Active angiogenesis-preventing immunetherapy
SUBSTANCE: invention refers to medicine and concerns angiogenesis-preventing immunotherapy. Invention substance includes immunogenic compositions for treatment of disorders associated with angiogenesis intensification, containing oligonucleotides, coding polypeptides VEGFR2, introduced as a part of plasmid or viral vectors, as well as polypeptides VEGFR2, oligonucleotides, coding autologous VEGF with damaged function of receptor activation, polypeptides VEGF and their combinations. Immunogenic compositions can be used for treatment of malignant neoplasms and metastasises, at benign neoplasm and chronic inflammatory and autoimmune diseases. Advantage of invention lies in humoral and cellular immunity induction by means of specified compositions.
EFFECT: development of effective method angiogenesis preventive immunotherapy.
19 cl, 11 ex, 7 tbl
The present invention relates to the field of biotechnology and pharmaceutical industry, in particular for active immunization targeted effect on the molecules that determine the development of blood vessels.
The formation of new blood vessels from pre-existing is called angiogenesis. This phenomenon is widely regulated by the balance of Pro - and antiangiogenic factors. The disease, caused by the induction of Pro-angiogenic factors and the formation of new blood vessels in abnormal form, include: (a) cancer (both primary tumors and their metastases), (b) acute and chronic inflammatory diseases, such as asthma, respiratory distress syndrome, endometriosis, atherosclerosis, and tissue swelling, (C) infectious diseases such as hepatitis and Kaposi's sarcoma, (d) autoimmune diseases, such as diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and (e) other diseases and disorders such as diabetic retinopathy and retinopathy of the newborn, rejection of transplanted organ, macular degeneration, neovascular glaucoma, hemangioma and angiofibroma (Carmelliet P. y R.K. Jain, Nature 407:249, 2000; Kuwano M. et al., Intern. Med. 40:565, 2001). A potentially attractive method of treatment of many of these diseases is the inhibition of the activity of Pro-angiogenic factors stimulate the abnormal formation of blood vessels, by neutralizing these factors or their receptors or elimination of sources of production of such factors.
Endothelial growth factors vessels form a family of molecules that induce a specific and direct the formation of new blood vessels (Leung Science 246:1306, 1989; Klagsburn M., Annual Rev. Physiol. 33:217, 1991). This family includes the factor of vascular permeability, also known as endothelial growth factor vascular VPF/VEGF (now called VEGF-A), growth factor PlGF placental, platelet-derived growth factors PDGF-A and PDGF-B and four other new molecules, structurally and functionally related to VEGF-A, called VEGF-B/VRF, VEGF-C/VRP, VEGD-D/FlGF and VEGF-E. (B. Olofsson et al., PNAS USA 13:2576, 1996; V. Joukov et al., EMBO J. 15:290, 1996; Yamada Y. et al., Genomics 42:483, 1997; Ogawa, S. et al., J. Biol. Chem. 273:31273, 1998).
VEGF-A is homodimeric glycoprotein formed by two subunits length of 23 kDa (Ferrara N. et al., Biochem. Biophys. Res. Sarnico. 165:198, 1989), from which the result of differential RNA splicing is possible to allocate five Monomeric isoforms. These isoforms include two isoforms, which remain associated with the cell membrane (VEGF 189 and VEGF 206), and three soluble isoforms (VEGF 121, VEGF-145 and VEGF-165). VEGF 165 isoform is most widespread in mammalian tissues except lung and the heart, where the predominant isoform of VEGF 189 (Neufeld, G. et al., Canc. Met. Rev. 15:153, 1995), and placenta, the de predominant expression of VEGF 121 (Shibuya M.A. et al., Adv. Canc. Res. 67:281, 1995).
VEGF-A is the most studied protein of this family and its modification described in the study of a large number of diseases. Overexpression of this factor is characteristic for tumors of different origin and localization and their metastases (Grunstein J. et al., Cancer Res. 59:1592, 1999), chronic inflammatory diseases, such as ulcerative colitis and Crohn's disease (S. Kanazawa et al., Am. J. Gastroenterol. 96:822, 2001), psoriasis (Detmar M. et al., J. Exp. Med. 180:1141, 1994), respiratory distress syndrome (Thickett D.R. et al., Am. J. Respir. Crit. Care Med. 164:1601, 2001), atherosclerosis (Celletti F.L. et al., Nat. Med, 7:425, 2001; Couffinhal, T. et al., Am. J. Pathol. 150: 1653, 1997), endometriosis (McLaren J. Hum. Reprod. Update 6:45, 2000), asthma (Hoshino M. et al., J. Allergy Clin. Immunol. 107:295, 2001), rheumatoid arthritis and osteoarthritis (Pufe T. et al., J. Rheumatol 28:1482, 2001), thyroiditis (Nagura, S. et al., Hum Pathol. 32:10, 2001), diabetic retinopathy and retinopathy of the newborn (Murata, T. et al., Lab. Invest. 74:819, 1996; Reynolds J.D., Paediatr Drugs 3:263, 2001), macular degeneration and glaucoma (Wells J.A. et al., Br. J. Ophthalmol. 80:363, 1996; Tripathi R.C. et al., Ophthalmology 105:232, 1998), tissue swelling (Kaner R.J. et al., Am. J. Respir. Cell Mol. Biol. 22:640, 2000; Ferrara N. Endocrinol. Rev. 13:18, 1992), obesity (C. Tonello et al., FEBS Lett. 442: 167, 1999), hemangioma (Wizigmann S. y Plate K.H. Histol. Histopathol., 11:1049, 1996), and synovial fluid of subjects suffering from arthritis (Bottomley M.J. et al., Clin. Exp. Immunol 119:182, 2000), and transplant rejection (Vasir B. et al., Transplantation 71:924, 2001). In the case of tumor cells expr serwisie three major isoforms of VEGF-A: 121, 165 and 189 grow faster in vivo; and in the final stages of development of most tumors, the expression of these factors is limited isoform VEGF 165 and in its absence by a combination of isoforms 121 and 189, which, not being additive, demonstrates cooperation, intensifying the tumor vascular network (Grunstein J. Mol. Cell Biol. 20:7282, 2000).
Factor PlGF, described in 1991, not able to induce proliferation of the endothelium in homodimeric form (D. Maglione et al., Proc. Natl. Acad. Sci. USA 88:9267, 1991, J. DiSalvo et al., J. Biol. Chem. 270: 7717, 1995). As a result of increasing concentrations of PlGF and, as a consequence, the gain of the signal transmitted VEGFR1, increases the response of endothelial cells to VEGF in the process of changing the angiogenic phenotype, due to certain pathologies (Carmeliet P. et al., Nat. Med. 7:575, 2001). The expression of PlGF induces vascularization meningiomas and gliomas man (M. Nomura et al., J. Neurooncol. 40:123, 1998). This molecule forms heterodimer with VEGF 165, with Pro-angiogenic activity, and overexpression of these factors, found in air-conditioned environments, some lines of tumor cells (Y. Cao et al., J. Biol. Chem. 271:3154, 1996), causes the development of rheumatoid arthritis and primary arthritis (Bottomley M.J. et al., Clin. Exp. Immunol. 119:182, 2000).
Overexpression of other members of the VEGF family, although it is less studied, is also associated with a number of pathologies. VEGF-B has the the breast tumors, ovary and kidney, melanoma and fibrosarcoma (Sowter H.M. et al., Lab. Invest. 77:607, 1997; Salven P. Am. J. Pathol. 153:103, 1998, Gunningham SP, et al., Cancer Res. 61:3206, 2001). Differential expression of isoforms of VEGF-B 167 in vitro was detected in tumor cells of different origin (Li X. et al., Growth Factors 19:49, 2001). VEGF-C and VEGF-D are involved in the regulation of lymphatic vessels (V. Joukov et al., EMBO J. 15: 290, 1996), and overexpression of VEGF-C is associated with tissue edema, tumors of the breast, lung, head and neck, esophagus and stomach, lymphoma, prostate cancer and metastatic sites (T. Kajita et al., Br. J. Cancer 85:255, 2001; Kitadi Y. et al., Int. J. Cancer 93:662, 2001; Hashimoto I. et al., Br. J. Cancer 85:93, 2001; Kinoshita J. et al., Breast Cancer Res. Treat 66:159, 2001; Ueda, M. et al., Gynecol. Oncol. 82:162, 2001; Salven P. Am. J. Pathol. 153:103, 1998; O-Charoenrat, P. et al., Cancer 92:556, 2001). Overexpression of VEGF-D in tumor cells causes an increase in the in vivo vascular network limpou in tumors and metastasis in the lymph nodes (Stacker S.A. et al., Nat. Med. 7:186, 2001; Marconcini L. et al., Proc. Natl. Acad. Sci. USA 96:9671, 1999).
Change the function of endothelial cells under the influence of the molecules of the family of VEGF-mediated binding of these molecules to receptors on cells of the tyrosine kinase class 3, which include: VEGFR1 (Flt1), VEGFR2 (KDR/Flk1) and VEGFR3 (Flt4) (Kaipainen A. J. Exp. Med. 178:2077, 1993). It is established that the N-terminal domain 2 is responsible for binding to ligands, stimulation of phosphorylation of the cytoplasmic domain and PE is edacho signal (Davis-Smyth T. et al., EMBO 15:4919, 1996).
The ligands identified for VEGFR1, include, in descending order of affinity of VEGF-A, PlGF and VEGF-B (Shibuya M. Int. J. Biochem. Cell Biol. 33:409, 2001). In endothelial cells, this receptor picks up circulating VEGF (H. Gille et al., EMBO J. 19: 4064, 2000). The binding of VEGF-A, VEGFR1, expressed in cells of hematopoietic lines, significantly activates the transcription factor NFκB cells-the precursors of dendritic cells and b - and T-lymphocytes. Last specified interaction determines the in vivo occurrence of adverse immunological balance, which reduces the maturation of dendritic cells and the fraction of T-lymphocytes, and the mentioned phenomenon is characteristic of subjects with immunosuppression and, in particular, cancer patients (Dikov M.M. et al., Canc. Res. 61:2015, 2001; Gabrilovich D. et al., Blood 92:4150, 1998). Overexpression of VEGFR1 is associated with psoriasis, endometrial cancer and malignant hepatoma (Detmar M. et al., J. Exp. Med. 180:1141, 1994; Ng IO Am. J. Clin. Patol. 116:838, 2001; Yokoyama Y. et al., Gynecol. Oncol. 77: 413, 2000).
