Combined therapy by using antiangiogenic agents and tnf-alpha

FIELD: medicine, combined therapy.

SUBSTANCE: invention relates to pharmaceutical composition for endothelial tumor treatment containing in therapeutically effective amount antiangiogenic agent such as polypeptide including RGD sequence namely cyclo(Arg-Gly-Asp-Dphe-NmeVal) and tumor necrosis factor TNFα. Also disclosed are kit containing pharmaceutical composition, application of abovementioned pharmaceutical composition and kit, and method for therapy.

EFFECT: agent for effective treatment of endothelial tumors.

15 cl, 16 ex, 8 dwg

The technical field of the invention

The invention relates to combination therapy for the treatment of tumors and metastasis of tumors, which provides for the introduction of antiangiogenic tools and tumor necrosis factor alpha (TNFαor a molecule having the biological activity of TNFαand, optionally together with other cytotoxic means, such as gamma interferon (IFNγ), or chemotherapeutics, such as cisplatin, or inhibitors of ErbB receptor, such as antibodies to EGFR. Method and pharmaceutical compositions containing these funds may have synergistic enhanced inhibitory effect on the proliferation of tumor cells of each individual therapeutic agents, providing a more effective treatment compared with the introduction of each individual component separately.

Background of invention:

Angiogenesis, which is also known as neovascularization, is the process of vascularization of tissue, which is characterized by involvement in the process of growth of developing new blood vessels in tissue. This process is mediated by infiltration of endothelial cells and smooth muscle cells. Suppose that the process is carried out in one of three ways: (1) vessels can grow from pre-existing vessel is in; (2) the development of blood vessels de novo can be accessed from precursor cells (formation and development of blood vessels); or (3) existing small vessels can increase in diameter (Blood and others, 1990, Bioch. Biophys. Acta 1032, 89. It is known that endothelial cells of blood vessels contain at least five RGD-dependent integrins, including the vitronectin receptor (α_vβ₃or α_vβ₅), receptors for collagen type I and IV, laminin receptor, the receptor for fibronectin/laminin/collagen and fibronectin receptor (Davis and others, 1993, J. Cell. Biochem. 51, 206). It is also known that smooth muscle cells contain at least six RGD-dependent integrins, including α_vβ₃and α_vβ₅.

Angiogenesis is an important process in the neonatal growth, but also plays a significant role in wound healing and in the pathogenesis of a variety of clinically important diseases, including tissue inflammation, arthritis, psoriasis, cancer, diabetic retinopathy, macular degeneration and other neovascular eye diseases. These diseases associated with angiogenesis, also referred to as angiogenic diseases (Folkman and others, 1987, Science 235, 442).

Inhibition of cell adhesion in vitro using monoclonal antibodies, immunospecificity to various α and βintegrin subunits, occurs with the involvement of the receptor for vitronectin α_vβ₃in the adhesion of different cell types, including endothelial cells of the microvessels (Davis and others, 1993, J. Cell. Biol. 51, 206).

Integrins are a class of cellular receptors known to bind extracellular matrix proteins and thus mediate interactions between cells and between cells and the extracellular matrix, resulting in adhesion of cells. The integrin receptors are a family of proteins that share structural characteristics, namely non-covalent heterodimeric glycoprotein complex formed α and β subunits. For the vitronectin receptor, named so due to the fact that it preferably is initially associated with vitronectin, I now know that it includes three different integrin identified as α_vβ₁that α_vβ₃and α_vβ₅. α_vβ₁binds fibronectin and vitronectin. α_vβ₃binds many ligands, including fibrin, fibrinogen, laminin, thrombospondin, vitronectin and von Willebrand factor. α_vβ₅binds vitronectin. Obviously, there are different integrins with different biological functions, as well as different integrins and subunit them who have joint biological specificity. One important part of recognition ligand for many integrins is Tripeptide sequence arginine-glycine-aspartic acid (RGD). RGD was detected in all the ligands mentioned above, for integranova vitronectin receptor.

This RGD site recognition can be simulated linear and cyclic (poly)peptides, which include the RGD sequence. Such RGD peptides known as inhibitors or antagonists, respectively, the functions of the integrin. It is important to note that, however, depending on the sequence and structure of the RGD peptide specificity of the inhibitory effects may vary depending on the specificity of the target integrins. A variety of RGD polypeptide integrins different specificity are described, for example, Cheresh, and others, 1989, Cell 58, 945, Aumailley and others, 1991, FEBS Letts. 291, 50, and in numerous patent applications and patents (for example, US patents 4,517,686, 4,578,079, 4,589,881, 4,614,517, 4,661,111, 4,792,525; EP 0770622).

The formation of new blood vessels, or angiogenesis, plays a key role in the growth of malignant tumors, and therefore considerable interest in the development of substances that inhibit angiogenesis (see, for example, Holmgren and others, 1995, Nature Medicine 1, 149; Folkman, 1995, Nature Medicine 1, 27; O′Reilly and others, 1994, Cell 79, 315). The antagonists of integrin α_vβ 3for the inhibition of angiogenesis is known in methods of inhibiting the growth of solid tumors by reducing the blood supply to solid tumors (see, for example, US patents 5,753,230 and US 5,766,591, which describes the antagonists α_vβ₃such as synthetic polypeptides, monoclonal antibodies and mimetics α_vβ₃that bind the receptor α_vβ₃and inhibit angiogenesis). Methods and compositions for inhibition α_vβ₅-mediated angiogenesis in tissues using vitronectin receptor antagonists α_vβ₅described in WO 97/45447.

Angiogenesis is characterized by invasion, migration and proliferation of endothelial cells, this process depends on the interaction of cells with components of the extracellular matrix. In this situation, the integrin receptors between the cell and the matrix mediates the proliferation and migration of cells. It was shown that the adhesion receptors of the endothelial integrin α_vβ₃play a key role, being specific vascular targets during antiangiogenic treatment (Brooks and others, 1994, Science 264, 569; Friedlander and others, 1995, Science 270). The involvement of integrins α_vβ₃vessels in angiogenesis has been demonstrated in several models in vivo, in which the development of new blood vessels in Proc. of splanirovannyh human tumors completely inhibited or with the systemic administration of peptide antagonists of integrin α _vβ₃and α_vβ₅as described above, or, alternatively, by using antibodies LM609 to α_vβ₃(Brooks and others, 1994, Cell 79, 1157; ATS HB 9537). This antibody blocks the receptor integrin α_vβ₃, activation of which natural ligands inhibits apoptosis of proliferating angiogenic vascular cells and thus violates the formation of the newly developing blood vessels, which is essential for proliferation of tumors. However, recently it was found that melanoma cells are capable of forming pautinopodobnoj patterns of blood vessels even in the absence of endothelial cells (Barinaga 1999, Science 285, 14), as a consequence, the tumor can be tolerant of some antiangiogenic drugs, which are effective only in the presence of endothelial tissue.

A variety of molecules that stimulate proliferation, migration, and aggregation of the endothelium include VEGF, Ang1 and bFGF, and represent the natural factors of survival. VEGF (factor vascular endothelial growth) was identified as a selective angiogenic growth factor that can stimulate the mitosis of endothelial cells. Suggest that VEGF, in particular, is a major mediator of angiogenesis in the primary tumor and in ischemic ocular diseases. VEGF is a glycosilated (MB: 46000) and is a specific angiogenic factor vascular endothelial cells (Ferrara and others, 1992, Endocrin. Rev., 13, 18) and factor vascular permeability (Senger and others, 1986, Cancer Res., 465629)with high affinity associated with membranosvyazannye receptors with tyrosinekinase activity (Jakeman and others, 1992, J.Clin. Invest., 89, 244). The material obtained by biopsy of human cancers is increased mRNA expression of VEGF in malignant cells and mRNA of VEGF receptor in adjacent endothelial cells. The maximum expression of VEGF detected in tumor sites adjacent to areas of necrosis of blood vessels (see review by Thomas and others, 1996, J.Biol. Chem. 271(2), 603; Folkman, 1995, Nature Medicine 1, 27). In the application WO 97/45447 described the involvement of integrin α_vβ₅in the process of formation of new blood vessels, which, in particular, is induced by VEGF, EGF and TGF-αand found that the antagonist α_vβ₅can inhibit the angiogenesis induced by VEGF. For effective antitumor therapy can also be used as a target the VEGF receptor to inhibition of angiogenesis, using monoclonal antibodies (Witte and others, 1998, Cancer Metastasis Rev. 17(2), 155). It is known that MAb DC-101 inhibits angiogenesis of tumor cells.

Tyrosine kinase is a class of enzymes that catalyze the transfer of the final phosphate adenosine state on tyrosine residues in protein substrates. I believe that tyrosine kinase via substrate phosphorylation play a crucial role in signal transmission in a variety of cellular functions. Although the exact mechanisms of signal transmission is still unclear, for tyrosinekinase it was shown that they are important factors involved in cell proliferation, carcinogenesis and cell differentiation.

Tyrosine kinase can be divided into tyrosine kinase receptor type and precepting type. Both types of tyrosinekinase - receptor and preceding type is involved in transmission of signals in cells, leading to many pathogenic conditions, including cancer, psoriasis and hyperimmune responses. Many tyrosine kinase involved in cell growth and angiogenesis.

Tyrosine kinase receptor type are extracellular, transmembrane and intracellular part, while tyrosine kinase precepting type are entirely intracellular. Tyrosine kinase-related receptors are transmembrane proteins, which include the extracellular ligand-binding domain, a transmembrane sequence, and a cytoplasmic tyrosine kinase domain. Tyrosine kinase receptor type include a large number of transmembrane receptors with diverse biological activity. In fact, identified different subfamilies tyrosinekinase receptor type. These include tyrosine kinase receptors of fibroblast growth factor (FGF)receptors growth factor epidermal (EGF) family of the main class and ErbB receptors growth factor derived platelets (PDGF). This also includes receptors for nerve growth factor (NGF)receptor-dependent brain-derived neurotrophic factor (BDNF) and receptors of neurotrophin-3 (NT-3) and the receptors of neurotrophin-4 (NT-4).

One subfamily of tyrosine kinase receptor type, designated as the subfamily HER or ErbB includes EGFR (ErbB1), HER2 (ErbB2 or p185neu), HER3, (ErbB3) and HER4 (ErbB4 or tyro2). The ligands of this receptor subfamily include growth factor epithelium (EGF), TGF-α, amphiregulin, HB-EGF, betacellulin and heregulin. The PDGF subfamily includes the FLK family, which includes the internal domain receptor kinase (KDR).

EGFR encoded erbB1 gene, involved and is one of the reasons of occurrence of malignant tumors in humans. In particular, the enhanced expression of EGFR is observed in breast cancer, bladder, lung, head, neck and stomach, as well as in glioblastomas. Enhanced expression of the receptor EGFR is often associated with enhanced production of the EGFR ligand, transforming growth factor alpha (TGF-α), in such tumor cells occurs through activation of the receptor when p is power autocrine stimulation (Baselga and Mendelsohn, Pharmac. Ther. 64:127-154 (1994)). The EGF receptor is a transmembrane glycoprotein, which has a molecular weight of 170,000, and was found in many types of epithelial cells. It is activated by at least three ligands, EGF, TGF-α (transforming growth factor alpha) and amphiregulin. It was shown that the growth factor epidermal (EGF), and transforming growth factor alpha (TGF-α) associated with the EGF receptor and lead to cell proliferation and growth of tumors. These growth factors are not associated with HER2 (Ulrich and Schlesinger, 1990, Cell 61, 203). In contrast, some families of growth factors that doziruut dimerization of the receptor due to their dimeric nature (e.g., PDGF), Monomeric growth factors, such EGF include two binding site to their receptors, and, therefore, can cross-link two adjacent EGF receptor (Lemmon and others, 1997, EMBO J. 16, 281). Dimerization of the receptor is required for stimulation of the intrinsic catalytic activity and to autophosphorylate receptors of growth factors. It should be noted that the receptor protein tyrosine kinase (PTKs) are able to undergo both Homo-and heterodimerization.

It has been shown that antibodies to the EGF receptor, although block the binding of EGF and TGF-α with a receptor capable of inhibiting the proliferation of tumor cells. Taking into account these findings,identified many mouse and rat monoclonal antibodies to the EGF receptor and studied for their ability to inhibit tumor cell growth in vitro and in vivo (Modjtahedi and Dean, 1994, J.Oncology 4, 277). Humanitariannet monoclinal antibody 425 (hMAb 425, US 5.558,864; EP 0531472) and chimeric monoclonal antibody 225 (cMAb 225, US 4,943,533 and EP 0359282), both to the EGF receptor, revealed efficacy in clinical studies. It was shown that the antibody S inhibits EGF-mediated tumor cell growth in vitro and inhibits formation of human tumors in vivo at messerstich mice. In addition, antibodies find, first of all, a synergistic effect with certain chemotherapeutic agents (e.g., doxorubicin, adriamycin, Taxol, and cisplatin) and destroy human tumors in vivo in mouse models of xenotransplantation. Ye and others (1999, Oncogene 18, 731) showed that human cancer cells of the ovary can successfully be treated by a combination of both cMab 225 and gumanitarnogo MAb 4D5 to the HER2 receptor.

