate to use them in accordance with the present invention and/or in accordance with currently available methods. As was shown in the present invention, SEQ ID NO: 1 refers to the BlyS polypeptide man full length of 285 amino acids. SEQ ID NO: 20 refers to a polynucleotide sequence BlyS person. Amino acid residues 134-285 in SEQ ID NO: 1 include the soluble isoforms of the BLyS polypeptide, which in some embodiments, implementation of the present invention is the isoform used in the conjugated polypeptides of the present invention. BLyS mouse (polypeptide SEQ ID NO: 2; nucleotide SEQ ID NO: 3) or other orthologues BLyS also included in the scope of the present invention. Functional equivalents of BLyS may also be used in combination conjugated polypeptides with directional effect of the present invention. For example, in the present invention are considered polypeptides that retain BLyS activity, which are characterized by at least 80%sequence homology, at least about 85%homology sequence at least 90%sequence homology with any of SEQ ID NO: 1 or SEQ ID NO: 2. In another specific embodiment, the present invention amino acid sequence BLyS comprises at least note the RNC 40, at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least 250, or at least about 275 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, functional equivalents include at least the D-E loop, involved in the recognition of the receptor (see Orenet al., Nat Struct Biol. 2002 Apr; 9(4): 288-92).
In some embodiments, can be used BLyS wild type, but in other embodiments can be used mutant BLyS. Examples of mutants BLyS include a mutant with a mutation in Cys146 (Chenet al.,2002; 2004; 2005) and in specific embodiments, the mutation can occur in alanine or valine. Mutants BLyS used in the present invention, also retain the ability to achieve In cells, in particular retain the ability to bind at least one BLyS receptor. Mutant BLyS may have increased any activity BLyS compared to wild type, including the ability to bind to B-cell, such as the ability to bind to BLyS receptor.
BLyS polypeptides with directed action according to the present invention is directed to cells that Express the receptor for BLyS, such as TNFRSF13B/TACI (SEQ ID NO: 4), TNFRSF17/BCMA (SEQ ID NO: 5) and TNFRSF13C/BFFR (SEQ ID NO: 6). In some embodiments, implementation of the present invention conjugated to the polypeptides of the present invention can specifically bind to a cell expressing a functional equivalent of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, implementation of the present invention is believed that cells expressing BLyS receptor, may be associated with abnormal proliferation of b-cells. For any specialist in this area with an average level of knowledge it is clear that BLyS may form dimers or trimers in order to participate in the recognition of the receptor. You should also understand that protein-containing composition conjugated polypeptides described in the present description, will also contribute to the formation of dimers BLyS, trimers or any other multimeric protein complexes.
In some embodiments, implementation of the present invention, the BLyS polypeptide is attached to the molecule. In preferred embodiments, the pacing is covalent. In additional embodiments, the implementation of the present invention indicated molecule is a cytotoxic agent, a drug, a chemotherapeutic agent, a radioactive isotope, a Pro-apoptotic agent, an antiangiogenic agent, a hormone, a cytokine, a growth factor, a peptide, a protein, an antibiotic, EN zymes is (such for example, as Granzyme B or Granzyme A), antibody, Fab fragment antibodies, visualisasi agent, nucleic acid or antigen. These molecules are given only as examples. Molecules included in the scope of the present invention include virtually any molecule that can be attached to a BLyS polypeptide, and can then be introduced into the human body. In preferred embodiments, the implementation of the present invention Pro-apoptotic agent is, for example, gramicidin, magainin, mellitin, defensin or cecropin. In other preferred embodiments, the implementation of the present invention antiangiogenic agent represents thrombospondin, angiostatin, endostatin or a factor derived from the pigment epithelium. In other preferred embodiments, the implementation of the present invention indicated cytokine represents, for example, interleukin-1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte-macrophage colony-stimulating factor). These examples are illustrative only and in no way exclude other Pro-apoptotic agents, antiangiogenic agents or cytokines known in this field.
Some embodiments of the present invention relate to recombinant peptide is th toxin gelonin (shown as SEQ ID NO: 7), which is described in U.S. patent No.5631348 and which is included in the present description by reference. Recombinant toxin gelonin of the present invention can be any part or fragment of SEQ ID NO: 7, which retains the toxin activity. In some embodiments, implementation of the present invention gelonin includes balances 110-210 of SEQ ID NO: 7. In other specific embodiments, the amino acid sequence r-gelonin includes at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, or at least 250 consecutive amino acids of SEQ ID NO: 7. Other compounds of the present invention include recombinant toxin gelonin, which preserves the nuclear section of the toxin, except that it contains at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive amino acid residues of SEQ ID NO: 7 in addition to the specified nuclear site of the toxin. In other embodiments, implementation of the present invention gelonin includes SEQ ID NO: 21 (the access number in GenBank No.P33186). In some embodiments, implementation of the present invention can be used rGel wild type, but in other embodiments can be used mutant rGel. In specific embodiments, the implementation of this izopet is of molecule rGel receive in accordance with the application for U.S. patent No: 10/074 596, submitted February 12, 2002, which is given in the present description by reference in full.
In one of the embodiments of the present invention representative conjugated polypeptide has the sequence of SEQ ID NO: 8. See as an example polynucleotide encoding specified conjugated polypeptide shown as SEQ ID NO: 9. Another variant of implementation of the present invention refers to a representative anywherefrom the polypeptide sequence SEQ ID NO: 10. Polynucleotide encoding specified conjugated polypeptide shown as SEQ ID NO: 11.
Other embodiments of the present invention relates to a cytotoxic agent, which may represent, for example, abrin, dodecandra, trichosanthin, trichokirin, Brodin, antiviral protein from Mirabilis, a protein, a ribosome inactivating barley (BRIP), antiviral proteins of lacunosa (PAP), saporin, Ruffini and/or MOMORDICA.
In some aspects of the present invention, the polypeptides of the present invention can be conjugated. Mostly these conjugated polypeptides contain at least five amino acids in length. In some embodiments, implementation of the present invention conjugated to the polypeptides have a length, for example, from about 5 to about 10 amino acids, from when is Erno 5 to about 50 amino acids, from about 5 to about 100 amino acids, from about 5 to about 150 amino acids, from about 5 to about 500 amino acids, from about 5 to about 1000 amino acids or from about 5 to about 5000 amino acids.
The conjugation of polypeptides of the present invention can be achieved by using appropriate methods, including, for example, chemical conjugation and genetic conjugation", in which the recombinant fused protein comprising a BLyS polypeptide functionally associated with cytotoxic peptide. Consider the conjugation with BLyS polypeptide includes the conjugation of the N-terminal site of the protein (within the first 100 amino acids), inner segment (between N-terminal and C-terminal regions) and/or C-terminal site of the protein (within the last 100 amino acids). Further detail of the method of conjugation considered for use in the present invention.
Conjugated polypeptides of the present invention can be expressed in exogenous mode. In a specific embodiment of the present invention the polypeptide comprises fused polypeptide or chimeric polypeptide. The chimeric polypeptide includes all or a discrete part of two or more polypeptides. A discrete portion of a polypeptide refers to an amino acid segment that contains identificarea function or activity. Protein is a chimeric protein in which the first polypeptide or a portion of the first polypeptide is linked to type the end-to-end with a second polypeptide or the second polypeptide.
The present invention also relates to a method of treatment of an individual with a b-cell proliferative disorder involving the introduction of a specified individual a therapeutically effective amount of one or more molecules of the present invention, including conjugated polypeptides. The term "cell proliferative disorder" denotes malignant as well as non-malignant cell population, which often differ from the surrounding tissue both morphologically and genotypic. Cytotoxic polypeptide can be connected to a BLyS polypeptide in a variety of ways, known to specialists in this field and are described in more detail below.
Examples of b-cell proliferative disorders, the treatment of which can be carried out in accordance with the present invention, include at least the following: b-cell chronic lymphocytic leukemia/small lymphocytic lymphoma when prolymphocyte leukemia cells, immunocytoma/lymphoplasmacytoid lymphoma (+/- macroglobulinemia Waldenstrom), lymphoma mantle cell, lymphoma b-cells in the border zone in lymphodenoctomy, associated with mucous (MALT type lymphoma b-cells in the border zone of the spleen (+/- villous lymphocytes), leukemia retikulez, diffuse lymphoma, large B-cell mediastinal (thymus) lymphoma large b-cells, intravascular lymphoma large B-cell lymphoma Burkitt, myeloma plasma cells (multiple myeloma), a monoclonal gammopathy or gammopathy of unknown origin (MGUS), indolent myeloma, myeloma of Smoldering, osteosclerotic myeloma (POEMS syndrome), leukemia plasma cells, " non-secretory myeloma, plasmacytoma, solitary plasmacytoma of bone, extramedullary plasmacytoma, macroglobulinemia Waldenstrom, a disease caused by an abnormality of the heavy chain (HCD), a disease associated with deposition of immunoglobulin, systemic pathology of the light chain and primary amyloidosis.
In some aspects, the present invention compositions and methods related to b-cell proliferative disorders that are resistant to treatment. Specified disorder may be initially resistant to treatment or specified disorder may become resistant to treatment as a result of initial or subsequent treatment.
Other embodiments of the present invention relate to compositions of the present invention for the prevention and/or treatment of at least one the first symptom of b-cell proliferative disorder. In a specific embodiment of the present invention the composition of the present invention are used for the prevention and/or treatment of autoimmune disorders, such as arthritis or systemic lupus erythematosus. As for prophylactic use, you can achieve complete prevention of the development of the symptom or its appearance may be delayed. With regard to the application for treatment, the symptom can be eliminated completely or partially reduced.
In other aspects of the implementation of the present invention describes methods and compositions used in relation to an individual who passes a different treatment for the same b-cell proliferative disorders, and who will undergo another treatment or who is already undergoing this treatment. Can be viewed any additional therapy, where particular variants of implementation of the present invention additional therapy includes radiation, chemotherapy, surgery, gene therapy, immunotherapy, hormonal therapy or a combination of both.
In specific embodiments, the invention proposes a method of treatment of an individual with a b-cell proliferative disorder, including the introduction of the indicated patient a therapeutically effective amount of the composition, t is coy as conjugated polypeptide, comprising a BLyS polypeptide conjugated with a cytotoxic polypeptide, in particular embodiments, the method also includes an introduction to the individual an agent that increases the expression of BLyS receptor.
In additional embodiments of the invention, the BLyS receptor can be introduced into the cell the owner and expressed in a cell host, which allows BLyS to affect cells outside its natural tropism. In specific embodiments, the implementation of the present invention the expression of polynucleotide containing a nucleic acid sequence which encodes a conjugate BLyS (such as protein) of the present invention, is regulated by tissue-specific promoter. For example, a molecule of BLyS receptor, such as SEQ ID NO: 4 (gene sequence SEQ ID NO: 12), administered using a suitable vector into the cell-the owner of the liver. The BLyS receptor expression is controlled by a promoter that is specific for hepatoma. Thus, the effect of BLyS-conjugated polypeptides can be directed to cells for the treatment of hepatoma. In other embodiments, implementation of the present invention conjugated to polypeptides can be associated with a visualizing agent to track progress targeting polypeptide in the patient's body.
In additional embodiments, the implementation of the infusion is his invention proposes a method of selective targeting to the cell, expressing BLyS receptor, comprising contacting the specified cells conjugated with a polypeptide containing the BLyS polypeptide conjugated with a cytotoxic peptide.
In another embodiment, the present invention proposes a method of monitoring therapy of b-cell proliferative disorders, comprising the administration to a patient a therapeutically effective amount of conjugated polypeptide containing the BLyS polypeptide conjugated with a cytotoxic polypeptide and visualizing agent.
The present invention relates to multipolarity compositions in which more than one polypeptide is present in the form of a specific individual compounds. Thus, the BLyS protein can be, for example, attached to the toxin gelonin, for example to the second, third, fourth, fifth, sixth, seventh or more polypeptide.
In other embodiments, implementation of the present invention provides kits for the treatment and/or prevention of b-cell proliferative disorders containing composition comprising a BLyS polypeptide conjugated to another molecule, such as, for example, a cytotoxic agent or a pharmaceutical agent. In specific embodiments, the implementation of the composition comprises a protein rGel and BLyS and/or polynucleotide, its encoding.
Therefore clicks the zoom, in embodiments implementing the present invention features a composition comprising a BLyS polypeptide conjugated with an additional molecule comprising a molecule that is not identical to the BLyS polypeptide. In certain additional aspects, the molecule is not a homologue of BlyS. In a particular aspect of the present invention, an additional molecule contains, for example, a pharmaceutical agent, a chelate or a cytotoxic agent. The components of the composition can be included in the composition of the fused protein, or may be, for example, chemically conjugated. The composition of the present invention may contain a recombinant polypeptide and/or polynucleotide encoding the recombinant polypeptide. In particular aspects, the composition also contains a radioactive agent, cellular imaging agent, or both agents. This composition may be included in a pharmaceutically acceptable carrier.
In a particular aspect of the BlyS polypeptide, the polypeptide includes b-cell directed the domain that contains the loop-D-E for recognition receptor that includes all or only the functional part of the sequence SEQ ID NO: 1 or SEQ ID NO: 2 and/or contains a functional equivalent BLyS, where the sequence specified functional equivalent has at least 80% homology to SEQ ID NO: 1 or SEQ ID NO: 2.
In those the variants of implementation of the present invention, where use of a cytotoxic agent, specified cytotoxic agent may be of any suitable type, while in some embodiments, the cytotoxic agent comprises a peptide, polypeptide or small molecule. In specific aspects of the cytotoxic peptide includes a peptide gelonin that in variants of implementation, are given by way of example, is a 5' position with respect to the BLyS polypeptide. In specific embodiments, the implementation of the present invention the peptide Galanin contains SEQ ID NO: 7. In other specific embodiments of the invention the peptide Galanin contains amino acid residues 110-210
sequence SEQ ID NO: 7.
In specific embodiments, the implementation of the present invention, the cytotoxic agent is selected from the group consisting, for example, ricin A, diphtheria toxin, abrin, dodecandra, trichosanthin, trichokirin, brodina, the anti-virus agent of Mirabilis, protein, ribosome inactivating barley (BRIP), antiviral protein from lacunosa (PAP), saporin, luffin, Pseudomonas exotoxin and MOMORDICA.
In some aspects of the implementation of the present invention, this composition contains, for example, SEQ ID NO: 8 and/or SEQ ID NO: 10.
In a particular aspect of the present invention is considered a host cell containing composition of the present invention. In the other concr is the shaft aspects of the present invention features a dedicated polynucleotide, encoding at least part of the composition of the present invention, including the whole composition. In specific embodiments, the implementation of polynucleotide contains SEQ ID NO: 9 or SEQ ID NO: 11.
In additional embodiments, the implementation of the present invention proposes a method of treatment of an individual with a b-cell proliferative disorder, comprising the administration to a patient a therapeutically effective amount of a composition comprising a BLyS polypeptide conjugated with an additional molecule, such as, for example, the cytotoxic agent. This method can also include the introduction of the individual agent, which, for example, increases the expression of BLyS receptor. In specific aspects of the implementation of the present invention BLyS selected from the group consisting of TNFRSF13B/TACI (SEQ ID NO: 4), TNFRSF17/BCMA (SEQ ID NO: 5) and TNFRSF13C/BAFFR (SEQ ID NO: 6).
In one of the embodiments of the present invention proposes a method of selective targeting to the cell expressing the receptor for BLyS comprising contacting the cell with a composition comprising a BLyS polypeptide conjugated with a cytotoxic agent.
In additional embodiments, the implementation of the present invention proposes a method of monitoring the treatment of an individual with a b-cell proliferative disorder involving the introduction of a specified individual Ter is piticescu effective amount of the composition, containing the BLyS polypeptide conjugated with a cytotoxic agent, and the visualization agent.
The above outlines the features and technical advantages of the present invention for clarity, the following detailed description of the present invention. Additional characteristics and advantages of the present invention described form below claimed under this invention the objects. Specialists in this field it is clear that the concept and described specific embodiments of can be used as the basis for subsequent changes or the development of other structures to achieve the same goals and objectives of the present invention. For specialists in this sphere, it is also clear that such equivalent structure design will not go beyond and to change the essence of the present invention described in the present description. New features believed to be characteristic of the present invention from the point of view of organization, and methods of implementation, in combination with other additional objects and features can be better understood from the following description, which is most fully present invention in combination with the above figures. These features are given in explicit form, however, each of p is izlagaemykh figures given for the purpose of illustration and descriptive purpose and they should not be construed as limiting the scope of the present invention.
DESCRIPTION of FIGURES
For a more complete understanding of the present invention are described below figures for a better understanding of the description.
Figure 1 shows the approximate orientation of BLyS and rGel.
In Fig. 2 shows a representative DNA sequence (SEQ ID NO: 9) and protein sequence (SEQ ID NO: 8) fused toxin BLyS/rGel.
Figure 3 shows a representative DNA sequence (SEQ ID NO: 11) and protein sequence (SEQ ID NO: 10) fused toxin rGel/BLyS.
Figure 4 illustrates the design of a representative merged toxin rGel/BLyS. The obtained fused toxin rGel/BLyS containing rGel at N-end, followed by peptide G4S attached to the BLyS molecule using PCR according to the method of overlapping splicing extensions. Recombinant design DNA rGel/BLyS injected into the sites of the restriction enzymes Kpn I and Xho I in the vector pET-32a with obtaining expression vector pET32rGel/BLyS.
Figure 5 shows the procedure for cleaning the slit toxin rGel/BLyS. According to the results of staining of Kumasi blue when conducting electrophoresis in SDS-PAGE merged toxin rGel/BLyS indexMrfor rGel/BLyS is set KD, showing the molar ratio of 1:1 BLyS and rGel (left panel). The results of Western blotting using antiglomerular antibodies or anti-BLyS antibodies show that merged toxin rGel/BLyS contains the toxin and BLyS in f the th toxin ( right panel).
Figure 6 demonstrated inhibitory activity in a cell-free system for protein synthesis fused toxin rGel/BLyS. To determine n-glycosidase activity component rGel the merged toxin rGel/BLyS this material is added to test for broadcastin vitrousing a test to assess the inclusion of [3H]-latinae, isolated rabbit reticulocytes. Compare inhibition curves for fused toxin rGel/BLyS and native rGel.
In Fig. 7 illustrates the results of comparison of rGel/BLyS and merged toxin BLyS/rGel against cell line cells of the mantle JEKO. Cell line cells of the mantle JEKO seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add to the wells in a fourfold repetition rGel, rGel/BLyS or BLyS/rGel. After 96 hours to each well, add 75 ál of XTT-labeled mixture, after which the cells incubated for another 4 hours. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 8A-8M are shown curves dose-response for fused toxin rGel/BLyS against various tumor cell lines: Jurkat (8A), KBM-5 (8B), THP-1 (8C), HL-60 (8D), IM-9 (8E), MM1.S (8F), 1.R (8G), RPMI18226 (8H), 8226/LR-5 (8I), JEKO (8J), SP53 (8K), Mino (8L) and Granta (8M). Cells thirteen tumor cell lines were seeded (with a density of 5·103/well) in wells of 96-Lu is full-time microtiter tablets with a flat bottom and add to the wells in a fourfold repetition rGel or rGel/BLyS. After 96 hours to each well, add 75 ál of XTT-labeled mixture, after which the cells incubated for another 4 hours. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 9 illustrates the specificity merged toxin rGel/BLyS for cell line cells of the mantle JEKO expressing BLyS receptors. Cell line cells of the mantle JEKO seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add to the wells in a fourfold repetition BLyS, rGel, CTP/rGel and rGel/BLyS. After 96 hours to each well, add 75 ál of XTT-labeled mixture, after which the cells incubated for another 4 hours. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 10 shows curves dose-response for fused toxin rGel/BLyS against dexamethasone-sensitive (MM1.S) and dexamethasone-resistant (MM1.R) cells cell lines of multiple myeloma. Cells cell lines MM1.S and MM1.R seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add to the wells in a fourfold repetition rGel, Dex or rGel/BLyS. After 96 hours to each well, add 75 ál of XTT-labeled mixture, after which the cells incubated for another 4 hours. Determine pohlad the tion on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 11 illustrates the maximum tolerated dose (MTD) for rGel/BLyS. To obtain values for MTD rGel/BLyS different concentrations of rGel/BLyS is injected to Balb/C mice for 5 consecutive days intravenously into the tail vein and determine the body weight and the number of surviving mice.
In Fig. 12 illustrates the specificity of rGel/BLyS to cells expressing the receptor for BLyS (Fig. 14A). Activity by binding with the receptor fragment of BLyS component in the rGel/BLyS determined by ELISA method using intact cells JeKo-1 and HL-60 (Fig. 14B). To determine the specific activity of rGel/BLyS against three cell lines lymphoma mantle cell (MCL)expressing one or more BLyS receptors, cells, cell lines JeKo-1 MCL seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add to the wells in a fourfold repetition BLyS, rGel, CTP/rGel or rGel/BLyS. After 96 hours to each well add 50 ál of XTT-labeled mixture, after which the cells incubated for another 4 hours or over night. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 13A-13B illustrates the competitive inhibition under the action of rGel/BLyS on cells JeKo-1. To perform the test in a competitive inhibition of cell JeKo-1 sow (with a density of 5×103/well) in the unki 96-well microtiter tablets with a flat bottom (Becton Dickinson) and conduct pre-treatment with 1 nm BLyS, 50 nm BLyS (Fig. 15A), 10 μg/ml BAFF-R:Fc, 10 μg/ml TACI:Fc or 10 μg/ml BCMA:Fc (Fig. 15V) for 2 hours and then added to wells in a fourfold repetition rGel, BLyS or rGel/BLyS. After 96 hours to each well add 50 ál of XTT-labeled mixture (Roche), after which the cells incubated for another 4 hours or over night. On the spectrophotometer to determine the absorbance at a wavelength of 450 nm using a counter for ELISA.