Receptor VEGFR2 (KDR/Flk1) mediates the biological activity of VEGF-A, and also binds VEGF-C and VEGF-D. This receptor is differentially expressed in activated endothelium and in some cell lines of tumor origin, where it creates an autocrine way with secreted factor VEGF. Regardless of participation in the above pathologies, which is due to light what expressia its ligands, overexpression of VEGFR2 is directly related to the development of endometrial cancer (Giatromanolaki A. et al., Cancer, 92:2569, 2001), malignant mesothelioma (L. Strizzi et al., J. Pathol. 193:468, 2001), astrocytomas (Carroll R.S. et al., Cancer 86:1335, 1999), primary breast cancer (Kranz A, et al., Int. J. Cancer 84:293, 1999), gastric cancer of the intestinal type (Takahashi Y. et al., Clin. Cancer Res. 2:1679, 1996), glioblastoma multiforme, anaplastic oligodendrogliomas and necrotic ependymomas (Chan, A.S. et al., Am. J. Surg. Pathol. 22:816, 1998). Overexpression of KDR is also associated with autosomal disease VHL and hemangioblastomas (Wizigmann-Voos, S. et al., Cancer Res. 55:1358, 1995), the development of diabetic retinopathy (Ishibashi T. Jpn. J. Ophthalmol. 44:324, 2000) and in combination with overexpression of Flt-1 is related to allergic reactions of the delayed type (Brown, L.F. et al., J. Immunol. 154:2801, 1995).
Lymphangiogenesis-mediated VEGF-C and VEGF-D occurs as a result of binding of these factors to the receptor FLT4 or VEGFR3, expressed in the endothelium of lymphatic vessels. In some cases, even in the absence of overexpression of these ligands, overexpression of the receptor is directly related to adverse prognosis in the group of pathologies, including diabetic retinopathy (Smith G. Br. J. Ophthalmol. 1999 Apr; 83(4):486-94), chronic inflammation and ulcers (Paavonen K. et al., Am. J. Pathol. 156:1499, 2000), the formation of lymph node metastasis and the development of RA is and breast cancer (Gunningham SP, Clin. Cancer Res. 6: 4278, 2000; Valtola, R. et al., Am. J. Pathol. 154:1381, 1999), tumors of the nasopharynx and squamous cell carcinoma (A. Saaristo et al., Am. J. Pathol, 157:7, 2000; M. Moriyama et al., Oral Oncol, 33:369, 1997). In addition, overexpression of VEGFR3 is a clear indicator of Kaposi's sarcoma, hemangioendothelioma of Dabska and skin lymphangiomatosis (Folpe A.L. et al., Mod. Pathol. 13:180, 2000; A. Lymboussaki et al., Am. J. Pathol. 153:395, 1998).
Currently identified two receptor for VEGF, called NRP1 and NRP2. These receptors belong to the family of neuropilin (NRP) and act as used for some specific isoforms of VEGF protein family: VEGF-A145, VEGF-A165, VEGF-B167and PlGF1, increasing their mitogenic ability. Expression of NRP1 is an indicator of prostate cancer is related to increased angiogenesis in melanomas and apoptosis in the case of breast cancer (A. Latil et al., Int. J. Cancer 89:167, 2000; Lacal P.M. J. Invest. Dermatol. 115:1000, 2000; Bachelder R.E. Cancer Res. 61:5736, 2001). Coordinated overexpression of NRP1, KDR and VEGF-A165associated with the proliferation of vascular fibrous tissue in the case of diabetic retinopathy and rheumatoid arthritis (S. Ishida et al., Invest. Ophthalmol. Vis. Sci. 41:1649, 2000; Ikeda, M. et al., J. Pathol. 191:426, 2000). Receptor NRP2 sverkhekspressiya in osteosarcoma, where it stimulates angiogenesis and tumor growth (Handa A. et al., Int. J. Oncol. 17:291, 2000).
The basis of most treatment methods aimed the and inhibition of angiogenesis, especially if cancer is blocking molecules VEGF family and their receptors, and to achieve this goal in a clinical trial use: (1) a monoclonal antibody that blocks VEGF or a receptor KDR, (2) inhibitors of metalloproteinases, such as neovastat and prinomastat, (3) VEGF inhibitors, such as thalidomide, suramin, troponin I, IFN-α and neovastat, (4) VEGF receptor blockers, such as SU5416, FTK787 and SU6668, (5) inducers of apoptosis of tumor endothelium, such as endostatin and CA4-R, and (6) ribozymes, which reduces the expression of VEGF or VEGF receptors (angiozyme). Due to the high homology existing between human VEGF, its receptors KDR and Flt-1 and homologs of the mouse (respectively ˜90%, 81% and 89%), usually using different animal models for the preclinical evaluation of the efficacy of antiangiogenic compounds acting on this system (Hicklin D.J. et al., DDT 6: 517, 2001).
Passive antibodies to VEGF or VEGFR successfully investigated at different stages of clinical trials involving human subjects (Hicklin D.J. et al., DDT 6:517, 2001). Humanitariannet monoclonal antibody A against VEGF (Genentech, San Francisco, United States) is undergoing phase III clinical trials for the treatment of tumors of the colon, breast, kidney and lung (Kim K.J. et al., Nature 362:841, 1993; Boersig C. R&D Directions, Oct. 7:44, 2001). In particular, it was established monoclonal antibody to the receptor KDR (IC-1C11, ImClone), which recognizes the N-terminal extracellular domain of this receptor and inhibits proliferation and migration of leukemic human cells, increasing the survival rate xenotransplantion mice. Currently, the effects of the antibodies examined in subjects with metastatic colon cancer (S. Dias et al., J. Clin. Invest. 106:511, 2000). In the above tests demonstrated the absence of associated harmful effects in the introduction discusses monoclonal antibodies.
A new treatment method not previously used to block neoangiogenesis is specific active immunotherapy (SAI). When cancer treatment method use SAI antigens, such as peptides, proteins or DNA, in combination with an acceptable adjuvants. Methods SAI is directed to stimulation of the immune response humoral type (activation of b-lymphocytes) and cell type (activation of T cells-helper cells, cytotoxic lymphocytes and natural killer cells) in dendritic cells, which are professional antigen presenting cells in sit I and MHC II (Bystryn J.C., Medscape Hematology-Oncology 4:1, 2001; Parker K.C. et al., J. Immunol 152:163, 1994; F.O. Nestle et al., Nature Medicine 7:761, 2001; Timmerman J.M., Annual Review Medicine 50:507, 1999).
Specific active immunotherapy is emerging experimental and clinical studies with interesting features p is imeneniya in Oncology, where has completed more than 60 clinical trials according to the method of SAI, and the specified number currently exceeds the number of clinical trials using monoclonal antibodies. In the case of cancer antigens used as immunogens for SAI, selected based on their physiological suitability, the complexity of the replacement process phenotypic drift of the tumor (Bodey B. et al., Anticancer Research 20:2665, 2000) and specific connection with the growth and development of tumor tissues.
In addition, in the treatment of cancer by the method of SAI preferably identify the antigens expressed in tumors of different types, which may increase the number of indications for the use of the same vaccine preparation. Examples of such antigens are the carcinoembryonic antigen (CEA), HER2-neu, telomerase person and gangliosides (Greener M., Mol. Med. Today 6:257, 2000; J. Rice et al., J. Immunol. 167:1558, 2001; Carr, A. et al., Melanoma Res. 11:219, 2001; J.L. Murray et al., Semin. Oncol. 27:71, 2000).
In the case of human tumors VEGF sverkhekspressiya in the tumor compartment (Ferrara N., Curr. Top. Environ. Immunol. 237:1, 1999), while high levels of VEGF and its receptors found in the tumor vascular network (R.A. Brekken, J. Control Release 74:173, 2001). Stromal cells also produce VEGF under the influence of transformed cells, resulting in even after the removal of tumor cells from the subjects retained high levels of VEGF. The presence of VEGF and its receptor who has practical value for predicting and determining the stage of development of prostate tumors, cervical and breast cancer (George D.J. et al., Clin. Cancer Res. 7:1932, 2001; Dobbs, S.P. et al., Br. J. Cancer 76:1410, 1997; Callagy G. et al., Appl. Immunohistochem. Mol. Morphol. 8:104, 2000). On the other hand, VEGF is also in the group of soluble factors, which together with other cytokines, such as IL-10, TNF-α and TGF-β (Ohm J.E. y Carbone D.P., Immunol. Res. 23:263, 2001), may be involved in immunosuppression characteristic of cancer patients (K. Staveley et al., Proc. Natl. Acad. Sci. USA 95:1178, 1998; Lee K.H. et al., J. Immunol. 161:4183, 1998). Immunosuppressive effect, apparently caused by binding with the receptor Flt1 (Gabrilovich D. et al., Blood 92:4150, 1998).
The present invention describes the use SAI for the treatment of experimental tumors caused by molecules of the VEGF family and their receptors. Achieved antitumor activity may be due to at least four different mechanisms and their possible combinations: (a) direct destruction of cancer and stromal cells that produce VEGF, cytotoxic lymphocytes, (b) lysis of endothelial cells of tumor vessels due to the capture or neutralization of circulating VEGF using antibodies, (C) direct destruction of endothelial cells expressing VEGF receptors, cytotoxic lymphocytes or complimentative antibodies, (d) activation of the local immune response due to the capture or neutralization of circulating VEGF and subsequent device is in its immunosuppressive action.
These treatments can be used to prevent the formation of metastases or reduce the likelihood of their occurrence, to reduce or eliminate primary tumors in therapy of first or second order in combination with other anticancer drugs or without the use of such assets.
Active immunization aimed at the suppression of the VEGF family and their receptors, may also be effective in the implementation of monotherapy or combination therapy of acute and chronic inflammatory diseases (asthma, respiratory distress syndrome, endometriosis, atherosclerosis, tissue swelling), infectious diseases (hepatitis, Kaposi's sarcoma), autoimmune diseases (diabetes, psoriasis, rheumatoid arthritis, thyroiditis, synovitis), diabetic retinopathy and retinopathy of the newborn, rejection of transplanted organ, macular degeneration, neovascular glaucoma, hemangiomas, angiofibromas, and other diseases.
Detailed description of the invention
In accordance with the present invention the introduction of in vivo oligonucleotide sequences that encode proteins of the VEGF family and their receptors, used or fragments and polypeptide variants induces cellular and humoral immune response with achievement of antiangiogenic and protivoopujolevami.