Another representative of the family of ErbB, HER2 (ErbB2 or RPI) was originally identified as the product of the transforming gene for neuroblastoma in rats subjected to chemotherapy. An activated form of the neu proto-oncogene occurs due to a point mutation (valine for glutamic acid) in the transmembrane site kadurugamuwa protein. Amplification of the human homolog of neu is observed in breast cancer and ovarian cancers and correlates with poor prognosis (Slamon and others, Science, 235: 177-182 (187); Slamon and others, Science, 244:707-712 (1989); US 4,968,603). ErbB2 (HER2) has a molecular weight of about 185,000, and has significant homology to the EGF receptor (HER1), despite the fact that the specific ligand for HER2 has still not been definitively identified.

The antibody 4D5 receptor HER2 additionally detects the sensitivity to the cytotoxic action of TNFα in cell lines of breast tumors that sverkhekspressiya ErbB2 (patent US 5,677,171). Recombinant humanitariannet modification of the murine antibody 4D5 to ErbB2 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; US 5,821,337) has clinical activity in patients with metastatic breast tumors that sverkhekspressiya ErbB2, which previously received intensive anti-cancer therapy (Baselga and others, J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® was obtained when testing in 1998 for the treatment of patients with metastatic breast cancer, with tumors sverkhekspressiya ErbB2 protein.

TNFα refers to a large family of molecules that includes important cytokines, such as Fas ligand, CD40 ligand, TRAIL, lymphotoxin and others (Locksley and others, 2001, Cell 104:487-501). In addition, he is released from different cell types, TNFα there is also bound to the cell membrane, in molecular form in the cells, and this form is also the mediator of many biological deystviya, that TNFα has some value in normal development and physiology; however, he reveals the pernicious and destructive effects in many tissues under different conditions (Tracey and others, Ann. Rev. Med 1994; 45:491). Painful conditions in which TNFα plays a significant role include septic shock, cancer cachexia, rheumatoid arthritis, etc.

First TNFα man was cleared in 1985 (see Aggarwal and others, J.Biol. Chem. 1985, 260, 2345-2354). Shortly after this was completed molecular cloning of cDNA and cloned TNF TNF locus person (Pennica and other, Nature 1984, 312, 124-729; Wang and others, Nature 1985, 313, 803-806). TNFα represents the trimeric polypeptide with a molecular weight of 17 kDa, which is mainly produced by macrophages. The first peptide is expressed as a transmembrane protein with a molecular weight of 26 kDa, which is cleaved and released subunit 17 kDa after proteolytic cleavage. TNFα produced, as a rule, a variety of cells such as activated macrophages and fibroblasts. It was reported that TNFα induced by many different factors. It was also reported that TNFα also participates, directly or indirectly, in various diseases, such as infectious diseases, autoimmune diseases such as systemic lupus erythematosus (SLE), and arthritis, AIDS, sepsis is some other types of infections.

TNFα and inflammatory response, infection and tissue damage stimulates a cascade of biochemical changes that initiate the beginning of a complicated reactions of the immune system, which together represent an inflammatory response. The development of this response is based, at least in part, on the local blood vessels to dilate or increase vascular permeability and activation of the endothelium of blood vessels, which allows white blood calves effectively circulate and migrate to the affected area, and thus increases the probability of binding to and destroying any antigens. I believe that the endothelium of the blood vessels can then be activated or inflamed. Normally, inflammation is a desirable immune response to various unforeseen impacts, and is essentially characterized by a rapid onset and is of short duration (acute inflammation). However, his prolonged or uncontrolled activity (chronic inflammation) has a detrimental impact on the body and plays a role in the pathogenesis of certain immunological diseases such as septic shock, rheumatoid arthritis, inflammatory bowel disease and congestive heart failure (see "TNF and the superfamily of TNF receptors" in "cytokines and receptors C is Takenov", edited by Bona and Revillard, Harvard Academic Publishers, Amsterdam 2000, p.118-148).

TNFαas well as many other cytokines, secreted by macrophages shortly after the initiation of the inflammatory response and induces coagulation, increases the permeability of blood vessels and activates the expression of adhesion molecules in endothelial cells of blood vessels.

TNFα is never fully useful or completely harmful to the host. Thus, TNFα is an effective modulator of the function of endothelial cells. Depending on the condition of the vessels it causes inflammation by inducing the activation of endothelial cells and survival, or it causes necrosis of the tissue by inducing aporosa endothelial cells and destruction of blood vessels (Pober, J.S., Pathol Biol (Paris) 46, 159-163 (1998); Aggarwal, & Natarajan, Eur. Cytokine Netw. 7, 93-124 (1996)). Many intracellular transmission of signals mediated by these two different responses (Wallach and others, Annual Review of Immunology 17, 331-367 (1999)), but the transmission of extracellular signals, determines which of the two effects of TNFα is - survival or death are unclear.

Preferably the ratio between his generation and the regulation is a guarantee that the owner is able to respond effectively to defeat microorganisms without risk to their health. Being a mediator is asplenia, TNFα helps the body in its fight against bacterial infections and tissue damage through repeated immunization of the appropriate immune response. However, its overproduction leads to chronic inflammation, has a detrimental impact on the body and plays an important role in the pathogenesis of certain diseases.

IFNγ is a powerful amplifier of TNFα (Dealtry and others, Eur Immunol, 17, 689-693, (1987)). If TNFα causes apoptosis of cells, activation of NF-κB, a transcription factor that promotes the survival of cells, can inhibit apoptosis, which is induced by TNFα (Van Antwerp and others, Science 274, 787-789 (1996)).

TNFα participates in the transfer of a variety of cellular signals that lead to cellular responses, such as proliferation, activation, differentiation, but also and programmed cell death. The transmission of cellular signals to TNF(can be divided into early responses, such as activation of kinases, phosphatases, lipases, proteases and transcription factors, and late responses, and thus more indirect responses, such as the violation of the electron transport chain in mitochondria, production of radicals, generation of nitric oxide and the release of various substances. Many of the early response of cells, such as the restoration of the death domain containing transition be the key, activation of NF_KB or activation of caspase, also initiated by binding of other members of the family of TNF ligands to their respective receptors. Accordingly, molecules such as lymphotoxin, Fas ligand or TRAIL may interact with excess TNF (Grell and Clauss, I.c.).

Integrin-mediated adhesion to extracellular matrix (ECM) is essential for the survival of most cells, including endothelial cells. For example, integrin, vascular α_vβ₃promotes proliferation and survival of angiogenic endothelial cells and antagonists α_vβ₃induce apoptosis of angiogenic endothelial cells and inhibit angiogenesis (Brooks and others, Cell 79, 1157-1164 (1994). Some biochemical reactions associated with integrin-mediated cell survival, including the activation of PI 3-K/AKT (Khwaja and others, Embo Journal 16, 2783-2793 (1997)) and NF-κ (Scatena and others, J.Cell Biol 141, 1083-1093 (1998)) were identified as routes of transmission signals. In addition to integrins, adhesion molecules between cells MEETING-1 and VE Cadherin also contribute to the survival of endothelial cells (Bird and others J Cell Sci 112, 1989-1997 (1999); Carmeliet and other Cell 98, 147-157 (1999)).

TNF is cytotoxic for some lines of tumor cells, but most of them are almost not influenced by the growth process. Therefore, it is unlikely that eooperative effects of TNF in some animal models (Balkwill and others, Cancer Res. 46: 3990-3993 (1986)) is due to a direct effect of cytokines on tumor cells. In some studies it has been shown that indirect mechanisms of the host involved in the regression of the tumor, which is initiated by TNF (Manda and others, Cancer Res. 47: 3707-3711 (1987)). Accumulating data suggests that hemorrhagic necrosis of tumors, which is called TNF, initiated in endothelial cells the level of intratumoral vessels (Havell, etc., J. Exp.Med. 167: 1967-1985 (1988)).

The results of clinical trials of TNF in cancer patients are, in General, disappointing (review Haranaka, J.Biol. Response Mod. 7: 525-534 (1988)). Usually antitumor activity of TNF limited by significant side effects. One approach to limiting the adverse effects of TNF is receiving TNF mutants that detect each activity of the TNF receptor 1-specific type or variety of pharmacodynamic properties (Brouckaert and others, Circ. Shock 43: 185-190 (1994); Eggermont, Anticancer Res. 18: 3899-3905 (1998); Lucas and others, Int. J.Cancer 15: 543-549 (2001)). In recent years there have been advances in the treatment of patients who are suffering from melanomas or sarcomas of the extremities. Significant positive results can be obtained using isolated perfusion. Excessive doses of TNF until 4 mg used in combination with cytotoxic drugs or IFN (Lienard and others, J.Clin. Oncol. 10:52-60 (1992)). Local response Rea the tion, including a strong softening and redness of the tumor, associated with a strong inflammatory response, similar to those mediated TNF antitumor effects in mouse systems.

It was shown that such treatment of patients with metastatic melanoma of the extremities selectively destroys a tumor vascular network, but does not damage the rest of intact vessels. This effect is associated with the suppression induced by TNF and IFNγ, integranova αVβ3-function in endothelial cells in vitro and induction of apoptosis of endothelial cells in vivo (Ruegg and other, Nature Med 4, 408-414 (1998)). These results indicate that TNF in combination with additional therapies may be very clinically effective in the treatment of certain tumors, provided that can be controlled systemic toxicity.

The present invention describes that the molecules involved in angiogenesis, such as integrins, in modulating the activity of TNFαcan be used directly in the clinical use of TNFα as anti-cancer tool. Joint introduction of antiangiogenic funds with TNFαpreferably antagonists of integrin may selectively increase the sensitivity of receptors angiogenesis-related endothelial cells against apoptotic the banking activity of TNF, resulting in increased destruction of tumor vessels. In addition, this combination therapy may help to reduce the doses of TNF to reduce systemic side effects of TNF.

The invention

The present invention for the first time describes a new concept in the treatment of tumors, which provides for the introduction of individuals means that blocks or inhibits angiogenesis, together with TNFα, mutants of TNF or TNF-like molecules. Optional composition in accordance with this invention includes additional therapeutically active compounds, preferably selected from the group including cytotoxic tools, chemotherapeutic agents and inhibitors or antagonists of the receptor family of tyrosine kinase ErbB, such as described below in more detail. Thus, the invention relates to pharmaceutical compositions containing, preferably, antiangiogenic tools, integrin antagonists (receptor) and TNFα, mutant TNF or TNF-like molecules in therapeutically effective amounts. In particular, the invention relates to pharmaceutical compositions containing linear or cyclic RGD-peptides and TNFα not necessarily in conjunction with IFNγ. A preferred composition in accordance with the invention includes a cyclic peptide cyclo-(Arg-Gly-Asp-DPe-NMe-Val), TNFα and IFNγ. In accordance with the present invention these therapeutic active agents can also be represented in the form of a pharmaceutical kit comprising a package containing one or more antiangiogenic funds, TNFαand, optionally, one or more cytotoxic /chemotherapeutic agents/ anti-ErbB tools in a single package or in separate containers. The invention relates in particular to a combined therapy involving the application and introduction of, respectively, two or more molecules, where at least one molecule has inhibitory activity against angiogenesis and the other is a TNFα. In addition, the invention relates to combination therapy, involving the introduction of only one (merged) molecules with antiangiogenic activity and the activity of TNFαand, optionally together with one or more cytotoxic/chemotherapeutic agents. For example, a protein consisting essentially of cyclo-(Arg-Gly-Asp-DPhe-NMe-Val), which is directly attached to TNFαor through a linker molecule may be administered to the patient. Another example is the antibody to the integrin, such as LM609, as described below, which is attached to the end of its Fc-region to the TNFα. An additional example is bespecifically titulo, attached to TNFαin which one part is specific for the integrin receptor or VEGF receptor and the other part is specific for the EGF receptor.

In most cases, the introduction may be followed by radiation therapy, while radiation therapy can be carried out basically at the same time or before or after administration of the drug. The introduction of various tools in combination therapy in accordance with the invention can also be carried out basically at the same time or sequentially. Tumors that on the surfaces of their cells bear receptors involved in the development of blood vessels of a tumor can be successfully treated with combined therapy in accordance with the present invention.

It is known that tumors use alternative ways for its development and growth. If one path is blocked, they often have the ability to switch to another path by expression and other receptors and other transmission signals. Therefore, pharmaceutical compositions in accordance with the present invention can block several possible strategies for the development of the tumor and, therefore, to provide various advantages. The combination in accordance with the present invention are useful for the treatment and prevention of tumors, tumor-like violations is s, neoplasms and metastasis of tumors, which is described below in more detail. Preferably a combination of various means of the present invention are combined at low doses, these doses are lower than the doses commonly used in medicine. The advantage of lower doses of the compounds, compositions, tools, and therapies of the present invention that introduces individuals, is to reduce the number of adverse effects associated with administration of high doses. For example, at lower doses of chemotherapeutic drugs, such as methotrexate, there is a reduction in the frequency and degree of nausea and vomiting compared with the observed with the introduction of higher doses. By reducing the number of side effects improves the quality of life of cancer patients. An additional advantage of reducing the number of side effects is increased compliance sick regime and regimen, reducing the number of hospitalizations for treatment of side effects, and reduction of injection pain medications needed to treat pain associated with side effects. Alternatively, the methods and combinations of the present invention can also provide maximum therapeutic efficacy at the higher doses.