In Fig. 14A-14C shows the effects of rGel/BLyS in apoptosis (14A) and the type of cells JeKo-1 under the microscope after treatment. Cells JeKo-1 was treated with 100 PM BLyS, 100 PM rGel or 100 PM rGel/BLyS. After 96 hours to assess the presence in the cells of JeKo-1 apoptosis when conducting staining by TUNEL method (Fig. 14V). The number of apoptotic cells counted in randomly selected fields (magnification × 200) and expressed in percentage. To identify the effect of rGel/BLyS in apoptosis of cells JeKo-1 and Granta 519 seeded with a density of 5×105/cells in wells of a 24-hole tablet and then treated with 100 PM BLyS, 100 PM rGel or 100 PM rGel/BLyS. After treatment, cells are harvested, washed, and are lysed in 0.2 ml of buffer for lizirovania. Cell lysates (50 µg) fractionary when conducting electrophoresis in 8-15% SDS-PAGE and electrophoretic transfer to nitrocellulose membrane Immobilon-p Membrane block and then probe the various antibodies (Fig. 14C). Use secondary antic the La, conjugated with horseradish peroxidase, to visualize immunoreactive proteins using a detection reagent ECL. Actin used as a control for protein loading.
DETAILED description of the INVENTION
In the context of the present description, the term "other" can mean at least a second or more. Several embodiments of the present invention can comprise or consist essentially of one or more items, stages of implementation techniques and/or methods of the present invention. It is believed that any method or composition described in the present description may be implemented with respect to any other method or composition described herein.
The term "conjugated" in the context of the present description refers to some variants of implementation of the present invention, in which a joining BLyS molecule to molecule cytotoxic agent. The specified connection can be carried out by any appropriate techniques from those known in this field, while in some aspects the joining occurs by recombination using the linker, etc. In specific aspects of the use of ion Association, such as, for example, using the avidin-bitenova St is zi.
II. The present invention
The present invention relates to compositions that are directionally affect the abnormally proliferating b-cells, which demonstrate the pristutstvii receptors for BLyS polypeptides. Normally proliferating B-cells and other cell types that do not Express the receptors for BLyS. The polypeptides of the present invention include a BLyS polypeptide, which serves as the directional domain and a cytotoxic peptide, which reduces or eliminates the proliferation of one or more target cells. Such polypeptides have a specific cytotoxic activity against abnormally proliferating b-cells and therefore useful for the treatment of any B-cell proliferative disorder. How to create and use of such polypeptides in therapy are described below.
III. Proteinopathies connection BLys
The present invention relates to conjugated polypeptides with directional effects, in particular to such polypeptides, which have a therapeutic effect for the individual. In some embodiments, the present invention relates to new compositions comprising proteinopathies molecules. In some embodiments, it is believed that these proteinopathies connections can be modified by deletions, Sames the treatment or addition of amino acid residues. In specific embodiments, BLyS include a mutation that does not affect the activity of the receptor binding BLyS. This mutation may be present in polynucleotide, encoding a BLyS molecule. Representative mutants BLyS include mutation Cys146 (Chenet al., 2002; 2004; 2005) and in specific embodiments, these mutations affect alanine or valine. Mutants BLyS used in the present invention retain the ability to directionally affect B-cells, for example, retain the ability to bind at least one BLyS receptor. In the mutant BLyS can be increased any activity in comparison with BLyS wild type, including the ability to bind to B-cell, for example, BLyS receptor.
In addition, proteinopathies connection may include a molecule of the amino acids comprising more than one polypeptide units. In the context of the present description, the term "proteinopathy molecule", "proteinopathy composition", "proteinopathies connection", "proteinopathy chain" or "proteinopathy material" generally refers without limitation to a peptide comprising from about 3 to about 100 amino acids, to a polypeptide comprising more than 100 amino acids and the polypeptide comprising more than about 200 amino acids, or to the endogenous sequence of the full length, streaming with gene. All of these you who e terms including the word "proteinopathy"may be used interchangeably. Moreover, these terms are equal can be applied to conjugated polypeptide or protein conjugates.
In some embodiments, the size of at least one proteinopathy molecules may comprise without limitation about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or more residues in the molecule of the amino acids and include any range, which can be obtained based on the specified values.
Accordingly, the term "proteinopathy composition" refers to the sequence of amino acid molecules, comprising at least one of the 20 common amino acids in natural proteins or at least one modified or unusual amino acid, including, without limitation, amino acids listed below in table 1.
Modified and unusual amino acids
|Reduction||Amino acid||Reduction||Am nakikita|
|Bala||β-alanine, β-aminopropionic acid||AHyl||Allowedactions|
|4Abu||4-aminobutyric acid, piperidino acid||4Hyp||4-hydroxyproline|
|Aib||2-aminoadamantane acid||MeGly||N-methylglycine, sarcosine|
In the context of the present description, the term "amino acid molecule" refers to any amino acid derivative of the amino acid or mimetic amino acids, which is obvious to any person skilled in the art. In some embodiments, the remains proteinopathy molecules are sequential and h shall include any molecule, other than amino acids, which would violate the sequence of residues in the molecule of the amino acids. In other embodiments, the sequence may include one or more fragments of molecules other than amino acids. In specific embodiments, the sequence of residues in proteinopathy molecule may be interrupted by one or more fragments of molecules other than amino acids.
Aimed conjugated polypeptides of the present invention may contain deletions and/or substitutions of amino acids; thus, proteins with a deletion of the proteins with substitution and proteins with a deletion and substitution are aimed conjugated polypeptides. In some embodiments, implementation of the present invention indicated aimed conjugated polypeptides may also include an insert or added amino acids, such as, for example, the linkers. The term "directed merged deleteriously protein" is characterized by the loss of one or more residues of the native protein, but has specificity and/or activity of a native protein.
Options for substitution or replacement typically include the substitution of one amino acid to another in one or more sites in the protein and can be designed to modulate one or more properties of the polypeptide, in particular to improve the performance efficiency or specificity. Substitution of this kind are preferably conservative, that is, one amino acid is replaced with another amino acid of similar size and charge. Conservative substitutions are known in this field and include, for example, substitution of alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to Proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
In addition to deletions or substitutions will be directed protein may contain insertions of residues, which typically represent the addition of at least one residue of the polypeptide. Such an additive may include the insertion of a targeted peptide or polypeptide, or just one of balance. Terminal insert, called fused proteins, described below.
The term "biologically functional equivalent" is well-known in this field and will be further defined in more detail. Accordingly, sequences that have the ome 70% and about 80% or from about 81% to about 90%, as well as in the range from about 91% to about 99% of amino acids that are identical or functionally equivalent to the amino acids in the native polypeptide, are included in the present invention, provided that they retained the biological activity of the protein. Directed protein may represent a biologically functional equivalent of its natural partner. For example, it can represent a functional equivalent from the point of view of ability to bind with the receptor. In other embodiments, the conjugated polypeptides of the present invention can have a greater affinity to their receptors than their native counterparts.
The term "functionally equivalent codon" in the context of the present description refers to codons that encode the same amino acid, for example, as in the case of the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see table 2 below).
TABLE of CODONS
|Alanine||Ala||A||GCAGCC GCG GCU|
|Aspartic acid||Asp||D||GAC GAU|
|Glutamic acid||Glu||E||GAA GAG|
|Glycine||Gly||G||GGA GGC GGG GGU|
|Isoleucine||Ile||I||AUA AUC AUU|
|Leucine||Leu||L||UUA UUG CUC CUA CUG CUU|
|Proline||Pro||P||CCA CCC CCG CCU|
|Arginine||Arg||R||AGA AGG CGA CGC CGG CGU|
|Serine||Ser||S||AGC AGU UCA UCC UCG UCU|
|Threonine||Thr||T||ACA ACC ACG ACU|
|Valine||Val||V||GUC GUA GUG GUU|
It should also be understood that amino acid sequences and nucleic acid sequence may include additional residues, such as additional N - or the-terminal amino acids or 5'or 3'sequences, and, in fact, represent the sequence given in this list, provided that this sequence satisfies the above criteria for the preservation of the biological activity of the protein, in the case where a protein expression. The addition of terminal sequences particularly applies to sequences of nucleic acids, which may include, for example, various non-coding sequences flanking one of the 3'- or 5'-coding parts of the site, or may include various internal sequences, such as introns, which are known to lie within genes.
Below is a description of various aspects of the replacement of amino acids in the protein with the creation of equivalent or even superior molecules of the second generation. For example, some amino acids can be substituted for other amino acids in a protein structure without appreciable loss of the ability to interactive linking to such structures as, for example, the binding sites with the molecules of the substrate. Since it is the interactive capacity and nature of a protein determine biological and functional activity of the protein, some amino acid substitutions can be made in a protein sequence and the corresponding coding DNA sequences, and so is e less can be obtained protein with similar properties. Thus, the inventors believe that can be made various changes in the DNA sequence of genes without appreciable loss of their biological applicability or activity, as described below. Table 2 shows the codons that encode a particular amino acid.
With the introduction of such changes should be taken into account hydropathicity index of amino acids. The importance hydropathical index of amino acids in the characteristic of the interactive biological function of a protein are well known in the art (Kyte &Doolittle, 1982). It is believed that the relative hydropathicity the nature of the amino acids is the contribution of the secondary structure of the resulting protein, which, in turn, determines the nature of the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.
This area is also known that the substitution within the amino acids can be efficiently carried out on the basis of hydrophilicity. In U.S. patent No. 4 554 101 described in the present description by reference, indicates that the highest local average hydrophilicity of a protein, determined by the hydrophilicity of the adjacent amino acids, correlates with a corresponding biological property of the protein. As described in detail in U.S. patent No. 4 554 101, amino acid residues at voiny following indicators hydrophilicity: arginine (+3,0); lysine (+3,0); aspartate (+3,0±1); glutamate (+3,0 ±1); serine (+3,0); asparagine (+0,2); glutamine (+0,2); glycine (0); threonine (-0,4); Proline at (- 0.5±1); alanine (-0,5); a histidine at (- 0.5); cysteine (-1,0); methionine (-1,3); valine (-1,5); leucine (-1,8); isoleucine (-1,8); tyrosine (-2,3); phenylalanine (-2,5); tryptophan (-3,4).
It should be understood that an amino acid can be substituted by another amino acid having a similar hydrophilicity index, and this will create a biologically equivalent and immunologically equivalent protein. In carrying out such substitutions preferably the amino acid substitution, for which the hydrophilic indices are within ±2, and especially preferred the specified index within ± 1, and if these figures are within ±0,5, they are even more preferred.
As mentioned above, amino acid substitutions, typically based on the relative similarity of the substituents in the side chain of amino acids, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Representative of substitution, which take into account the various above-listed characteristics, known to specialists in this area and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
IV. Conjugated polypeptides
The present invention also is tositsa conjugated to polypeptides, such as translated proteins, polypeptides and peptides, generally of the monoclonal type, that are associated with at least one agent with the formation of the conjugate. Conjugated polypeptides of the present invention include chemical conjugation and genetic conjugation", such as in the case of a recombinant fused protein. It should also be understood that the polypeptides of the present invention can be synthesizedde novousing techniques known to experts in this field.
A. Peptide synthesis
Conjugated polypeptides of the present invention can be synthesized. Methods of peptide synthesis known to specialists in this field (Bodanczkyet al., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984; The Proteins, 1976). Appropriate protective groups suitable for use in such synthesis methods can be found in the above papers, as well as in the manual of protective group (Protective Groups in Organic Chemistry, 1973). These synthesis methods include the sequential addition of one or more amino acid residues or suitably substituted amino acid residues to a growing peptide chain. Typically, amino or carboxyl group of the first amino acid residue is subjected to a protection using a suitable selectively removable protective gr is PPI. Use a different selectively removable protective group for amino acids containing reactive side group, such as lysine.
When using as an example of a method of solid-phase synthesis of protected or derivatizing amino acid attached to an inert solid substrate through her unprotected carboxyl or amino group. The protective group for amino group or carboxyl group is then selectively removed and the next amino acid in the sequence that is complementary (amino or carboxyl) group, a protected appropriately added and subjected to reaction with the residue that is already attached to the solid substrate. The protective group for amino group or carboxyl group is then removed from the specified newly added amino acid residue and contribute accordingly the following protected amino acid (and so on). After all the desired amino acids are linked in the proper sequence, remove any remaining terminal and lateral protective group (as well as a solid substrate), sequentially or simultaneously, with the final peptide. The peptides of the present invention preferably do not contain basiliani or methylbenzylamine amino acids. Such fragments of the protective group may be used in order to find during synthesis, but they should be removed before using the peptides. May be required additional reactions described in various manuals for the formation of intramolecular bonds, to maintain the desired conformation.
Many of the peptides or polypeptides can be connected using biologically redundant communication, such as selectively cleaved the linker or amino acid sequence. For example, discusses the peptide linkers that include a biodegradable enzyme site, preferably localized or active in the tumor environment. Representative forms of such peptide linkers include forms, cleaved by urokinase, plasmin, thrombin, factor IXa, factor Xa or metalloproteinases, such as collagenase, gelatinase or stromelysin. Alternative peptides or polypeptides can be combined with adjuvant.
Amino acids, such as selectively degradable linkers, synthetic linkers, or other amino acid sequences, can be used for separating proteinopathy fragments. Additionally, in light of the fact well-known fact that many types of linkers that contain a disulfide bond, can be used for conjugation toxin fragment intended by the agent, some the e linkers may be preferable in comparison with other linkers, based on different pharmacological characteristics and abilities. For example, linkers that contain a disulfide bond, which is a spatial "hidden", is preferred due to their greater stability in vivo, which prevents the release of toxin fragment to bind to the site of action. In addition, some of the advantages of the present invention can be identified using any of a variety toxin fragments comprising gelonin and deglycosylated a chain of ricin.
It should be borne in mind as a basic guideline that any biochemical cross-linking, the linker, suitable for use in the present invention may also be used in this context and can be considered additional linkers.
Cross linking reagents used for the formation of a molecular bridge ties that tie together functional groups of two different molecules, such as stabilizing and coagulating agent. To bind two different proteins in sequential mode can be used heterobifunctional cross-linking linkers, which eliminate the undesirable formation of homopolymer.
It is believed that cross-linking linkers can be used in combination with Halekulani protein of the present invention. Bifunctional cross linking R the agents are widely used for a variety of purposes, including obtaining an affine matrices for modification and stabilization of various structures, identification of binding sites and structural studies. In the context of the present invention, such cross-linking, the linker can be used to stabilize the polypeptide or to make it more useful as a therapeutic agent, for example, by improving the ability of the modified protein to directed impact or improve the overall efficiency. Cross-linking linkers can be cleaved, for example, such as disulfide bonds, acid-sensitive linkers, and others. It was shown that homobifunctional reagents, which contain two identical functional groups, are highly effective in inducing the formation of linkage between identical and different macromolecules or subunits of the macromolecule and binding polypeptide with a specific binding sites on the respective partner molecules. Heterobifunctional reagents contain two different functional groups. By combining advantages of various reactively two different functional groups, the process of crosslinking can be controlled and selectively and sequentially. Bifunctional cross-linking reagents can be divided according to their functional specificity is Rupp, for example groups, specific for amino groups, sulfhydryl, guanidino, indole and carboxyl groups. Of these reagents, aimed at free amino groups, especially widely used due to their commercial availability, ease of synthesis and mild reaction conditions under which they are used. Many heterobifunctional cross-linking reagents contain a primary aminorityview group and terracciano group.
In another example, describes heterobifunctional cross-linking reagents and methods of using such cross-linking reagents (U.S. patent No.5889155 specifically described in the present description by reference in full). Cross-linking reagents contain Association nucleophilic hydrazide residue with electrophilic maleimide balance, allowing the binding of, for example, aldehydes with free thiols. Cross-linking reagent can be modified in such a way that he will be joining various functional groups, and in this case it will be useful for cross-linking of proteins and sugars. In cases where a particular polypeptide, such as gelonin, does not contain in its native sequence residue, suitable for this cross-linking reagent in the primary sequence can be introduced by conservative substitutions of amino acids of the genetic method and or methods of synthesis.
In this area there are several methods that are suitable for attachment or conjugation of the polypeptide with its conjugate fragment. Some methods of attaching involve the use of metalheadneko complex using, for example, an organic chelating agent such as an anhydride diethylenetriaminepentaacetic acid (DTPA); etilendiamintetrauksusnoy acid; N-chloro-p-toluensulfonate; and/or tetrachloro-3α-6α-diphenylpicryl-3, attached to the polypeptide (U.S. patent NoNo.4472509 and 4938948, each of which is given in the present description by reference in full.). The polypeptides may also interact with the enzyme in the presence of a binding agent, such as glutaraldehyde or periodate. Conjugates with fluoresceine markers get in the presence of the above binding agents or by reaction with isothiocyanates. In U.S. patent No.4938948 describes the visualization of breast tumors using monoclonal antibodies, where the detected visualisatie fragments associated with the polypeptide with the use of linkers, such as methyl-p-hydroxybenzamide or N-Succinimidyl-3-(4-hydroxyphenyl)propionate.
C. Visualizeus agents
In some aspects of the present invention, the BLyS polypeptide is subjected to conjugation at least one of visua soudem agent. Non-limiting examples visualizeus agents that can be conjugated to polypeptides include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, molecules that determine photoaffinity, colored particles or ligands, such as Biotin.
In this area there are many suitable visualizeus agents, and how they attach to the antibodies (see, for example, U.S. patent NoNo.5021236; 4938948 and 4472509, each of which is given in the present description by reference). Used visualizeus fragments may represent a paramagnetic ions; radioactive isotopes; fluorochromes; substances identified by NMR; x-ray visualization.
In the case of paramagnetic ions as an example ions such as ions of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), Nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), gadolinium is especially the preferred. Ions used in other contexts, such as x-ray visualization, include, without limitation, the lanthanum (III), gold (III), lead (II) and especially bismuth (III).
In the case of radioactive isotopes, skin is utilized for therapeutic and/or diagnostic purposes, you can mark astatin211,14carbon,51chrome,36chlorine,57cobalt,58cobalt, copper67,152Eu, gallium67,3hydrogen, iodine123, iodine125, iodine131, indium111,59iron,32phosphorus, rhenium186, rhenium188,75selenium,35sulfur, technetium99mand/or yttrium90.125I often preferred for use in some embodiments of implementation of the present invention, and technetium99mand/or indium111also often preferred due to their low energy and suitability for detection in a wide range. Radioactively labeled monoclonal antibodies of the present invention can be obtained in accordance with known in this field techniques. For example, monoclonal antibodies can be subjected to jodirovannuju when interacting with iodide of sodium and/or potassium and chemical oxidant such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Intermediate functional groups which often are used to bind radioisotopes which exist as a combination of metal ions with the polypeptide, include diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
In the number of fluorescent labels that can and shall be used as conjugates, includes Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, isothiocyanate fluorescein, HEX, 6-JOE, Oregon green 488, Oregon green 500, Oregon green 514, Pacific blue, REG, rhodamine green, rhodamine red, renografin, ROX, TAMRA, TET, tetramethylrhodamine and/or Texas red.
Can also be used molecules containing sidegroup to form covalent bonds with proteins through reactive mitrinovi intermediates, which are formed under the action of low-intensity ultraviolet light (Potter & Haley, 1983). In particular, use 2 - and 8-azidoanilide purine nucleotides as site-directed fatsondo to identify the nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Athertonet al., 1985). 2 - and 8-azithromiacin used for mapping nucleotidebinding domains of purified proteins (Khatoonet al., 1989; Kinget al., 1989; and Dholakiaet al., 1989) and can be used as binding agents.
V. Therapeutic agents
In a particular aspect of the present invention discussed herein BLyS polypeptides are subjected to conjugation to another molecule that can act as a therapeutic agent. In specific aspects, therapeutic agent may be a cytotoxic agent, a radioactive is stop, a small molecule chemotherapeutic agent, a Pro-apoptotic agent, a natural product, antibody, cytokine, chemokine, an angiogenic inhibitor, a regulator of programmed cell death, etc.
Some examples of therapeutic agents are discussed in the following text.
A. Cytotoxic agents
The present invention relates to the use of the cytotoxic activity of oriented molecules, and in particular embodiments, the implementation of the present invention the cytotoxic activity can be viewed in the context of the toxin. Any toxin is acceptable for use in the present invention, provided that it does not interfere with the action of directional fragment conjugated polypeptide (for example), and provided that it at least slows down, if not completely inhibited, proliferation of the target cells.
Toxins with inhibitory activity against ribosomes (RIT) are powerful inhibitors of protein synthesis in eukaryotes. The enzymatic domain of these proteins acts as a cytotoxic n-glycosidase, which is able to inactivate the catalytically active ribosomes, because it has the ability to enter into the intracellular space. This process is accompanied by the cleavage of n-glycosidic bonds adenine at position 4324 in 28sr that leads to neopatrimonial ribosomes apparently, due to the destruction of the binding site with elongation factors. RIT, which were isolated from bacteria that are prevalent in higher plants. In plants there are two types RIT: toxins type I contain single-stranded polypeptide possessing inhibitory activity of ribosomes and toxins type II comprise a circuit comparable to that of the protein of type I, which is linked by a disulfide bond to a B chain with the site-binding ability. Examples RIT type I include gelonin, dodecandra, trichosanthin, trichokirin, Brodin, antiviral protein from Mirabilis, a protein, a ribosome inactivating protein of barley (BRIP), antiviral proteins of lacunosa (PAP), saporin, Ruffini and MOMORDICA. The type II toxins include ricin and abrin. Toxins can be conjugated to or subjected to expression in the form of a fused protein with any of the polypeptides described in the present description.