Immunogenic polypeptide of the origin of interest to the present invention and fragments thereof can be isolated from natural sources or obtained by synthesis or by recombinant DNA. These polypeptides can also be obtained by fusion with proteins with known adjuvant activity, such as RC (R. Silva et al., US 5286484 y EP 0474313), or by covalent binding to proteins prior. Another acceptable way is to obtain a natural polypeptide, his mutated or modified variants and their fragments as part of the loops, existing or missing in bacterial proteins, such as AMR that are part of immunostimulatory drugs, in this particular case, VSSP (R. Perez et al., US 5788985 y 6149921). In addition, you can get polypeptide immunogen expressed on the surface of viral particles (HbsAg, VP2 of parvovirus etc) and associated with specific peptides that are targeted cells or organs, inducing an immune response (CTLA4, Fc-segment Ig etc), or proteins, can increase the biological distribution like VP22.
The main natural sources of proteins of interest to the present invention, expressed primarily in placenta, activated endothelially the cells and tumor cells. the mRNA of these cells or tissues are used to obtain complementary DNA (cDNA) with known methods. Extracted RNA is used as template for amplification using the polymerase synthesis reaction chain (PCR) cDNA corresponding to the selected antigen. In each case used the seed match the characteristics of the vector, which is expected to be this cDNA, formerly known sequences of interest protein. Alternative and preferably, in the case of receptors, the amplified PCR, which are the major antigens used in the present invention, the coding region amplified in two or more overlapping fragments. These fragments include General site ligating used for the Assembly of intact DNA from its fragments.
Alternative cloning of interest antigens is a selection of commercially available DNA libraries derived from the endothelium of human or tumors of the same origin. Sometimes it may be desirable to mutate some antigens, which is the object of the present invention, in order to avoid induction of angiogenesis induced by vaccination, especially in the case of the VEGF family. These mutations are preferably present on the binding sites of receptors previously described is in the scientific literature. To achieve this goal are creating a seed, which cover both ends of the desired molecule, and the PCR products are used as matrix to obtain the mutated molecules. Data mutated variants do not have biological activity, but reproduce the immunogenic properties of the selected antigen.
The cDNA molecules obtained by the methods described above is introduced into an appropriate vector, representing a virus, plasmid, bacterial artificial chromosome or the like. This vector carries the elements necessary for the expression of the gene in target cells, as well as other items necessary for producing vector in the cellular system of the host, depending on its nature. DNA molecules according to the invention can contain one or more genes of interest, formed by one or more nucleic acids (cDNA gdnc, synthetic or semi-synthetic DNA and the like), which are the result of transcription or translation (depending on need) in target cells form products having therapeutic/vaccine value.
Gene therapeutic vaccine product according to the invention is normally controlled by the promoter of transcription, which operates in the target cell or organism (mammal), and 3'-is Onaway area which generates the signals required for the termination and polyadenylation of mRNA in interest product, expressing this gene. The promoter may be the natural promoter of the gene or heterologous promoter that is transcriptionally active in the target cell. The promoter may be derived from eukaryotic or viral cells. You can use any eukaryotic promoter or a selected sequence that stimulates or suppresses the transcription of a gene, providing specific or non-specific, inducible or pendullum, strong or weak action. In addition, the promoter region can be modified by introduction of an activator or induction sequences, providing tissue-specific or preferred expression of the gene.
In addition, the gene of interest may contain a signal sequence required for subcellular localization, which allows you to modify the localization or secretion of the synthesized gene into the cell where it is expressed, or in any other place. Specified gene may also contain a sequence encoding a region of specific binding with a ligand, characteristic of immune tissue, which is directly related to the site of occurrence Rea the tion, providing therapeutic/vaccine steps.
In addition, the gene of interest may precede the coding sequence necessary for replication of the mRNA, resulting in the amplification of mRNA in the target cell, amplifying the expression of the indicated gene and together with therapeutic/vaccine action according to the invention. This mechanism of replication may be the origin of the alpha virus (Schlesinger, S., Expert Opin. Biol. Ther. 1:177, 2001), in particular, it can be isolated from virus Sindbis or Semliki, or similar. In this particular case, the transcription of the gene of interest controls subgenomic promoter, which provides amplification of mRNA in target cells after internalization of molecules according to the invention. In addition, vector-based DNA may contain sequences that allow for replication of the molecules, which is the object of the present invention, in mammalian cells. This allows you to increase the levels of expression and/or therapeutic/vaccine action (Collings, A., Vaccine 18:4601, 1999).
Vector-based DNA can be purified by standard methods used for purification of plasmid DNA. These methods include treatment in the density gradient of cesium chloride or in the presence of ethidium bromide, alternative application jonoob the built in speakers or any other ion exchanger or separation of DNA molecules (Ferreira G.N. et al., Trends Biotechnol. 18:380, 2000).
The present invention relates to the use of vectors based on the plasmid DNA, preferably belonging to the family PAEC compact vectors for DNA immunization and gene therapy human (Herrera et al., Biochem. Biophys. Res. Commu. 279:548, 2000). This family includes the vectors PAEC-K6 (access number AJ278712), PAEC-M7 (access number AJ278713), PAEC-Δ2 (access number AJ278714), PAEC-SPE (access number AJ278715) and PAEC-SPT (access number AJ278716). These vectors contain only the essential elements necessary for expression of interest product in mammalian cells, including human cells, and the unit of replication of Escherichia coli. The transcription unit is formed pregradnim promoter of the human cytomegalovirus (CMV), a universal site multicameraframe to insert the interest of product and sequences for termination of transcription and polyadenylation isolated from virus 40 monkeys (SV40). The unit of replication vector contains the gene for resistance to kanamycin (Tn903) and the origin of replication pUC19 (ColE1) to ensure you are receiving multiple copies and breeding bacteria, bearing interest plasmid.
The present invention further relates to the use of vectors based on the plasmid DNA, preferably belonging to the family of RMAE compact vectors for DNA immunization h the rights. These vectors contain the same functional elements in bacteria as vectors of a series of PAEC, and pretani CMV and site multicameraframe. In addition, these vectors are synthetic and synthetic intron sequence for termination of transcription and polyadenylation isolated from β-globin rabbit. It is known that sequences similar to the above sequence, it is possible to achieve higher levels of expression of the cloned gene (Norman J.A. et al., Vaccine 15:801, 1997). The vectors of this series further include consecutive repetitions of immunostimulatory sequences (CpG-fragments), which stimulate the natural immune system in mice and humans with subsequent activation of humoral and cellular responses against the interest of the molecule (Krieg A.M., Vaccine 19:618, 2001).
Immunization with the recombinant virus (adenovirus, adeno-associated virus, vaccinia virus, poxvirus chicken poxvirus Canaries and similar) is a strong cytotoxic response of cells in the host. For the introduction of interest sequence in the recombinant virus using vectors containing the integrating sequence and promoters that are specifically designed for each type of viruses. This method is also in the scope of the present from which bretania, preferably use poxvirus chickens and vector pFP67xgpt. Vector pFP67xgpt used for cloning of genes under the control of a strong early/late synthetic promoter between open reading frames 6 and 7 BamHI fragment length 11,2 TPN poxvirus Chicks FP9. The indicated plasmid also contains Ecogpt controlled by the promoter of the vaccinia virus RK, which is used to identify recombinant virus.
Another variant of implementation of the present invention relates to immunization with proteins of the VEGF family and their receptors and/or used. The cDNA molecules obtained as described above, clone in the vector for expression in viruses, yeast, phage, plants or cells of higher organisms to obtain protein variants of antigens after the sequence has been tested traditional methods of automatic sequencing. It has been described several expressing vectors intended for the production of recombinant proteins. These vectors contain at least one sequence that controls the expression of functionally related DNA sequence or expressed fragment. Examples of sequences that are suitable for controlling expression, include the lac system, the trp, tac, and trc, the promotor region and the main operator lambda is yeah, controlling the surface area fd protein, the glycolytic promoters of yeast (for example, 3-phosphoglyceraldehyde), the promoters of acid phosphatase of yeast (e.g., Pho5), yeast promoters sparavalo alpha-factor and promoters isolated from polyoma, adenovirus, retrovirus, virus of monkeys (e.g., early/late promoters of SV40), and other known sequences that regulate gene expression in prokaryotic and eukaryotic cells, viruses, and their combinations.
The hosts used for replication of these vectors and the production of recombinant proteins, which is the object of the present invention include prokaryotic and eukaryotic cells. Prokaryotic cells include E. coli (DHI, MRCI, HB101, W3110, SG-936, X1776, X2282, DH5a), Pseudomonas, Bacillus subtilis, Streptomices and others. Eukaryotic cells include yeast, fungi, insect cells, animal cells (such as COS-7 and Cho cells, plant cell, tissue culture and the like. After expression in the selected system and appropriate environment polypeptides or peptides can be isolated by known methods.
The use of adjuvants
Even if it is established that vaccination deproteinizing DNA or proteins is effective in some animal models, it is unknown, how will react to the way treatment is ia according to the invention actors having tumors or autoimmune diseases. To stimulate the immune response DNA-containing or protein-based vaccines can be combined with the previously described immunopotentiators, such as mineral salts (eg, aluminum hydroxide, aluminum phosphate, calcium phosphate), Immunostimulants, such as cytokines (eg, IL-2, IL-12, GM-CSF, IFN-α, IFN-γ, IL-18), molecules (such as CD40, CD154, the invariant chain of MHC type I, LFA3), saponins (e.g., QS21), derived MDP, oligonucleotides CpG, LPS, MPL and polyphosphazene, lipid particles, such as emulsions (for example, beta-blockers, SAF, MF59), liposomes, virosomes, ikonami, chelators, adjuvants in the form of microparticles, such as PLG microparticles, poloxamer viral type (e.g., HBcAg, HCcAg, HBsAg) and the bacterial type (i.e VSSP, OPC) and mucous adjuvants, such as thermo-labile enterotoxin (LT), cholera enterotoxin and mutant toxins (for example, LTK63 and LTR72), microparticles and polymerized liposomes. In the case of vaccination DNA of interest, the antigen can be expressed together with some of the above molecules immunopotentiators in bitestrenos vector.
The experiments, described in detail in the examples, show that DNA can be ecovalence associated with some of these particles and that the use of such mixtures omensetter concentration, necessary to achieve antitumor response, which is similar to the reactions that occur at higher doses deproteinizing DNA.