The combination in accordance with the invention reveal unexpected synergistic effective is. With the introduction of combination drugs is really on the decline and decay of tumors in clinical trials, while no significant adverse drug interactions.

In more detail, the invention relates to:

pharmaceutical compositions containing a therapeutically effective amount of at least (i) one-angiogenic agent and (ii) tumor necrosis factor alpha (TNFα) or a molecule having the biological activity of TNFαand, optionally together with a pharmaceutically acceptable carrier, excipient or diluent;

- corresponding pharmaceutical compositions, in which the specified angiogenic agent is an inhibitor/antagonist of integrin (receptor) or an inhibitor/antagonist of VEGF (receptor);

- corresponding pharmaceutical compositions, in which the specified inhibitor/antagonist of the integrin receptor is a linear or cyclic peptide, which contains the RGD;

- corresponding pharmaceutical compositions, in which the specified peptide, which contains the RGD is a cyclo-(Arg-Gly-Asp-DPhe-NMeVal);

- corresponding pharmaceutical compositions, in which the specified angiogenic agent is an antibody or its immunotherapeutics the active fragment, which binds to the integrin receptor or VEGF receptor;

- appropriate pharmaceutical composition, which is specified as an antiangiogenic agent and TNFα linked together into one merged molecules;

- corresponding pharmaceutical compositions, which optionally contains at least one cytotoxic and/or chemotherapeutic agent;

- appropriate pharmaceutical composition, which indicated the cytotoxic agent is a gamma-interferon (IFNγ) and/or other effective cytokine;

- corresponding pharmaceutical compositions, in which the specified chemotherapeutic compound selected from the group comprising cisplatin, doxorubicin, gemcitabine, docetaxel, paclitaxel (Taxol), bleomycin;

- corresponding pharmaceutical compositions, which optionally contains an inhibitor or antagonist of the receptor ErbB family of tyrosine kinase;

- corresponding pharmaceutical compositions, in which the specified inhibitor is an antibody to EGFR, antibody to HER2 or immunoturbidimetry fragment;

- pharmaceutical kit, which includes a package containing (i) at least one antiangiogenic agent, preferably an inhibitor/antagonist of the integrin receptor, (ii) TNFα and long the flax (iii) additional cytotoxic and/or chemotherapeutic agent;

- the corresponding preferred pharmaceutical kit containing

(i) cyclo(Arg-Gly-Asp-DPhe-NMeVal), (ii) TNFα and (iii) IFNγ and optionally (iii) an additional cytotoxic and/or chemotherapeutic agent and/or inhibitor or antagonist of the receptor ErbB family of tyrosine kinase;

- corresponding pharmaceutical kit which includes the pharmaceutically active funds are kept in separate containers in the specified packaging;

- the use of a pharmaceutical composition as described above and in the claims, for the preparation of a medicinal product or composition of the medicinal product for the treatment of tumors and metastases of tumors; and

- method of treating tumors or metastasis of tumors in animals, which provides for the introduction of specified individuals simultaneously or sequentially a therapeutically effective pharmaceutical compositions, as described above.

A detailed description of the invention

Unless otherwise specified, the terms and phrases used in this invention have the meanings and definitions stated below. In addition, these definitions and values describe the invention in more detail, including the preferred options for implementation.

"Biological molecule" includes a natural or synthetic molecule having, as a rule, the molecular weight of b is the larger than approximately 300, preferred are poly - and oligosaccharides, oligo - and polypeptides, proteins, peptides, poly - and oligonucleotides, as well as glycosylated derivatives of lipids. As a rule, biological molecules include immunotherapy, especially antibodies or their fragments, or functional derivatives of these antibodies or fragments, including fused proteins.

"Receptor" or "receptor molecule" is a soluble or membrane-associated/associated protein or glycoprotein that contains one or more domains, which bind ligands with the formation of the complex receptor-ligand. Upon binding with the ligand, which may be an agonist or antagonist, the activation or inactivation of the receptor and can be initiated or blocked path signal.

By "ligand" or "ligand-receptor" is meant a natural or synthetic compound which binds to the receptor molecule with the formation of the complex receptor-ligand. Under the concept of ligand fall agonists, antagonists, and compounds with partial agonistici/antagonistic action. In accordance with the characteristic features of this invention the term includes, first of all, TNF-like ligands.

The term "TNFα" as used in this invention includes, the EU is not specified, all types of TNF molecules and molecules having biological activity of TNFαincluding natural and synthetic, peptide or ones TNF mutants, variants or TNF-like ligands. Preferably the term includes natural peptide TNFα.

"Agonist" or "agonist receptor" is a natural or synthetic compound which binds to the receptor with the formation of a complex of receptor-agonist to activate the specified receptor and complex receptor agonist, respectively, initialized transmission signal, or any other biological process.

By "antagonist" or "antagonist of the receptor" refers to a natural or synthetic compound which has the opposite to the agonist biological activity. The antagonist binds to the receptor and blocks the activation of the receptor by the agonist by the competition with the agonist for the receptor. The antagonist is determined by its ability to block the action of the agonist. Receptor antagonist can be an antibody or immunotherapy effective fragment. Preferred antagonists in accordance with the present invention is shown and discussed below.

The term "therapeutically effective" or "therapeutically effective amount" refers to the quantity of the medicinal product, effective for the treatment of diseases or disorders in a mammal. In the case of malignant disease, a therapeutically effective quantity of a drug may decrease the number of cancer cells; reduce the tumor size; inhibit (e.g., to slow the spread and preferably stop) infiltration of cancer cells into peripheral organs; inhibit (e.g., to slow the spread and preferably stop) metastasis of tumors; inhibit, to slow, tumor growth; and/or reduce the spread of one or more symptoms associated with cancer. In this respect, the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. When cancer therapy effectiveness, for example, can be determined by measuring the time of disease progression (TTP) and/or measurement response (RR).

The concept of "effective immunotherapy" refers to the biological molecules that trigger an immune response in a mammal. More specifically, refers to molecules that can recognize and bind antigen. Typically, antibodies, antibody fragments and fused proteins, antibodies, which include their antigennegative participation is key (complementary determining areas CDRs), are immunotherapeutical.

The term "prodrug" as used in the present invention refers to a precursor or derivative form of the pharmaceutically active substance that is less cytotoxic against tumor cells compared to the original drug and are able to be activated under the influence of enzymes or to become more active in its original form (see, e.g., "Prodrugs in cancer chemotherapy", Biochemical Society Transactions, 14, str-382, 615 second meeting Belfast (1986)).

"Antiangiogenic agent" refers to a natural or synthetic compound that blocks or to some extent prevents the development of blood vessels. Antiangiogenic molecule may, for example, be a biologically active molecule that binds and blocks the angiogenic growth factor or growth factor receptor. Preferred angiogenic molecule when it binds to the receptor, preferably an integrin receptor or VEGF receptor. The concept in accordance with the invention also includes Prodrugs of the specified antiangiogenic tools.

There are many molecules that have different structure and original plot, which defines antiangiogenic properties. More preferred classes of funds, which inhibit the if block angiogenesis, which are suitable in accordance with the present invention, are, for example:

(i) antimitoticescoy tools, such as fluorouracil, mitomycin-C, Taxol;

(ii) the metabolites of estrogen, such as 2-methoxyestradiol;

(iii) inhibitors of matrix metalloproteinases (MMP), which inhibit canceallations (metalloprotease) (for example, batimastat, CC 16, TIMPs, minocycline, GM6001, or described in the "Inhibition of matrix metalloproteinases: therapeutic applications" (Golub, Yearbook of the new York Academy of Sciences, Toma; Grunewald, Zucker (ed), 1999);

(iv) a multifunctional antiangiogenic tools and factors such as IFNα (patents US 4,530,901; 4,503,035; 5,231,176); fragments of angiostatin and plasminogen (for example, kringle 1-4, kringle 5, kringle 1-3 (O′'reilly, M.S., and others, Cell (Cambridge, Mass.) 79(2): 315-328, 1994; Cao and others, J.Biol. Chem. 271: 29461-29467, 1996; Cao and others, J. Biol. Chem 272: 22924-22928, 1997); endostatin (O′Reilly, M.S. et al., Cell 88(2), 277, 1997 and WO 97/15666), thrombospondin (TSP-1; Frazier, 1991, Curr Opin Cell Biol 3(5):792); platelet factor 4 (PF4);

(v) inhibitors of the plasminogen activator/urokinase;

(vi) antagonists of the receptor for urokinase;

(vii) heparinase;

(viii) analogues fumagillin, such as TNP-470;

(ix) the tyrosine kinase inhibitors, such as SUI 01 (many of above - and below antagonist of ErbB receptor (antagonist of EGFR/HER2) are tyrosine kinase inhibitors, and m is able to demonstrate therefore, the blocking activity against EGF receptor, resulting in inhibition of tumor growth, as well as antiangiogenic activity, resulting in inhibition of the development of blood vessels and endothelial cells, respectively);

(x) suramin and analogues.;

(xi) angiostatic steroids;

(xii) antagonists of VEGF and bFGF;

(xiii) the VEGF receptor antagonists, such as antibodies to VEGF receptor (DC-101);

(xiv) flk-1 and flt-1 antagonists;

(xv) inhibitors of cyclooxygenase-11 such as COX-II;

(xvi) integrin antagonists and antagonists of the integrin receptor, such as αv antagonists and antagonists αv receptor, for example, antibodies to the receptor αv and RGD peptides. Preferred in accordance with the present invention are antagonists of the integrin (receptor).

The term "antagonists /inhibitors of integrin" or "antagonists/ inhibitors of integrin receptor" refers to a natural or synthetic molecule that blocks and inhibits the integrin receptor. In some cases, the term includes antagonists to the ligands of these receptors integrin (such as for α_vβ₃: vitronectin, fibrin, fibrinogen, von Willebrand factor, thrombospondin, laminin; α_vβ₅: vitronectin; α_vβ₁: fibronectin and vetrone the tin; for α_vβ₆: fibronectin).

Preferred in accordance with the invention are antagonists of the integrin receptors. The integrin antagonists (receptor) can be a natural or synthetic peptides, dipeptide, peptidomimetics, antibodies, such as antibodies or their functional fragments, or immunoconjugate (fused proteins). Preferred in accordance with the invention, inhibitors of integrin are those that are associated with receptor αv integrins (e.g. α_vβ₃that α_vβ₅that α_vβ₆and subclasses).

Preferred integrin inhibitors are α_vantagonists, and, in particular, α_vβ₃antagonists. Preferred α_vantagonists in accordance with the invention are RGD peptides, peptidomimetics (ones) antagonists and antibodies to integranova receptors, such as antibodies, blocking α_vthe receptors.

Typical non-immunological antagonists α_vβ₃described in research in patents US 5,753,230 and US 5,766,591. Preferred antagonists are linear and cyclic peptides that contain the RGD. Cyclic peptides are generally more stable and have increased peri the house half-life in serum. The most preferred integrin antagonists according to the invention, however, is cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (EMD 121974, Cilengitide®, Merck KgaA, Germany; EP 0770622), which is an effective blocker of receptor integrin α_vβ₃that α_vβ₁that α_vβ₆that α_vβ₅that α_IIbβ₃.