As part of the present invention, it should be noted that acceptable such toxins as the a chain of ricin (Burbage, 1997), diphtheria toxin A (Massudaet al., 1997; Lidor, 1997), subunit And pertussis toxin, subunit And enterotoxinE. colithe subunit And the cholera toxin and the C-terminal site of the toxinPseudomonas. It was shown that transfection of plasmids containing regulated gene for the circuit And diphtheria toxin in fused protein, possesses cytotoxicity for rakovi the cells. Other toxins, which are considered as useful for the implementation of the present invention include abrin, A/B thermolabile toxins, botulinum toxin, toxins Helix pomatia, breadfruit or jackfruit, agglutinin peanuts, it nigra, Tetanus, Ulex and Viscumin.
It is believed that any of the above therapeutic agents can be anywhereman with the polypeptides of the present invention. In some cases it is preferable to carry out recombinant expression of chimeric proteins, including toxin of other proteins. In other cases it may be desirable to carry out chemical conjugation of compounds based on small molecules with converted polypeptides internalization given in this description.
In specific embodiments, the specified toxin comprises a mutation that still allows the molecule to exhibit cytotoxic activity. This mutation may be present in polynucleotide, encoding a toxin.
B. Radioactive pharmaceuticals
Many different radioactive substances, including radioactive isotopes, can be used in cancer therapy. Examples of radioactive isotopes used for therapeutic purposes include, for example, astatin211,51chrome,36chlorine,57cobalt,58cobalt, copper67,152the Euro is s, gallium67, iodine123, iodine125, iodine131, indium111,59iron,32phosphorus, rhenium186, rhenium188,75selenium,35sulfur, technetium99m, yttrium90, Lu177, samarium153the holmium166and anemone225.
C. Himikofarmatsevticheskom drugs
The term "chemotherapy" refers to the use of medicines for the treatment of cancer. The term "chemotherapeutic agent" is used to refer to compounds or compositions that are administered during the treatment of cancer. One of the subtypes of chemotherapy known as biochemotherapy, involves combining chemotherapy with biological therapy.
Chemotherapeutic agents include without limitation 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, this drug called cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, agents that bind to the estrogen receptor, etoposide (VP16), transferase inhibitor farnesyl-protein, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosamine, plicamycin, procarbazine, raloxifene, tamoxifen, Taxol, temozolomide (aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative of the above compounds. Listed agents or drugs are distributed by the ka is egorian depending on the manner of the manifestation of their activity within the cell, for example, at what stage of the cell cycle they have an impact. Alternative such agent can be characterized by its ability to directly transversely to sew with DNA intercalated into DNA or to induce chromosomal or mitotic aberrations by affecting the synthesis of nucleic acids. The majority of chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, inhibitors of mitosis and nitrosoanatabine, hormonal agents, and other agents, and any analogs, derivatives or variants of the above agents.
Chemotherapeutic agents and methods of administration, dosages, etc. are well-known experts in this field (see, for example, Goodman & Gilman''s "The Pharmacological Basis of Therapeutics" and in the manual Remington "Remington''s Pharmaceutical Sciences", where these works are included in the present description with reference to specific sections as a reference) and can be used in the present invention in light of the above description. Some variation in dosage will be needed depending on the condition of the individual subject treated. The person responsible for the introduction, should in any case to determine the appropriate dose for a particular individual. Examples of specific chimiotherapie the political agents and dosing schedules are also described in this text. And of course, all of these dosages, the agents described in the present description is only representative and not limiting, and other doses or agents can be used by specialists in this field in relation to a specific patient or use case. Any dosage, lying between these points, or any range derived from them, are also expected to be possible for use in the present invention.
1. Alkylating agents
Alkylating agents include, but not limited to busulfan, chlorambucil, cisplatin, cyclophosphamid (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen) and melphalan. In particular aspects can be used troglitazone for the treatment of cancer in combination with one or more specified alkylating agents, some of which are discussed below.
Antimetabolites include, without limitation 5-florouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate. Purine analogs and related compounds include, without limitation mercaptopurine (6-mercaptopurine; 6-MP), tioguanin (6-tioguanin; TG) and pentostatin (2-deoxycoformycin). Mercaptopurine use in acute lymphocytic, acute granulocytic and chronic granulocytic leukemia. Tioguanin used in the treatment of such cancers, acostly granulocytic leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia. Pentostatin used in such cancers, like leukemia retikulez, mushroom avium and chronic lymphocytic leukemia. Inhibitors of mitosis include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, Taxol, vinblastine, vincristine and vinorelbine. Epipodophyllotoxin include compounds such as teniposide and VP16. Taxaide include without limitation compounds such as docetaxel and paclitaxel. The alkaloids of belladonna include compounds such as vinblastine (VLB) and vincristine.
3. Antitumor antibiotics
Antitumor antibiotics possess antimicrobial and cytotoxic activity. These drugs also interact with DNA by chemical inhibition of enzymes and mitosis or by changing cell membranes. These agents are not specific to a particular phase of the cell cycle because they operate on all phases of the cell cycle. Thus, they are widely used in various types of cancer. Examples of antitumor antibiotics include, without limitation bleomycin, dactinomycin, daunorubicin, doxorubicin (adriamycin), plicamycin (mithramycin) and idarubicin. Widely used in clinical trials for treatment of neoplasm specified connected what I mostly administered intravenously by bolus injection or orally.
Corticosteroid hormones are considered as chemotherapeutic agents, when they are used to destroy or slow the growth of cancer cells. Corticosteroid hormones can increase the efficacy of other chemotherapeutic agents, and in this regard, they are often used in combined therapies. Prednisone and dexamethasone are examples of corticosteroid hormones.
Progestins, such as Caproic of peroxidation, acetate medroxyprogesterone and acetate megestrol use with endometrial cancer and breast cancer. Estrogens, such as diethylstilbestrol and ethinyl estradiol is used in such cancers as breast cancer and prostate cancer. Antiestrogens, such as tamoxifen, used to treat such cancers as breast cancer. Androgens, such as testosterone propionate and fluoxymesterone, used in the treatment of breast cancer. Antiandrogens, such as flutamide, used in the treatment of prostate cancer. Analogues of gonadotropin-releasing hormone, such as leuprolide, used in the treatment of prostate cancer.
5. Other agents
Some chemotherapeutic agents are not to be classified in the above categories from the point of view of their activity. They include the ez limitations of the coordination complexes of platinum, anthracenedione, substituted urea, derivatives of methylhydrazine, suppressor of adrenocorticotropic hormone, amsacrine, L-asparaginase and tretinoin.
D. Natural products
Natural products mainly refer to compounds that were originally isolated from a natural source and are identified by their pharmacological activity. Such compounds, their analogs and derivatives can be isolated from a natural source, can be chemically synthesized or obtained by recombinant techniques in accordance with known this area procedure. Natural products include the group of compounds as inhibitors of mitosis, antitumor antibiotics, enzymes and biological response modifiers.
Inhibitors of mitosis include plant alkaloids and other natural agents that can inhibit either protein synthesis necessary for cell division, or mitosis. They operate on a specific phase of the cell cycle. Inhibitors of mitosis include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, Taxol, vinblastine, vincristine and vinorelbine.
Taxaide are a class of closely related compounds isolated from the bark of the ash tree Taxus brevifolia. Taxaide include without limitation compounds such as docetaxel and paclitaxel. Paclitaxel binds to the I-tubulin (website, the remote, which is used Vinca alkaloids) and accelerates the Assembly of microtubules.
Alkaloid herb belong to this type of plant alkaloids that have been identified as having pharmaceutical activity. They include compounds such as vinblastine (VLB) and vincristine.
That is, the Mimetics of the peptides
Another option for obtaining polypeptides of the present invention refers to the use of peptide mimetics. Mimetics are peptideatlas molecules that mimic the secondary structure elements of the protein (see, e.g., Johnson et al., "Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, New York (1993), where this work is given in the present description by reference). The basic principle underlying the use of peptide mimetics is the understanding that the peptide skeleton proteins exist just for orientation of the side chains of amino acids, so as to facilitate molecular interactions, such as the interaction between antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles can be used to produce molecules of the second generation, with many of the natural properties of the designed peptides according to the present description, but the change is significant or even improved characteristics.
In some embodiments, implementation of the present invention there is a need to create antibodies against the identified targeted peptides or their receptors. The corresponding directional peptide or receptor or parts thereof can be combined, linked, attached, conjugated or chemically-linked to one or more agents through the use of linkers, polylinkers or derivatives of amino acids. This process may be carried out in such a way that it creates bespecifically or multivalent composition or vaccine. It is also assumed that the methods of obtaining these compositions known to specialists in this field and should be suitable for the introduction of man, there must be pharmaceutically acceptable. Preferred agents include carriers, which are hemocyanin fromMegathura crenulata(KLH) or bovine serum albumin (BSA).
The term "antibody" in the context of the present description is used to denote any antitelephone molecule that contains antigennegative the site and includes fragments or antibody-based test, such as Fab', Fab, F(ab')2 antibodies with one domain (DAB), Fv, scFv (single-chain Fv), etc. methods of obtaining and using different antibodies on the basis of certain structures and fragment is known to specialists in this field. How to create and characteristics of the antibodies are also known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; where this work is given in the present description by reference).
G. cytokines and chemokines
In some embodiments, implementation of the present invention, it may be desirable to associate specific bioactive agents with one or more directional peptides for mistaway delivery to the organ or tissue. Such agents include without limitation, cytokines, chemokines, Pro-apoptotic factors and angiogenic factors. The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines include lymphokines, Monokini, growth factors and traditional polypeptide hormones. The group of cytokines are growth hormones such as human growth hormone, N-metionine derived human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor cells; prostaglandin; fibroblast growth factor; prolactin; placental the th lactogenic, OB protein; tumor necrosis factor alpha and beta; Mullerian-inhibiting substance; the peptide associated with the mouse gonadotropin, inhibin; activin; factor vascular endothelial growth; integrin; thrombopoietin (TPO); nerve growth factors cells, such as NGF-β; platelet growth factor; transforming growth factors (TGF)such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and -γ; colony stimulating factors (CSF)such as macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF); interleukins (IL-1, IL-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT. In the context of the present description, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
Can be used cytokines with stimulating activity, or cytokines, which have ratingaverage activity, and in specific embodiments, the composition of the present invention includes one of them.
Chemokines mainly act as chemoattractant for leisure studies at the ment of effector cells to the site of expression of chemokines. It may be useful to conduct gene expression of specific chemokine in combination with, for example, the genome of the cytokine to enhance recruitment of other components of the immune system in the treatment site. Chemokines include, without limitation RANTES, MCAF, MIPI-alpha, MIPI-β and IP-10. For specialists in this field it is clear that some cytokines also have chemoattractive effect and can be classified in the group of chemokines.
N. The regulators of programmed cell death
Apoptosis or programmed cell death is a process required for normal embryonic development, maintenance of homeostasis in adult tissues and suppression of carcinogenesis (Kerr et al., 1972). It has been shown that members of the family proteins Bcl-2 and ICE-like proteases are important regulators and effectors of apoptosis in other systems. Bcl-2 protein identified in conjunction with follicular lymphoma, plays an important role in the control of apoptosis and increased survival of cells in response to various apoptotic stimuli (bakhshi for et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). Evolutionary conservative Bcl-2 protein is known at present as the representative of a family of related proteins that can be separated into categories agonist of cell death or antagonists of cell death.
After the discovery of Bcl-2 was is rendered, that Bcl-2 acts in the direction of suppression of cell death initiated by many stimuli. In addition, at the present time, it is obvious that there is a family of proteins Bcl-2 proteins regulate cell death, which share structural features and homology in sequence. It has been shown that various members of the specified collection possess similar functions to Bcl-2 (e.g., BclXL, BclW, Bcls-S, Mcl-1, A1, Bfl-1) or counteract the function of Bcl-2 and accelerate cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
Representative apoptotic agents include TNF, any caspase, including caspase-3, caspase-7, caspase-6, caspase-9 or caspase 10a/b, for example, any Grasim, including Grasim a or Grasim B.
Non-limiting examples of Pro-apoptotic agents, are included in the scope of the present invention include gramicidin, magainin, mellitin, defensin, cecropin, or a combination or mixture.
I. Angiogenic inhibitors
In some embodiments, implementation of the present invention may be a problem with the introduction of targeted peptides attached to antiangiogenic agents, such as angiotensin, laminine peptides, fibronectin peptides, inhibitors of plasminogen activator inhibitors of tissue metalloproteinases, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, the 2nd is toxictrail, proliferin-related protein, carboxamidates, SM, marimastat, polysulphate of pentosan, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin AND PNU145156E, 16K fragment of prolactin, linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutan, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.
Specialists in this field can refer to the Remington ("Remington''s Pharmaceutical Sciences" 15ththEdition, chapter 33) and in particular to pages 624-652. Some variation in dosage will be needed depending on the condition of the individual subject treated. The person responsible for the introduction, should in any case to determine the appropriate dosage for each individual patient. In addition, for introducing people drugs must meet the criteria of sterility, progenote and General safety, and purity in accordance with the biological standards of the Federal Agency of the U.S. drug administration (FDA Office of Biologics standards).
Dose any of these therapeutic agents can be determined by the person skilled in the art. In some embodiments, the appropriate dose can be from about 0.1 mg/kg to about 0.3 mg/kg or from about 1.5 mg/m2to about 2 mg/m2. Al is ternative the appropriate dose may be dose, equal to about 0.1 mg/m2approximately 0,12 mg/m2approximately 0.14 mg/m2approximately 0.15 mg/m2approximately 0.2 mg/m2about 0.25 mg/m2approximately 0.5 mg/m2approximately 1.0 mg/m2approximately 1.2 mg/m2approximately 1.4 mg/m2approximately 1.5 mg/m2about 2.0 mg/m2approximately 2.5 mg/m2approximately 5.0 mg/m2approximately 6 mg/m2approximately 8 mg/m2approximately 9 mg/m2and about 10 mg/m2.
VI. Slit proteins
Other variants of the present invention relates to fused proteins. These molecules contain mostly whole or a substantial part of the targeted peptide attached to the N - or C-end of the second polypeptide or protein, all or parts of them. For example, the merger may involve the leader sequences from other species for the implementation of the recombinant expression of the protein in a heterologous host. Other merge options include adding immunologically active domain, such as the determinants of antibodies, for easier cleaning fused protein. The inclusion of a cleavage site at the junction of the merged fragments or next to it will facilitate the removal of foreign polypeptide after purification. Other useful options include merge linking functional domains, such as active sites of enzymes, the house is by glycosylation, the cellular target signals or transmembrane sites. In preferred embodiments, the slit proteins of the present invention include a directional peptide attached to a therapeutic protein or peptide. Examples of proteins or peptides that can be included in the composition of the fused protein, include cytotoxic proteins, cytosine proteins, Pro-apoptotic agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments of antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins, and binding proteins. These examples should not be construed as limiting and it should be understood that the scope of the present invention may be incorporated virtually any protein or peptide that can be embedded in a fused protein comprising a directional peptide. How to create fused proteins known to specialists in this field. Such proteins can be obtained, for example, by chemical connection with the use of bifunctional cross-linking reagents in the synthesis ofde novofull fused protein, or by joining a DNA sequence that encodes a directional peptide to a DNA sequence that encodes a second peptide or protein, followed by expression of the intact fused protein.
VII. Sintet the ical peptides
Due to its relatively small size designed peptides of the present invention can be synthesized in solution or on a solid substrate in accordance with conventional procedures. Currently, various commercially available automated synthesizers that can be used in the framework of the known methods. See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each of which is given in the present description by reference. Short peptide sequences typically ranging in size from about 6 to about 35-50 amino acids can be synthesized through such methods. An alternative may be to use the technology on the basis of recombinant DNA in which the nucleotide sequence that encodes the peptide of the present invention is inserted into an expression vector and then transformed or transferout in the appropriate cell the owner and cultivated under conditions suitable for the expression.
VIII. Purification of proteins
Because some embodiments of the present invention include recombinant proteins, the present invention in some of its variants refers to the use of techniques and methods purification of proteins, including recombinant proteins. Mainly these techniques include n the same level of gross fractionation of the cellular environment on the polypeptide and polypeptide not faction. By separating the polypeptide from other proteins of interest, the polypeptide can then be subjected to purification using the methods of chromatography and electrophoresis to achieve partial or complete purification (or purification to homogeneity). Analytical methods that are particularly suitable for obtaining pure peptide are ion-exchange chromatography, displacement chromatography, polyacrylamide gel electrophoresis, isoelectric focusing. A particularly effective method of purification protein is a fast liquid chromatography of proteins or even HPLC. In addition, the conditions under which such methods are carried out can affect characteristics such as functional activity of the purified molecules.
Some aspects of the present invention relate to the treatment and in specific embodiments, highly significant purification of the encoded protein or peptide. The term "purified protein or peptide" in the context of the present description is used to denote a composition isolated from other components, where the specified protein or peptide is purified to some extent relative to its state in which it is obtained from a natural source. In this regard, a purified protein or peptide also refers to a protein or peptide free from the environment components, in which the n is found in natural form. This so-called in the description of the substantially purified protein or peptide.
Basically, the term "purified" refers to a protein or peptide composition that has been subjected to fractionation for the purposes remove various other components, and where the specified composition essentially retains its biological activity. In that case, when using the term "substantially purified", this designation refers to compositions in which the specified protein or peptide is a major component of this composition, for example, is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.2%, and approximately 99.4%of the approximately 99.6%and about 99.8 percent and about 99.9 percent or more of protein in a given composition.
Professionals in this field are known various methods of quantitative assessment of the degree of purification of the protein or peptide that may be applicable to the present invention. These techniques include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides in a particular faction in the analysis using electrophoresis in SDS/PAGE. The preferred method of estimating the purity of the fraction is the calculation of the specific activity of the fraction, to compare it with specific activity of the source e is stricta, with the subsequent calculation of purity, expressed in the text of this description in the form of parameter expansion treatment. The actual units used to indicate the magnitude of the activity, will of course depend on the specific selected for use in the methods of testing the purity and demonstrates whether the expressed protein or peptide detectable activity.
Various techniques suitable for use in protein purification well known to specialists in this field. They include, for example, precipitation with ammonium sulfate, the use of PEG, antibodies and the like or the use of heat denaturation, followed by centrifugation; the various stages of the chromatography was carried out, such as ion exchange chromatography, gel filtration, chromatography with phase reversal, chromatography on hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. Is in accordance with well-known in this field representations that the order of conducting the various purification stages can be changed, or that some stages may be omitted, and the method will still be reasonably acceptable to obtain essentially purified protein or peptide.
Basically there is no fundamental need for protein and the and the peptide was delivered always in their most purified state. Actually is for some variants of implementation of the present invention that can be used products, the degree of purification which is less than it can be characterized by the term "significant". Partial purification can be achieved using fewer stages when they are combined, or when using different forms of the same General pattern of cleaning. For example, it is recognized that the cation-exchange column chromatography carried out on the apparatus for HPLC, will generally lead to greater "multiplicity" cleaning than the same methodology, but using the chromatography was carried out at low pressure. Ways that demonstrate a lower degree of relative purification may have advantages based full selection of protein product or preserve the activity of expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of electrophoresis in SDS/PAGE (Capaldiet al., 1977). In this regard, it is considered that the values of the apparent molecular weight of purified or partially purified products expression can vary, if the electrophoresis is carried out in different conditions.
We also consider the use of a peptide tag in combination with the methods and compositions according to the present izopet the tion. The specified tag has the advantage that it allows the interaction between the two polypeptides. Part of one of the polypeptides that are involved in the interaction, can be used as a label. For example, the binding site of the glutathione-S-transferase (GST) may function as a label, so the beads with glutathione can be used for enrichment of compounds containing GST tag. Can be used epitope tag from the plot, amino acids which are recognized by an antibody or T-cell. The specific label may be encoded by a nucleic acid segment that is functionally associated with a segment of a nucleic acid encoding a modified protein, so protein is encoded by a nucleic acid molecule. Other suitable fused proteins include fusion with β-galactosidase, ubication, hexaglycine (6xHis), etc.
IX. The nucleic acid molecules
In one aspect of the present invention is used, the nucleic acid molecule encoding a conjugated polypeptide of the present invention.
A. Polynucleotide encoding conjugated polypeptides
The present invention relates to polynucleotides isolated from cells that do not contain the total genomic DNA and that are capable of expression of all or part of a protein or merged polyp is Chida according to the present invention. Polynucleotide can encode the native protein, that can be manipulated in order to achieve the encoding target fused protein. For example, polynucleotide can encode multiple pieces, such as the polypeptide gelonin, which is covalently attached to the target BLyS polypeptide. In the context of the present description, the term "polynucleotide" refers to a nucleic acid molecule that has been separated from the total genomic nucleic acid. In this regard, the phrase "polynucleotide encoding a polypeptide" refers to a segment of DNA that spans the allocated sequence encoding the polypeptide, or which is purified from total genomic DNA of the mammal or in particular person. In this regard, for example, in the case where the present application is related to a function or activity gelonin, the phrase "native polypeptide gelonida" or "fusion polypeptide on the basis of gelonin", which is encoded by polynucleotides means that polynucleotide encodes a molecule that has activity gelonin.