Introduction to the mammal
Vaccine preparations according to the invention is administered for therapeutic purposes, to a mammal, preferably a human, in a pharmaceutically acceptable dose of the following ways: through the mucous membrane, subcutaneously, intramuscularly, intraperitoneally, lymph, topically and by inhalation. These drugs can be introduced into the interstitial space of tissues, including muscle, skin, brain, lung, liver, bone marrow, spleen, thymus, heart, lymph nodes, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testis, ovary, uterus, rectum, eye, gland, and connective tissue. Expression vectors designed for transfer of oligonucleotides, preferably directed in differentiated somatic cells, although it may be directed in undifferentiated or less differentiated cells such as skin fibroblasts and pluripotent cells of the blood.
Immunogen in the required doses can be entered in pharmaceutically acceptable carriers, not having toxic or therapeutic activity. Examples of such carriers include ion exchangers, alumina, stearate aluminum is tion, lecithin, serum proteins, such as albumin, buffer solutions such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides with saturated fatty acids of vegetable origin, water, salts or electrolytes, such as Protamine sulfate, intrigejosa, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, substances based on cellulose and polyethylene glycol. In the present invention as carriers of vaccines preferably used phosphate buffers.
Used proteins and peptides can be covalently or ecovalence conjugated with molecules known as carriers, acting like adjuvants. Such molecules are KLH, p64K, OPC (Musacchio A. et al., Vaccine 19; 3692, 2001) and VSSP. The combination deproteinizing DNA, viral vectors and protein immunogen is an alternative that is also in the scope of the present invention. The use of plasmid DNA allows you to create drugs, containing vaccine, one or several interest of the molecules. Thus, the molecules according to the invention can be introduced in accordance with the scheme of vaccination, using a combination of vectors of different types (option re-stimulation induction through DNA, proteins, viral vectors).
Etc) the market-based DNA can be administered to a subject or as specified by the vectors can be modified cells of the host in vivo or ex vivo. The last route of administration may be combined with the introduction using sitespecifically recombination or immunization method of somatic transgenesis, which directs the expression of the vector in certain cells. In addition, as carriers for the transfer vectors based on DNA in vivo can be used as bacterial hosts.
Molecules carrying the genes according to the invention, can be used in the form deproteinizing DNA or in combination with different vectors: chemical/biochemical/biological, natural/synthetic, or recombinant. These molecules may be linked or combined with a cationic peptides, firming molecules (e.g., PEG, PEI), nuclear localizing peptides (NLP), etc. these molecules can also enter together with cations that can form precipitates DNA in the composition of the liposomal preparations, in which these molecules add up to merge with the membrane, and synthetic vectors lipid type or educated cationic polymers (e.g., DOGS or DOTMA). For the introduction of vectors based on DNA can also be used chimeric proteins that are able to condense DNA and mediate the transfer of the formed complex, and selective endocytosis mediated by specific cells. DNA molecules carrying therapeutic/vaccine genes according to the SNO invention, can be used for gene transfer into cells by physical migration techniques such as particle bombardment, electroporation (in vitro, in vivo or ex vivo), or directly in vivo by topical application, inhalation of finely ground substances, etc. Live vectors include adenoviruses or the same owners who had received molecule according to the invention.
Acceptable dose of polypeptides and/or oligonucleotides can be determined taking into account various parameters, in particular, depending on the gene or protein, entered as immunogen, the method of administration, intended for the treatment of the disease, the treatment period and in the case of oligonucleotides from the vector used for immunization. Changing patterns of use or the route of administration compared to described in the following examples does not imply going beyond the scope of the present invention, allowing to optimize the immunization scheme to achieve the best response.
The present invention has the advantage compared with passive immunotherapy, which is the last stage of clinical trials using such molecules as targets. Compared with passive transfer of immunity resulting from the introduction of monoclonal antibodies (for example, EN is Itel against VEGF) immunization, made with protein or oligonucleotide induces endogenous production of antibodies, as well as the proliferation and multiplication of specific cytotoxic CD8+lymphocytes.
An advantage of the present invention in comparison with therapies aimed at blocking system VEGF-VEGFR, is that these methods only reduce the levels of circulating VEGF or block KDR. The proposed method, in addition to achieving the above steps, also destroys the source of VEGF (i.e. tumor cells and associated stroma) and/or cells expressing these receptors (the endothelium of the tumor and some tumor cells). In previous work performed in this area, described only the humoral response as the main component of the observed actions. Without limiting the scope of the present invention to a specific mechanism, the examples show that, in addition to the specific humoral response, a vaccine composition capable of causing the reaction of CD8+cells, which interacts with the humoral response; and in the case of tumors, the combination of both reactions are able to exert anti-tumor effect observed in example 9.
It is quite possible that the response of cytotoxic cells is mediated by the recognition of some of the peptides are shown in tables 1 and 2. In these tables is not what the quiet peptide segments, which can be related to the reaction cells, aimed at the selected target in the VEGF family and their receptors and can be used. This information was obtained by performing computer analyses of publicly available databases in the NIH and the Institute of Heidelberg (http://bimas.dcrt.nih.gov/molbio/hla_bind and www.bmi-heidelberg.com/scripts/MHCServer.dll/home.htm), using BIMAS software and SYFPHEITI. The flagged peptides and other sequences isolated from interest antigens, can be used for active immunotherapy of the above diseases in the form of monotherapy or combination therapy separately or as part of molecules possessing properties of adjuvants. These peptides can also be used to obtain the vaccine in the form of oligonucleotide options.
Methods of inhibiting angiogenesis and associated pathologies include administration to a mammal by any means effective amount of DNA or protein of some molecules described in this invention, using some previously described immunopotentiation or adjuvants. This mammal is preferably a human.
Irreversible and unregulated development of blood vessels is characteristic of a number of diseases. System, including family factors VEGF, its receptors and can be used, shorecrest arowana in many pathologies, as was described above. Methods of treatment according to the invention allow to obtain effective results in the treatment of diseases such as (a) cancer (primary tumor and metastases), (b) acute and chronic inflammatory diseases, such as asthma, respiratory distress syndrome, endometriosis, atherosclerosis, and tissue swelling, (C) infectious diseases such as hepatitis and Kaposi's sarcoma, (d) autoimmune diseases, such as diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and (e) other diseases and disorders such as diabetic retinopathy and retinopathy of the newborn, rejection of transplanted body, macular degeneration, neovascular glaucoma, hemangioma and angiofibroma.
In the case of cancer vaccination immunogenum according to the invention allows to obtain effective results in the treatment of carcinomas, sarcomas, and vascularizing tumors. Some examples of tumors that can be treated with the proposed methods include epidermoid tumors, squamous cell tumors, affecting the head and neck, tumors colorectal, prostate, breast, lung (including small and large cell), pancreas, thyroid, ovary and liver. These methods are also effective in the treatment of other types of tumors, such as Kaposi's sarcoma, neoplasia Central nervous system (neuroblastoma, capillary hemangioma, meningioma and metastases in the brain), melanomas, carcinomas of the kidney and gastrointestinal tract, rhabdomyosarcoma, glioblastoma, and leiomyosarcoma.
In particular, the use of VEGF-A and/or receptors VEGFR-1 and VEGFR-2 as an immunogen may be effective in the treatment of tumors of different origin and localization and their metastasis, hemangioma, endometriosis, tissue edema, chronic inflammatory diseases, such as ulcerative colitis and Crohn's disease, atherosclerosis, rheumatoid arthritis and osteoarthritis, arthritis, psoriasis, respiratory distress syndrome, asthma, thyroid, diabetic retinopathy and retinopathy of the newborn, macular degeneration and glaucoma, autosomal VHL disease, obesity and exclusion of some of the transplanted organs. On the other hand, the reaction against PlGF useful in the case of rheumatoid arthritis and for the treatment of primary arthritis.
The use of VEGF-B as immunogen is useful in the case of tumors of the breast, ovary and kidney, as well as melanoma and fibrosarcoma. The use of VEGF-C and its receptor VEGFR-3 allows you to get effective results in the treatment of edema, diabetic retinopathy, chronic inflammation, ulcers, tumors of the breast, lung, head and neck, esophagus, stomach, lymphoid tissue, to depict athelney gland, metastatic nodules, Kaposi's sarcoma, hemangioendothelioma type Dabska and skin lymphangiomatosis. Immunization VEGF-D can be used specifically for the treatment of metastatic lymph nodes.
The application can be used NRP1 and NRP2 to immunize mammals is useful for treating, in particular, proliferation of vascular fibrous tissue in the case of prostate cancer, melanoma, osteosarcoma, metastatic breast cancer, diabetic retinopathy and rheumatoid arthritis.
Studies based on passive immunotherapy by injection of antibodies, showed that the combination of antibodies against VEGF-A and KDR is more effective in models of syngeneic tumors. Thus, the use of two or more immunogens according to the invention is particularly effective results in the inhibition of angiogenesis and tumor growth. These immunogene you can enter individually or in pairs, using bicistronic vectors described ways. In addition, vaccine compositions according to the invention can be used in conjunction with a medical or chemotherapeutic agents designated for the treatment of specific diseases, with the simultaneous or sequential administration.
The results presented below indicate that antiangiogenic and FR is loopwhole reaction mediated by a combination of humoral and cellular responses. In particular, VEGF and its receptor are involved in the maturation process of dendritic cells and affect cell precursor b - and T-lymphocytes. In example 10 shows that the proposed method of treatment, a reduction in VEGF levels in the serum also helps to normalize the ratio of b - and T-lymphocytes and Mature dendritic cells. This effect contributes to the presentation of tumor antigens in MHC I, which improves the quality and intensity of antitumor immune responses aimed not only at the immunogen, but also to other tumor-associated, tumor-specific and sverhagressivny antigens in the tumor.