Suitable peptide antagonists, and coworkers peptide (ones) antagonists α_vβ₃/α_vβ₅/α_vβ₆receptor integrin described in the scientific and patent literature. For example, they are referred to Hoekstra and Poulter, 1998, Curr. Med. Chem. 5, 195; WO 95/32710; WO 95/37655; WO 97/01540; WO 97/37655; WO 97/45137; WO 97/41844; WO 98/08840; WO 98/18460; WO 98/18461; WO 98/25892; WO 98/31359; WO 98/30542; WO 99/15506; WO 99/15507; WO 99/31061; WO 00/06169; EP 0853084; EP 0854140; EP 0854145; US 5,780,426; and US 6,048,861. Patents that describe benzazepine and related benzodiazepines and benzocycloheptene, which are antagonists of the integrin receptor α_vβ₃that are also suitable for use in accordance with the present invention, include WO 96/00574, WO 96/00730, WO 96/06087, WO 96/26190, WO 97/24119, WO 97/24122, WO 97/24124, WO 98/15278, WO 99/05107, WO 99/06049, WO 99/15170, WO 99/15178, WO 97/34865, WO 97/01540, WO 98/30542, WO 99/11626 and WO 99/15508. Other antagonists of the integrin receptor, characterized by limited changes in the structure of the main to LCA, described in WO 98/08840; WO 99/30709; WO 99/30713; WO 99/31099; WO 00/09503; US 5,919,792; US 5,925,655; US 5,981,546; and US 6,017,926. In the US 6,048,861 and WO 00/72801 describes several derivatives Romanovyh acids, which are strong antagonists of the receptor integrin α_vβ₃. Other chemical small molecule antagonists of integrin (main antagonists of vitronectin) are described in WO 00/38665. Other antagonists of receptor α_vβ₃it is shown that they are effective inhibitors of angiogenesis. For example, a synthetic receptor antagonists, such as (S)-10,11-dihydro-3-[3-(pyridine-2-ylamino)-1-propyloxy]-5H-dibenzo[a, d]cycloheptene-10-acetic acid (known as SB-265123) were studied in different model systems in mammals. (Keenan and others, 1998, Bioorg. Med. Chem. Lett. 8(22), 3171; Ward and others, 1999, Drug Metab. Dispos. 27(11), 1232). Research to identify antagonists of integrin suitable for use as an antagonist, as described, for example, Smith and others, 1990, J.Biol. Chem. 265, 12267, and in the cited patent literature. Antibodies to the integrin receptor is also well known. Suitable monoclonal antibodies to the integrin (e.g., α_vβ₃that α_vβ₅that α_vβ₆) can be modified to improve their antigennegative fragment, F(ab)₂, Fab, and designed Fv or single-chain antibodies is. A suitable and preferably applied monoclonal antibody to the integrin receptor α_vβ₃was identified as LM609 (Brooks and others, 1994, Cell 79, 1157; ATS HB 9537). Effective specific antibody to α_vβ₅, P1F6, described in WO 97/45447, and it is also preferred in accordance with this invention. Also acceptable selective α_vβ₆the antibody is MAb 14D9.F8 (WO 99/37683, DSM ACC2331, Merck KGaA, Germany), and MAb 17.E6 (EP 0719859, DSM ACC2160, Merck KGaA), which are selective to α_v-chain of integrin receptors. Other suitable antibody to the integrin is commercially available Vitraxin®.

"Angiogenic growth factor or growth factor receptor" is a factor or a receptor that when activated, promotes growth and development of blood vessels. Typically, this group includes the growth factor vascular endothelial (VEGF) and its receptor.

The term "antibody" or "immunoglobulin" is used here in a broad sense and, in particular, refers to intact monoclonal antibodies, polyclonal antibodies, mnogomillionnym antibodies (for example, bespecifically antibodies)formed from at least two intact antibodies, and fragments of antibodies, provided that they are necessary for biological activity. As a rule, posetietljam heterodontidae, which consist of two or more antibodies or their fragments with different binding specificity, United together.

Depending on the amino acid sequences of their constant plots of intact antibodies can be assigned to a different class of antibodies (immunoglobulins)". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into "subclasses (isotypes), e.g., IgG1, lgG2, IgG3, lgG4, IgA, and lgA2. The constant domains of the heavy chain, which correspond to the different classes of antibodies are called α, δ, ε, γ and μ respectively. Preferably, the main class of antibody in accordance with the invention is an IgG, more IgG1 and lgG2.

Antibodies are glycoproteins, which have a molecular weight of approximately 150,000 and consist of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies in the heavy chains of antibodies of different isotypes. Each heavy and light chain also has regularly joined in space Mezhdunarodnye disulfide bridges. Each heavy chain has at one end a variable domain (VH)followed by several postenrichment. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. Constant domain of the light chain is combined with the first constant domain of the heavy chain variable domain and light chain combined with the variable domain of the heavy chain. I believe that specific amino acid residues form a surface between the variable domains of light and heavy chains. The "light chains" of antibodies from many vertebrate species can be assigned to one of two clearly distinct types, called Kappa (κ) and lambda (λ), on the basis of amino acid sequences of their constant domains.

The term "monoclonal antibody", as used here, refers to an antibody obtained from a population essentially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, which occur in natural conditions that may be present in a single quantity. Monoclonal antibodies are highly specific to a single antigenic site. In addition, unlike drugs polyclonal antibodies, which include different antibodies against different determinants (epitopes), each monoclonal antibody is associated with a separate determinate the antigen. In connection with their specificrecommendations antibodies have the advantage, as can be synthesized without contamination from other antibodies. Methods for obtaining monoclonal antibodies, including hybridoma method described by Kohler and Milstein (1975, Nature 256, 495) and in "Technology of monoclonal antibodies, fabrication and characterization of hybrid rodents and humans" (1985, Ed. by Burdon and others, Laboratory techniques in biochemistry and molecular biology, volume 13, scientific publishing house Elsevier, Amsterdam), or can be obtained using well known methods of recombinant DNA (see, for example, US patent 4,816,567). Monoclonal antibodies can also be isolated from libraries of phage antibodies using, for example, techniques described in Clackson and others, Nature, 352:624-628 (1991) and Marks etc., J. Mol. Biol., 222:58, 1-597 (1991).

The term "chimeric antibody" refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a specific antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they identify the necessary biological activity (for example: US patent 4,816,567; Morrisn and others, Proc. Nat. Acad. Sci. USA, 81:6851-6855 (1984)). Methods of obtaining chimeric and humanized antibodies are also known in the art. For example, such methods of obtaining chimeric antibodies are described in patents in the name of the Boss (Celltech) and Cabilly (Genentech) (patents US 4,816,397; US 4,816,567).

"Humanized antibodies" are a form of inhuman (e.g., rodents) chimeric antibodies, which include the minimal sequence derived from a nonhuman immunoglobulin. Mainly, humanized antibodies are human immunoglobulins (recipient antibody)in which hypervariable residues plot (CDRs) of the recipient are replaced remnants of the hypervariable area nonhuman species (donor antibody)such as mouse, rat, rabbit or nonhuman APE, which has the desired specificity, affinity and functional activity. In some cases, wireframe plot (FR) residues of the human immunoglobulin are replaced by corresponding residues of nonhuman origin. Furthermore, humanized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are carried out to facilitate further purification of antibodies. Basically, humanitariannet antibody can contain is usesto all at least one, and typically two, variable domain, in which all or substantially all of the hypervariable loops correspond to those of nonhuman immunoglobulin and all or substantially all of the FRs are those of a sequence of a human immunoglobulin. Humanitariannet antibody optionally also may contain at least part of the permanent plot (Fc) of an immunoglobulin, typically this part is part of a human immunoglobulin. Methods for obtaining humanized antibodies are described, for example. Winter (US 5,225,539) and Boss (Celltech, US 4,816,397).

The concept of "variable" or "FR" refers to the fact that certain parts of the variable domains differ considerably according to their sequence in antibodies and used for binding and specificity of each individual antibodies with his particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable sites variable domains in the light chain and heavy chain. The most highly conserved areas of variable domains are called the frame sections (FRs). The variable domains of the natural heavy and light chains each comprising four FRs (FR1-FR4), which is significant is Noah extent have β -folded configuration, connected by three hypervariable regions that form a binding loop, and in some cases partially form β-folded structure. Hypervariable sites in each chain are held together directly close using the FRs and, with the hypervariable sections of the other chain, contribute to the formation of antigennegative site of antibodies (see Kabat, etc., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but perform different effector functions, such as participation of the antibody in antibody-dependent cellular toxicity (ADCC).

The term "hypervariable area" or "CDR" when used in the invention, refers to the amino acid residues of an antibody which are responsible for binding to the antigen. Hypervariable area generally includes amino acid residues from a "complementary determining plot" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk . Mol. Biol. 196:901-917 (1987)).

Wireframe plot" or "FR" residues are those variable domain residues, which are residues from a hypervariable area, as defined in the present invention.

"Fragments of antibodies include a portion of an intact antibody, preferably containing antigennegative or variable plot. Examples of fragments of antibodies include Fab, Fab′, F(ab′)2, Fv and Fc fragments, ditella, linear antibodies, single-stranded molecule of the antibody; and mnogotarifnye antibodies formed from a fragment(s) of antibodies. "Intact" antibody is an antibody containing antigennegative variable section and a constant domain (CL) light chain and the constant domain of the heavy chain CN, CH2 and CH3. Preferably, inactive antibody is one or more effector functions.

The cleavage of antibodies with papain formed two identical antigenspecific fragment, called "Fab" fragments, each of them includes one antigennegative plot and CL and SN plot, and the remainder of the "Fc" fragment, whose name represents its ability to rapid crystallization.

"Fc" part of the antibody typically comprises CH2, CH3, and hinge area IgG1 or lgG2 the main class of the antibody. The hinge area is a group of approximately 15 aminokislotnykh residues, which connects SN plot with the CH2-CH3 regions.

When processing pepsin produces F(ab′)2 fragment that has two antigenspecific site and retains the ability to cross-linking antigen. "Fv" is the minimum antibody fragment which contains a complete plot of antigen recognition and binding site of the antigen. This site consists of a dimer of one heavy chain and one variable domain of the light chain, which are tightly ecovalence attached. In this configuration, three hypervariable segment (CDRs) of each variable domain interact with the formation antigennegative area on the surface of the VH-VL dimer. Together, the six hypervariable sections define antigennegative specificity of the antibodies. However, even a single variable domain (or half of an Fv, which includes only three hypervariable site specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity compared with the whole angiogenesis plot. Fab fragment also includes the constant domain of the light chain and the first constant domain (SN) the heavy chain. "Fab'fragments differ from Fab fragments by the addition of a few residues to the carboxyl end of the domain heavy chain SN, including one or more cysteines with loop is the first site of the antibody. F(ab′)2 fragments of antibodies does not necessarily form a pair of Fab fragments in the presence of cysteine in the loop between them. Other chemical binding fragments of antibodies are also known (see, for example, Hermanson, Technology bioconjugates. Academic Press, 1996; US 4,342,566). "Single-chain Fv" or "scFv fragments of antibodies include V, and V domains of antibodies, in which these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide includes polypeptide linker between the VH and VL domains which enables scFv to acquire the necessary for binding of the antigen structure. Single-chain FV antibodies are known, for example, from Plückthun The Pharmacology of Monoclonal Antibodies, vol 113, edited by Rosenburg and Moore, Springer-Verlag. New York. str-315 (1994)), WO 93/16185; US 5,571,894; US 5,587,458; Huston and others (1988, Proc. Natl. Acad. Sci. 85, 5879) or Skerra and Plueckthun (1988, Science 240,1038).

The concept of "detele" refers to small fragments of the antibodies with two antihistamine areas, these fragments comprise variable heavy domain (V) associated with variable light domain (V) in an identical polypeptide chains (V - V). Using a linker, which is very short for pair formation between the two domains are identical circuits, the domains form a pair with complementary domains of another chain and form two antigenspecific plot. More detail of ditel described, for example, EP 404,097; WO 93/11161.

The concept of "immunoconjugate" refers to an antibody or immunoglobulin, respectively, or its immunologically effective fragment, which is connected covalent bond with immunologically inactive molecule. Preferably this attached portion is a peptide or protein, which can be glycosylated. Specify the second molecule, which is not an antibody, can join With the end of the constant heavy chain antibody or to the N-to the ends of the variable light and/or heavy chains. Attached parts may be connected by means of a linker molecule, which is, as a rule, a polypeptide that includes 3-15 amino acid residues. Immunoconjugate in accordance with the invention preferably include fused proteins containing immunoglobulin or immunotherapy effective fragment of an angiogenic receptor, preferably to the integrin or the VEGF receptor and TNFα or a fused protein containing essentially TNFα and IFNγ or other acceptable cytokine that is attached to its N-end-to-end immunoglobulin, preferably in its Fc part.

The term "fused protein" refers to a natural or synthetic molecule that includes one or more immunotherapy is ineffective (not antibodies) proteins or peptides with different specificity, which are not necessarily linked together using a linker molecule. Fused protein according to the invention can be a molecule that consists of, for example, cyclo-(Arg-Gly-Asp-DPhe-NMeVal), is condensed with TNFα and/or IFNγ.

"Heterodontidae" represent two or more antibodies or binding FR is gment antibodies which are connected together, each of which has a different binding specificity. Heterodontidae can be obtained by combining the conjugation of two or more antibodies or fragments of antibodies. Preferably heterodontidae include cross-linked Fab/Fab' fragments. For the conjugation of antibodies can be used different binding or cross-linking agents. Examples include protein a, carbimide, M-Succinimidyl-3-acetyl-thioacetate (SATA) and N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Karpovsky and others (1984) J.EXP. Med. 160, 1686; Liu and others (1985) Proc. Natl. Acad. Sci. USA 82, 8648). Other similar methods described Paulus, Behring Inst. Mitt., №.78, 118 (1985); Brennan and others (1985) Science 30 m:81 or Glennie and others (1987) J. Immunol. 139, 2367. Another way is to use α-phenylenedimaleimide (αPDM) to link the three Fab' fragments (WO 91/03493). Mnogotarifnye antibodies in the context of this invention are also acceptable and can be obtained, for example, in accordance with the study WO 94/13804 and WO 98/50431.