In the context of the present description, the term "DNA segment" refers to a DNA molecule that has been separated from the total genomic DNA of a particular species. In this regard, the segment of DNA that encodes a polypeptide, refers to a segment of DNA that contains the coding for the polypeptide sequence of wild tee is a, polymorphic or mutant sequence, already separated from the total genomic DNA of the mammal or in particular person or peeled from her. The term "DNA segment" includes one or more polypeptides, the DNA segments that are smaller than the polypeptide and recombinant vectors, including, for example, plasmids, Comedy, phage, viruses, etc.
The term "cDNA" is used to refer to DNA, obtained using messenger RNA (mRNA) as the matrix. The advantage of using cDNA unlike genomic DNA or DNA polymerized on the basis of genomic, reprezentirovannoe or partially protestirovanny matrix RNA, is that cDNA mainly includes coding sequences for the corresponding protein. There may be cases when the preferred full or partial genomic sequence, for example, when the optimal expression requires non-coding areas or when non-coding areas, such as introns, are being targeted in the framework of the antisense strategy.
It is also believed that the specific polypeptide of this type may be represented by natural variants that have slightly different nucleic acid sequence, but, nevertheless, they encode the same protein (see table 1).
Similarly Poliny eatig, containing the gene for the selected or purified polypeptide wild-type, polymorphic or mutant polypeptide, refers to a segment of DNA that includes a sequence encoding a polypeptide wild-type, polymorphic or mutant polypeptide and, in certain aspects, regulatory sequences, substantially separated from other natural genes or coding sequences for proteins. In this context, the term "gene" is used to simplify the notation of the functional unit that encodes a protein, polypeptide or peptide. Obviously the experts in this field, the specified functional unit includes genomic sequences, cDNA sequence, and smaller engineered gene segments that Express, or may be adapted for the expression of proteins, polypeptides, domains, peptides, conjugated polypeptides and mutants. Nucleic acid containing all or part of the native or modified polypeptide may contain a continuous sequence of nucleic acid encoding all or part of the polypeptide shown below length: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 20, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1110, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000 or more nucleotides, nucleosides, or base pairs. It is believed that one molecule can encode polynucleotide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different polypeptides (whole or partial).
The present invention in specific embodiments, implementation refers to the selected DNA segments and recombinant vectors incorporating DNA sequences that encode a polypeptide with directed action or fused to a peptide that includes within its amino acid sequence the sequence of consecutive amino acids corresponding to or essentially corresponding native polypeptide. Thus, the selected segment of DNA or a vector containing a DNA segment may encode, for example, fused polypeptide-based gelonin, which has an inactivating activity against ribosomes and specificity of the native polypeptide gelonin and which is functionally associated with BLyS polypeptide, for example SEQ ID NO:4 or SEQ ID NO:5. The term "recombinant" may be used in combination with the polypeptide or the designation of a specific polypeptide, obtained on the basis of a molecule of nucleic acid, which was used for manipulation in vitroor which is a product of replication of such a molecule.
The nucleic acid segments used in the present invention, regardless of the length of the coding sequence can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional sites for enzymes, multiple cloning sites and the like, so that the total length can vary greatly. In this regard, it should be assumed that it can be used a fragment of the nucleic acid of practically any length, while the total length preferably being limited in the case of obtaining and using the procedures of the strategy of recombinant DNA.
It should be assumed that the structure of nucleic acids of the present invention can encode a polypeptide is full length, from any source, or can encode truncated version of the polypeptide, for example the truncated polypeptide gelonin, so that the transcript encoding the plot will be a truncated version. The truncated transcript can then be subjected to a broadcast with the formation of a truncated protein. An alternative sequence of the nucleic acid can encode a sequence of the polypeptide is full length, with g teologiczne coding sequences, for example, to facilitate purification of the polypeptide, transport, secretion, post-translational modification, or to achieve a therapeutic effect, such as target impact or effectiveness. As noted above, to a modified sequence that encodes a polypeptide, can be added to a label or other heterologous polypeptide, where the term "heterologous" refers to a polypeptide that differs from the polypeptide, which is regarded as a modified polypeptide.
In a non-limiting example, one or more structures of nucleic acids may be made so that they will contain a continuous strand of nucleotides that is identical or complementary to a specific gene, such as gene toxin gelonin. The design of the nucleic acid may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 50000, 100000, 250000, 500000, 750000, at least 1,000,000 nucleotides in length, and can also include the design of a larger size, up to the size of the chromosome, including the following sizes (including all intermediate lengths and intermediate ranges), which ultimately leads to the production of such structures of nucleic acids, as, for example, an artificial chromosome, yeast, and is known to specialists in this field. It should be understood that the terms "intermediate lengths and intermediate ranges" in the context of the present description denotes any length or range that includes or is located between these values (i.e., all integers, including the specified values and in between).
In the context of the present description, the term "DNA segment" refers to a DNA molecule that has been separated from the total genomic DNA from an organism of a particular species. In this regard, the segment of DNA that encodes a polypeptide, refers to a segment of DNA that contains the sequence encoding the polypeptide of wild-type, polymorphic or mutant polypeptide that is already separated or purified after its release from total genomic DNA of the mammal or a specific person. In this context, the term "DNA segment" is used to refer to one or more polypeptides, the segments of DNA, less than that required for the polypeptide, and also recombinant vectors, including, for example, plasmids, Comedy, phage, viruses, etc. In the context of the present description segments of DNA relate to biologically functional equivalent proteins or peptides, such as the modified functional equivalent of toxin gelonin or modified functional equivalent of BLyS. Such sequences can is to occur as a result of the redundancy of codons and functional equivalence, which are known to occur naturally in the sequences of nucleic acids and proteins that they encode. Alternative functionally equivalent proteins or peptides may be obtained through recombinant strategy DNA, which can be introduced by changes in the protein structure, taking into account the characteristics of the exchanged amino acids. Changes programmed by man may be introduced by methods site-directed mutagenesis, for example, to enhance the cytotoxicity of the protein or increase the effectiveness of any treatment Protocol using a protein.
Native and modified polypeptides can be encoded by a nucleic acid molecule included in the vector. The term "vector" is used to refer to nucleic acid molecules that perform the function of the media, which can be integrated sequence of nucleic acid to introduce it in a cage, where it will be replicated. The sequence of the nucleic acid can be "exogenous", where this term means that it is foreign to the cell into which the vector is introduced, or that the specified sequence homologous sequence present in the cell, but is in a position in the nucleic acid of the cell host, where succession is alnost are not usually found. Vectors include plasmids, Comedy, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). Specialist with the average level in this area will be able to construct a vector using standard recombinant techniques, which are described in Sambrook et al. and Ausubel et al. (Sambrooket al., 1989 and Ausubelet al., 1996), which are listed in nastoem description as a reference. Vector, except that it will encode a modified polypeptide, such as a modified gelonin, can encode unmodified polypeptide sequence, such as a fragment of a label or a targeted molecule. Used vectors that encode such fused proteins include pIN vectors (Inouyeet al., 1985), the vectors encoding the his-tag heavy, and pGEX vectors used to obtain soluble fused proteins on the basis of the glutathione-S-transferase (GST), with the later purification and separation or cleavage. Directed molecule is a molecule that sends the modified polypeptide to a specific organ, tissue, cell, or to another position in the body of the individual.
The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least part of the gene about the ukta, capable of transcription. In some cases, RNA molecules next broadcast with the formation of a protein, polypeptide or peptide. In other cases, these sequences are not translated, for example, in the formation of antisense molecules or ribozymes. The expression vectors can contain a variety of "control sequences", where this term refers to nucleic acid sequences necessary for the transcription and possibly translation operatively linked coding sequence in the organism of a particular owner. Vectors and expression vectors, in addition to control sequences that direct transcription and translation, can contain a nucleic acid sequence that performs other functions, which will be described below.
1. Promoters and enhancers
The term "promoter" refers to a control sequence that represents a portion of a nucleic acid sequence, where the initiation and control the speed of transcription. It may contain genetic elements with which regulatory proteins and molecules may contact, such as RNA polymerase and other transcription factors. The phrase "functionally located", "functionally linked", "controlled by" and "under the control of transcription" who appoints, the promoter is in a correct functional location and/or orientation relative to the nucleic acid sequence to control the initiation of transcription and/or expression of this sequence. The promoter may be used in combination with the term "enhancer", but may not be used in combination with the specified term, where this term refers to CIS-active regulatory sequence involved in the activation of transcription of the nucleic acid sequence.
The promoter can be a promoter naturally associated with the gene or sequence, and can be obtained by selection of the 5' non-coding sequences located against the direction of reading information from the coding segment and/or exon. Such a promoter can be considered as "endogenous". Similarly enhancer may constitute such enhancer, which in a natural state is associated with a nucleic acid sequence, located either in the direction of reading of the information or against reading information from the sequence. Alternative some benefits may be determined by the location of the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to the prom the Torah, do not normally associated with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancer refers to the enhancer, which normally is not associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters and enhancers isolated from any other prokaryotic, viral, or eukaryotic cells, and promoters or enhancers "non-natural", that is, containing different elements or different regulatory sites of transcription and/or mutations that alter expression. In addition to obtaining nucleic acid sequences for promoters or enhancers in the synthesis of the above sequence can be obtained by the methods of recombinant cloning and/or nucleic acid amplification, including PCRTMin combination with the compositions given in this description (see U.S. patent NoNo.4683202, 5928906, each of which is incorporated into this description by reference). In addition, it is believed that also can be used control sequences directing transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc.
Once Eesa, it may be important to use the promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism selected for expression. Specialists in the field of molecular biology is mostly known variants of use of promoters, enhancers, and combinations of cell types for expression of the protein (see, for example, the work of Sambrook (Sambrooket al., 1989), cited in the present description by reference). The promoters may be constitutive, tissue-specific, inducible and/or used under appropriate conditions for directing high level expression of the embedded segment of DNA that can be useful for large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.
Identity tissue-specific promoters or members, as well as the tests for characteristics of their activity known to specialists in this field. Examples of such areas include LIMK2 gene of the person (Nomotoet al., 1999), gene receptor 2 somatostatin (Krauset al., 1998), gene component that communicates with retinoic acid epididymus mouse (Lareyreet al., 1999), gene human CD4 (Zhao-Emonetet al., 1998), A2-mouse (XI) collagen (Tsumakiet al, 1998), gene dopamine receptor D1A (Leeet al., 1997), gene insulin-like fact the growth RA II (Wu et al., 1997), molecule-1 adhesion with trombozitarnoe endothelial cell (Almendroet al., 1996).
Promoters that may be considered in the context of use in the expression of conjugated polypeptides, such as fused proteins of the present invention, is shown below in table 3.
The promoters and/or enhancers
|Heavy chain immunoglobulin||Banerjiet al., 1983; Gilleset al., 1983; Grosscheldet al., 1985; Atchinsonet al., 1986, 1987; Imleret al., 1987; Wienbergeret al., 1984; Kiledjianet al., 1988; Portonet al., 1990|
|Light chain immunoglobulin||Queenet al., 1983; Picardet al., 1984|
|Receptor T-cell||Liriaet al., 1987; Winotoet al., 1989; Redondo et al., 1990|
|HLA DQ and/or DQ β||Sullivanet al., 1987|
|β-interferon||Goodbournet al., 1986; Fujitaet al., 1987; Goodbournet al., 1988|
|Interleukin-2||Greeneet al., 1989|
|Retz is ptor interleukin-2||Greeneet al., 1989; Linet al., 1990|
|MHC class II 5||Kochet al., 1989|
|MHC class II HLA-Dra||Shermanet al., 1989|
|β-actin||Kawamotoet al., 1988; Nget al., 1989|
|Muscle creatine kinase (MCK)||Jayneset al., 1988; Horlicket al., 1989; Johnsonet al., 1989|
|Prealbumin (Transthyretin)||Costaet al., 1988|
|Elastase I||Ornitzet al., 1987|
|Metallothionein (MTII)||Karinet al., 1987; Culottaet al., 1989|
|Collagenase||Pinkertet al., 1987; Angelet al., 1987|
|Albumin||Pinkertet al., 1987; Troncheet al., 1989, 1990|
|α-fetoprotein||Godboutet al., 1988; Campereet al., 1989|
|γ-globin||Bodineet al., 1987; Perez-Stableet al., 1990|
|β-globin||Trudelet al., 1987|
|c-fos||Cohenet al., 1987|
|c-HA-ras||Triesmanet al., 1986; Deschampset al., 1985|
|Insulin||Edlundet al., 1985|
|Molecule adhesion of neural cells (NCAM)||Hirschet al., 1990|
|α1-antitripsin||Larimeret al., 1990|
|H2B (TH2B) histone||Hwanget al., 1990|
|The mouse collagen and/or collagen type I||Ripeet al., 1989|
|Glucose-regulated proteins (GRP94 and GRP78)||Changet al., 1989|
|Rat growth hormone||Latsenet al., 1986|
|Serum amyloid a person A (SAA)||Edbrookeet al., 1989|
|Troponin I (TN I)||Yutzeyet al., 1989|
|Platelet-derived growth factor (PDGF)||Pechet al., 1989|
|Muscular dystrophy DuChene||Klamutet al., 1990|
|SV40||Banerjiet al., 1981; Moreauet al., 981; Sleighet al., 1985; Firaket al., 1986; Herret al., 1986; Imbraet al., 1986; Kadeschet al., 1986; Wanget al., 1986; Ondeket al., 1987; Kuhlet al., 1987; Schaffneret al., 1988|
|Polyoma||Swartzendruberet al., 1975; Vasseuret al., 1980; Katinkaet al., 1980, 1981; Tyndellet al., 1981; Dandoloet al., 1983; Villierset al., 1984; Henet al., 1986; Satakeet al., 1988; Campbell and/or Villarrealet al., 1988|
|Retroviruses||Kriegleret al., 1982; Levinsonet al., 1982; Kriegleret al., 1983, 1984a, b, 1988; Boszeet al., 1986; Miksiceket al., 1986; Celanderet al., 1987; Thiesenet al., 1988; Celanderet al., 1988; Choiet al., 1988; Reismanet al., 1989|
|Papillomavirus||Campoet al., 1983; Luskyet al., 1983; Spandidos and/or Wilkieet al., 1983; Spalholzet al., 1985; Luskuet al., 1986; Cripeet al., 1987; Glosset al., 1987; Hirochikaet al., 1987; Stephenset al., 1987|
|Hepatitis b virus||Bullaet al., 1986; Jameelet al., 1986; Shaulet al., 1987; Spandauet al., 1988; Vanniceet al., 1988|
|The human immunodeficiency virus||Muesinget al., 1987; Hauberet al., 1988; Jakobovitset al., 1988; Fenget al., 1988; Takebeet al., 1988; Rosenet al., 1988; Berkhoutet al., 1989; Laspiaet al., 1989; Sharpet al., 1989; Braddocket al., 1989|
|Cytomegalovirus (CMV)||Weberet al., 1984; Boshartet al., 1985; Foeckinget al., 1986|
|Virus simian leukemia Gibbon||Holbrooket al., 1987; Quinnet al., 1989|
2. The initiation signals and internal binding sites of the ribosome
For efficient translation of coding sequences may also need specific signal initiation. These signals include the initiation codon ATG or neighboring sequences. May require exogenous control signals broadcast, including the initiation codon ATG. The person skilled in the art is able to identify the need and provide the necessary signals. It is also well known that the initiation codon must be "within the reading frame so that the frame reading desired coding sequence guaranteed translation of the entire insert. Exogenous control signals broadcast and initiation codons can be either of natural origin or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate enhancer elements, transcription.
In some embodiments, implementation of the present invention can be used items, including internal sites of entry of ribosomes (IRES), to create multigene, or polycistronic messengers. IRES ale the options can bypass dependent on a 5' methylated Cap broadcast within a liposomal model translation and begin translation at internal sites (Pelletier and Sonenberg), 1988). Were described IRES elements from two members of the family of picornaviruses (poliovirus virus encephalomyocarditis) (Pelletier and Sonenberg, 1988), as well as IRES elements from the messenger of mammal (Macejak and Sarnow, 1991). IRES elements may be associated with heterologous open reading frames. Multiple open reading frames can be transcribed together, each of which is separated by the IRES, creating a polycistronic messengers. Due to the IRES element, each open reading frame is available for ribosomes to effectively broadcast. Many genes can be efficiently expressed using a single promoter/enhancer for transcription of one of the messenger (see U.S. patent No. 5 925 565 and 5 935 819, each of which is given in the present description by reference).
3. Multiple cloning sites
Vectors can include a multiple cloning site (MCS), which represents a portion of a nucleic acid that contains the sites for a variety of enzymes, each of which can be used in the framework of standard recombinant technology for splitting vector (see Carbonelliet al., 1999, Levensonet al., 1998, and Cocea, 1997, where these works are given in the present description by reference). The phrase "splitting restriction enzyme" refers to catalytic the splitting of molecules of the nucleic acid enzyme, which functions only in certain positions in the molecule of nucleic acid. Many restriction enzymes currently commercially available. The use of such enzymes is well known to specialists in this field. Often the vector is subjected to linearization or fragment using restriction enzymes that cut the area within MCS, thereby allowing exogenous sequences to ligitamate with the vector. The term "ligation" refers to the process of forming fosfolipidnyh relations between the two fragments of nucleic acids, which can come into contact with each other, but may not be contiguous. Methods relating to the use of enzymes and ligation reactions, known to experts in the field of recombinant technology.
4. Splicing sites
A large part of the transcribed molecules of eukaryotic RNA is subjected to RNA splicing to remove introns from the primary transcript. In the case of vectors containing eukaryotic genomic sequences, may require donor and/or acceptor splicing sites to ensure appropriate processing of the transcript to protein expression (see Chandleret al., 1997, where this work is given in the present description by reference).
5. Arminius signals
The vectors or constructs of the present invention generally include at least one termination signal. The term "termination signal" or "terminator" includes DNA sequences involved in specific termination RNA transcript under the action of RNA polymerase. Thus, in some embodiments, implementation of the present invention considers the termination signal, which terminates the production of RNA transcripts. The terminator may be necessaryin vivoto achieve desirable levels of messenger.
In eukaryotic systems, the plot of the terminator may also include specific DNA sequences that allow for site-specific cleavage of a new transcript in order to open for interaction site of polyadenylation. This gives the signal specialized endogenous polymerase to add serious length of about 200 a residues (poly) to 3'-end of the transcript. RNA molecules, modified the poly-a fragment, apparently, more stable and transmitted more efficiently. Thus, in other embodiments related to eukaryotes, it is preferable that the terminator has included the signal for the cleavage of RNA, and even more preferably, the termination signal has accelerated the polyadenylation of messenger. The terminator and/or website Paul is adenalineage can perform the functions of the elements, acting towards increased levels of messenger and/or minimize read from one cassette to the other sequences.
Terminators considered for use in the context of the present invention relate to any known transcription terminator from the number given in the present description or known to specialists in this field, including, for example, without limitation, the termination sequence of genes, such as, for example, the terminator bovine growth hormone termination or viral sequences, such as, for example, the SV40 terminator. In some embodiments, implementation of the present invention the termination signal lost transcribed or translated sequence, for example, due to truncation of the sequence.
6. The polyadenylation signals
In the expression, especially in eukaryotic expression usually involves the polyadenylation signal for the appropriate polyadenylation of the transcript. The nature of the polyadenylation signal is not considered as decisive for the successful implementation of the present invention and/or may use any such sequence. Preferred options include the SV40 polyadenylation signal and/or a polyadenylation signal of bovine growth hormone, convenient the La and/or known, they function well in various target cells. The polyadenylation may increase the stability of the transcript or can facilitate cytoplasmic transport.
7. Origin replication
Vector for propagation in cell host may contain one or more sites original replication (often called abbreviated as "ori"), which represent a specific sequence of nucleic acid, where replication is initiated. An alternative can be used alone can replicate the sequence (ARS), if a host cell is a yeast cell.
8. Markers suitable for screening of breeding
In some embodiments, implementation of the present invention, the cells containing the construct nucleic acid of the present invention, can be identifiedin vitroorin vivodue to the inclusion of a marker in the expression vector. Such markers will bring in the cell identified in different ways changes, allowing easy identification of cells containing the expression vector. Basically breeding marker is a marker which brings the property, which allows you to make selection. Positive selective marker is a marker, the presence of which it is possible to choose it, have a look at the negative marker is a marker, the presence of which does not allow a selection. An example of a positive marker is a drug resistance marker.
Usually the inclusion of marker selection for medicinal tool allows you to clone and identify transformants, for example, it may be genes that give resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol, representing a useful breeding markers. In the present invention in addition to markers that affect the phenotype, which allow the separation of transformants using appropriate conditions, we also consider the use of other types of markers, including appropriate screening markers such as green fluorescent protein (GFP), the identification of which is based on the results of the colorimetric analysis. Alternative for screening can be used enzymes such as thymidine kinase of herpes simplex virus (tk) or chloramphenicol-acetyltransferase (CAT). For professionals in the field understand how to use immunological markers, and possibly in combination with the methods of analysis based on fluorescence sorter (FACS analysis). It is believed that the nature of the used marker is not the decisive factor, as long as it was capable of expressional the Xia simultaneously with the nucleic acid, encoding the desired gene product. Specialists in this area there are also other examples of breeding markers and markers that are suitable for screening.