Cloning and transient expression of antigens
VEGF man, its isoforms and functional mutants
The VEGF isoforms cloned using polymerase reaction synthesis chain (PCR)using as template cDNA, obtained from the previously selected mRNA cell line CaSki (ATSS CRL 1550), in accordance with the manufacturer's instructions (Perkin-Elmer) and a dose of SEQ ID NO:1 and SEQ ID NO:2. Bands corresponding to the products of amplification isoforms of VEGF 121, 165 and 189, cut from 2% agarose gels. Strip hydrolyzed in the endonucleases BamHI and EcoRI, cDNA isoforms of VEGF was purified and independently cloned in the vector RA is WithΔ 2 (a patented vector CIGB). The resulting plasmids sequenced, were not detected mutations with amino acid sequences reported EMBL (www.embl-heidelberg.de for cloned isoforms. cDNA corresponding to the isoforms of VEGF, and then cloned Kpnl/EcoRV in the vector RMEΔ5, which, along with other characteristics different from PAECΔ2 the presence of 5'-end of immunostimulatory CpG sites.
cDNA of VEGF variant with no binding receptor KDR (VEGFKDR(-)) was obtained by direct mutagenesis previously cloned isoforms of VEGF121in accordance with the description given Siemeister, G. and others (G. Siemeister et al., J. Biol. Chem. 273: 11115, 1998).
Mutated variant was obtained by means of PCR using the following basic forms:
(A) amplification of the 5'-end fragment (315 BP) using seed sequences of SEQ ID NO:3 and SEQ ID NO:4;
(C) amplification of the 3'-terminal fragment (93 BP) using seed sequences of SEQ ID NO:5 and SEQ ID NO:6.
Amplificatoare thus the fragments were purified as described above and used in equimolar concentrations as a matrix for merge using PCR with the use of nucleating corresponding to the sequences SEQ ID NO:7 and SEQ ID NO:8. The obtained cDNA containing the mutation, hydrolyzed BamHI/EcoRI, purified and cloned in the vector PAEC&x00394; 2. Introduced mutations were checked by sequencing, and DNA corresponding to VEGFKDR(-)was subclinically Kpnl/EcoRV in vector RMEΔ5, the result of which was received vector RMEΔ5 VEGFKDR(-).
Plasmids used for transfection and for vaccination of animals, was purified in conditions without endotoxin in accordance with the description given Whalen R. and others (Whalen, R.G. y Davis H.L., Clin. Immunol. Immunopathol. 75:1, 1995). DNA was purified using system QIAGEN without endotoxin, in accordance with the manufacturer's instructions, after which the DNA was subjected to a second precipitation. Finally, the DNA was dissolved in saline phosphate buffer (PBS) without endotoxin (Sigma, USA) to a final concentration of 4 mg/ml
1.2. The VEGF receptor human (KDR/Flk1)
cDNA encoding the extracellular domain of the receptor KDR of VEGF (KDR1-3), a transmembrane and intracellular domain of this receptor (KDR TC), were obtained with RT-PCR using mRNA line of endothelial cells HUVEC (Clonetic, USA) and processed human VEGF (Sigma) and heparin (Sigma).
In the case of the extracellular domains 1-3 used seed correspond to the sequences SEQ ID NO:9 and SEQ ID NO:10. Amplificatory fragment (943 BP) hydrolyzed in the endonucleases BamHI and EcoRI, after which cDNA encoding the domains 1-3 KDR, was purified and cloned in the vector PAECΔ2. Positive clones identified by restriction analysis is om, were checked by sequencing of the corresponding DNA. cDNA corresponding to KDR 1-3, subclinically Kpnl/EcoRV already described in the vector RMEΔ5 (RMEΔ5 KDR1-3).
Transmembrane and cytosolic region of the receptor cloned two-stage method. To insert the first segment of the used seed corresponding to SEQ ID NO:11 and SEQ ID NO:12. The segment length of 747 BP hydrolyzed XbaI/BglII, the product was cloned in the vector RME pre-hydrolyzed by the same enzyme, the result of which was obtained plasmid PMAE5 KDR 747. The indicated plasmid hydrolyzed BglII/NotI to insert the rest of carboxykinase fragment length 1091 BP, which was amplified using the seed corresponding to the sequences SEQ ID NO:13 and SEQ ID NO:14. Positive clones identified by restriction analysis and verified by DNA sequencing and were then given the name rmae KDR C.
1.2.1. Cloning transmembrane and cytosolic regions of KDR in viral vector
Transmembrane and cytosolic region of the VEGF receptor (KDR) was cloned in the poxvirus chickens, using the seed corresponding to the sequences SEQ ID NO:15 and SEQ ID NO:16. Fragment length 953 digested hydrolyzed by enzymes StuI/SmaI, the product was cloned in the vector pFP67xgpt pre-hydrolyzed by the same enzyme. In the same vector SmaI/BamHI, put the rest of the fragment length 919 P.N., which is amplified from the original cDNA using the seed corresponding to the sequences SEQ ID NO:17 and SEQ ID NO:18. Positive clones identified by restriction analysis and verified by DNA sequencing and were then given the name pFP67xgpt KDR C.
Poxvirus chickens (FWPV) replicated in fibroblasts of chicken embryo (CEF) in DMEM containing 2% fetal calf serum (FBS). Clone pFP67xgpt KDR C transfusional using lipofectin (Gibco BRL, Grand Island, USA), CEF, which previously infected weakened strain FP9. After 24 hours was added to fresh medium and cells were cultured for 3-4 days. Then the cells three times froze and thawed. Recombinant viruses expressing the gene encoding the enzyme Ecogpt, was purified in the electoral environment, using mikofenolna acid (25 μg/ml), xanthine (250 μg/ml) and gipoksantin (15 μg/ml) (MIN). The correct inclusion of a gene in the recombinant viruses were tested using PCR. Recombinant viruses are called FPKDRgpt, non-recombinant viruses were used as negative control strain FP.
Expression of antigens in vivo
To confirm the potential of the structures created for the expression of proteins in vivo, these constructs were injected with mice 57BL6 in the quadriceps muscle of the thigh (3 thinking is in the group).
1. RMEΔ5-VEGF121(10 and 50 μg/mouse) in PBS pH 7,2
2. RMEΔ5-VEGF165(10 and 50 μg/mouse) in PBS pH 7,2
3. RMEΔ5-VEGF189(10 and 50 μg/mouse) in PBS pH 7,2
4. RMEΔ5-VEGFKDR(-)(10 and 50 μg/mouse) in PBS pH 7,2
5. RMEΔ5-KDR 1-3 (10 and 50 μg/mouse) in PBS pH 7,2
6. rmae KDR C (10 and 50 μg/mouse) in PBS pH 7,2
7. FPKDRgpt (2,5*107CFU) in PBS, pH 7,2
8. PBS, pH 7,2 (negative control group)
At 48 hours after injection, animals were slaughtered and completely deleted the muscles, in which an injection was used. Part muscle tissue is homogenized in the presence of protease inhibitors and non-ionic detergents. The presence of VEGF in the protein extracts were analyzed by dot blotting and Western blotting, using a polyclonal antibody that recognizes all isoforms of human VEGF (sc-152G), in accordance with the described methods. RNA was extracted from the rest of the muscle tissue using TRI-reagent (Sigma). A total of 20 μg of RNA obtained in each experiment were purified by electrophoresis in 1% agarose gel containing formaldehyde. RNA was transferred to nylon filter (HYBOND) and hybridisable with cDNA isoforms of VEGF 121, labeled ATP32that recognizes all isoforms of VEGF, or with cDNA of the receptor KDR, labeled similarly. In both cases, the filters are re-hybridisierung cDNA corresponding structural gene: glyceralde the guide-3-phosphatedehydrogenase (GAPDH). In all analyzed structures identified band corresponding to human VEGF, and cloned fragments of the receptor KDR.
Experiments to create immunity in vivo by vaccination with plasmid containing the gene fragments of factor receptor KDR VEGF
Groups consisting of 10 mice 57BL/6, were vaccinated or not vaccinated in accordance with the following options:
1. RMEΔ5-KDR 1-3 (1, 10, 50 and 100 µg/mouse) in PBS pH 7,2
2. rmae KDR C (1, 10, 50 and 100 µg/mouse) in PBS pH 7,2
3. FPKDRgpt (2,5*107SOME)
4. PBS, pH 7,2 (negative control group)
5. FP (2,5*107SOME) (negative control group 3)
In all experiments, mice were immunized by intramuscular injection (im) in a total volume of 50 µl in the rear left leg. All animals were immunized again after 15 days in accordance with the original scheme of immunization. Control contamination of the tumor was made thirty days after the last immunization by subcutaneous (sc) injection of 104melanoma cells B16-F10 (ATS, CRL-6475) all the animals in the right part of the abdomen. Tumor growth was controlled, producing three measurements per week until the animal's death.
In mice immunized with plasmid RMEΔ5-KDR 1-3, a decrease in tumor size was observed at a dose of 50 and 100 μg DNA/mouse, while op is hol was significantly less than in animals in the negative control group (table 3). The results of the survival analysis performed on the 33rd day, showed a significant increase (compared with negative control group) this parameter in animals immunized with the indicated DNA at doses of 50 and 100 µg/mouse, compared with unimmunized mice (group PBS pH 7,2). In the case of the use RMEΔ5-KDR C (table 3) significant reduction of tumor volume was observed at all four used doses, and the survival rate was increased at a dose of 100 to 10 μg/animal. When using viral vectors was observed reduced tumor volume and increased survival in conditions similar to the application of design FPKDRgpt (table 3), compared with the corresponding negative control group (group of mice immunized with the vector without insert FPgpt).