"Effector functions" antibodies refer to such biological activities, which are determined by the Fc site (natural sequence Fc plot or the amino acid sequence of various Fc plots) antibodies. Examples of effector functions of antibodies include cytotoxicity-dependent complement, binding of Fc with what eception, antibody-dependent cytotoxicity mediated by cells (ADCC); phagocytosis; inhibitory regulation of receptors on the cell surface (for example, receptor β-cells), etc.

The concept of "ADCC" (antibody-dependent cytotoxicity mediated by cells) refers to the mediated cell reaction in which nonspecific cytotoxic cells that Express Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils and macrophages), recognize bound antibody on the target cell and subsequently cause lysis of the target cells. Primary cells mediating ADCC, NK cells, Express only FcγRIII, whereas monocytes Express FcγRI, FcγRII and FcγRIII. To assess ADCC activity of a molecule of interest, can be applied study ADCC in vitro, such as described in the prior art (patents US 5,500,362; US 5,821,337). Suitable effector cells for such studies include the mononuclear cells of peripheral blood (RVMS) and natural killer (NK) cells.

"Effector cells" are leukocytes which Express one or more FcRs and perform effector functions. Preferably, the cells Express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC, are m nonnuclear cells of peripheral blood (RVMS), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.

The concept of "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc site antibodies. Preferred FcR is the natural sequence human FcR. Moreover, a preferred FcR is such that binds IgG antibody (a gamma receptor) and includes receptors of the subclasses FcγRI, FcγRII, and FcγRIII, including allelic variants and alternative strengthened forms of these receptors. Reviews regarding FcRs see, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).

The term "cytokine" is a generic term for proteins, which are released one population of cells and act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and normal polypeptide hormones. The cytokines are growth hormones such as human growth hormone, N-nationally human growth hormone and growth hormone in cattle; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prolactin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); a growth factor for hepatocytes; fibroblast growth factor; prolactin; lactogenic p is acenti; mouse gonadotropin-associated peptide; inhibin; activin; growth factor vascular endothelial (VEGF); integrin; thrombopoietin (TPO); nerve growth factors such as NGFβ; platelet growth factor; transforming growth factors (TGFs)such as TGFα and TGFβ; erythropoietin (EPO), interferons such as IFNα, IFNβ and IFNγ; colony stimulating factors such as M-CSP, GM-CSF and G-CSF; interleukins, such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; TNFα or TNFβ. Preferred cytokines in accordance with the invention are interferons and TNFα.

The term "cytotoxic agent"as used here, refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The concept also includes radioactive isotopes, chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal organisms or fragments thereof. The concept may also include representatives of the family of cytokines, preferably IFNγ.

The term "chemotherapeutic agent" or "anti-cancer agent" includes chemicals that have anti-tumor action, for example, prevent the development, maturation or proliferation of tumor cells, directly acting on the entrances of the tumor, for example, by cytostatic or cytotoxic actions, and indirectly by acting through mechanisms such as the modification of the biological response. Acceptable chemotherapeutic agents in accordance with the invention preferably represent a natural or synthetic chemical compounds, but also biological molecules such as proteins, polypeptides, etc. There are many anticancer agents that are commercially available, are investigated in the clinical setting and in clinical conditions that may be included in this invention for the treatment of tumors/neoplasms in combination therapy with TNFα and antiangiogenic means, as indicated above, optionally with other means, such as antagonists of EGF receptor. You should pay attention to the fact that chemotherapeutic agents can be optionally together with the above combination medicines. Examples of chemotherapeutic agents are alkylating agents such as nitrogen mustards, ethyleneimine compounds, alkyl sulphonates and other compounds with an alkylating action, such as nitrosamine, cisplatin and dacarbazine; antimetabolites, such as folic acid antagonists of purine or pyrimidine; inhibitors of mitosis, for example, al is alaidy periwinkle and derivatives podofillotoksina; cytotoxic antibiotics and derivatives camptothecin. Preferred chemotherapeutic agents or chemotherapy include amifostine (ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine HCl (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustin (CCNU), doxorubicin (adriamycin), lipotoxicity (doxil), gemcitabine (Gemzar), daunorubicin, lepidonotothen (daunoxome), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (Taxol), docetaxel (Taxotere), aldeslakin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11,10-hydroxy-7-ethyl-camptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydrosilation, ifosfamide, idarubitsin, mesna, alpha-interferon, beta-interferon, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, tioguanin, thiotepa, 5-[bis-(2-chloroethyl)amino]uracil, vinorelbine, chlorambucil, and combinations thereof.

The most preferred chemotherapeutic means in accordance with the invention are cisplatin, gemcitabine, doxorubicin, paclitaxel (Taxol) and bleomycin.

The concept of "cancer" and "tumor" refer or about icyhot physiological condition in mammals, which is typically characterized by unregulated cell growth. Using the pharmaceutical compositions in accordance with the present invention can prevent the development of tumors may be treated tumors in the mammary gland, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovarian, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, cervix, and liver. More specific tumor selected from the group including adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hamartoma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma. In more detail, the tumor is selected from the group including gralow lentiginous melanoma, senile keratoses, adenocarcinoma, adenomatosnuu carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, tumor astrocytes, carcinoma of the Bartholin glands, basal cell carcinoma, alveolae cell carcinoma, capillary, carcinoid tumor, carcinoma, carcinosarcoma, cavernous, cholangio-carcinoma, chondrosarcoma, papilloma/sarcoma chorioidea plexus, renal cell R is K, cystadenoma, tumor endodermally cavity, endometrial hyperplasia, endometrial sarcoma, adenocarcinoma of the endometrium, ependymal, epithelioid, abnormal Ewing sarcoma, fibroblastic, focal nodular hyperplasia, ulcerogenic adenoma of the pancreas, tumors of germ cells, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, adenoma liver adenomatosis of the liver, hepatic cell carcinoma, insulinoma, intraepithelial the neoplasia, brainwas ion this form may cell neoplasia, invasive this form may cell carcinoma, both carcinoma, leiomyosarcoma, limited malignant melanoma, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial, metastatic carcinoma, slizeobrazujushchej squamous cell carcinoma, neuroblastoma, neuroepithelial adenocarcinoma nodular melanoma, asanovic-cell carcinoma, oligodendroglial, osteosarcoma, pancreatic polypeptide, papillary serous adenocarcinoma, pinealocyte, pituitary tumors, plasmacytoma, false sarcoma, blastoma lungs, kidney cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell lung cancer, magadalene carcinoma, a tumor secreting somat is a statin, squamous carcinoma, squamous cell carcinoma, submesothelial, superficial spreading melanoma, undifferentiated carcinoma, uveal melanoma, papillary carcinoma, vipoma, well-differentiated carcinoma and adenosarcoma kidney (Wilms tumor).

"ErbB receptor" is a receptor protein tyrosine kinase which belongs to the family of ErbB receptors and includes receptors of the EGFR(ErbB1), ErbB2, ErbB3 and ErbB4, and other representatives of this family, which will be identified in the future. The ErbB receptor in most cases can include the extracellular domain that can bind an ErbB ligand; lipophilic transmembrane domain; conservative intracellular tyrosine kinase domain; and carboxykinase signal domain, which contains several tyrosine residues that can be phosphorylated. The ErbB receptor may represent a "natural sequence" ErbB receptor or variant amino-acid sequence. Preferably the ErbB receptor is a natural sequence of the receptor ErbB person. ErbB1 refers to genes coding for the protein product of the EGFR. More preferred is the EGF receptor (HER1). The expression "ErbB1" and "HER1" are used interchangeably in the present invention and belong to a protein HER1 person. The expression "ErbB2" and "HER2" cable is replaceable used in the present invention and belong to the protein HER2 person. Preferred in accordance with this invention are ErbB1 receptors (EGFR).

"The ErbB ligand is a polypeptide that binds to and/or activates ErbB receptor. ErbB ligands that bind EGFR include EGF, TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin.

The term "antagonist/inhibitor of ErbB receptor" refers to a natural or synthetic molecule, which binds and blocks or inhibits ErbB receptor. Thus, by blocking receptor antagonist inhibits the binding ErbB ligand (agonist) and the activated complex of agonist/ligand with the receptor. ErbB antagonists can be to HER1 (EGFR or HER2. Preferred antagonists of the invention are antagonists to the EGF receptor (EGFR, HER1). Antagonist of ErbB receptor can be an antibody or immunotherapy effective fragment or nekanoniceskie molecule such as a peptide, polypeptide protein. Also included are chemical molecules, however, preferred antagonists in accordance with the invention are antibodies to EGFR and antibodies to HER2. Preferred antibodies according to the invention are antibodies to Neg and antibodies to New, more preferably antibodies to Herl. Preferred antibodies to Her1 represent MAb 425, preferably humanitariannet MAb 425 (hMAb 425, S 5,558,864; EP 0531472) and chimeric MAb 225 (cMAb 225, US 4,943,533 and EP 0359 282). Most preferred is a monoclonal antibody h425, which has a high efficiency with drug monotherapy in combination with reduced undesirable side effects. The most preferred antibody to HER2 HERCEPTIN is®manufactured by Genentech/Roche. Effective antagonists of EGF receptor in accordance with the invention can also include natural or synthetic chemical compounds. As examples of the preferred molecules of this type can lead organic compounds, ORGANOMETALLIC compounds, salts of organic and ORGANOMETALLIC compounds.

Examples of antagonists HER2 receptor are heteroaryl compounds substituted by sterila (US 5,656,655); bis - mono-and/or bicyclic aryl heteroaryl, carbocyclic and heterocorallia connection (US 5,646,153); tricyclic pyrimidine compounds (US 5,679,683); derivatives of hintline possessing inhibitory activity against receptor tyrosine kinase (US 5,616,582); heteroarylboronic or heteroarylboronic connection (US 5,196,446); the connection, designated as 6-(2,6-dichlorophenyl)-2-(4-(2-diethyl-aminoethoxy) phenylamino)-8-methyl-8H-pyrido(2,3)-5-pyrimidine-7-he (Panek and others, 1997, J.Pharmacol. Exp. Therap. 283,1433) inhibiting family is eception EGFR, DERIVED and FGFR.

"Radiation therapy": Tumors that can be treated by the pharmaceutical compositions in accordance with the invention, may optionally be subjected to treatment with radiation or radioactive drugs. The radiation source can be external or internal to the patient, which is treatable. If the source is external to the patient, it is a treatment known as external beam radiotherapy (EBRT). If the radiation source is internal to the patient, such a treatment called brachytherapy (BT). Some typical radioactive atoms, which may be used include radium, cesium-137, and iridium-192, americium-241 and gold-198, cobalt-57; copper-67; technetium-99; iodide-123; iodide-131; and indium-111. You can also mark means in accordance with the invention with radioactive isotopes. Today, radiation therapy is a standard treatment for control neudalimye or inoperable tumors and/or metastasis of tumors. Improved results are observed when the combination of radiotherapy with chemotherapy. Radiation therapy is based on the principle that radiation in high doses in radiation areas causes death reproductive cells into the tumor and normal tissues. The resulting radiation dose is determined largely on the basis of the absorbed dose (rad), time and fractionation, and can be determined by the oncologist. The amount of ionizing radiation received by a patient depends on a variety of reasons, but the two most important considerations are the localization of the tumor in relation to other critical structures or organs of the body, and the extent of tumor spread. The preferred course of treatment for a patient who undergoes radiation therapy, is the treatment for a period of 5-6 weeks to a total dose of 50-60 Gy, which the patient is irradiated in the form of single daily fractions of 1.8-2.0 Gy, 5 days a week. Gr represents a reduction from gray and refers to a dose of 100 rad. In a preferred embodiment, the invention provides the joint effects when tumors in humans are treated with antagonist of angiogenesis and TNFα/IFNγ and irradiation. In other words, the inhibition of tumor growth when exposed to these compounds is enhanced in combination with radiation and/or chemotherapeutic agents. Radiation therapy may not be applied in accordance with the invention. It is recommended, and is preferred in cases in which the patient cannot be put sufficient funds in accordance with the invention.

"Pharmaceutical treatment: the Method according to the invention includes the various effects of a practical embodiment of the invention in stages. For example, the funds in accordance with the invention can be administered simultaneously, sequentially or separately. In addition, funds may be entered separately in the time span of about 3 weeks between injections, for example, mainly from the introduction immediately after the introduction of the first means until approximately 3 weeks after the introduction of the first means. The method can be carried out after the surgical intervention. Alternative surgical intervention can be performed during the interval between introduction of the first active means and the second active means. An example of this method is the combination of this method with surgical removal of the tumor. Treatment in accordance with the method generally includes the introduction of therapeutic compositions by one or more cycles of administration. For example, if the simultaneous introduction of a therapeutic composition that includes both tools, entered during the period from about 2 days to about 3 weeks per cycle. After that, the treatment cycle may be repeated, if necessary, considering the condition of the patient. Similarly, if there is a successive introduction, the introduction of each individual therapeutic substances is installed on osnovannogo period of time. The interval between cycles can vary from 0 up to 2 months.