9. Cell host
In the context of the present invention, the terms "cell", "cell line" and "cell culture" may be used interchangeably. All of these terms also include their offspring, which represents any one offspring, and all his subsequent generation. It should be understood that all such generation does not have to be identical due to the presence of weak or irreversible mutations. In the context of the expression sequence heterologous nucleic acid, the term "a host cell refers to a prokaryotic or eukaryotic cell, and includes any subject transformation of organisms that are capable of replicating a vector and/or to Express the heterologous gene encoded by a vector. A host cell can be and was used as a recipient for vectors. A host cell may be "transliterowany" or "transformed", where the term refers to the process by which exogenous nucleic acid, such as a modified sequence encoding a protein that is transferred or injected into the cell owner. Transformed cell includes the primary to edu-individual and its offspring.
Cell host may be derived from prokaryotes or eukaryotes, including yeast cells, insect cells and mammalian cells, depending on what the desired effect: replication of the vector or expression of part or all nucleic acid sequence encoded by the vector. Currently, there are many cell lines and cultures for use as a host cell, which can be obtained from the American type culture Collection (American Type Culture Collection, ATCC), which is an organization that focuses on maintaining living cultures and genetic materials (www.atcc.org). Appropriate host organism can be determined by the person skilled in the art based on the vector skeleton and taking into account the desired result. Plasmid or cosmid, for example, can be introduced into prokaryotic cell-host for replication of many vectors. Bacterial cells used as host cells for replication and/or expression of viruses, include DH5α, JM109, and KC8, as well as many commercially available bacterial host cells, such as competent cells SURE® Competent Cells and the so-called Golden cage SOLOPACKTMGold Cells (STRATAGEN®, La Jolla). Alternate as host cells for phage viruses can be used in bacterial cells, such as E coliLE392. Appropriate yeast cells includeSaccharomyces cerevisiae, Saccharomyces pombeandPichia pastoris.
Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat 293, Cos, CHO, Saos, and PC12. Specialists in this field is known and there are many cells of different host cell types and from different types of organisms. Similarly can be used as a viral vector in combination with either eukaryotic or prokaryotic cell host, especially a cell, which allows the replication or expression vector.
Some vectors may be used sequences that allow them to be replicated and/or expressed in prokaryotic and eukaryotic cells. For the specialist in this area is also known under which conditions should be hatching any of the above host cells for their maintenance and for replication of the vector. The obvious and well-known techniques and conditions that may allow large-scale production of vectors, and obtaining nucleic acids encoded by the vectors, and the corresponding polypeptides, proteins or peptides.
10. Expression system
There are various expression systems that include at least part or all of the above composition. The system is recuriting and/or eukaryotic type can be used within the present invention to obtain sequences of nucleic acids or the corresponding polypeptides, proteins and peptides. Many such systems are commercially available and widely used.
System based on insect cells/baculovirus can be used for the production of high levels of protein in the expression of heterologous segment of nucleic acid, for example as described in U.S. patent No. 5 871 985 and 4 879 236, each of which is incorporated into this description by reference, which may be purchased under the trade name MaxBac® 2.0 from Invitrogen®and under the trade name BacPackTMBaculovirus Expression System from Clontech®.
In addition to the above-described expression systems other examples of expression systems of the present invention include the inducible expression system in mammalian cells STRATAGENE®'s COMPLETE CONTROLTMand the expression system in the cells ofE. coli. pET. Another option inducible expression system available from INVITROGEN®, which contains the T-RexTM(tetracycline-regulated expression), which is the inducible expression system in mammalian cells using CMV full length. The company INVITROGEN® also provides an expression system in yeast cells, called expression System in cellsPichia methanolica, which was developed to achieve high-level production of recombinant proteins in methylotrophic Pichia methanolica. Professionals in this field know how to make the expression vector, such as the design of the expression, to obtain a nucleic acid sequence or the corresponding polypeptide, protein or peptide.
11. Viral vectors
There are many ways in which the expression vectors can be integrated into the cells. In some embodiments, implementation of the present invention, the expression vector comprises a viral or genetically engineered vector, derived from a viral genome. The first viruses used as a viral gene vectors were DNA viruses including papovaviruses (simian virus 40, a virus bovine papilloma and virus polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). They have a relatively low capacity for sequences of foreign DNA and are characterized by a limited range of host organisms. In addition, their carcinogenic potential and cytopathic effects in permissive cells is of concern in terms of their security. They can only adapt to 8 KB of foreign genetic material, but can be easily introduced into a variety of cell lines and in laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986). Retroviruses are a group of viruses with odnosa the targeted RNA, characterized by the ability to convert their RNA to double-stranded DNA in infected cells. They can also be used as vectors. Other viral vectors can also be used as a design expression according to the present invention. Can be used with vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Couparet al., 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses. They have some useful features for their application in various mammalian cells (Friedman, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Couparet al., 1988; Horwich et al., 1990).
Molecules of the present invention, that is, protein conjugates, considered in the context of their therapeutic use, are molecules which retain the specificity of BLyS part in combination with cytotoxic potential toxin or therapeutic agent.
In the framework of the present invention, it is assumed that the it polypeptides are introduced in therapeutically effective amounts to a patient, if necessary. The term "therapeutically effective" is used in this context in relation to the number of connections required to improve a symptom, as is aliowance with the disease. For example, in the treatment of cancer, the compound, which to some extent reduces cancer or stops any symptom of cancer, will be considered therapeutically effective. For example, the weakening of cancer can be a inhibition of angiogenesis of cancer cells and/or tissue inhibition or delay of cell growth, increased cell death, or both. A therapeutically effective amount of the compound is not considered as the amount required to cure the disease, but it is proposed to treat the condition.
The present invention addresses the polypeptides, which are useful for the treatment of b-cell chronic lymphocytic leukemia/small lymphocytic lymphoma when prolymphocyte leukemia cells, immunocytoma/lymphoplasmacytoid lymphoma (+/- macroglobulinemia Waldenstrom), lymphoma mantle cell, lymphoma b-cells in the border zone in the lymphoid tissue associated with mucous (MALT type lymphoma b-cells in the border zone of the spleen (+/- villous lymphocytes), leukemic reticulata, diffuse lymphoma, large B-cell mediastinal (thymus) lymphomas of large b-cells, intravascular lymphoma In large-cell lymphoma Burkitt, myeloma plasma cells (multiple myeloma), monoclonal gammopathy or gammopathy Ney the aqueous Genesis (MGUS), indolently myeloma, myeloma of Smoldering, osteosclerotic myeloma (POEMS syndrome), leukemia plasma cells, " non-secretory myeloma, plasmacytoma, solitary, a cancer of the bone, extramedullary plasmacytoma, macroglobulinemia Waldenstrom, illness, caused by an abnormality of the heavy chain (HCD), diseases associated with deposition of immunoglobulin, systemic pathology of the light chain and primary amyloidosis.
In preferred embodiments, the implementation of the present invention discussed herein, the polypeptides are used to treat people.
XI. Combined treatment/therapy cancer
To improve the efficiency of the conjugated polypeptide of the present invention or the coding of its design expression, it may be desirable to combine these compositions with other agents effective in the treatment of b-cell proliferative disorders such as anti-cancer agents. In fact, in specific embodiments, the conjugated polypeptides of the present invention are used in combination with one or more chemotherapeutic agents, which give the effectiveness of the chemotherapeutic agent in respect of cells, including sensitive or resistant cell. Conjugated polypeptides, by themselves or in combination with one or more of terapeutiche the Kimi agents can be administered to an individual with a b-cell proliferative disorder, in addition to other cancer therapies, such as radiation, surgery, gene therapy, etc.
The term "cancer" in relation to the agent indicates the agent is capable of negatively influencing the manifestation of cancer in an individual, for example, due to the destruction of cancer cells, including apoptosis of cancer cells, by reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply of the tumor or cancer cells, strengthen the immune response against cancer cells or a tumor, by preventing or inhibiting the progression of cancer, or increasing the length of time of survival of an individual with cancer. In a more General case, these other tracks will be offered in a combined combination in amounts effective to kill cells or inhibiting their proliferation. This process may involve the simultaneous contact of cells with the construction of the expression and one or more agents or one or more factors. This can be achieved by contact of the cell with a single composition or pharmacological drug that includes both agents, or by contact of the cell with two distinct compositions or drugs at the same time, where one HDMI is tion includes the design expression, and the other includes one or more second agents.
Resistance of tumor cells to chemotherapy and radiation therapy is a major problem in clinical Oncology. One of the goals of research in the field of cancer treatment is to find ways to increase the effectiveness of chemo - and radiotherapy by combining them with another form of therapy. For example, gene timedancing (HS-tK) from herpes simplex virus during delivery to brain tumors vector system based retrovirus successfully induces sensitivity to the antiviral agent ganciclovir (Culver,et al., 1992). In the present invention is that the conjugated polypeptides can be used in combination with chemotherapy, radiation therapy, gene therapy or immunotherapy treatments, in addition to other Pro-apoptotic agents or agents that regulate the cell cycle.
This therapy may precede other kind of treatment or it may follow another kind of treatment at certain intervals of time, ranging from minutes to weeks. In those embodiments, the implementation of the present invention, when another agent and conjugated polypeptide is delivered into the cell individually should primarily provide guarantees that will not be too great a period of time the Yeni between each delivery, so that the agent and the construction of the expression could be helpful in the combined effect on the cell. In such cases, it is believed that you can contact the cell with both kinds of functional components for about 12-24 hours, more preferably, within about 6-12 hours. In some situations, it may be desirable to significantly increase the duration of the treatment, keeping, however, a period of from several days(2, 3, 4, 5, 6 or 7) to several weeks(1, 2, 3, 4, 5, 6, 7 or 8) between the respective introductions.
Can be used in various combinations, where treatment of the conjugated polypeptide designated as a, And therapy using a secondary agent, such as radiation therapy or chemotherapy, is indicated as "B":
A/In a/A B/A/B B/B/A A/A/B A/B a/B B/A/A A/B/B/B B/A/B/B
In/B/B/A B/B/A/B A/A/B/B A/B A/B A/B a/B a/A B/B/A/A
In/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
Introduction to the patient therapeutic expression constructs of the present invention are carried out by the main protocols used for administration of chemotherapeutic drugs, taking into account the toxicity, if any, of this vector. It is expected that the cycles of treatment, if necessary, will be repeated. It is considered that can be used by various standard therapies, as well as surgical intervention, in combination with those described what Apia hyperproliferative cell disorders.
Cancer treatment also includes a variety of concomitant therapies using both chemical drugs and regimens based on irradiation. Combined chemotherapy includes, for example, the use of CIS-platinum (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, hlorambuzila, busulfan, nitrosamine, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, agents that bind with the estrogen receptor, Taxol, gemcitabine, navelbine, inhibitors farnesyl-proteincenter, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the above agents.
B. Radiation therapy
Other factors that cause DNA damage and are widely used include well-known types, such as γ-rays, x-rays and/or the directed delivery of radioisotopes to tumor cells. Also considered other forms of factors that damage DNA, such as microwaves and UV-irradiation. It is highly likely that these factors affect a large range of DNA damage, the precursors of DNA replication and DNA repair, and Assembly and support is the integrity of chromosomes. Ranges dosage of x-rays range from daily doses of 50-200 x-rays for a long period of time (3-4 weeks) to single doses ranging from 2000 to 6000 x-ray. Ranges dosing of radioactive isotopes vary widely and depend on the half-life of the isotope, capacity and type of radiation as well as from their absorption cells neoplasm.
The terms "contacting" and "open to influence," as applied to the cells used to describe the process by which therapeutic design and chemotherapeutic or radiotherapeutic agent is delivered to a target cell or are placed in close proximity to the target cells. For the destruction of cells or stasis both agents are delivered into the cell in such total amount, which is effective for the destruction of cells or to prevent its division.
Immunotherapy drugs in General is based on the use of immune effector cells and molecules that are directed at the cancer cells and destroy them. Immune effector may represent, for example, an antibody specific for a marker on the surface of tumor cells. One antibody can serve as effector therapy, or can be used for recruiting other cells to achieve actually th destruction of cells. The antibody may also be conjugated to a drug or toxin (chemotherapeutic drug, a radionuclide, a chain of ricin, cholera toxin, pertussis toxin, and the like) and just function as directed agent. Alternative effector may be a lymphocyte, characterized in that the surface of the molecule interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T-cells and NK-cells.
Thus, immunotherapy can be used in combination therapy as part of, for example, in combination with gene therapy. Below we discuss the main strategy of combined therapy. Basically, the tumor cell must carry some token that can be affected, i.e. it is not present on most other cells. There are many tumor markers and each of them can be used in the context of the present invention. The most commonly used tumor markers include carcinoembryonic antigen, antigen-specific prostate antigen associated with a tumor of the urinary tract, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, cially antigen Lewis, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.
In another the embodiment of the present invention, the secondary treatment is a gene therapy, in which therapeutic polynucleotide administered before, after or simultaneously with the conjugated polypeptide of the present invention. Shipping conjugated polypeptide in combination with a second vector encoding one of these gene products, has a combined anti-hyperproliferative effect on target tissues. An alternative may be to use a single vector encoding both genes. The present invention relates to a variety of proteins, some of which will be described below.
1. Inducers of cell proliferation
Proteins that induce cell proliferation, also can be divided in different categories depending on their function. A common characteristic of all these proteins is their ability to regulate cell proliferation. For example, the form of PDGF, as sis oncogene, is a secretory growth factor. Oncogenes rarely arise from genes encoding growth factors, and now sis is the only known risk factor in the growth of natural origin. In one embodiment of the present invention it is believed that antisense mRNA which is directed against a specific inducer of cell proliferation, is used to prevent the expression of the inducer of cell proliferation.
Proteins EMS, ErbA, ErbB, and neu represents the t of the receptors of growth factors. Mutations of these receptors lead to loss of function of regulation. For example, point mutations affecting the transmembrane domain protein receptor Neu, leads to the formation of neu antigen. ErbA oncogene is derived from intracellular receptor for thyroid hormone. It is believed that the modified oncogenic ErbA receptor competes with the endogenous receptor for thyroid hormone, causing uncontrolled growth.
The largest class of oncogenes includes proteins signal transduction (e.g., Src, Abl and Ras). Protein Src is a cytoplasmic proteincontaining, transformation from which the proto-oncogene into an oncogene occurs in some cases through mutations in the remainder of tyrosine 527. While the transformation of GTP-asnago ras protein from a proto-oncogene into an oncogene occurs in one example, through the mutation of valine to glycine at amino acid 12 in the sequence, reducing the GTP-asnow activity of ras.
Proteins of the Jun, Fos and Myc are proteins that both transcription factors have a direct effect on nuclear functions.
2. Inhibitors of cell proliferation
The function of tumor suppressor oncogenes is the excessive inhibition of cell proliferation. Inactivation of these genes disrupts their inhibitory activity, leading to unregulated cell proliferation. The following describes the tumor suppressor p53, p16 and C-CAM.
High levels of mutant p53 detected in many cells transformed by chemical carcinogens, ultraviolet radiation and certain viruses. The p53 gene is a frequent target for mutational inactivation in a large number of human tumors and, as shown, represents the most frequently mutated gene in many types of human cancer. It is mutated in more than 50% of NSCLC cases in humans (Hollsteinet al,. 1991) and in a wide range of other tumors.
The p53 gene encodes a phosphoprotein consisting of 393 amino acids, which can form complexes with host proteins, such as large T antigen and E1B. This protein is found in normal tissues and cells, but in lower concentrations than in transformed cells and tumor tissue.
Gene p53 wild type known as an important regulator of growth in cells of different types. Missense mutations is common to the p53 gene and required for the manifestation of the transforming ability of the oncogene. A single genetic change, created by point mutations can generate carcinogenic potential of p53. However, unlike other oncogene point mutation in p53 occurs in at least 30 separate codons, often dominant alleles that several changes in cellular dryer is a type without reduction of homozygosity. Additionally, many of the dominant negative alleles, apparently, are characterized by tolerance for the body and are held in the germ line. Different mutant alleles, as can be seen, manifestations vary from minimal dysfunction to strongly penetrating dominant negative alleles (Weinberg, 1991).
Another inhibitor of cell proliferation is p16. The basic processes of transition between stages of the cell cycle in eukaryotes run cyclin-dependent kinases, or cdks. One of CDK, cyclin-dependent kinase 4 (CDK4) regulates the promoting of the cell cycle from G1. The activity of this enzyme is associated with the phosphorylation of Rb at the late G1 phase. The activity of CDK4 is controlled by an activating subunit, the cyclin D-type and inhibitory subunit p16INK4, which biochemically characterized as a protein that can specifically bind and inhibit CDK4, and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al., 1995). As p16INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation protein Rb. It is also known that p16 regulates the function of CDK6.
p16INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes p16B, p19, p21WAF1 and p27KIP1. P16INK4 gene was mapped to 9p21, on romosome site in many types of tumors often deleterows. Homozygous deletions and mutations in the gene p16INK4 often found in cell lines of human tumor. This observation leads us to conclude that the gene 16INK4 is a tumor suppressor gene. However, this interpretation is difficult to reconcile with the fact that the frequency of changes to the p16INK4 gene significantly lower in primary rekultivirovannyh tumors than in cultured cell lines (Caldaset al., 1994; Chenget al., 1994; Hussussianet al., 1994; Kambet al., 1994; Kambet al., 1994; Moriet al., 1994; Okamotoet al., 1994; Noboriet al., 1995; Orlowet al., 1994; Arapet al., 1995). Restoration of function p16INK4 wild type by transfection of a plasmid expression vector reduces the formation of colonies of the same cell lines human cancer (Okamoto, 1994; Arap, 1995).
Other genes that may be used in accordance with the present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, fused genes p27/p16, fused genes p21/p27, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF or their receptors) and MCC.
3. The regulators of programmed cell death
Apoptosis or programmed cell death is a mandatory process for normal is embrionalno development maintain homeostasis in adult tissues and suppression of carcinogenesis (Kerret al., 1972). It has been shown that proteins of the Bcl2 family and ICE-like proteases are important regulators and effectors of apoptosis in other systems. Bcl2 proteins discovered in Association with follicular lymphoma, play an important role in the control of apoptosis and increased survival of cells in response to various apoptotic stimuli (bakhshi foret al., 1985; Cleary and Sklar, 1985; Clearyet al., 1986; Tsujimotoet al., 1985; Tsujimoto and Croce, 1986). Currently, evolutionary conservative Bcl2 protein is considered as representative of the family of related proteins that can be classified as agonists death or antagonists of cell death.
After the opening of Bcl2 has been shown that this protein acts in the direction of suppression of cell death triggered by a variety of stimuli. In addition, at the present time, it is obvious that there is a whole family of Bcl2 proteins, regulators of cell death, which share structural features and are characterized by homology in sequence. It has been shown that various members of this family or have functions similar to Bcl2 (e.g., BclXL, BclW, BclS, Mcl-1, A1, Bfl-1)or inhibit the function of BCL-2 and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
Approximately 60% of persons with cancer are R the importance of the type of surgical intervention, which includes preventive, diagnostic or aimed at determining the stage of healing or palliative surgery. Healing surgery is a kind of impact on cancer, which can be used in combination with other therapies, such as treatment of the present invention, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. The chimeric molecules of the present invention can be used as neoadjuvant surgical strategy in order to reduce the size of the tumor before resection or can be used as a post-surgical adjuvant strategy in order to sterilize the surgical site after removal of part or all of a tumor.
Healing surgery includes resection, in which cancerous tissue completely or partially physically remove, cut, and/or destroy. Resection of the tumor refers to the physical removal of at least part of the tumor. In addition to tumor resection surgical treatment methods includes laser surgery, cryosurgery, electrosurgery and surgery, controlled under the microscope (Mohs' surgery). In addition, it is believed that the present invention can be used in conjunction with the removal of superficial cancers, Petrakov or incidental to the of richest normal tissues.
When excision of part or the whole of the cancer cells, tissue, or tumor in the body can be formed cavity. The treatment may be carried out in the framework of the additional anti-cancer therapy by perfusion, direct injection or local application to this area. This treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 or 5 weeks, or every 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also be carried out using various dosages.
F. Other agents
It is believed that other agents can be used in conjunction with the present invention to improve therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that act in the direction of positive regulation of cell surface receptors and GAP joints, cytostatic agents, and agents that affect the differentiation, inhibitors of cell adhesion, or agents that increase the sensitivity hyperproliferation of cells to inducers of apoptosis. Immunomodulatory agents include tumor necrosis factor, interferon-alpha, beta and gamma; IL-2 and other cytokines; F42K and other analogues of cytokines or MIP-1, MIP-beta, MCP-1, RANTES, and other chemokines. It is also believed that positive regulation of cellular receptors the surface or their ligands, such as Fas/Fas ligand, DR4 or DR5/TRAIL, will contribute to the apoptosis-inducing ability of the present invention through the creation of autocrine or paracrine effect on hyperproliferative cells. The increase in intercellular signaling functions due to the growth in the number of GAP joints will strengthen antilipidperoxidative effects in relation to neighbouring populations hyperproliferating cells. In other embodiments, use of the cytotoxic agent or the agent acting on the differentiation, in combination with the present invention to enhance antihypercholesterolemic the effectiveness of treatment. Inhibitors of cell adhesion are considered as agents that increase the efficiency of the present invention. Examples of inhibitors of cell adhesion include inhibitors of focal adhesion kinase (FAK) and lovastatin. In addition, it is considered that can be used with other agents that increase the sensitivity hyperproliferative cells to apoptosis, such as antibody s, in combination with the present invention to improve the efficiency of treatment.