Tumor volume and survival rate of mice immunized with fragments of the gene of the receptor (KDR) and VEGF
|Group||[Μg DNA]||Tumor volume (mm3) Day 24||Survival (43rd day)|
|pMAE5Δ5-KDR 1-3||100||424,0 ± 199,2 (***)||(***)|
|50||756,32 ± 435,9 (***)||(**)|
|10||1024,2 ± 397,1 (*)||(ns)|
|1||1334,2 ± 620,7 (ns)||(ns)|
|pMAE5Δ5-KDR C||100||404,23 ± 200,0 (***)||(***)|
|50||633,2 ± 365,2 (***)||(***)|
|10||924,3 ± 437,1 (**)||(*)|
|1||1114,2 ± 665,7 (*)||(ns)|
|FPKDRgpt||2,5*107SOME||304,23 ± to 152.0 (***)||(***)|
|FPgpt||2,5*107SOME||1891,0 ± 726,0 (ns)||(ns)|
|PBS pH 7,2||-||1785,0 ± 826,0||-|
|Note: tumor volume specified as the average value of ± standard deviation (SD) of measurements of the animals, performed in each group; statistical comparisons were made using unidirectional method ANOVA and post-test, Bonferroni. Survival was determined on the basis of the level of statistical significance using criteria logarithmic distribution to compare each group with the control group is on the specified day. Statistical significance is indicated as ns, p ≤ 0,05 without statistical significance; *, p ≤ 0,05; **, p ≤ 0,01; ***, p ≤ 0,001.|
Experiments to create immunity in vivo by vaccination with plasmids containing isoforms of VEGF and mutated variant
Groups consisting of 10 mice 57BL/6, were vaccinated or not vaccinated in accordance with the following options:
1. PAECΔ2-VEGF121(1, 10, 50 and 100 µg/mouse) in PBS pH 7,2
2. RMEΔ5-VEGF121(1, 10, 50 and 100 µg/mouse) in PBS pH 7,2
3. RMEΔ5-VEGF165(1, 10, 50 and 100 µg/mouse) in PBS pH 7,2
4. RMEΔ5-VEGF189(1, 10, 50 and 100 µg/mouse) in PBS pH 7,2
5. RMEΔ5-VEGFKDR(-)(1, 10, 50 and 100 µg/mouse) in PBS pH 7,2
6. PBS, pH 7,2 (negative control group)
In all experiments, mice were immunized by intramuscular injection (im) in a total volume of 50 µl in the rear left leg. All animals were immunized again after 15 days in accordance with the original scheme of immunization. Control contamination of the tumor was made thirty days after the last immunization by subcutaneous injection of 104melanoma cells B16-F10 (ATS, CRL-6475) all the animals in the right part of the abdomen. Tumor growth was controlled, producing three measurements per week until the animal's death.
In those cases, when mice were immunized is preteensyoung DNA in the vector series PAEC at a dose of 100 μg/animal, slowed down the growth of tumors compared to negative control group (table 4). When using options, including vector series RMEΔ5 from the 5'end of the CpG sites irrespective of the isoform of VEGF, tumor size was significantly decreased in the groups of mice immunized with DNA at doses of 10, 50 or 100 µg, compared with the negative control group. In the case of mutated variants RMEΔ5 VEGFKDR(-)a significant decrease in tumor size was achieved at a dose similar to that used for immunization RMEΔ5-VEGF121.
The results of the survival analysis performed on the 43rd day, showed a significant increase (compared with negative control group) this indicator in animals immunized with options RMEΔ5-VEGF121, RMEΔ5-VEGF165, RMEΔ5-VEGF189and RMEΔ5-VEGFKDR(-), at a dose of 50 and 100 μg/animal (table 4).
Tumor volume and survival rate of mice immunized with different options deproteinizing DNA containing different isoforms of the VEGF gene and the mutated version
|Group||[Μg DNA]||Tumor volume (mm3) Day 24||Survival(43rd day)|
|PAECΔ2-VEGF121||100||991,5 ± 354 (*)||ns|
|50||1429,2 ± 396 (ns)||ns|
|10||1506,6 ± 442 (ns)||ns|
|1||1660,5 ± 456 (ns)||ns|
|PMAE5Δ5-VEGF121||100||645,0 ± 215 (***)||***|
|50||850,1 ± 463 (***)||***|
|10||992,1 ± 410 (*)||ns|
|1||1560,3 ± 598 (ns)||ns|
|PMAE5Δ5-VEGF165||100||799,2 ± 335 (***)||***|
|50||916,6 ± 390 (**)||**|
|10||1000,5 ± 662 (*)||ns|
|1||1845,3 ± 450 (ns)||ns|
|PMAE5Δ5-VEGF189||100||790,1 ± 235 (***)||***|
|50||996,5 ± 255 (*)||**|
|10||1050,2 ± 362 (*)||ns|
|1||1670,2 ± 408 (ns)||ns|
|pMAE5Δ5-VEGFKDR(-)||100||550,1 ± 335 (***)||***|
|50||894,7 ± 408 (**)||***|
|10||991,8 ± 362 (*)||ns|
|1||1489,3 ± 510 (ns)||ns|
|PBS pH 7,2||0||1673,9 ± 712|
|Note: tumor volume specified as the average value of ± standard deviation (SD) of measurements of the animals, performed in each group; statistical comparisons were made using unidirectional method ANOVA and post-test, Bonferroni. Survival was determined on the basis of the level of statistical significance using criteria logarithmic distribution to compare each group with the control group on the specified day. Statistical significance is indicated as ns, p ≤ 0,05 without statistical significance; *, p ≤ 0,05; **, p ≤ 0,01; ***, p ≤ 0,001.|
Experiments to create immunity in vivo by immunization RMEΔ5-VEGF121and RMEΔ5-KDR 1-3 in a model of collagen-induced arthritis
The group, consisting of the s of 20 mice, were vaccinated or not vaccinated in accordance with the following options:
1. RMEΔ5-VEGF121(50 μg of DNA/mouse) in PBS pH 7,2
2. RMEΔ5-KDR 1-3 (50 μg of DNA/mouse) in PBS pH 7,2
3. PBS, pH 7,2 (negative control group)
In all experiments, mice were immunized by intramuscular injection (im) in a total volume of 50 µl in the rear left leg. All animals were immunized again after 15 days in accordance with the initial immunization scheme.
On the 5th day caused autoimmune arthritis by immunization with chicken collagen type II (Sigma) in accordance with the model described by Campbell and others (Campbell, I.K. et al., Eur. J. Immunol. 30:1568, 2000). The specified immunization was repeated on the 26th day. Each mouse was daily assessed the status of all four limbs on the basis of the indicator lesions arthritis, what did 0-3 puncture each limb for signs of erythema (1), inflammation (2) or immobility (3) if the maximum assessment, equal to 12. Mice appeared clinical symptoms develop arthritis on the 23rd day after immunization, with the highest incidence was found in 50 days. Table 5 shows the incidence of arthritis in animals in the different experimental groups. On the 40th and 55th day, there was significant reduction in the incidence of arthritis in vaccinated groups (1 and 2) comparedwith the control group.
The incidence of arthritis in certain days (40 and 55)
|Group||40-day morbidity||55th day morbidity|
|1||20/8 (40%)||20/9 (45%)|
|2||20/6 (30%)||20/12 (60%)|
|3||20/10 (50%)||20/14 (70%)|
Antiangiogenic effect of vaccination in vivo
Groups of 15 mice were vaccinated or not vaccinated in accordance with the following options:
1. RMEΔ5-VEGF121(50 μg of DNA/mouse) in PBS pH 7,2
2. RMEΔ5-KDR 1-3 (50 μg of DNA/mouse) in PBS pH 7,2
3. rmae KDR C (50 μg/mouse) in PBS pH 7,2
4. PBS, pH 7,2 (negative control group)
In all experiments, mice 57L/6 were immunized by intramuscular injection (im) in a total volume of 50 µl in the rear left leg. All animals were immunized again after 15 days in accordance with the original scheme of immunization. Thirty days after the last immunization, the animals were evaluated by the degree of development of blood vessels in vivo using Matrigel similarly described Coughlin M.C. and others (Coughlin M.C. et al., J. Clin. Invest. 101:1441, 1998). Previously vaccinated animals were divided in groups of animals in each in the middle of the abdomen were subcutaneously injected with 500 μl of Matrigel (Becton Dickinson and Co., Franklin Lakes, New Jersey, USA)containing:
1. VEGF 50 ng/ml, heparin 50 IU/ml
2. 105melanoma cells B16-F10
3. Saline phosphate buffer (PBS)
Six days after immunization, animals were killed and extracted a tube of Matrigel. The hemoglobin content in the tubes were analyzed according to the manufacturer's instructions (kit of reagents Drabkina; Sigma Diagnostics Co., St. Louis, Missouri, USA). Vaccination with plasmid encoding VEGF or its receptor KDR, significantly inhibited (p<0.001) in the development of blood vessels induced by VEGF, but also induced more complex systems, such as tumor cells.
Getting immunogen by non-covalent binding RMEΔ5-VEGF121with different adjuvants
Different immunostimulatory funds were used in a mixture with the design RMEΔ5-VEGF121in accordance with the following method. Protein Opc isolated from the outer membrane of Neisseria meningitidis, was purified by the method Musacchio and others (Musacchio et al., Vaccine, 67:751, 1997). 50 µg/ml RMEΔ5-VEGF121was added to 10 μg/ml protein ORS careful when shaking the mixture with acid pH. The resulting complex were dialyzed overnight in PBS without endotoxin with a pH of 7.2 (Sigma). The level of protein Association LFS-plasmid DNA (LFS-RMEΔ5-VEGF121) was checked by visualizing the DNA used is eat 1% agarose gel. More than 50% of the plasmid DNA was associated with protein LFS.
In combination with the interest of plasmid DNA used very small particles (VSSP) complex proteins of the outer membrane (OMRS) Neisseria meningitides, provided by the Center of molecular immunology (R. Perez et al., applications for U.S. patent No. 5788985 and 6149921). VSSP (1 mg) were incubated with 5 mg RMEΔ5-VEGF121during the night, with careful stirring. The resulting substance for a long time were dialyzed in PBS without endotoxin, pH of 7.2 (Sigma). The level of Association VSSP-plasmid DNA (VSSP-pMAE5Δ5-VEGF121) was checked by visualization of DNA using 1% agarose gel. More than 50% of the plasmid DNA was associated with particles VSSP.
Nuclear antigens of hepatitis C and hepatitis b (HCcAg and HBcAg) in the form of particles were obtained in accordance with the description given in the scientific literature (Lorenzo L.J. et al., Biochem. Biophys. Res. Commun. 281:962, 2001). 1 mg of antigen was mixed with 5 mg of plasmid and incubated over night. Levels of Association HCcAg or HBcAg-plasmid DNA (respectively HCcAg-pMAE5Δ5-VEGF121and HBcAg-pMAE5Δ5-VEGF121) was checked by visualization of DNA using 1% agarose gel. More than 50% of the DNA was associated with particles of antigen in each experiment.