The means according to the present invention can be administered parenterally by injection or by gradual infusion over a period of time. Despite the fact that the tissue undergoing treatment can usually be achieved by a system for the body of the introduction, and moreover, the most commonly used to treat intravenous administration of therapeutic drugs, other tissues and delivery are also considered, when the probability that the target tissue containing target molecules. Thus, the tools of the present invention can be vnutriglazna, intravenously, intraperitoneally, intramuscularly, subcutaneously, vnutripolostno, percutaneous, by orthotopic injection or infusion, and can also be delivered via peristalsis. Therapeutic compositions contain, for example, the integrin antagonist according to this invention, and is usually administered intravenously, for example, in the form of injection with a single dose. Therapeutic compositions of the present invention contain a physiologically tolerated carrier together with an appropriate tool, as described here, are dissolved or dispergirovany in it as an active ingredient. As used in this invention, the term "pharmaceutically acceptable" refers to compositions, carriers, rest is ritilan and reagents, which are substances that allow the administration to a mammal without undesirable physiological effects such as nausea, dizziness, gastric upset and the like. The preparation of a pharmaceutical composition that contains active ingredients dissolved or dispergirovannom, it is clear in the art and should not be limited on the basis of the composition. Typically such compositions are prepared in the form of injectable, liquid solutions or suspensions, however, solid forms suitable for solutions or suspensions may also be prepared in the form of a solution before use. The compositions can also be emulsified. The active ingredient may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in therapeutic methods described herein. Suitable fillers are, for example, water, saline, dextrose, glycerol, ethanol and the like and combinations thereof. In addition, if necessary, the composition may include minor amounts of auxiliary substances such as wetting or emulsifying agents, buffering agents and the like which enhance the effectiveness of the active ingredient. therapeutic composition is according to the present invention can include pharmaceutically acceptable salts of their components. Pharmaceutically acceptable salts include acid additive salts (formed with free amino groups of the polypeptide), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic acids as acetic, tartaric, almond and such. Salts formed free carboxyl group, can also be derived inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like, More preferred are salts with HCl, if the composition of the applied cyclic polypeptide antagonists αv. Physiologically tolerant carriers are well known in the art. Examples of liquid carriers are sterile aqueous solutions that do not contain other substances in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate buffer and saline. In addition, water carriers may include more than one buffer salt, as well as salts such as the chlorides of sodium and potassium, dextrose, polyethylene glycol and other dissolved substances. The Jew is their composition can also include a liquid phase in addition to and excluding water. Examples of such additional liquid phases are glycerin, vegetable oils, such as cottonseed oil, and oil-water emulsion.

Typically a therapeutically effective amount of immunotherapy, such as antibodies, blocking the integrin receptor or antibody fragment or conjugate antibodies or antibodies that block VEGF receptor, fragment or conjugate represents a number, which when introduced into physiologically tolerate composition is sufficient to achieve a plasma concentration of from about 0.01 microgram (μg) per milliliter (ml) to about 100 μg/ml, preferably from about 1 μg/ml to about 5 μg/ml, and usually about 5 μg/ml, the Dosage may be assigned differently from approximately 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, more preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more daily doses for one or several days. If immunotherapy tool is presented in the form of a fragment of a monoclonal antibody or conjugate, the amount can easily be determined based on the weight of the fragment or conjugate, which corresponds to the weight of the whole antibody. Preferably the I molar concentration in plasma is approximately 2 micromol (μm) to about 5 mmol (mm) and preferably from about 100 μm to 1 mm antagonist antibodies. A therapeutically effective amount in accordance with the present invention, which is Neimanis therapeutic peptide or proteiny polypeptide (e.g., TNFα, IFNγ) or other biological molecule with similar sizes, usually represents a number of polypeptide, which when introduced into physiologically tolerate composition is sufficient to achieve a plasma concentration from about 0.1 microgram (ug) per milliliter (ml) to about 200 μg/ml, preferably from about 1 μg/ml to about 150 μg/ml On the basis of the polypeptide having the weight approximately 500 grams per mole, the preferred molar concentration in plasma is approximately 2 micromol (μm) to about 5 mmol (mm) and preferably about 100 μm - 1 mm antagonist polypeptide. The usual dosage of active agent that is preferably a chemical antagonist or (chemical) chemotherapeutic agent in accordance with the invention (any of the immunotherapy agent, nor deimante.samaviciute peptide/protein) is a 10-1000 mg, preferably about 20-200 mg, and more preferably 50-100 mg per kilogram of body weight per day.

The pharmaceutical compositions according to the invention can is activated and treatment of individuals means, that reduce or give the possibility to avoid the side effects associated with combination therapy in accordance with the present invention (the"supplemental therapy"), including, but not limited to, such means, for example, which reduces the toxic effect of anticancer drugs, such as inhibitors of bone resorption, the cardioprotective funds. These additional means to prevent or reduce the degree of nausea and vomiting caused by chemotherapy, radiotherapy or surgery, or reduce the percentage of infections associated with the introduction of mielosupressivne anti-cancer drugs. Additional means are well known in the art.

Immunotherapy in accordance with the invention can optionally be administered with auxiliary facilities, similar to the BCG and stimulants of the immune system. In addition, the composition may include immunotherapy or chemotherapy agents containing cytotoxic effective radioactively labeled isotopes, or other cytotoxic agents such as cytotoxic peptides (such as cytokines or cytotoxic drug and the like. The term "pharmaceutical kit for treating tumors or metastasis of a tumor refers to the UPA the information, and, as a rule, the instructions for use of the reagents in the treatment of tumors and metastasis of tumors. The reagent kit according to the invention generally presents a therapeutic composition as described in the invention, and therefore, can be represented in a variety of forms suitable for distribution in the set. Such forms may include liquid, powder, tablets, suspensions and similar compositions for the intended antagonist and/or the fused protein according to this invention. The reagents can be placed in separate containers, suitable for separate introduction in accordance with these methods, or alternatively may be located in the composition in a single container in the package. Packaging may include a sufficient quantity for one or more dosages of Regents in accordance with the methods of treatment described herein. Set according to this invention also includes an "instructions for use" substances contained in the package.

Description of figures

Figure 1. The formation of HUVEC spheroids and survival does not require binding of the integrin. (a) Blocking anti-VE-cadherin (75) mAb or CA²⁺-decrease (EDTA, EDTA/Ca²⁺) inhibits formation of HUVEC spheroids, while blocking mAbs against integrin α₁(Lia1/2), α₅(SAM-1), α_Vβ₃

Figure 2. Integratability adhesion protects HUVEC from TNF-induced apoptosis. (a) YoPro-1 absorption: exposure to TNF (T) and TNF/IFNγ (T1) does not cause YoPro-1 staining in related fibronectin HUVEC, while causes severe YoPro-1 staining in HUVEC spheroids that is suppressed by caspase inhibitors BOG and ZVAD. TNF±IFNγ (T1). C, untreated culture. (b) Demonstration of the activation of caspase-3 and release of PARP (arrow) using Western blotting in the treated TNE/IFNα (T1) spheroids, but not associated with fibronectin HUVEC. C, untreated cultures (C, d) Curves viability of HUVEC, subjects the effects of TNFTNF/IFNγor control sera (About). (e) the Viability of HUVEC, cultured on immobilized antibodies (imAbs) to α1α_vβ₃and α4 integrins (O/I) in the absence (empty symbols) or presence (filled symbols) TNF/IFNγ.

Figure 3. Induced TNF activation of NF-κdoes not require binding integrin (a) Western blotting and (b) analysis of changes in electrophoretic mobility (EMSA) demonstrated couples who lelou the kinetics of I-KB phosphorylation (Piκ In), I-κdegradation (I-κ) and NF-κnuclear translocation (EMSA) associated with fibronectin HUVEC and spheroids exposed to TNF/IFNγ. (C) Flowing cytometrics analysis indicates identical induction of expression of ICAM-1 surfaces of cells on fibronectin and cultures of HUVEC spheroids, subjects the effects of TNF (----) or TNF/IFNγ―). (...) raw cells.

Expression of the RESOURCES-1 is shown as a control.

Figure 4. Activation of Akt is essential for the survival of HUVEC and requires binding integrin. (a) Detection of phosphorylated (Pi-Akt) and total Akt (Akt) associated with fibronectin and HUVEC spheroid cultures stimulated TNF/IFNγ during this time. (b) Left panel: inhibitors of PI-3 kinase wortmannin (W) and LY294002 (LY) increase the sensitivity associated with fibronectin to HUVEC TNF (T) and TNF/IFNγ(T1)-induced apoptosis. Dead cells were visualized by staining YoPro-1. Right panel: survival curves associated with fibronectin HUVEC exposed to LY294002TNF/IFNγ (Δ), or LY294002/TNF/IFNγ. (A) untreated culture. (C) a Significant activity of PI-3 kinase (R*) and Akt (Aktmp), but not wild-type Akt (Aktwt) or control plasmid (pBS), contributed to the survival of spheroids, subjects the effects of TNF or TNF/IFN (). (A) untreated culture. Inserts are running cytometrics analysis of EGFP fluorescence transfection cells (% positive cells), (d) HUVEC elektroporcelany with a control plasmid (pBS) or constitutive active Akt (Akt*) and infected with AdΔNIκB or AdLacZ, cultivated as associated with fibronectin monolayer or spheroids in the absence () or presence of TNF (T) or TNF/IFNγ (T1). Apoptotic cells were identified by staining YoPro-1. Viable associated with fibronectin cells were stained crystal violet, (e) HUVEC were electroporative with a control plasmid (empty symbols) or pAktmp (filled symbols) and infected AdANI-κor AdLacZand were cultured on fibronectin in the presence of different concentrations of TNF and living related cells was determined by measuring optical density (OP) colored crystal violet holes. (f) were Analyzed by flow cytometry the expression of ICAM-1 in untreated HUVEC (...), or HUVEC treated with TNF (-----) and TNF/LY294002 (―) (left panel), as well as HUVEC infected with AdΔNI-kV (middle panel) or AdLacZ and exposed to TNF (------) and TNF/IFNγ (―).

Figure 5. (a-C) Analysis using Western nl is tting Pi-Akt, MDM2, p53, Pi-FKHR/FRKHL1 (a), and Pi-MEK, Pi-p38 and Pi-JNK and Pi-ERK in fibronectin and HUVEC spheroid cultures exposed to TNF/IFNγ during this time. The amount of Akt, FKHR, MEK, p38, ERK and JNK protein shows about the same amount of total protein. Spheroid cultures are characterized by the lack of phosphorylation of Akt and FKHR/FKRL1, elevated levels of p53 and enhanced phosphorylation of MEK, p38, ERK and JNK in response to TNF/IFNγ compared with associated with fibronectin cells.

Figure 6. The decrease in binding integrin enhances the cytotoxicity of TNF in vitro. (a) HUVEC were cultured on fibronectin or PLL for 16 hours in the absence () or presence of TNF (T) or TNF/IFNγ (T1). Apoptosis and survival of adhered cells was determined by staining with YoPro-1 and crystal violet, respectively. (b) EMD121974 destroyed α_Vβ₃-mediated HUVEC adhesion to gelatinbut not α5β1 component α₅β₁/α_Vβ₃-mediated adhesion to fibronectin. The control peptide EMD135981 was ineffective (unfilled symbols). (C) HUVEC were cultured on fibronectin in the absence () or presence of TNF/IFNγ (T1), and EMD121974 EMD135981, as indicated. Cells undergoing apoptosis, and adhered cells was determined PU is eating staining with YoPro-1 and contrast microscopy, respectively. (d) survival Curves HUVBC experiment presented in panel C. Without peptide; EMD121974; EMD135981. Raw culture - unfilled symbols. Cultures treated with TNF/IFNγ, - shaded characters. (e) survival Curves HUVEC, elektroborudovaniya with Aktmp (empty symbols) or pBS (filled symbols), and cultured on fibronectin and obrabotannykh TNF/IFN γ withoutor in the presence of peptides EMD121974or EMD135981. Aktmp prevents cell death caused by the combined effects of EMD121974 and TNF/IFN.

Figure 7. The decrease in binding integrin enhances the cytotoxicity of TNF in vivo. BN rats, which have BN-175 isogenic soft tissue sarcoma were treated EMD121974, TNFor EMD121974/TNFusing ILP techniques. (O) falsely treated rats. Tumor growth was determined for 6 days after ILP. The results are presented as values of tumor volume ± SCO (n=6). Small fragments of isogenic soft tissue sarcoma BN-175 implanted in the right hind paw of male BN rats, and treatment was started after reaching the tumor sizes in diameter 12-14 mm (Manusama and others, Oncol. Rep.6, 13-177. (1999)). Then the femoral artery and vein were canliterally elastically tube and collaterals was tied with a rope. Perfusion was carried out for 30 min with 5 ml Heamaccel® (2.4 ml/min), under which the medicinal product was administered as bolus (EMD121974, 500 mcg, the final concentration in the perfusion solution 170 ΜM; TNF, 50 mcg). The perfusion solution was saturated with oxygen and a leg kept at 38-39°in a warm mattress. Rats, which were injected EMD121974 using perfusion, also systemically injected peptide 2 hours before and 3 hours after ILP (100 mg/kg intraperitoneally). Tumor diameter was measured in two directions with a compass and tumor volume (V) was calculated by the formula (V=0,4)(A²XB, where a represents the maximum diameter and a is the diameter perpendicular to it). The group was treated with 6 rats. Local and General side effects were assessed as described (Manusama and others, Oncol. Rep.6, 173-177. (1999)).