Hormone therapy can also be used in combination with the present invention, or in combination with any other form of cancer treatment, as described earlier. The use of hormones may be applicable in the treatment of some types of cancer, such as breast cancer, before the test cancer, ovarian or cervical cancer, to reduce the level of certain hormones, such as testosterone or estrogen, or block their effects. This type of treatment is often used in combination with at least one other cancer therapy as a treatment option or with the aim of reducing the risk of developing metastasis.
XII. Pharmaceutical compositions and routes of administration
The present invention relates to nucleic acid molecules coding for fused proteins. In some embodiments, the pharmaceutical composition is administered to the individual. Various aspects of the present invention relate to conducting effective amount of the aqueous composition. Such compositions mostly dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Additionally, such compositions can be introduced in combination with other types of treatment depending on the disease or condition to be treated. Treatment of lymphoma may include the introduction of a chemotherapeutic drug, radiation therapy, immunotherapy, or the introduction of hormones.
Specialists in this field is well known how to apply the method of gene delivery in situations ofin vivoandex vivo. In the case of viral vectors usually prepare the viral vector used for storage. Depending on the type of virus and its titer of the patient can be the ü delivered 1 - 100, 10 to 50, 100 or 1000 or up to 1·104, 1·105, 1·106, 1·107, 1·108, 1·109, 1·1010, 1·1011or 1·1012viral particles. Similar figures can be obtained by extrapolation for liposomal or other non-viral compositions based on a comparison of the relative efficiencies of absorption. Below is a description of the composition, such as a pharmaceutically acceptable composition.
The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities that do not cause an adverse, allergic or other adverse reactions in the introduction of an animal or person that is acceptable in a particular situation. In the context of the present description, the phrase "pharmaceutically acceptable carrier" includes any and all solvents, dispersion of the environment for cover, bacterial and antifungal agents, isotonic agents, and agents that delay absorption, etc. the Use of such media and agents for pharmaceutical active substances is well known in this field. Except in those cases where any conventional medium or agent is incompatible with the data of the active ingredients, discusses the use of the above components in therapeutic compositions. Auxiliary active ingredients, such as contradictory, the same agents, can also be included in the composition.
The active compounds according to the present invention can be manufactured in a form suitable for parenteral administration, for example, a composition for injection by intravenous, intramuscular, intrathecal, subcutaneous, or even intraperitoneal administration. Obtaining aqueous composition that contains one or more compounds that increase the expression of MHC molecules of class I, it will be clear to experts in this field in light of the above description. In a typical case, such compositions can be manufactured in the form of injectable preparations, in the form of liquid solutions or suspensions; can be also obtained solid forms suitable for use for the manufacture of a solution or suspension adding liquid prior to injection; and, furthermore, the preparations can also be emulsified.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be obtained in aqueous form with appropriate Association with a surface-active substance, such as hydroxypropylcellulose. Dispersions can be prepared in glycerol, liquid polyethylene glycols and their mixtures and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganism is C.
The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions, compositions, including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the preparation of sterile solutions or dispersions immediately before injection. In all cases, the resulting form must be sterile and must be liquefied to such an extent that it can be easily injected. It must be stable under conditions of manufacture and storage and must contain the preservatives that prevent contamination by microorganisms, such as bacteria and fungi.
The active compounds can be introduced into the composition in a neutral or salt form. Pharmaceutically acceptable salts include a salt additive acid (formed on the basis of free amino groups of the protein), salt, obtained using inorganic acids, such as, for example, hydrochloric acid or phosphoric acid, or such organic acids as acetic, oxalic, tartaric, almond acid and the like, Salts formed with the free carboxyl groups can be derived from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamine, wasted is h, procaine, etc.
The carrier may be a solvent or medium for dispersion containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures and vegetable oil. Appropriate fluidity can be maintained, for example, when using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, or by use of surfactants. To prevent the exposure of microorganisms can be achieved through the use of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like In many cases it is preferable to include agents that promote isotonicity, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved through the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions prepared with the inclusion of the active compounds in the required amount in an appropriate solvent, in combination with various other the above agents, if necessary, followed by sterile filtration. Basically dispersion is produced by on the treatment of the various sterilized active ingredients into a sterile medium, which contains the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders used for the manufacture of sterile injectable solutions, the preferred methods of obtaining are vacuum drying and drying methods during freezing, which lead to the formation of a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution.
Introduction therapeutic compositions of the present invention may be implemented using any known way, the main thing is that the target tissue was available with this method of introduction. In those cases, when the present invention uses a viral vector, a major challenge will be achieving the desired localization of heterogeneous sequences contained in the vector. Methods of administration include oral, nasal, transbukkalno, rectal, vaginal or local way of introduction. Alternative introduction can be performed orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions are within the norm introduced in the form of pharmaceutically acceptable compositions, which include physiologically acceptable carriers, buffers or other excipients. For the treatment of the s pathology of the lungs is considered aerosol delivery to the lungs. The amount of aerosol is from about 0.01 ml to 0.5 ml Similarly, the preferred method of treatment of diseases associated with colon rectum, may be the use of enemas. The volume of the contents of the enema is from about 1 ml to 100 ml Direct intratumoral injection is the preferred method, whereas continuous intratumoral perfusion is a more particular embodiment of the invention.
In some embodiments, implementation of the present invention it may be desirable to provide continuous delivery of therapeutic compositions in the body of the individual. In the case of intravenous or intra-arterial routes of administration such delivery is carried out using a drip system. For local injections used re-introduction. When different strategies provides the use of compositions with delayed release, which provides the supply limited, but regular amounts of therapeutic agent over an extended period of time. In the case of domestic make, it may be preferable to continuous perfusion in the right plot, for example, using a viral vector containing segment heterologous nucleic acid. This introduction can be achieved by posioperating catheterization, R is de cases, followed by continuous introduction of therapeutic agent. The duration of perfusion determined by the attending physician taking into account indicators of a particular patient and a particular situation, at this specified period of time varies from 1-2 hours up to 2-6 hours, to about 6-10 hours to about 10-24 hours, to about 1-2 days to 1-2 weeks or longer. Basically, the dose of therapeutic composition is delivered in continuous perfusion will be equivalent dose, administered by single or multiple injections, adjusted for the time period during which the conduct injection. However, it is believed that the perfusion can be achieved with higher doses.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first make isotonic by the addition of sufficient saline or glucose. These particular aqueous solutions are especially acceptable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. This might include the use of sterile water environment that will be understandable for specialists in this area in light of the above description. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of liquid for subcutaneous injection, or John is projected in the proposed site of infusion, (see, for example, Remington''s Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the individual subject treated. The person responsible for the introduction, should be in any case to determine the appropriate dose for a given individual.
An effective amount of therapeutic composition is determined on the basis of the intended purpose. The term "unit dose" or "dosage" refers to physically discrete units suitable for use by an individual, where each unit contains a given number of therapeutic compositions calculated with the aim of producing the desired response in accordance with the above discussion, in conjunction with this introduction, that is, with a suitable method of administration and mode of treatment. The quantity to be the introduction from the point of view of the number of introduction, and the standard dose, depends on the desired level of attainable protection.
The exact number of therapeutic compositions are also determined by the attending physician and will vary for each individual. Factors affecting dose include physical and clinical status of the patient, route of administration, the intended purpose of the treatment (relieving symptoms or cure), as well as the effectiveness, stability, and toxicity of specific therapeutic agents.p> After manufacturing solutions imposed in a way that should be compatible with the composition of the dosage, and in such a quantity that is therapeutically effective. The composition can easily be introduced in a wide range of dosage forms such as injectable solutions described above, but can also be used capsule with a modified release pharmaceutical agents, etc.
In the context of the present description, the term "introduction ofin vitro" refers to the manipulation performed on cells taken from the body of animals, including, without limitation, cells in culture. The term "doingex vivo" refers to cells that are subjected to manipulationin vitroand then injected into the body of the living animal. The term "introduction ofin vivoincludes all manipulations produced by cells within the animal.
In some aspects of the present invention the composition may be injectedin vitro, ex vivoandin vivo. In some embodiments, the introduction ofin vitroconstruction of expression encoding a modified protein may be transducible in a cage of a host. The transduced cells can then be used for analysis ofin vitroor an alternative to the introduction ofin vivo.
In U.S. patent NoNo.4690915 and 5199942, where both documents are incorporated into this description by reference, set forth the methods of manipulation is ex vivowith mononuclear blood cells and bone marrow cells for use in therapeutic purposes.
The present invention also seen the introduction ofin vivodiscussed in the compositions. These examples include, without limitation transduction in the bladder epithelium by intravesicularly catheterization of the bladder (Bass, 1995) and transduction in liver cells by infusion of appropriate transducers compositions via the portal vein using a catheter (Bao, 1996). Additional examples include direct injection into the tumor transducers compositions of the present invention and either intranasal or intrathecal (Dong, 1996) instillation transducers compositions for transduction in lung cells.
According to the present invention the administration can be intravenous, intradermally, intraarterially, intraperitoneally, inside, intracranial, intra-articular, inside the prostate, vnutriplevralno intratrahealno, intranasal, vitreous body, intrawaginalno, rectal, local way, inside the tumor, intramuscularly, intraperitoneally, subcutaneously, vnutrivaginalno, mucous, intrapericardially, orally, topically, locally, and using aerosol injection, injection, infusion, continuous infusion, lo is ilizovano perfusion zone, environmental target cells, either directly or through a catheter and/or lavage.
XIII. The kits of the present invention
Any of the above in the present description of the compositions can be included in the set. In the framework of non-limiting example, the conjugate BLyS and optional additional agent can be included in the kit. Thus, the kits include a suitable container conjugate BLyS and optional additional agent of the present invention.
Kits can include an appropriate aliquot of the conjugate BLyS and optional additional agent compositions of the present invention, labeled or unlabeled. The components of the kit can be packaged in the form of the drug in aqueous medium or in dried form. The shape of the containers in the data sets mainly include at least one vial, test tube, flask, bottle, syringe or other container, into which may be placed in the specified component and preferably corresponding to the selected quantity. In the case when the set includes more than one component, the specified set mainly includes the second, third or other additional container, inside of which can be separately introduced additional components. In addition, the bottle can be introduced by various combinations of components. On the ora of the present invention also typically include a device for maintaining a container conjugate BLyS, additional agent or any other agent in a certain combination for commercial applications. Such containers can include containers for injection molded or flat containers, inside which is placed the desired vials.
Therapeutic kits of the present invention are kits comprising the conjugate BLyS, which contain mostly in the appropriate container, its pharmaceutically acceptable composition. The specified set may include one container device and/or can contain multiple container device for each connection.
In the case when the components of the kit are available in one and/or more liquid solutions, the specified liquid solution is an aqueous solution, with a sterile aqueous solution is particularly preferred. One or more compositions of the conjugate BLyS can be made in the form of a composition in the composition of the syringe tube. In this case, the container device may itself represent a syringe, pipette, and/or other similar device, which may be selected composition and applied to the affected area of the body, inetservices in the body of the animal and/or applied to or mixed with the other components of the set.
In addition, the components of the specified collection can be offered in the form od the CSO or more dried powders. In the case when reagents and/or components are available in the form of a dry powder, the powder can be recovered by adding a suitable solvent. In one embodiment, may also be offered the solvent in the composition of another container of the device. These kits can also include a second container device for maintaining a sterile pharmaceutically acceptable buffer and/or other diluent.
The kits of the present invention typically also include a device for storing bottles in a compact ensemble for commercial applications, such as, for example, vials for injection and/or molded plastic containers in which you can store the desired vials.
Regardless of the number and/or type of containers, the kits of the present invention may also include and/or be packaged with the tool needed for the injection/administration and/or to put the finished composition in the body of the animal and/or for introduction into the body of the animal. Such an instrument may be a syringe, pipette, forceps and/or any permitted for medical delivery media.
Thus, in specific embodiments, the implementation of the present invention, the composition is included in a pharmaceutically acceptable carrier and/or correspondingly Turaida for delivery to the individual.
The following examples are included to demonstrate preferred embodiments of the present invention. For the person skilled in the art it is obvious that the methods described in the examples below, cover the techniques that have been identified by the applicant as a well-functioning in the practice of implementation of the present invention and can therefore be considered as constituting the preferred ways of its practical application. However, for the person skilled in the art will be apparent in light of the above description that there may be a lot of changes in specific ways disclosed explicitly in the description, but still allow you to achieve the same or similar result without derogating from the principles and scope of the present invention.
DEVELOPMENT of RECOMBINANT POLYPEPTIDE GELONIN/BLyS
cDNA encoding a BLys human and recombinant gelonin, merge together in the PCR by the method of overlapping splicing extension (OE-PCR (Higuchiet al., 1988) using BLyS and recombinant DNA gelonin as matrices. Orientation And (BLys-rGel) or orientation (rGel-BLys) fused protein according to the method of OE-PCR using the full coding of the site and recombinant BLys gelonin as matrices for DNA amplification is otdelnyh gene fragments. To construct a BLys-rGel spend amplification against the direction of reading information OE-PCR fragment encoding the cleavage site by enterokinase, and using enzymesKPN Ispend amplification from the N-terminal segment BLys gene using oligonucleotide primers PET BLys (5'-GGCGGAAGCGGTACCGACGACGACGACAAGGCCGTTCAGGGTCCA-3' SEQ ID NO: 12) and BLys Bac Link (5'-GCTCCCGCCTCCCCCCAGCAGTTTCAATGC-3' SEQ ID NO: 13). Amplified joining fragment OE-PCR in the direction of reading information encoding G4S linker and enzyme restrictionXHo Iof the recombinant gene gelonin using oligonucleotide primers Link RGel (5'-GGGGGAGGCGGGAGCGGCCTGGACACCGTG-3' SEQ ID NO: 14) and RGel Bac, (5'-GCTCGTGTCGACCTCGAGTCATTATTTAGGATCTTTATC-3' SEQ ID NO: 15). Fragments of OE-PCR against the direction and in the direction of reading information then subjected to a rearrangement in the form of a fused gene BLys-rGel full length, encoding a protein by introducing an additional stage PCR using pairs of oligonucleotide primers PET For BLys and RGel Bac flanking 5'- and 3'-ends (see above description). The final PCR fragment clean and assalut using restriction endonucleasesKPN IandXho Iand then clone into the expression vector pET-32a (Novagen, uses the T7 promoter for controlling transcription of the built-fused gene. The correctness of the design fused gene BLys-Gel confirmed by DNA sequencing before protein expression. In the case of orientation B for RGel-BLys use the above procedure for manipulation of DNA using oligonucleotide primers PET Gel For (5'-AGCCCAGATCTGGGTACCGACGACGACGACAAGGGCCTGGACACCGTGAGC-3' SEQ ID NO: 16); RGel Bac Link (5'-GCTCCCGCCTCCCCCTTTAGGATCTTT-3' SEQ ID NO: 17) for the fragment against the direction of reading of the information and BLys For (5'-GGGGGAGGCGGGAGCGCCGTTCAGGGTCCA-3' SEQ ID NO: 18); BLys Rev (5'-GCCGTCGACCTCGAGTCATTACAGCAGTTTCAATGC-3' SEQ ID NO: 19) for the slice direction reading information. The final fragment fused gene amplified by introducing an additional stage PCR using oligonucleotide primers PET Gel For and BLys Rev. Then design transformed into a strain ofEschrechia coliAD494(DE3)pLysS for expression of the fused protein.
CHEMICAL CONJUGATION WITH RECOMBINANT BLYS GALANINA AND PURIFICATION of CHEMICAL CONJUGATE BLYS/RGEL
Receive recombinant gelonin (rGel), containing an additional cysteine residue for site-specific conjugation, as described earlier, and carry out conjugation with BLys when using SPDP as described in the literature (Rosenblumet al., 1991; Mujooet al., 1995; Rosenblumet al., 1996). Chemical conjugate BLys/rGel purified using fast liquid protein chromatography was carried out (Pharmacia, New York, NY), which combines gel filtration (S-200) and affinity chromatography (Blue Sepharose). The purity of the conjugate BLys/rGel evaluate when conducting electro is oresa in SDS-PAGE and the results of Western blot analysis.
The STUDY of CYTOTOXICITY
In the following table 4 summarizes the results of various studies of cytotoxicity, carried out using a fused protein rGel/BLys.
Expression of BLys, BAFF-R, TACI, BCMA and comparative values of IR590for rGel/BLys against
cell lines of different types
|Cell type||BLyS||BAFF-R||TACI||BCMA||IR50 (nm)||Index|
|Jurkat||acute T-cell leukemia||+||++||-||-||3000||1500||2,0||KBM-5||myeloid leukemia||+||+||-||-||250||70||3,6|
|THP-1||acute monocytic leukemia||+||++||-||-||110||30||3,7|
|HL-60||acute promyelocytic leukemia||+||++||-||-||1000||300||3,3|
|RPMI 8226||plasmacytoma myeloma||+||++||+||+||280||10||28|
|JEKO||lymphoma mantle cell||+||+++||+||+||200||0,002||100000|
|SP53||lymphoma mantle cell||+++||+||+||60||0,001||60000|
|Mino||lymphoma mantle cell||+||+++||+||+||35||0,005||7000|
|Granta||lymphoma mantle cell||+||++||+||+||1500||700||2,1|
|BCL-1||In the mouse lymphoma||N.D.||N.D.||N.D.||N.D.||150||0,0008||187500|
|* The index of achieving the target corresponds IR50for rGel/IR50for rGel/BLys.|
N.D. = denotes the situation in which the determination was not conducted.
In Fig. 1 shows the orientation of BLyS and rGel. In Fig. 2 shows representative is sledovatelnot DNA (SEQ ID NO: 9) and protein (SEQ ID NO: 8) representative merged toxin BLyS/rGel, and in Fig. 3 shows a representative DNA sequence (SEQ ID NO: 11) and protein (SEQ ID NO: 10) representative merged toxin rGel/BLyS. In Fig. 4 illustrates the construction of a representative merged toxin rGel/BLyS.
In Fig. 5 shows the purification of the fused toxin rGel/BLyS. As can be seen by the results of staining of Kumasi dye in the analysis by electrophoresis in SDS-PAGE merged toxin rGel/BLyS, the indexMrfor rGel/BLyS is 45 kDa, which indicates that the molar ratio of BLyS and rGel equal to 1:1 (left panel). The results of Western blotting using antibodies against gelonin or antibodies against BLyS show that merged toxin rGel/BLyS contains the toxin component and component BLyS the merged toxin (right panel).
In Fig. 7 shows the results of comparison of the cytotoxic activity of merged toxin rGel/BLyS and BLyS/rGel against cell line cells of the mantle JEKO. Cells cell lines mantle JEKO seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add rGel, rGel/BLyS or BLyS/rGel in wells fourfold repetition. After 96 hours to each well, add 75 ál Templ-labeled mixture, after which the cells incubated for another 4 hours. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 8A-8M are shown kr the new dependence "dose-response" for merged toxin rGel/BLyS against various tumor cell lines: Jurkat (8A), KBM-5 (8B), THP-1 (8C), HL-60 (8D), IM-9 (8E), MM1.S (8F), MM1.R (8G), RPMI18226 (8H), 8226/LR-5 (8I), JEKO (8J), SP53 (8K), Mino (8L) and Gratna (8M). Cells thirteen tumor cell lines were seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add to the wells rGel or rGel/BLyS in a fourfold repetition. After 96 hours to each well, add 75 ál Templ-labeled mixture, after which the cells incubated for another 4 hours. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 9 illustrates the specificity merged toxin rGel/BLyS against cells in cell lines mantle JEKO expressing BLyS receptors. Cells cell lines mantle JEKO seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add to the wells of BLyS, rGel, CTP/rGel and rGel/BLyS in a fourfold repetition. After 96 hours to each well, add 75 ál Templ-labeled mixture, after which the cells incubated for another 4 hours. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 6 illustrates inhibiting activity of merged toxin rGel/BLyS in a cell-free system for protein synthesis. For evaluation of the n-glycosidic activity rGel component in the merged toxin rGel/BLyS this material is added to the system under study broadcast the tree in vitrowith the use of [3H]-leucine to evaluate the inclusion of isolated rabbit reticulocytes. Compare inhibition curves for fused toxin rGel/BLyS and native rGel.
In Fig. 10 shows the curves of dependence "dose-response" for merged toxin rGel/BLyS compared to cell lines of multiple myeloma, sensitive to dexamethasone (MM1.S) and resistant to dexamethasone (MM1.R). Cells cell lines MM1.S and MM1.R seeded (with a density of 5·103/well) in wells of 96-well microtiter tablets with a flat bottom and add to the wells rGel, Dex or rGel/BLyS in a fourfold repetition. After 96 hours to each well, add 75 ál Templ-labeled mixture, after which the cells incubated for another 4 hours. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA.
In Fig. 11 illustrates the maximum tolerance dose (MTD) in relation to the rGel/BLyS. To obtain values for MTD rGel/BLyS mice Balb/C mice injected intravenously into the tail vein of varying concentrations of rGel/BLyS for 5 consecutive days and determine the body weight and the number of surviving mice.