Experiments to create immunity in vivo by using the design RMEΔ5-VEGF121and adjuvant is, causing an immune response
Groups consisting of 10 mice 57BL/6, were vaccinated or not vaccinated in accordance with the following options:
1. RMEΔ5-VEGF121(1, 10 and 50 μg of DNA/mouse) in PBS pH 7,2
2. LFS-RMEΔ5-VEGF121(1, 10 and 50 μg of DNA/mouse)
3. VSSP-RMEΔ5-VEGF121(1, 10 and 50 μg of DNA/mouse)
4. HBcAg-RMEΔ5-VEGF121(1, 10 and 50 μg of DNA/mouse)
5. HCcAg-RMEΔ5-VEGF121(1, 10 and 50 μg of DNA/mouse)
6. PBS, pH 7,2 (negative control group 1)
7. ORS (negative control group 2)
8. VSSP (negative control group 3)
9. HBcAg (negative control group 4)
10. HCcAg (negative control group 5)
Immunization, infection control tumor volume measurements of the tumor was performed similar to the previous example. Options vaccine in doses equal to 10 μg DNA/mouse or above, slowed tumor growth compared to the negative control groups (table 6). Significantly higher survival rate compared with the same period in the corresponding control group was observed in animals immunized with VEGF gene, associated or not associated with LFS, VSSP, HCcAg and HBcAg, which was used as immunopotentiating media. All options with the carrier provided a significantly higher survival rate with which avanyu with an appropriate control group at doses of 10 μg/mouse and higher while option representing deproteinizing DNA vector RMEΔ5-VEGF121, called the results significantly different from the negative control group at a dose of 50 μg/mouse (table 6).
Tumor volume and survival in mice immunized with different boosting tools
|Group||[Μg DNA]||Tumor volume (mm3) Day 24||Survival (43rd day)|
|pMAE5Δ5-VEGF||50||1050,9 ± 689 (**)||ns|
|10||1229,0 ± 596 (*)||ns|
|1||1895,3 ± 596 (ns)||ns|
|OpC-pMAE5Δ5-VEGF||50||960,6 ± 456 (**)||**|
|10||1100,5 ± 615 (**)||*|
|1||1654,8 ± 663 (ns)||ns|
|VSSP-pMAE5Δ5-VEGF||50||884,6 ± 410 (***)||**|
|10||1002,3 ± 598 (**)||*|
|1||1532,7 ± 745 (ns)||ns|
|HBcAg-pMAE5Δ5-VEGF||50||950,1 ± 570 (**)||**|
|10||1230,5 ± 662 (*)||*|
|1||1867,2 ± 652 (ns)||ns|
|HCcAg-pMAE5Δ5-VEGF||50||950,1 ± 570 (**)||**|
|10||1230,5 ± 662 (*)||*|
|1||1867,2 ± 652 (ns)||ns|
|OpC (5 μg/mouse/dose)||5 mcg||2059,0 ± 687 (ns)||ns|
|VSSP||2156,0 ± 759 (ns)||ns|
|1998,2 ± 798 (ns)||ns|
|1897,0 ± 812 (ns)||ns|
|PBS pH 7,2||2073,0 ± 816 (ns)||ns|
|Note: tumor volume specified as the average value of ± standard deviation (SD) of measurements of the animals, performed in each group; statistical comparisons were made using unidirectional method ANOVA and post-test, Bonferroni. Survival was determined on the basis of the level statistices the th significance using criteria logarithmic distribution to compare each group with the control group on the specified day. Statistical significance is indicated as ns, p ≤ 0,05 without statistical significance; *, p ≤ 0,05; **, p ≤ 0,01; ***, p ≤ 0,001.|
Experiment in creating immunity in vivo using VEGF in the form of protein
VEGF165 (20 μg/mouse) with complete or incomplete adjuvant's adjuvant;
complete and incomplete beta-blockers (negative control group).
Antigen VEGF165was obtained from a commercial source (Sigma) with a purity of more than 97%. Mice were immunized via intraperitoneal injection, using the full beta-blockers (Sigma), and re-immunized after 15 and 30 days in the same way, but using the incomplete beta-blockers. Control contamination of the tumor and the tumor volume was produced similarly to the previous example.
In the group immunized with VEGF, there was a significant decrease of tumor volume and increased survival compared with unimmunized control group. The achieved effect was similar to the one found in the previous examples using DNA VEGF.
Experiments to create immunity in vivo in C57BL/6 mice with severe combined immunodeficiency (SCID)
Mice C57BL/6 immunizable mcg doses RMEΔ 5-VEGF121DNA/mouse as described in example 5 were immunized or not. Mice were killed after 45 days after the first immunization. CD8+, CD4+ and b cells of these mice were separated using magnetic beads (Dynabeads, USA) according to the manufacturer's instructions.
Of C57BL/6 mice at the age of six weeks with SCID in groups consisting of 10 animals, were subjected to immunohistologically therapy, including the introduction of the following combinations of previously deleted lymphocytes.
Group 1: CD8+ T-lymphocytes and CD4+ T-lymphocytes obtained from mice immunized with DNA RMEΔ5-VEGF121. B-lymphocytes were not restored.
Group 2: b-lymphocytes and CD4+ T-lymphocytes immunized mice and CD8+ T-lymphocytes unimmunized mice.
Group 3: b-lymphocytes, CD8+ T-lymphocytes and CD4+ T-lymphocytes immunized mice used in this experiment as a positive control tools.
Group 4: b-lymphocytes, CD8+ T-lymphocytes and CD4+ T-lymphocytes unimmunized mice used in this experiment as a negative control tools.
Mice with SCID subjected immunohistologically therapy, infected by means of subcutaneous injection of 104melanoma cells B16-F10. Tumor growth was controlled, producing three measurements per week until the animal's death. Levels of antibodies against VEGF analysis is believed laboratory method ELISA. 96-well plates were incubated overnight with 0.5 μg/ml VEGF165 (Sigma). The wells were blocked with a solution of PBS with 1% BSA (BDH, UK) and then incubated with several dilutions of animal serum. The culture was washed with PBS 0.05% tween was added obtained commercially polyclonal antibody against mouse IgG (Sigma, A0168). The signal amplified in the presence of commercial substrate orthophenylphenol (OPD, Sigma).
Table 7 summarizes the indicators of tumor volume (day 24) and survival (the 40th day) mice from different groups, infected tumor. Starting from the 15th day after immunohistologically therapy, the animals in groups 1-3 were observed reduction in tumor size compared with group 4, which was introduced lymphocytes unimmunized mice. Thus, effects, stimulating the immune system of the immunized mice, which decreases the size of the tumor associated with humoral and cellular responses, and the last mentioned reaction refers to the cytotoxic type (CTL) due to the lack of antibodies against VEGF in group 1. However, in experimental conditions, the survival rate increased only in group 3 (b - and T-lymphocytes immunized mice) compared with the other groups (table 7). In animals with no reactions - or T-cell type CTL (respectively, group 1 is 2), which were subjected to incomplete immunohistologically therapy, the survival rate did not differ from the similar index in the negative control group. The results show that the combination of humoral and cellular responses (group 4) has a synergistic effect, which can cause an effective response that can increase the survival rate of mice infected with a tumor.
Tumor volume and survival in mice with SCID subjected to reductive immunotherapy using lymphocytes of mice immunized RMEΔ5-VEGF121
|Group||Mouse donor lymphocytes injected mice 57BL/6 SCID||Tumor volume (day 24)||Survival (the 40th day)|
|Note: mice-donors were immunized with 50 μg doses RMEΔ5-VEGF DNA/mouse were immunized or not. Tumor volume is specified by its mean value ± standard deviation (SD) of measurements of the animals, performed in each group; statistical comparisons were made using unidirectional method ANOVA and post-test, Bonferroni. Survival was determined on the basis of the level of statistical significance using criteria logarithmic distribution to compare each group with the control group on the specified day. Statistical significance is indicated as ns, p ≤ 0,05 without statistical significance; *, p ≤ 0,05; **, p ≤ 0,01; ***, p ≤ 0,001.|
Immunological recovery in the immune response, determined by the reduction of circulating VEGF
For groups of 15 female mice 57BL/6, intramuscularly were injected with the following options:
1. RMEΔ5-VEGF121(50 μg/mouse) in PBS pH 7,2
2. PBS, pH 7,2
In all experiments, mice were immunized is by intramuscular injection (im) in a total volume of 50 µl in the rear left leg. All animals were immunized again after 15 days in accordance with the original scheme of immunization. Thirty days after the last immunization 5 randomly selected animals from each group were killed for analysis of immunological status immunized and control animals, as well as toxic effects of vaccination on the organs and tissues using macroscopic and histological analyses.
The rest of the animals in all groups were subcutaneously injected with 104melanoma cells B16-F10 in the right part of the abdomen. After 15 and 30 days after injection of tumor cells were killed on 5 mice from each group and made the above findings.
In the studied animals was not observed toxic effects at the macroscopic level, and histopathological analysis revealed no damage to any of the bodies in the study through 30 days after the last immunization. Immunological analysis included: (1) determination of VEGF levels in mice serum; (2) the content of T - and b-lymphocytes and Mature dendritic cells in the spleen and bronchial auxiliary and inguinal lymph nodes.
Analysis of VEGF levels (set R&D for determination of VEGF in mice) in the serum of unimmunized mice showed that with increasing time of development of the tumor levels of VEGF in savored is increased in accordance with increase in tumor size over time. In groups of mice immunized against human VEGF was detected a significant reduction (p<0,001 method ANOVA, post-test, Bonferroni) levels of VEGF in mice, which remained after 30 days of infection control tumor.
The immune system of animals succumbing in each time period analyzed, examining the ratio of populations of cells in the lymph nodes and spleen as described Gabrilovich and others (Gabrilovich D. et al., Blood 92:4150, 1998). To perform these studies used commercial monoclonal antibodies that recognize molecules CD3, CD19, CD11c and CD86 (B7-2) (Pharmingen), labeled fluoresceinisothiocyanate (FITC) and phycoerythrin (PE), which allowed to visualize the population of cells using a flow cytometer (FACS). The results are shown in table 8.
A brief overview of the results of FACS analysis of populations of cells based on surface markers
|Group (days)||The total number of cells||The fraction enriched in dendritic cell|
|Lymph nodes||Spleen||Lymph nodes||Spleen|
|A. Neiman the new||CD 19||CD 3||CD 19||CD 3||CD-11c/B7-2||CD-11c/B7-2|
|Unimmunized (30 days)||8%||86%||38,1%||40,8%||60%||62,4%|
|After infection, tumor (60 days)||20,1%||60,5%||3,8%||11,4%||32,8%||10,2%|
|Century Immunized||CD 19||CD 3||CD 19||CD 3||CD-11c/B7-2||CD-11c/B7-2|
|Immunized (30 days)||to 7.2%||87,3%||40%||39%||58,6%||60,3%|
|After infection, tumor (60 days)||10,9%||80,1%||25,4%||34%||53,5%||52,9%|
|Note: the above values show the percentage of positive cells from the total number of cells calculated by the method of quantitative determination.|
Analyses of populations of lymphoid cells and maturation of dendritic cells in animals 30 days after immunization show that vaccination with DNA VEGF does not cause any changes in the immune status of the animal. However, after 30 days the village is e implantation of tumors in unvaccinated animals observed decrease in the ratio of T-lymphocytes/lymphocytes (CD3/CD19) as in the lymph nodes, and in the spleen compared with the ratio that occurred before infection tumor. In addition, the spleen there is a significant decrease in the number of lymphoid cells. These animals also showed a reduction in the number of Mature dendritic cells in lymph nodes and spleen. In the group of mice vaccinated with DNA VEGF showed significant recovery in all indicators, which can be correlated with decreased levels of VEGF in serum, detected in animals in this group.