Figure 8. The decrease in binding integrin enhances cytotoxicity induced TNF, TRAIL and FasL in vitro. HUVEC were cultured over night at covered with fibronectin microtiter tablets in the absence (control) or in the presence of EMD121974 (300 μm), TNF (200 ng/ml), FasL (200 ng/ml), TRAIL (200 ng/ml), LIGHT (200 ng/ml) and IFN γ (330 ng/ml)as indicated. Viability was determined using the MST analysis.

In more detail, the invention can be described using the following example is in:

Example 1: Integratability adhesion of endothelial cells against TNFα-induced apoptosis.

HUVEC formation of spheroids and survival does not require integrins

To study the effect of binding integrin on TNF-induced apoptosis, we determined the conditions under which endothelial cells can be cultured without integrisanjem adhesion. Suspension of single endothelial cells quickly die due to anoikis (apoptosis-induced separation of cells) (Meredith and others, Mol. Biol Cell. 4, 953-961 (1993)), which is an obstacle for further analysis. However, the seeding of endothelial cells of the umbilical vein of a person (HUVEC) at high density (1,0×106 cells/ml) in wells coated with BSA, the formation of multicellular spheroids within 2-4 hours and may persist for 24 hours depending on the VE-cadherin and independently of any detectable influence from integrins. Inhibition of VE-catherinebell activity by blocking monoclonal antibody (mAb), or by decreasing the CA-Mg²⁺using EDTA blocks the formation of spheroids, whereas inhibitory mAbs in relation to α₂that α₃that α₅that α₆that β₁that α_Vβ₃or α_Vβ₅the integrins, blocking peptides based on the RGD and blocking against the REPUBLIC of THE M-1 mAb, alone or in combination, do not affect HUVEC spheroids (Figa, and data not shown).

To determine the effect of spheroid culture on cell viability spheroids and associated with fibronectin HUVEC were collected in the interval between 6 and 72 hours of culture growth on the tablet, serially diluted and were further cultured for 48 hours before determined the relative number of cells. The shift to the left or smoothing dissolution curve shows the reduction of viability. After 6, 12, 16 and 24 hours after cultivation on the tablet, the viability of HUVEC recovered from spheroid cultures was comparable with such associated fibronectin cultures, but after 36 hours it progressively decreased (Fig.1b after 16 hours, and data not shown). Taken together, these results indicate that HUVEC can form spheroids, which are viable for 24 hours in the absence integrisanjem adhesion.

Example 2: Adhesion to fibronectin protects HUVEC from TNF-induced apoptosis.

To explore the fact that modulate whether integrins TNF-mediated apoptosis, we cultured HUVEC on fibronectin (integratability adhesion) or as spheroids (intevenezuela adhesion) in the absence or in the presence of TNF (200 ng/ml) and IFNγ (ng/ml), amplifier cytotoxicity of TNF (Dealtry and others, Eur. J. Immunol. 17, 689-693 (1987)). The effects of TNF±IFNγ HUVEC monolayers on fibronectin ("fibronectine HUVEC") does not cause increase of apoptosis, as evidenced by the absence of absorption YoPro-1 (Idziorek, etc., J. Immunol. Methods 185, 249-258 (1995)), the binding surfaces of cells annexin V, activation of caspase-3 and degradation of its substrate PARP (Figa, 2b and data not shown). In contrast, treatment of spheroids TNF±IFNγ causes increased absorption of YoPro-1 (the gain is suppressed by caspase inhibitors BOG, Z-VAD, IETD and DVED), DNA fragmentation, activation of caspase-3 and decomposition of PARP (Figa, 2b and data not shown). To explore the impact of TNF±IFNγ on the survival of cells we determined the viability of untreated and treated cultures. The action of TNF±IFNγ fibronectine HUVEC did not affect cell viability (Figs). Processing spheroids TNF caused the death of more than 80% of the cells and the combined treatment of TNF/IFNγ caused the death of all cells (Fig.2d). Processing only one IFNγ was not cytotoxic (data not shown). HUVEC stick to immobilizovannogo to fibronectin using α_Vβ₃and α₅β₁integrins (Rüegg and other, Nature Med. 4, 408-414 (1998)). To examine the individual contribution of these integrins in the survival of cells on fibronectin we cool what was rivervale HUVEC on immobilized on plastic mAbs (imAbs) to α Vβ3, α1, α5 and α4 integrins. Immobilized anti-αvβ3, anti-α5 and antiα1 mAbs protects HUVEC from TNF-induced death, while anti-α4 mAbs such actions do not detect (File and data not shown). Based on these results, we concluded that αVβ3 and αVβ1 integrin-mediated adhesion suppresses TNF-induced apoptosis, and in its absence increases the sensitivity to HUVEC TNF and caspase-mediated apopse.

Example 2: Integratability signaling protects endothelial cells from TNFα-induced apoptosis.

TNF activation of NF-κdoes not require binding integrin

Activation of nuclear factor-κB (NF-κC) promotes the survival of cells exposed to TNF (Beg & Baltimore, Science 274, 782-784; Van Antwerp and others, Science 274, 787-789 (1996)). As the adhesion of cells through integrins activates NF-κ (Scatena and others, J, Cell Biol. 141, 1083-1093 (1998)), we studied whether the sensitivity of the spheroids, the induction of TNF apoptosis due to the lack of activation of NF-κB. Activation of NF-κwas estimated by measuring I-κIn phosphorylation and degradation, NF-κnuclear translocation and expression of the surfaces of cells ICAM-1, NF-κ-inducible gene (Collins and others, Faseb J. 9, 899-909. (1995)), in spheroids and fibronectine HUVEC cultures subjects effects of TNF± IFNγ. We did not observe significant differences in I-κIn the phosphorylation and degradation, NF-κnuclear translocation or the expression of ICAM-1 (Figa-C), suggesting that TNF-iduciary apoptosis in cultures of HUVEC spheroids is not associated with impaired activation of NF-κC.

Example 3: Activation due to the binding of integrin and is essential for survival of cells.

Then analyzed the activation of Akt/RKV, activating kinase proteins using TNF, which contributes to the survival of endothelial cells (Madge & Pober, J.Biol. Chem. 275, 15458-15465. (2000)). The main Akt phosphorylation in fibronectin-related HUVEC was increased when exposed to TNF/IFNγthat is consistent with constitutive and TNF-induced Akt activation. In contrast, there were no signs of Akt phosphorylation in untreated spheroids and the impact of TNF/IFNcaused only weak phosphorylation (Figa). To assess the significance of activated Akt to the survival of HUVEC we were treated fibronectine cells wortmannin and LY294002, two pharmacological inhibitors phosphoinositide-3 (PI-3) kinase, an activator of the initial Akt activation processes (Kandel, & Hay, Exp. Cell Res. 253, 210-229. (1999)). We also expressed a constitutive active form of Akt (Aktmp) and the catalytic subunit of PI-3 kinase (R*) in spheroids. Processing wortmannin and LY294002 vyzyvayushaya apoptosis and reduces survival fibronectins cells when exposed to TNF± IFNγ (Fig. 4b), while Aktmp and R*, but not wild type Akt (Aktwt) or control plasmid (pBS), protects the spheroids from apoptosis induced by TNF±IFNγ (Pigs). Based on these results, we concluded that activation of Akt is essential for the survival of HUVEC exposed to TNF±IFNγand that both main and TNF-induzirovanna activation of Akt depends on the binding integrin.

Example 4: the Survival of treated HUVEC TNF requires the activation of Akt and NF-κC.

Aktmp suppresses TNF-induced apoptosis in spheroids in the presence of activated NF-κB. We also studied whether activation of both signal transduction pathways NF-κand Akt is necessary for survival, or rather the activation exclusively Akt. We blocked the activation of NF-B in cells that Express constitutively active Akt (Aktmp) by HUVEC infection by adenovirus that expresses non-biodegradable 1-κAdΔNIκwhich prevents IkB-NFκB dissociation (Brown and others, Science 267, 1485-1488. (1995)). AdΔNIκincreases the sensitivity of the fibronectin-related HUVEC to TMF±IFNγ-induced apoptosis and is not affected Aktmp. Control electroporation (pBS) or infected with adenovirus (AdLacZ) also has no effect. AdΔNIκalso increases the sensitivity of spheroids, which sverkhekspressiya Aktmp, TNF±IFNγ-inducido nomu apoptosis (Fig.4d). To determine the fact whether Akt to protect from the effects of TNF in low doses in HUVEC in the absence of NF-κactivation adhered monolayers of wt and HUVEC, which sverkhekspressiya Aktmp, were infected with AdΔNIκand were exposed to TNF (0,33-100 ng/ml). AdΔNIκincreased sensitivity HUVEC apoptosis (TNF>0.1 ng/ml), but Aktmp had no protective effect on such HUVECS even at such low doses of TNF (Fige). Moreover, LY294002 and wortmannin not inhibited induced TNF expression of ICAM-1, which indicates that NF-κIn activation in HUVEC does not require signaling through Akt (Fig.4f and data not shown), and is consistent with the induction of ICAM-1 in the spheroids (see figure 3C). In contrast, infection of HUVEC AdΔNIκinhibits expression of ICAM-1 in response to TNF±IFNγ (Fig.4f). Taken together, these results indicate that HUVEC survival when exposed to TNF±IFN γ requires joint activation of Akt and NF-κC.

Example 5: Binding of integrin promotes activation of FKHR and MDM2 and inhibits the phosphorylation of MEK, R and JNK

Antiapoptotic effect of Akt was originally attributed it to the phosphorylation and inhibition of caspase-9 and Bad (Datta and others, Genes Dev. 13, 2905-2927. (1999)). Now, however, it was shown that Akt-dependent survival is associated with the phosphorylation and inhibition "forkhead" transcription facto is s (FKHR/FKHRL1) (Datta and others, Genes Dev. 13, 2905-2927. (1999); Brunet and others, Cell 96, 857-868. (1999)) and MDM2, p53 degradation (Mayo & Donner, Proc. Natl. Acad. Sci. USA 98, 11598-11603. (2001)), and suppression of the activation of protein kinases ERK, R and JNK (Estadio Rommel and others, Science 286, 1738-1741. (1999); Gratton and others, J. Biol. Chem. 276, 30359-30365. (2001); Park and others, J. Biol. Chem. 277, 2573-2578. (2002); Madge & Pober, J. Biol. Chem. 275, 15458-15465. (2000)). We investigated whether the lack of binding integrin and Akt signaling associated with rebuilding these transmission signals. We have determined the levels of MDM2, p53 and phosphorylation of FKHR/FRKHL1, MEK, R and JNK in related HUVEC and spheroids, subjects the effects of TNF/IFNγ. Such spheroids lacked the phosphorylation of FKHR/FKRL1, reduced levels of MDM2 and accumulation of p53 compared with fibronectine cells (Figa). In addition, the spheroids had increased primary and TNF/IFNγ-induced phosphorylation of MEK, P3 8 and JNK (Fig.5b).

These results are consistent with a role of Akt in maintaining survival through inhibition of FKHR/FKHRL1 by reducing the levels of p53 by suppressing the phosphorylation of MEK, R and JNK.

Example 6: Inhibition integrisanjem adhesion using compounds with small molecules enhances the sensitivity of endothelial cells to TNFα-induced apoptosis in vitro and in vivo.

The reduction of binding of integrin increases the sensitivity associated HUVEC apoptosis, induced TNF.

The increase is obsticales to TNF in decreased binding of the integrin is not unambiguous for spheroids: culturing HUVEC on poly-L-lysine (PLL), the substrate, which contributes integrisanjem adhesion (Bershadsky and others, Curr. Biol. 6, 1279-1289. (1996)) preserves the survival on the PLL, and the addition of TNF±IFN γ causes massive destruction (Figa), this loss is prevented by expression of Aktmp (not shown). In addition, we selectively inhibited integrin αVβ3 in HUVEC on fibronectin using EMD121974 ((cyclic (Arg-Gly-Asp-D-Phe-N-Me]-Val), which is antagonistic cyclopeptides α_Vβ₃/α_Vβ₅) (Dechantsreiter etc., J. Med. Chem. 42, 3033-3040. (1999))that does not affectcomponent α₅β₁/α_Vβ₃-dependent adhesion to fibronectin (Fig.6b). While neither TNF/IFNγnor EMD121974 separately had no effect on survival, the combined effect of TNF/IFNγ and EMD121974 (but not reinhabiting control peptide EMD135981) increased apoptosis and Department (Figs), and reduced survival (Fig.6d). Expression Aktmp defended fibronectine from HUVEC apoptosis induced by TNF, IFNγ and EMD121974 (Fige).