CONSTRUCTION, EXPRESSION AND PURIFICATION of the FUSED TOXIN rGel/BLyS
The authors present invention has created the design of the merge rGel/BLyS, orienting rGel on the N-end with the subsequent fragment G4S PE the Chida to join the BLyS molecule, using PCR according to the method of overlapping splicing extension (Ho et ak., 1989) (Fig. 4). The design of the merge then are ligated to the cleavage sites and kpni restriction sites XhoI in the vector pET-32a(+) and transformed into E. coli strain AD494 (DE3). rGel/BLyS Express and purified using affinity chromatography, including immobilizovannyi metal ion (Amersham). After enzymatic removal of a fragment of His fragment size of 20 kDa purified rGel/BLyS migrates during electrophoresis in SDS-PAGE in the form of a monomer with an estimated molecular weight of 5 kDa in reducing conditions. rGel/BLyS is also characterized by immunoreactivity with antibodies BLyS and rGel, thus demonstrating the presence of the design of the merge of both components: toxin and BLyS (figure 5).
The BIOLOGICAL ACTIVITY of R/GEL COMPONENT IN the MERGED TOXIN RGEL/BLYS
The biological activity of the toxin can be significantly weakened by conjugation or fusion with other proteins. To determine the n-glycosidic activity rGel component in the merged toxin rGel/BlyS this material adds a cell-free protein synthesis using test products luciferase. Comparing the curves for inhibition rGel/BLyS and native rGel. It was shown that the calculated values IR50for rGel/BLyS and rGel is 10 PM and 61 PM respectively. In this regard, the results obtained allow the designers to put, that the enzymatic activity of the rGel component in the rGel/BLyS is slightly higher than the free rGel (Fig. 6). Similar results were obtained for construction merge VEGF121and rGel. Glycosilated VEGF121/rGel, as shown, has a high specific activity in comparison with the toxin rGel (Veenendaalet al., 2002). The data show that multimerization rGel component may allow to achieve joint effects between merged molecules of toxin in some embodiments, implementation of the present invention.
EXPRESSION of BLyS, BAFF-R, TACI and BCMA AND REACTION TO RGEL/BLYS
The authors of the present invention investigated the expression profile of BLyS ligand and its three receptors according to the method of RT-PCR using a set of cell lines of leukemia, myeloma and lymphoma cells in the mantle. Thirteen cell lines (Jurkat, KBM-5, THP-1, HL-60, IM-9, MM1.S, MM1.R, RPMI8226, 8226/LR-5, Jeko-1, SP53, Mino and Gratna 519) Express BLyS and BAFF-R, whereas TACI and BCMA are only expressed in the 9 studied representative cell lines, except for the 4 cell lines leukemia (Jurkat, KBM-5, THP-1 and HL-60).
In the next step, the authors investigated whether there is a correlation between levels of expression of one or more BLyS receptors and sensitivity to rGel/BLyS. With this in mind, we conducted a comparative analysis of indicators IR50for rGel/BLyS against 13 Reprise the consultative cell lines, including lines of leukemia, myeloma and lymphoma cells mania. For each type of cells calculate the ratio IR50for rGel and rGel/BLyS. The aforementioned ratio (index of achieving targets) characterizes the ability of a component BLyS in the rGel/BLyS to mediate the delivery of the toxin component in the cytoplasm of target cells. As shown in table 4, cell lines Jurkat, KBM-5, THP-1, HL-60, IM-9, MM1.S, MM1.R, 8226/LR-5 and Gratna 519 exhibit index achieving the target in the range from 2 to 4, while 3 MCL cell lines (Jeko-1, SP53 and Mino)expressing BAFF-R, TACI and BCMA, were highly sensitive to the rGel/BLyS and showed the index of achieving the target in the range from 7,000 to 100,000. MCL cell line JeKo-1, as shown, is most sensitive to the rGel/BLyS (index achieving target = 100000). The authors of the present invention have not been able to establish a direct correlation between levels of expression of one or more BLyS receptors and sensitivity to rGel/BLyS.
BINDING ACTIVITY of RGEL/BLYS
Conduct a comparative analysis of the activity of BLyS component in the rGel/BLyS binding cells using intact cell lines JeKo-1 and HL-60. Merged toxin rGel/BLyS demonstrates a specific activity by binding with JeKo-1 cells expressing all three BLyS receptor, whereas rGel is not associated with JeKo-1 cells and HL-60. Interesting to note is, that rGel/BLyS is not associated with cells HL-60, expressing only BAFF-R, the evaluation by PCR (Fig. 12).
The SPECIFICITY of RGEL/BLYS
When assessing the specificity of rGel/BLyS against cells expressing BLyS receptor, the authors treated cells JeKo-1 by BLyS, free rGel, CTP/rGel (not B-cell directed chemical conjugate) or rGel/BLyS. BLyS itself promotes proliferation of cell growth, while not B-cell directed conjugate CTP/rGel index shows the IR50close to the corresponding figure for free rGel. While rGel/BLyS demonstrates a high cytotoxicity for cells JeKo-1 expressing three BLyS receptor (Fig. 9).
Pre-treatment with the use of BLyS demonstrates a shift in the curve of dependence "dose-response in cells JeKo-1, processed rGel/BLyS, but not in cells JeKo-1, rGel-treated (Fig. 13A). Advanced pre-processing BAFF-R:Fc, TACI:Fc or BCMA:Fc blocks the cytotoxic activity of rGel/BLyS in cells JeKo-1, but not in cells JeKo-1, rGel-treated and BLyS (Fig. 13B). The results demonstrate the fact that the cytotoxic effects of rGel/BLyS, apparently mediated BLyS receptors. In addition, it is obvious that any of the three receptors may be effective for its involvement in cellular cytotoxic effects of the fused protein.
The EXAMPLE 10
The INTERNALIZATION of RGEL/BLYS IN CELL LINES LYMPHOMA MANTLE CELL (MCL) JEKO-1
The internalization of rGel/BLyS detected using an anti-rGel antibody rabbit. Fragment rGel of rGel/BLyS detected in the cytoplasm and nucleus after 1-hour exposure with rGel/BLyS, which indicates the ability of the fused protein rGel/BLyS effectively communicate with cells by binding BLyS with BLyS receptors, for rapid internalization and delivery rGel toxin into the cytoplasm and the nucleus of cells JeKo-1.
The EFFECTS of RGEL/BLYS IN the WAY of APOPTOSIS
To identify, whether associated cytotoxic effect of rGel/BLyS with the mechanism of apoptosis, cells JeKo-1 was treated with 100 PM BLyS, 100 PM rGel or 100 PM rGel/BLyS. After 96 hours the cells JeKo-1 analyze when TUNEL staining to assess apoptosis. Cells JeKo-1, processed rGel/BLyS demonstrate in 34% of cases cell death from apoptosis, whereas rGel treatment does not induce apoptosis (Fig. 14A and Fig. 14V).
It is known that proteins series caspase is a Central mediator of the process of apoptosis. To identify, activate whether the caspase-3 in cells JeKo-1 at rGel/BLyS-induced cell death, the authors investigated the cleavage of caspase-3 and its substrate poly(ADP)-ribosomally (PARP). Processing BLyS or rGel has no effect on the cleavage of caspase-3 and PARP, whereas the processing of rGel/BLyS leads cher the C 96 hours to the cleavage of caspase-3 and PARP (Fig. 14C).
The RESULTS of the PRESENT INVENTION
BLyS is a binding growth factor, accelerating the development of peripheral b cells, and growth-stimulating effects of BLyS mediated by three receptors on the cell surface, designated as BAFF-R, TACI and BCMA (Thompsonet al., 2001; von Bulow and Bram, 1997; Laabiet al., 1992). Existing messages suggest that BLyS, apparently, is expressed differently in samples B-CLL, so can be described in many patients with congenital resistance to therapeutic agents. In this regard, the inventors chose BLyS as miseriway ligand for specific delivery rGel toxin to tumor cells expressing one or more BLyS receptors. The inventors chose the rGel/BLyS orientation, but not BLyS/rGel, for construction of a merger, because unpublished data it follows that the open-end of the BLyS molecule important for the trimerization and recognition receptor. Other studies using inactive structures merge BLyS/rGel confirmed this observation.
To identify potential correlations between the levels of cellular expression of BLyS receptor and sensitivity to rGel/BLyS the authors of the present invention investigated the levels of expression of BLyS and its three receptors and analyzed in a comparative perspective pok the indices IR 50for rGel/BLyS and rGel against various cell lines, including cell lines of leukemia, multiple myeloma, lymphoma cells of the mantle of human rights and cell lines In the mouse lymphoma. Cells cell lines In the mouse lymphoma BCL-1 exhibit the highest index of achieving the target (187500). Kanakaraj et al. (Kanakarajiet al., 2001) reported that cells BCL-1 contain 4800 binding sites/cell, whereas cells of the IM-9 contain 3200 binding sites/cell. The responsiveness of cells BCL-1 rGel/BLyS may also be associated with the total number of BLyS receptors. Three cell lines MCL (JeKo-1, SP53 and Mino)expressing BAFF-R, TACI and BCMA, have high sensitivity to rGel/BLyS and will show the index of achieving the target of 7,000 to 100,000, whereas cells cell lines Granta 519 show the index of achieving target 2 to 4, even when cells Granta 519 Express all three receptors for BLyS level, which roughly corresponds to the expression on the cells of JeKo-1, the estimation by the method of RT-PCR. It was shown that the cells of cell line JeKo-1 MCL are characterized by the highest sensitivity to rGel/BLyS (index achieving target = 100000). Among the cell lines MCL cells Granta 519 are characterized by very high resistance to rGel/BLyS and most rGel. The inventors were unable to detect a direct correlation between the level of expression of one or more prescriptions the Directors BLyS and sensitivity to rGel/BLyS and explored different cytotoxic mechanisms of rGel/BLyS in these two cell lines MCL to identify mechanisms which may be responsible for different cytotoxic effects. The inventors have shown rGel/BLyS can quickly internalization in the most sensitive cell line JeKo-1, but not Granta 519 (results not shown). This result indicates that rGel/BLyS may internalization into cells JeKo-1 after binding to BLyS receptors and deliver rGel toxin in the cell line JeKo-1 MCL expressing three BLyS receptor.
Lymphoma mantle cell (MCL) is a special type of B-cell non-Hodgkin lymphoma, which is characterized by a combination of morphologic, immunophenotypic and cytogenetic features and which expresses cyclin D1. Traditional cytotoxic therapy in this case is ineffective and the overall Outlook is quite unfavorable (Leonard,et al., 2001). MCL is the most resistant to therapy of b-cell non-Hodgkin lymphoma (Weisenburgeret al., 2000). Resistance MCL to the currently used method of chemotherapy indicates a need for new therapeutic approaches to the treatment of MCL. The results obtained indicate that the merged toxin rGel/BLyS is a great therapeutic agent, at least for the case of lymphoma cells in the mantle, i.e. lymphoma that is resistant to most currently available methods of treatment.
Multiple mie is Ohm (MM) is a B-cell neoplasia, which is characterized by clonal expansion of plasma cells in the bone marrow and which is not treatable even with the introduction of high doses of drugs within the traditional modes of treatment. Grinstein et al. (Greensteinet al., 2003) found three forms MM: MM1.S (dexamethasone-sensitive cells), MM1RE (early form of dexamethasone-resistant cells MM1.R) and MM1.RL (late form dexamethasone-resistant cells 1.R) to study the etiology of MM, effects of chemotherapeutic agents and the development of clinical resistance. Interestingly, the inventors have shown that sensitive to dexamethasone (MM1.S) and resistant to dexamethasone (MM1.R) cell lines are equally sensitive to rGel/BLyS (indicators IR50equal to 300 nm for rGel/BLyS). The inventors also showed that the original cells of multiple myeloma sensitive melphalan RPMI8226 and resistant to melphalan 8226/LR-5, are equally sensitive to rGel (200 against 280, respectively), but not to the rGel/BLyS (10 vs 110, respectively) (table 4). These observations suggest that cellular resistance to dexamethasone is not a result of the development of cross-resistance to fused toxin rGel/BLyS, whereas cellular resistance to melphalan appears in the result of the development of cross-resistance to melphalan. This is currently researches the signal function to detect the cytotoxic mechanism of rGel/BLyS in sensitive and drug-resistant drug cell lines.
Taken together, these findings indicate that rGel/BLyS is a good potential agent for the treatment of at least lymphoma cells of the mantle, and in specific embodiments, BLyS serves as misenyova ligand for the specific delivery of the toxin to cells expressing one or more receptors for BLyS.
REPRESENTATIVE MATERIALS AND METHODS
The present invention relates to the following representative materials and methods used for its implementation.
Reagents for PCR kit for RNA extraction and kits for carrying out reverse transcription (FROM)-PCR are available from Life Technologies, Inc. (Frederick, MD). The enzymes acquire from New England Biolabs (Beverly, MA). Kits for purification of DNA and RNA receive from the company Qiagen, Inc. (Valencia, CA). Bacterial strains, pET plasmids expression in bacteria and recombinant enterokinase (rEK) receive from Novagen (Madison, WI). Hi-Trap chelating resin HP and other types of resins for chromatography acquire from Amersham Bioscience (Uppsala, Sweden). Mouse monoclonal anti-PARP antibody (ATA), rabbit antibody against cycline D1, and goat antibody against β-actin receive from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody against active caspase-3 receive from BD Biosciences (San Jose, CA).Cell lines and cell culture
Doxorubicin-sensitive cell line multiple myeloma MM1.S and the same resistant cell line MM1.R were kindly provided by Dr. Varsha Gandhi (Dr.Varsha Gandhi (M.D. Anderson Cancer Center, Houston, TX)). Resistant to melphalan cell line plasmacytoma myeloma 8226/LR-5 was kindly provided by Dr. William Dalton (Dr. William Dalton (Arizona Cancer Center, Tucson)) (Philips,et al., 2003). Four cell lines lymphoma mantle cell (MCL) (JeKo-1, SP53, Mino and Granta 519) were kindly provided by Dr. Hashes Amin (Dr. Hesham Amin (M.D. Anderson Cancer Center, Houston, TX) (Kanakarajet al., 2001). Cell lines Jurkat, KBM-5, THP-1, HL-60, IM-9, RPMI 8226, MM1.S, MM1.R and JeK-1 grown in medium RPMI 1640 (ATCC Manassas, VA) with the addition of 10% V / V heat inactivated fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin. In medium RPMI 1640 add 5 μm of melphalan (ATCC) cell line RPMI8226/LR-5. Cell line Granta 519 grow in DMEM (Invitrogen, Grand Island, NY) with the addition of 10% V / V heat inactivated fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin. Cell lines SP53 and Mino are grown in RPMI medium 1640 (ATCC) with the addition of 20% V / V heat inactivated fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin.
The design merged toxin rGel/BLyS
Distinguish RNA from cells JeKo-1 and amplifies the shape cDNA, encoding a BlyS person, by the method of RT-PCR using the following primers: BLySf (5'→3'): GGAGAAGGCAACTCCAGTCAGAAC (SEQ ID NO: 22) and BLySr (5'→3'): GTCCATGTCTTTGGGGATGAATTG (SEQ ID NO: 23) (Schneideret al., 1999). The design DNA for fused protein rGel/BLyS create within the PCR by the method of overlapping splicing extension (OE-PCR) (Weisenburgeret al., 2000) using the full coding of the site and recombinant BLyS gelonin as matrices DNA for amplification of individual gene fragments. For constructing the structure of DNA corresponding to the slit protein rGel/BLyS, americasouth against the direction of reading information in the framework of OE-PCR fragment encoding enterokinase and the cleavage sites of restriction enzymes Kpn I, from the N-terminal part of recombinant gelonin using oligonucleotide primers PETgelfor (5'-AGCCCAGATCTGGGTACCGACGACGACGACAAGGGCCTGGACACCGTGAGC-3'; SEQ ID NO: 16); rGelbaclink (5'-GCTCCCGCCTCCCCCTTTAGGATCTTT-3'; SEQ ID NO: 17). Within the OE-PCR in the direction of the read data amplified joining fragment encoding the G4S linker and cleavage site of the restriction enzymeXHo I,from the BLyS gene using oligonucleotide primers BLySfor, (5'-GGGGGAGGCGGGAGCGCCGTTCAGGGTCCA-3', SEQ ID NO: 18) and BLySrev, (5'-GCCGTCGACCTCGAGTCATTACAGCAGTTTCAATGC-3', SEQ ID NO: 19). Fragments of OE-PCR against the direction and in the direction of reading information then subjected to a rearrangement in the form of a fused gene for rGel/BLyS full length for which no additional stage PCR using pairs of oligonucleotide primers PETgelfor and BLySrev, flanking 5'- and 3'-ends (see above description). The final PCR fragment clean and assalut using restriction endonucleasesKpn IandXho Iand then clone into the expression vector pET-32a (Novagen) using the T7 promoter for controlling transcription of the built-fused gene. Correct gene structures for fused toxin rGel/BLyS confirmed by DNA sequencing and the correct design of the merge transform in theEscherichia colistrain AD494 (DE3) for expression fused to a toxin.
Expression and induction of rGel/BLyS in E. coli
Bacterial colonies transformed with the plasmid containing the insert rGel/BLyS, cultivated in bouillon medium Luria containing 400 μg/ml of ampicillin and 30 μg/ml kanamycin, at 37°C overnight on a rocking chair with the speed of 235 rpm Then the bacterial culture was diluted in the ratio 1:100 in fresh LB medium containing antibiotics, and raise to A600= 0,8 at 37°C. then the culture was diluted in the ratio 1:1 with fresh medium KB plus 400 μg/ml of ampicillin and 30 μg/ml kanamycin and induce the expression of growth factor fused to a toxin rGel/BLyS at a temperature of 23°C by addition of 100 μm topropel-1-thio-β-D-galactopyranoside (IPTG) overnight. Cells are harvested by centrifugation, resuspended 40 mm Tris-HCl (pH 8) and frozen (-80°C).
The sight of the TKA rGel/BLyS
Frozen bacterial cells are thawed and are lysed by physical disruption (Bead Beaters, Biospec Product, Bartlesville, OK) at 4°C. Bacterial lysates objective ultracentrifugation at 40,000·g for 1.5 hours. The final NaCl concentration in the supernatant adjusted to 500 mm NaCl and then applied to a Hi-Trap chelating HP resin (Amersham)containing 200 mm Ni2SO4. The column was washed with 40 mm Tris-HCL (pH 8) and 500 mm NaCl containing 30 mm imidazole, and elute with 40 mm Tris-HCl (pH 8) and 500 mm NaCl, containing 300 mm imidazole. The fractions containing rGel/BLyS, unite and cialiswhat in the buffer for dialysis containing 20 mm Tris-HCl (pH of 7.4) and 150 mm NaCl. To remove the his-tag tag fused toxin rGel/BLyS containing his-tag label, left overnight at room temperature for cleavage by recombinant enterokinase (rEK, Novagen). Nonspecific protein his-tag and a label size of 20 kDa are removed by ion-exchange chromatography on sepharose (Q-Sepharose Fast Flow (Amersham)) and affinity chromatography on Blue-Sepharose CL-6B (Amersham). Purified samples of rGel/BLyS sterile filtered, selected aliquots and stored at 4°C.
Analysis of rGel/BLyS
To assess the presence of toxin rGel component and component BLyS the merged toxin rGel/BLyS target samples analyzed by electrophoresis in 12% SDS-PAGE in reducing conditions. Purified rGel/BLyS (5 g) the Department shall Aut electrophoresis in SDS-PAGE (8-15%) and electrophoretic transfer to PVDF membrane (Millipore Corporation, Bedford, MA) overnight at 4°C in buffer for transfer [25 mm Tris-HCl (pH 8,3), 190 mm glycine, 20% methanol]. The PVDF membrane blocking for 1 hour Tris-buffered saline (TBS)containing 5% skim milk, and then probe antibody rabbit against gelonin or goat antibody against BLyS. Use goat anti-rabbit or pork anticosti antibody conjugated with horseradish peroxidase (Bio-Red Laboratories, Herciles, CA)to visualize immunoreactive proteins in the dilution 1:4000, using a detection reagent ECL (Amersham Pharmacia Biotech Inc., Piscataway, NJ). BLyS or recombinant gelonin used as positive controls. Data is expressed as the average density of protein bands normalized to β-actin. The intensity of the bands determined quantitatively by using the histogram.
Inhibitory activity of rGel/BLyS in a cell-free system for protein synthesis
N-glycosidic inhibitory activity of recombinant rGel toxin assessed in comparison with the corresponding activity of the fused toxin rGel/BLyS. Assess toxin-induced inhibition of protein products in the test using lysate of rabbit reticulocytes according to the manufacturer's instructions (Promega, Madison, WI)as previously described (Hale, 2001).
The activity of rGel/BLyS binding with the receptor, BLyS
To assess actively linking the tee merged toxin rGel/BLyS with BLyS receptor cells JeKo-1 and HL-60 immobilized in the wells of 96-well plates, covered with a layer of poly-L-lysine (Becton Dickinson Labware, Franklin Lakes, NJ), with a density of 1·105cells/well. Tablets dehydrate, blocked by adding 3% BSA and then incubated with different concentrations of rGel/BLyS or rGel in dilution buffer (FBI, 0.1% tween-20, 0.1% BSA) for 2 hours at room temperature. After washing tablets incubated with rabbit polyclonal antibody against gelonin for 1 hour at room temperature, washed 4 times PBRT (mcg/ml in the FBI, 0.1% tween-20, 0.1% BSA) and then add to each well 100 μl of goat anti-rabbit IgG conjugated to peroxidase (1 mg/ml in dilution buffer; Vector, Burlingame, CA). Tablets incubated for 1 hour at room temperature, washed 4 times using PBRT and add to staining of the substrate tetramethylbenzidine (Sigma). Measure the absorbance at a wavelength of 450 nm using a device Spentra Max 3000 (Molecular Devices, Sunnyvale, CA).