1. Immunogenic composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes, and the composition contains oligonucleotides that encode polypeptides VEGFR2 or fragments thereof, administered as part of a plasmid or a viral vector, and optionally further comprises a pharmaceutically acceptable adjuvant.
2. Immunogenic composition according to claim 1, in which the oligonucleotides that encode the polypeptides VEGFR2 and fragments thereof, are autologous, heterologous or chimeric.
3. Immunogenic composition according to claim 1, in which the oligonucleotides encoding the polypeptides VEGFR2 and fragments thereof, obtained from the synthetic is practical, recombinant or natural sources.
4. Immunogenic composition according to claim 1, in which the adjuvant is selected from the group consisting of recombinant particles nuclear antigen hepatitis b, recombinant particles nuclear antigen hepatitis C, protein LFS and montanide ISA 51.
5. Immunogenic composition according to claim 1, in which the oligonucleotides encoding the polypeptides VEGFR2 and their fragments, introducing covalently or ecovalence associated with drugs VSSP isolated from the outer membrane of Neisseria meningitidis, for the treatment of disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes.
6. Immunogenic composition according to claim 1, in which the oligonucleotides encoding the polypeptides VEGFR2 and their fragments, introducing covalently or ecovalence associated with protein RK, for the treatment of disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes.
7. Immunogenic composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, slocate the public tumors and their metastases, in benign tumors and chronic inflammatory and autoimmune processes, and the composition contains a VEGFR2 polypeptide or fragments thereof and optionally further comprises a pharmaceutically acceptable adjuvant.
8. Immunogenic composition according to claim 7, in which the VEGFR2 polypeptide and fragments thereof are autologous, heterologous or chimeric.
9. Immunogenic composition according to claim 7, in which the VEGFR2 polypeptide and fragments thereof derived from synthetic, recombinant or natural sources.
10. Immunogenic composition according to claim 7, in which the adjuvant is selected from the group consisting of recombinant particles nuclear antigen hepatitis b, recombinant particles nuclear antigen hepatitis C, protein LFS and montanide ISA 51.
11. Immunogenic composition according to claim 7, in which the VEGFR2 polypeptide and fragments thereof is administered covalently or ecovalence associated with drugs VSSP isolated from the outer membrane of Neisseria meningitidis, for the treatment of disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes.
12. Immunogenic composition according to claim 7, in which the VEGFR2 polypeptide and fragments thereof is administered covalently or ecovalence SV is related protein RK, for the treatment of disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes.
13. Immunogenic composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes, and the composition contains oligonucleotides encoding autologous VEGF polypeptides with impaired ability to activate the receptor, administered as part of a plasmid or a viral vector, and optionally further comprises a pharmaceutically acceptable adjuvant.
14. Immunogenic composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes, and the composition comprises autologous VEGF polypeptides with impaired ability to activate the receptor, and optionally further comprises a pharmaceutically acceptable adjuvant.
15. Immunogene the composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes, and the composition contains oligonucleotides encoding autologous VEGF polypeptides with impaired ability to activate the receptor, and at least a molecule, described in claims 1 to 12, and optionally further comprises a pharmaceutically acceptable adjuvant.
16. Immunogenic composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes, and the composition comprises autologous VEGF polypeptides with impaired ability to activate the receptor and at least a molecule, described in claims 1 to 12, and optionally further comprises a pharmaceutically acceptable adjuvant.
17. Immunogenic composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes, to notice contains bicistronic vector, encoding VEGFR2 or its fragments, and VEGF polypeptide with impaired activation of the receptor, which is injected in the presence or in the composition of the VSSP, isolated from the outer membrane of Neisseria meningitidis, and which optionally further comprises a pharmaceutically acceptable adjuvant.
18. Immunogenic composition for treating disorders associated with increased angiogenesis that occurs when tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes, and the composition contains a protein consisting of the polypeptide VEGFR2 or their fragments and VEGF polypeptide with impaired activation of the receptor, which is injected in the presence or in the composition of the VSSP, isolated from the outer membrane of Neisseria meningitidis, and which optionally further comprises a pharmaceutically acceptable adjuvant.
19. How active vaccination, characterized in that the injected immunogenic composition according to claims 1 to 18 for the treatment of disorders associated with increased angiogenesis caused by tumors in humans, malignant tumors and their metastases, benign neoplasms and chronic inflammatory and autoimmune processes.
FIELD: biotechnology, molecular biology, proteins.
SUBSTANCE: invention relates to a method for preparing cytokines of class II and can be used in medicine. Prepared proteins zcyto20, zcyto22, zcyto24 and zcyto25 are the most relative with interferon-α at amino acid sequence level. Receptor of cytokines of class II represents a receptor for this family of proteins. Proteins can be prepared by recombinant way using a cell-host transformed with expression vector that comprises nucleic acids corresponding to proteins. Base on proteins xcyto20, xcyto21, zcyto22, zcyto24 and zcyto25 antiviral pharmaceutical composition and specific antibodies are prepared. Invention provides preparing the novel cytokine that stimulates cells of differentiation hemopoietic line and possesses the expressed antiviral activity.
EFFECT: valuable biological and medicinal properties of polypeptide, improved preparing method.
24 cl, 21 tbl, 32 ex
FIELD: gene engineering, in particular method for treatment of viral infections.
SUBSTANCE: protein ZCYTO21 has amino acid sequence which is nearly similar to amino acid sequence of interferon-α. Protein and antibodies thereto have antiviral activity and are useful in treatment of hepatitis B and C as well as other diseases.
EFFECT: new protein with antiviral activity.
71 cl, 1 dwg, 6 tbl, 7 ex
FIELD: biotechnology, medicine.
SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.
EFFECT: valuable properties of new cytokine.
18 cl, 5 tbl, 1 dwg, 55 ex
FIELD: technological processess.
SUBSTANCE: oligomeric polypeptide dimmers include ligand-binding leptyne domains. Domains are connected by means of flexible polypeptide linker molecules. Linker molecules might possibly include sites of sensitivity to proteases.
EFFECT: releases biologically active cytokines when injecting to human being or animal.
31 cl, 13 dwg
FIELD: genetic engineering, medicine.
SUBSTANCE: invention proposes chimeric protein (ANTH1) consisting of a sequence corresponding to 60 amino acids of N-terminal region of interleukin-2 and extracellular moiety of N-terminal moiety of human gamma-interferon alpha-chain that are bound each with other by peptide Ala-His-Met-Met. Protein ANTH1 possesses capacity to inhibit biological activity of gamma-interferon and/or interleukin-2 and therefore it can be used in preparing drugs designated for treatment of diseases mediated by activity of interleukin-2, gamma-interferon or both these cytokines, in particular, in treatment of cerebrospinal sclerosis, erythematosus lupus, proliferative arthritis, diabetes mellitus and others. Invention can be used in medical-biological industry.
EFFECT: valuable medicinal and biological properties of protein.
13 cl, 1 tbl, 10 dwg, 11 ex
FIELD: genetic engineering, medicine.
SUBSTANCE: invention relates to interleukin-20 receptors and can be used in treatment of inflammatory diseases. Soluble receptors for interleukin-20 (IL-20) are constructed consisting of two polypeptide subunits IL-20RA and IL-20RB. Subunits as components of IL-20 receptor are bound by disulfide bonds. Also, variants of IL-20 receptor are prepared wherein one subunit is fused with the constant region of immunoglobulin light chain, and another subunit is fused with the constant region of immunoglobulin heavy chain, and these chains of immunoglobulin are bound by a disulfide bond. Invention provides using new IL-20 receptors for down-regulation of IL-20.
EFFECT: valuable medicinal properties if IL-20 receptor.
9 cl, 8 dwg, 13 ex
FIELD: genetic engineering, pharmaceutical and medical-biological industry.
SUBSTANCE: invention proposes a chimeric sequence of nucleic acid encoding a fused polypeptide able to bind with the vessel endothelium growth factor (VEGF) and to inhibit its specific mitogenic effect. The fused polypeptide molecule comprises immunoglobulin-like domain 2 of VEGF-receptor Flt1, immunoglobulin-like domain 3 of VEGF-receptor Flk 1 or Flt4 and multimerizing component represented by either domain Fc IgG or heave chain IgG. By expression of the proposed chimeric sequence or its two successively joined copies in a host-cell a monomer or dimer of the fused polypeptide are prepared, respectively, that can be used for suppression of VEGF activity in mammals, in particular, in humans. New VEGF inhibitors differ from the known one by the improved pharmacokinetics.
EFFECT: improved preparing method, valuable biological properties of polypeptide.
23 cl, 67 dwg, 1 tbl, 35 ex
FIELD: medicine, oncology, biochemistry.
SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.
EFFECT: valuable medicinal properties of protein complexes.
13 cl, 40 dwg, 18 ex
FIELD: genetic engineering, proteins, medicine, pharmacy.
SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.
EFFECT: improved targeting method, valuable biological properties of fused protein.
10 cl, 5 dwg, 9 ex
FIELD: medicine; pharmacology.
SUBSTANCE: releasing peptides of growth hormone are described with formula (I): R1-А1-А2-А3-А4-А5-R2, where:А1 designates Aib, Apc or Inp; А2 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl) or D-Тrp; А3 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-2Ser(Bzl) or D-Trp; А4 designates 2Fua, Orn, 2Pal, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, 3Thi, Thr(Bzl); А5 designates Apc, Dab, Dap, Lys, Orn or deleted; R1 designates hydrogen; and R2 designates NH2; and their pharmaceutically acceptable salts.
EFFECT: pharmaceutical compositions and the methods of their application are presented.
25 cl, 1 tbl, 2 ex