Example 7: Reduced binding of integrin increases the sensitivity associated HUVEC apoptosis, induced by different ligands death of the TNF family of ligands.

Increased sensitivity to the proapoptotic action of signaling through death receptors decreasing binding integrin does not exhaust anchoveta TNF, but it is also seen when exposed to TRAIL and FasL in the presence of EMD121974 on HUVEC, cultured on fibronectin. LIGHT, ligand, which binds to receptors, reducing the area of death, does not detect synergies with αVβ5-blocking (Fig.7).

Example 8: EMD121974 increases the sensitivity set for the tumors to anticancer action of TNF

Angiogenic endothelial cells Express αVβ3 integrin and link αVβ3 promotes the survival of endothelial cells (Brooks and others, Cell 79, 1157-1164 (1994); Brooks and others, Science 264, 569-571 (1994)). Information that EMD121974 increases the sensitivity of endothelial cells to apoptosis induced by TNF in vitro, suggests that this compound may enhance the antitumor activity of TNF. To explore this hypothesis, we subjected the treatment of rats, which are isogenic soft tissue sarcoma BN175, which is extremely aggressive and vascularized tumors that are resistant to the cytotoxic action of TNF in vitro and in vivo (Manusama and others, Oncol. Rep.6, 173-177. (1999)). We applied the technique of isolated limb perfusion (ILP) for the introduction of TNF, EMD121974, or combinations thereof to the extremity swelling. Treatment of TNF or peptide separately had no effect on tumor growth. In contrast, the combined introduction of TNF and EMD121974 caused complete tumor regression in 50% of the LM is now in General a significant decrease in tumor growth (Fig). While in animals, subjects effects EMD121974/TNF was not observed local and General toxicity, which suggests that EMD121974 selectively increases the sensitivity of tumors in relation to the cytotoxicity of TNF. Because tumor cells BN175 are insensitive to TNF and does not Express active αVβ3 integrin, what svidelstvuet their weak adhesion to fibrinogen even in the presence of high concentrations of Mn²⁺and their low sensitivity to selective inhibitors αVβ3, such as EMD 121974 (unpublished study), we have concluded that effective synergistic antitumor effects are most probably due to the destruction of the tumor vascular network.

Taken together with our in vitro data, they are a strong confirmation of the importance of integrin αVβ3 compared to αVβ1 in this system to control the survival of the endothelium.

Example 9: Culturing HUVEC and electroporation

HUVEC were obtained and cultured as described previously (Ruegg and other, Nature Med 4, 408-414 (1998)) and used between passages 3 and 7. Full serum represented: M199 (Life technologies, Basel, Switzerland), 10% PCS (serum fetal cow) (Seromed, Berlin, Germany), 12 μg/ml of extract brain of cattle (Clonetics-Bio Whittaker, Walkersville, MD, USA), 10 ng/ml human recombinant the aqueous EGF (growth factor epidermal) (Peprotech, London, UK), 25 units/ml heparin, 1 mg/ml hydrocortisone (Sigma Chemie), 2 mm L-glutamine, 100 µg/ml streptomycin and 100 units/ml penicillin (Life Technologies). For electroporation HUVEC resuspendable in full serum, incubated on ice for 5 min with DNA (20 µg specific plasmid and 5 μg pEGFP-C1) and electroporative with the pulse generator for genetic transformation (Biorad, Glattbrugg, Switzerland). Elektrooborudovanie HUVEC were cultured for 48 hours before use. Approximately 80% of the cells expressed EGFP 40 hours after electroporation.

Example 10: the Formation of spheroids

HUVEC were collected in the result of cleavage by trypsin, resuspendable in full serum 1.0×10⁶cells/ml and were sown 1 ml/well in 12 Lunnoye tablets natkaniec culture (Evergreen Scientific, Los Angeles, CA, USA) pre-coated with 1% BSA. To study the aggregation 200 ál of cell suspension was inoculated in the wells of ELISA tablets coated with 1% BSA (Maxisorp II, NUNC, Rosklide, Denmark) alone or in the presence of mAbs (10 μg/ml), EDTA (5 mm) or CA /EDTA (10/5 mm). The formation of spheroids was assessed after 6 hours and 16 hours. Microsemi was done under a microscope Televal 31 (Carl Zeiss AG, Zurich, Switzerland).

Example 11: Morphological analysis of spheroids

To assess the morphology of the spheroids was inserted in Epon (Fluka Chemie) and thick sections were stained with 1% methylene/azurovym si is them. For immunological staining of frozen sections of spheroids were fixed in 4% (Fluka Chemis, Bucha, Switzerland) formaldehyde. After blocking with 1% BSA, the sections sequentially incubated for 1 hour with primer mAb (20 μg/ml) and cyan-labeled GaM-anticorodal (West Grove, PA, USA). For the TUNEL reaction frozen sections of spheroids were fixed in 4% paraformaldehyde and processed as described in (Ruegg and others, I.c.). Calculated staining of spheroids with iodide of propecia for total DNA content. The sections were observed in epifluorescence microscope (Axioskop, Carl Zeiss AG)equipped with a CCD camera (Photonic Science, Milham, UK) or using a laser confocal microscope (LSM 410, Carl Zeiss AG). The apoptosis index was determined by calculating the ratio between the green (TUNEL stained DNA fragments) and red (painted iodide of propedia total DNA) pixels.

The number of investigated spheroids per condition were: C, 31; T., 21; T1, 12. To identify apoptotic cells in culture, the DNA dye YoPro-1 (250 nm) was added to a culture or to the collected moving cells (Delhase, M., Li, N. & Karin, M. Kinase regulation in inflammatory response. Nature 406, 367-368. (2000)). Cultures were observed with an inverted fluorescence microscope (Leica DM IRB, Heerbrugg, Switzerland). For electron microscopy spheroids were fixed with 2.5% glutaraldehyde in 100 mm cacodylate buffer and subsequently recorded the 1% OsO ₄. Cells were obezvozhivani ethanol and put in Epon. Ultrathin sections were examined using a transmission electron microscope Philips CM10.

Example 12: the Survival of cells and proliferation.

For the survival of HUVEC spheroids on the tablet 1×10⁶cells/ml in covered with 1% BSA 24 mm holes, or adhered cells on the tablet 4×10⁵cells coated with fibronectin 3 µg/ml 35 mm holes tablets natkaniec cultures (Evergreen Scientific), were subjected to stimulation with TNFα (200 ng/ml=10⁴units/ml)± IFNγ (330 ng/ml=10⁴units/ml). Inhibitors of the kinase or EMD peptides were added 1 hour or 4 hours before stimulation, respectively, at the following concentrations: wortmannin, 100 nm; LY294002, 20 μm; EMD peptides, 300 μm. After 16 hours of culture, cells were collected using dissociation (5 min at 20°for spheroids) with 5 mm EDTA or 1x trypsin (for adhered cultures), washed, resuspendable in full serum up to 4×10⁵cells/ml, an aliquot of 100 µl/well in microtiter tablets tissue cultures (Falcon, Becton Dickinson) and were titrated 1:2 stages in three repetitions. The relative number of cells was assessed after 48 hours by measuring the conversion of MTT for 4 hours in culture. The results served as values OF at 540 nm (Packard Spectra Count, Meriden, CT, USA) and presented as values with three repetitions per well S.O.

Example 13: the Research division of cells

Maxisorp II ELISA tablets were covered with 1 µg/well of fibronectin or 0.5% gelatin overnight at 4°in phosphate-saline buffer solution PBS. Coated wells were washed and blocked with 1% BSA for 2 hours at 37°and washed before use. Added 2×10⁴cells/well in the main serum without FCS and quickly besieged by centrifugation (40×g). The cells were given the opportunity to adhesivity for 2 hours at 37°before the addition of peptides at different concentrations. After 2 hours, cells were washed with warm phosphate-saline buffer solution and attached cells were fixed in 2% paraformaldehyde, stained with 0.5% crystal violet (Sigma Chemie) and measured UP at 620 nm (Packard Spectra Count). The results served as values OF and presented in the form of values with two repetitions per well ± S.O. specific adhesion (=adhesion on ECM protein minus adhesion to BSA).

Example 14: Flow cytometry

Indirect immunological staining of HUVEC and the expression of EGFP was performed according to standard Protocol (Ruegg and others, I.c.). Dead cells were excluded by staining with iodide of propecia. All samples were analyzed using a FACScan II® and Cell Quest® computer software (Becton Dickinson, Mountain View CA, USA).

Example 15: a Study essentialcomponents mobility (EMSA)

Nuclear extracts from HUVEC (1×10 cells per condition) were prepared as described (Cai and others, J.Biot Chem 272, 96-101. (1997)), and incubated with synthetic donativum 31-dimensional oligonucleotides containing κsequences person with HIV long terminal repeat, labeled on the end [γ-32 P]ATP using T4 kinase. The binding of NF-κlabeled with P oligonucleotides was determined by PAGE and autoradiography.

Example 16: Western blot

50 μl of the supernatant of the cell lysate (1×10⁶250 μl of 2× Laemmli buffer) was dissolved with the aid of 7.5%is 12.5% SDS-PAGE and transferred using a wet blotting (Bio Pad) to Immobilon-P membranes (Millipore, Volketswil, Switzerland). Membrane sequentially incubated in 5% dry milk for 1 hour, with the original antibody over night at 4°and HRP-labeled GaM (Dako, Zug, Switzerland) for 1 hour. To determine used the ECL system (Amersham-Pharmacia Biotech). For re-probing the membranes were cleaned in 2% SDS, 50 mm Tris and 100 mm TOGETHER for 30 min at 50°C.

1. Pharmaceutical composition for the treatment of endothelial tumors or their metastases, containing a therapeutically effective amount of (i) at least one antiangiogenic agent, where the specified-angiogenic agent is a peptide, which contains the RGD, namely cyclo(Arg-Gly-Asp-DPhe-NMeVal), and (ii) the factor n is Crozat tumor-alpha (TNFα or a molecule having the biological activity of TNFαand, optionally together with a pharmaceutically acceptable carrier, excipient or solvent.

2. The pharmaceutical composition according to claim 1, which additionally contains at least one cytotoxic and/or chemotherapeutic agent.

3. The pharmaceutical composition according to claim 2, in which the specified cytotoxic agent is a gamma-interferon (IFN_γ).

4. The pharmaceutical composition according to claim 2, in which the specified chemotherapeutic compound selected from the group comprising cisplatin, doxorubicin, gemcitabine, docetaxel, paclitaxel (Taxol), bleomycin.

5. The pharmaceutical composition according to claim 1, containing

(i) cyclo (Arg-Gly-Asp-DPhe-NMeVal),

(ii) TNFα,

(iii) IFN_γ.

6. Pharmaceutical kit for the treatment of endothelial tumors or their metastases, including a package containing (i) at least one antiangiogenic agent, where the specified-angiogenic agent is a peptide, which contains the RGD, namely cyclo(Arg-Gly-Asp-DPhe-NMeVal), (ii) tumor necrosis factor alpha (TNFα) or a molecule having the biological activity of TNFαand optionally (iii) cytotoxic and/or chemotherapeutic agent.

7. The pharmaceutical kit according to claim 6, in which the specified qi is toksicheskoe tool is a gamma-interferon (IHFγ ).

8. The pharmaceutical kit according to claim 6, containing

(i) cyclo(Arg-Gly - Asp-DPhe-NMeVal),

(ii) TNFα,

(iii) IFNγ.

9. The pharmaceutical kit according to any one of p-8, in which the specified pharmaceutical active funds are kept in separate containers in the specified package.

10. The use of the pharmaceutical composition according to claim 1 or a pharmaceutical set of claim 6 for the preparation of a medicinal product or composition of the medicinal product for the treatment of tumors and metastases of tumors.

11. A method of treating endothelial tumors or their metastases specimens, which provides for the introduction of specified individuals simultaneously or sequentially a therapeutically effective amount of a cyclic peptide, which contains the RGD, namely cyclo(Arg-Gly-Asp-DPhe-NMeVal),

(ii) TNFα.

12. The method according to claim 11, which includes the introduction of more specified individuals therapeutically effective amount of a cytotoxic and/or chemotherapeutic agents.

13. The method according to item 12, which indicated the cytotoxic agent is an IFNγ.

14. The method according to item 12, in which the specified chemotherapeutic agent is chosen from the group comprising cisplatin, doxorubicin, gemcitabine, docetaxel, paclitaxel (Taxol), bleomycin.

15. The method according to claim 11, which provides for the introduction mentioned the sobi simultaneously or sequentially a therapeutically effective amount

(i) cyclo(Arg-Gly-Asp-DPhe-NMeVal),

(ii) TNFαand

(iii) IFNγ.