Detection of expression of BLyS, BAFF-R, TACI and BCMA in different cell lines
The expression of BLyS, BAFF-R, TACI and BCMA appreciate in 13 cell lines by the procedure of polymerase-cableway reaction with reverse transcriptase (analysis RT-PCR). Allocate the total RNA of 13 cell lines and used for the synthesis of the first chain cDNA, which, in turn, amplified by PCR method using specific primers designed deamplification human BLyS (313 BP) (Schneider et al., 1999), BAFF-R (256 BP) (Kernet al., 2004), TACI (196 BP) (Phillips,et al.2003) and BCMA (285 BP) (Phillips,et al., 2003). GADPH is used as a control.
Cytotoxic activity of rGel/BLyS and competitive inhibition of free BLyS or false receptors
For comparative evaluation of indicators IR50rGel/BLyS against 13 cell lines listed thirteen cell lines were seeded (with a density of 5·103cells/well) in wells of 96-well plates with flat bottom (Becton Dickinson) and in holes in the fourfold repetition adds itself rGel or rGel/BLyS. After 96 hours to each well add 50 ál of XTT-labeled mixture (Roche), after which the cells incubated for another night. Determine the absorption on the spectrophotometer at a wavelength of 450 nm using a counter for ELISA (Bio-Tek Instruments, Inc., Winooski, VT).
To assess the specificity of rGel/BLyS against cells expressing BLyS receptor, the authors treated cells JeKo-1 by BLyS, free rGel, CTP/rGel (not b-cell directed chemical conjugate), rGel/BLyS or environment that is added to the wells in a fourfold repetition. For the analysis of competitive inhibition of cell JeKo-1 sow (with a density of 5·103cells/well) in wells of 96-well microtiter tablets with a flat bottom (Becton Dickinson) and conduct pre-treatment with 1 nm BLyS, 50 nm BLyS, 10 μg/ml BAFF-R:Fc, 10 μg/ml TACI:Fc or 10 μg/ml BCMA:Fc in ECENA 2 hours and then in the hole in the fourfold repetition add rGel/BLyS or rGel.
The internalization of rGel/BLyS in cell lines JeKo-1 lymphoma mantle cell (MCL)
Cell line JeKo-1 MCL add to slides 16-hole camera, covered with lysine (Nunc, Rochester, NY)with a density of 1·104cells/well and treated with 100 nm rGel or 100 nm rGel/BLyS during different periods of time. The cells are then placed on slides using cytocentrifuge (Shandon, Pittsburg, PA), after which proteins associated with the cell surface, remove by 10-minute incubation with glycine buffer [500 mm NaCl, and 0.1 M glycine (pH 2,5)], neutralize for 5 minutes using 0.5 M Tris (pH of 7.4), washed quickly FBI and fixed in 3.7% formaldehyde (Sigma, St. Louis, MO) for 20 minutes at room temperature followed by rinsing in the FBI. The cells are then treated for 10 minutes to impact on their permeability using the FBI, containing 0.2% Triton X-100, washed three times by the FBI and block the FBI, containing 3% BSA, for 1 hour at room temperature. After a quick rinse FBI cells incubated with rabbit anti-rGel polyclonal antibody, diluted in the ratio 1:500 in the FBI, containing 0.1% tween-20 and 0.2% BSA, for 1 hour at room temperature. Cells are washed three times in the FBI, containing 0.1% tween-20 for 15 minutes and incubated with anti-rabbit IgG linked with FITC (Sigma), diluted 1:100, with the addition of 1 is kg/ml iodide of propecia (PI). After three washes using the FBI, containing 0.1% tween 20, the cells are again washed in by the FBI for 10 minutes and put on the environment DABCO. Next, the slides are analyzed using a laser scanning microscope Zeiss LSM510 (Carl Zeiss, Jena, Germany).
Detection of apoptosis
Apoptosis detected by the method of TdT-mediated dUTP labeled end gap (TUNEL test). For assessment of apoptosis cells JeKo-1 add to slides 16-hole camera, covered with polylysine (Nunc)with a density of 5·103cells/well and treated with 100 PM BLyS, 100 PM rGel, 100 PM rGel/BLyS or medium for 4 days. Pop-up cells are collected and attached to slides using cytocentrifuge (Shandon), dried and fixed with 3.7% formaldehyde (Sigma) for 20 minutes at room temperature and then quickly washed in the FBI. Next, cells treated for 10 minutes to impact on their permeability using the FBI, containing 0.2% Triton X-100 and 0.1 % sodium citrate, washed three times by the FBI and block the FBI, containing 3% BSA, for 1 hour at room temperature. Fixed cells stainedin situusing the kit to identify dead cells (Roche). After conducting the final stage of fast rinse slides contribute in an appropriate environment and analyzed under a fluorescent microscope.
Western blot EN is Liz
To study the effects of rGel/BLyS on the expression of cleaved caspase-3 and PARP, cells JeKo-1 were seeded with a density of 5·105cells/well 24-hole tablet and then treated with 100 PM BLyS, 100 PM rGel, 100 PM rGel/BLyS or environment. After 96 hours the cells are washed twice with phosphate-buffer solution (FBI) or are lysed on ice for 20 minutes in 0.3 ml of buffer for lizirovania (10 mm Tris-HCl, pH 8, 60 mm KCl, 1 mm EDTA, 1 mm DTT, and 0.2% NP-40). Cell lysates (50 μg) was separated by electrophoresis in SDS-PAGE (8-15%) and electrophoretic transfer to PVDF membranes (Millipore Corporation, Bedford, MA) overnight at 4°C in buffer for transfer [25 mm Tris-HCl (pH 8,3), 190 mm glycine, 20% methanol]. The PVDF membrane blocking for 1 hour Tris-buffered saline (TBS)containing 5% skim milk, and then probe a mouse monoclonal antibody (Antibodies) against PARP, rabbit polyclonal antibody against active caspase-3 or goat antibody against β-actin. Use goat antimurine/anti-rabbit antibody or pork anticosti antibody conjugated with horseradish peroxidase (Rio-Bad Laboratories, Herciles, CA)to visualize immunoreactive proteins, dilution 1:4000, using a detection reagent ECL (Amersham Pharmacia Biotech Inc., Piscataway, NJ). Data is expressed as the relative density of protein bands normalized to β-actin. The intensity of Yunosti bands determined quantitatively by using the histogram.
All patents and publications cited in the description, are indicative of the level of knowledge achieved to date in the field to which the invention relates. All patents and publications included in the list of references in the extent to which each specific publication specifically and individually corresponds to a specific reference.
PATENTS AND PATENT APPLICATIONS
U.S. patent No.5631348
U.S. patent No.6669938
U.S. patent No.RE37462
U.S. patent No.6750329
U.S. patent No.6599505
U.S. patent No.6214974
U.S. patent No.5624827
U.S. patent No.5053226
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Novak AJ, Bram RJ, Kay NE, Jelinek DF. By aberrant expression of B-lymphocyte stimulator by B chronic leukemia cells: a mechanism for survival. Blood. 2002; 100:2973-2979.
O'boyle KP, Colletti D, Mazurek C, et al. Potentiation of antiproliferative effects of monoclonal antibody Lym-1 immunoconjugate and Lym-1-gelonin on human Burkitt''s lymphoma cells with gamma-interferon and tumor necrosis factor. J Immunother Emphasis Tumor Immunol. 1995; 18:221-230.
Phillips TA, Ni J, Hunt JS. Cell-specific expression of B-lymphocyte (APRIL, BLyS)- and Th2 (CD30L/CD153)-promoting tumor necrosis factor superfamily ligands in human placentas. J Leukoc Biol. 2003; 74:81-87.
Riccobene TA, Miceli RC, Lincoln C, et al. Rapid and specific targeting of 1251-labeled B lymphocyte stimulator to lymphoid tissues and B cell tumors in mice. J Nucl Med. 2003; 44:422-433.
Rosenblum MG, Cheung LH, Liu Y, Marks JW. Design, expression, purification, and characterization, in vitro and in vivo, of an antimelanoma single-chain Fv antibody fused to the toxin gelonin. Cancer Res. 2003; 63:3995-4002.
Rosenblum MG, Murray JL, Cheung L, Rifkin R, Salmon S, Bartholomew R. A specific and potent immunotoxin composed of antibody ZME-018 and the plant toxin gelonin. Mol Biother. 1991; 3:6-13.
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Although the present invention and its advantages have been described in detail, it should be understood that the specified text can be entered various changes, substitutions or rearrangements without departure from the essence and scope of the present invention defined by the attached claims. In addition, the scope of the present invention is not limited to a particular variant of the method, device, process the sa production, compositions, means, methods and stages that are listed in the description. As it is obvious to experts in this field, based on the description of the present invention, the methods, devices, manufacturing process, composition, tools, techniques and stages, currently existing or that may be developed later and which perform essentially the same function and achieve essentially the same result as in the above respective embodiments of the invention according to the present description may be used in accordance with the present invention. In this connection the accompanying claims includes within its scope such methods, devices, manufacturing process, composition, means, methods, or stage.
1. Pharmaceutical composition for targeting cells bearing the receptor for BLyS, containing an effective amount of the fused protein consisting of a BLyS polypeptide, fused to cytotoxic polypeptide, where the cytotoxic polypeptide is located at the N end of the fused protein and the BLyS polypeptide is located at the end of the fused protein, and a pharmaceutically acceptable carrier.
2. The composition according to claim 1, wherein the BLyS polypeptide contains B-cell directed domain.
3. The composition according to claim 1, wherein the BLyS polypeptide contains D-E loop recognition receptor.
4. The composition according to the .2, wherein the BLyS polypeptide and a cytotoxic agent conjugated to a chemical.
5. The composition according to claim 2, characterized in that said cytotoxic agent contains a molecule gelonin.
6. The composition according to claim 2, characterized in that said cytotoxic agent is selected from the group consisting of ricin A, diphtheria toxin, abrin, dodecandra, trichosanthin, trichokirin, brodina, the anti-virus agent Mirabilis, protein, ribosome inactivating barley (BRIP), antiviral protein from lacunosa (PAP), saponin, luffin, exotoxin Pseuodomonas and MOMORDICA.
7. The method of treatment of an individual with a B-cell proliferative disorder involving the introduction of a specified individual a therapeutically effective amount of a composition according to any one of claims 1 to 6.
8. The method according to claim 7, characterized in that the said B-cell proliferative disorder selected from the group consisting of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma when prolymphocyte leukemia cells, immunocytoma/lymphoplasmacytoid lymphoma (+/- macroglobulinemia Waldenstrom), lymphoma mantle cell, lymphoma b-cells in the border zone in the lymphoid tissue associated with mucous (MALT type lymphoma b-cells in the border zone of the spleen (+/- villous lymphocytes), leukemic reticulata, diffuse lymphoma croup is s b-cells, mediastinal (thymus) lymphomas of large b-cells, intravascular lymphoma large B-cell lymphoma Burkitt, myeloma plasma cells (multiple myeloma), monoclonal gammopathy or gammopathy of unknown origin (MGUS), indolently myeloma, myeloma of Smoldering, osteosclerotic myeloma (POEMS syndrome), leukemia plasma cells not secreting myeloma, plasmacytoma, solitary, a cancer of the bone, extramedullary plasmacytoma, macroglobulinemia Waldenstrom, illness, caused by an abnormality of the heavy chain (HCD), diseases associated with deposition of immunoglobulin, systemic pathology light chain, primary amyloidosis, non-Hodgkin's lymphoma, systemic systemic lupus or arthritis.
9. The method of selective effects on cells expressing BLyS receptor, comprising contacting cells with an effective amount of a composition according to any one of claims 1 to 7.
10. Set containing composition according to any one of claims 1 to 7, placed in an acceptable container.
11. The set of claim 10, wherein the specified composition are selected appropriately for delivery to the individual.
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, in particular to obtaining protein complexes and can be used in medicine. A complex of biologically active recombinant protein is obtained, which is non-covalently bonded to polysialic acid which is inactive in aqueous solution at pH 4.4-4.7 which has prolonged therapeutic effect and has general formula:
where m=20-110; n=1-4; P is biologically active recombinant protein.
EFFECT: invention enables to obtain a complex of biologically active recombinant protein with polysialic acid, having stability and prolonged effect.
6 dwg, 2 tbl, 15 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to genetic and protein engineering and can be used in biomedical industry. A genetic make up is proposed, which codes a peptide in which two domains bonding the growth hormone (GH) receptor are bonded into a tandem by a "semirigid" or "rigid" linker, which consists of at least 1-4 copies of the A(EAAAK)A amino acid sequence.
EFFECT: as a result of expression of the nucleotide sequence coding the said tandem, GH-linker-GH polypeptides are obtained, which exhibit growth hormone receptor agonist properties, which determine the possibility of their use in medicinal agents for treating diseases related to the need to administer the growth hormone.
10 cl, 31 dwg, 5 ex
SUBSTANCE: invention is related to production of new hybrid polypeptide GST-CFP10 by microbiological synthesis with properties of species-specific protein-antigen CFP10 Mycobacterium tuberculosis, which may be used for early species-specific diagnostics of tuberculosis infection. Recombinant plasmid DNA pTB232 has been constructed, which codes hybrid polypeptide GST-CFP10 with properties of mycobacterial antigen CFP10, with average molecular weight (m.w.) 3.4 MDa and having size of 5257 p.n. Recombinant strain of bacteria E. coli BL21/pTB232 contains recombinant plasmid DNA pTB232, is producer of hybrid polypeptide GST-CFP 10 with properties of mycobacterial antigen CFP10 and is deposited in KM GNC VB "Vector" under number B-1027. Recombinant polypeptide GST-CFP10, produced with strain of bacteria E. coli BL21/pTB232, contains as protein-carrier N-end polypeptide fragment glutathione S-transferase S.j. (226 a.o. with m.w. of 26.3 kDa), joined via end site of thrombin hydrolysis (LVPR∧GS) with C-end polypeptide fragment of antigen CFP10 (100 a.o. with m.w. of 10.8 kDa) and has complete aminoacid sequence with length of 326 a.o. and m.w. of 37.1 kDa, given in text of description.
EFFECT: use of invention provides for the possibility to produce target highly pure hybrid polypeptide GST-CFP10 in preparative amounts with preservation of immunogenic properties of the latter.
3 cl, 4 dwg, 4 tbl, 6 ex
SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.
EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.
16 cl, 4 dwg, 1 tbl, 13 ex
SUBSTANCE: recombinant strain Escherichia coli is produced, which contains plasmid pHINS21 (Escherichia coli JM109/ pHINS21), defining synthesis of hybrid protein, made of N-terminal fragment of human gamma-interferon and human proinsulin, joined by peptide linker, which contains site of splitting with enterokinase (Asp4Lys). Yield of hybrid product that includes proinsulin, provided with new strain-producer, makes at least 30% of total amount of cell protein. Method is suggested for preparation of human proinsulin, including cultivation of strain-producer Escherichia coli JM109/pHINS21, separation of inclusion bodies and their dissolution, renaturation of hybrid protein and its cleaning with ion-exchange chromatography, splitting of hybrid protein with enterokinase or its catalytic subunit and cleaning of proinsulin by ion-exchange chromatography on sorbates with sulfprofile groups.
EFFECT: invention simplifies technological process for production of recombinant human proinsulin and improves conditions of its execution from the point of view of safety engineering.
4 cl, 4 dwg, 5 ex
SUBSTANCE: protein is constructed, which includes DNA-binding domain SSBTne from thermophile microorganism Termatoga neapolitana, connected to C-end of domain VirD2 from Agrobacterium tumefaciens, which is a signal of nuclear localisation.
EFFECT: efficient transport of transgene into cell nucleus.
6 dwg, 1 tbl
SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.
EFFECT: invention provides for preparation of single type of antibodies, which controls activity of NK cells of various type, provides for amplification of their cytotoxicity, which may find application in therapy, for increase of activity or cytotoxicity of NK cells in individuals without preliminary detection of HLA type in individual.
7 cl, 13 dwg, 4 tbl, 7 ex
SUBSTANCE: there is offered application of humanised fused protein for making a medicine used for stimulation of immune response and stabilisation of disease progressing in patients with GD2-positive tumours. The antibody contains antibody H14.18 caught with surface glycosphingolipid GD2 of human cells, and cytokine IL2. There is disclosed method of increase in ADCC and lysis activity of natural killers in cancer patients by introduction of the fused protein. The invention can be applied in GD2-overexpression cancer therapy.
EFFECT: application of the invention provides low-immunogenicity antibody.
2 cl, 8 dwg, 1 tbl, 2 ex
SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.
EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.
4 cl, 6 dwg, 22 tbl, 19 ex
FIELD: chemistry, medicine.
SUBSTANCE: claimed is novel hybrid protein CFP10-ESAT6 from M. tuberculosis, inducing reaction of hypersensitivity of delayed type with respect to M. tuberculosis. Chimeric NA, coding claimed protein is described. Described is method of obtaining claimed protein by cultivating cells of strain BL21(DE3) E.Coli, transformed with constructed recombinant expression vector on the basis of plasmid pET22b(+). Claimed is dosed medicinal form, containing claimed protein, for intracutaneous injection for diagnostics of tuberculosis infection.
EFFECT: high output of protein CFP10-ESAT6, which possesses specific immunogenicity.
8 cl, 2 dwg, 7 tbl, 3 ex
SUBSTANCE: the invention relates to producing new peptides and may be used for treatment and prophylaxis of cytokine-sensitive disorders. Peptides, having a size of 5 to 40 amino acids and arising from cytokines, are used in a vaccine for treatment and prophylaxis of autoimmune diseases, disseminated sclerosis, rheumatoid polyarthritis, psoriasis, autoimmune diabeteses, lupus, allergy, asthma, cancer and AIDS.
EFFECT: allows effective immunization of patients against said diseases while minimizing side effects.
11 cl, 2 dwg, 17 tbl
FIELD: medicine, biotechnology, pharmaceutical industry.
SUBSTANCE: method involves increasing part of the most active conformation of a glycosylated recombinant protein secreted by mammalian cell by its contact with a reagent for coupled oxidation-reduction. The proposed promotion method of the most active conformation of protein is used in a method for preparing a glycosylated recombinant protein in its the most active conformation. Configuration isomer of protein prepared by the indicated method for preparing a glycosylated recombinant protein in it's the most active conformation used in a method for preparing the protein composition for its administration to user and/or a patient or for consumption by user and/or patient. Using of the proposed invention provides enhancing activity of glycosylated protein prepared by a method of recombinant DNAs using a mammalian cell.
EFFECT: improved preparing method, valuable properties of protein.
27 cl, 9 dwg, 2 tbl, 3 ex
FIELD: biotechnology, preparative biochemistry.
SUBSTANCE: invention proposes a method for preparing the recombinant human tumor necrosis factor-aplha (TNF-alpha). Method involves culturing the strain-producer E. coli SG20050/pTNF311Δ, disruption of cells by ultrasonic oscillation, extraction of the end protein and 3-step chromatography purification procedure on DEAE-cellulose column in the linear gradient concentrations of NaCl at pH 8.0, on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 8.0 and on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 6.7. The recombinant TNF-alpha prepared by the proposed method shows the reduced content of impurity proteins, nucleic acids and lipopolysaccharides especially that provides its direct medicinal using. Invention can be used in medico-biological industry.
EFFECT: improved preparing method, enhanced quality of polypeptide.
2 tbl, 5 dwg, 2 ex
man, is able to induce the formation of neutralizing antibodies to human tnfdna, its coding, vector (options), a method of obtaining a vaccine tnf(options), method of testing for the presence of tnfthe way to test body fluids of a person, the method of diagnosis, method of treatment and prophylaxis medication for the treatment of" target="_blank">
SUBSTANCE: invention relates to a novel chemical compound and specifically to methyl ether of 2-cyano-3-oxo-18,19-dehydroglycyrrhet-1-inic acid of formula (I): .
EFFECT: obtaining compounds which can be used in medicine as a medicinal agent with anti-tumour activity.
11 ex, 1 tbl, 3 dwg
SUBSTANCE: disclosed are α- and β-crystalline forms of 5'-desoxy-N4-carbopentyloxy-5-fluorocytidine of formula (III) , their preparation method through crystallisation of the raw product from a suitable solvent and pharmaceutical compositions based on the said compounds, having anti-cancer activity. The solvent used when preparing the α-modification is an ester or a mixture of ester-containing solvents. The solvent used when preparing the β-modification is a mixture of water and alkanol or a mixture of tetrahydrofuran and diethyl ether or carbon tetrachloride.
EFFECT: obtaining compounds and pharmaceutical compositions based on the said compounds, having anticancer activity.
10 cl, 2 dwg, 7 ex
SUBSTANCE: present invention discloses pharmaceutical compositions based on telozolomide-8-carboxylate compounds of general formula I, as well as a method of preparing the said compositions. The disclosed composition has anti-tumour activity and can be especially useful during transdermal application.
EFFECT: obtaining compositions which, along with conventional pharmaceutical carriers, contain a component of acidic nature which gives the pharmaceutical composition high resistance to pH fluctuation.
16 cl, 2 tbl, 21 ex
SUBSTANCE: formula (I) compounds, radicals of which are defined in the formula of invention, are described. A pharmaceutical composition containing formula (I) compounds is also described.
EFFECT: obtaining compounds which have inhibitory activity on protein kinase MEK1/2 and are meant for use as a therapeutically active substance which is useful for treating MEK1/2 mediated diseases.
13 cl, 18 ex