Methods of treatment of tumors and metastases using a combination of antiangiogenic therapy and immunotherapy

 

The invention relates to medicine and relates to methods of treatment of tumors and metastases using a combination of antiangiogenic therapy and immunotherapy. The invention includes a method for the treatment of neuroectodermal and epithelial tumor in a patient, therapeutic composition for treatment of tumor cells with the use of an agent that inhibits anglophones and which antagonistav3and antitumor immunotherapy agent containing cytokine component cells-effector and the component Ig immunoglobulin polypeptide, aimed at associated with tumor antigen and containing a variable region that binds to associate with tumor target antigen. The advantage of the invention is the use for the treatment of tumors antiangiogenic therapy. 3 C. and 23 C.p. f-crystals, 4 Il., 1 PL.

Cross-reference to related applications

In this application claimed priority to Provisional application U.S. patent registration number 60/119721, filed February 12, 1999.

The Declaration of the rights of the state patent

Some described here issledovaniya States of America. The government of the United States of America may have certain rights in this invention.

The technical field,

This invention relates to methods for inhibition of primary tumors and metastases using a therapy based on the joint application of antiangiogenic and directed antitumor immunotherapy.

Background of the invention

The generation of new blood vessels, or angiogenesis, plays a key role in the growth of malignant diseases, and development of agents that inhibit angiogenesis is of great interest (see, for example, Holmgren, L., O'reilly, M. S. & Folkman, J. (1995) "Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression". Nature Medicine, 1, 149-153; Folkman, J. (1995) "Angiogenesis in cancer, vascular, rheumatoid and other disease". Nature Medicine, 1, 27-31; O'reilly, M. S., et al., (1994) "Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by Lewis lung carcinoma", Cell 79, 315-328; Kerbel, R. S. (1997) "A cancer therapy resistant to resistance, Nature, 390, 335-336; Boehm, T., et al., (1997) "Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance". Nature, 390, 404-7; and Volpert, O. V., et al., (1998) "A human fibro-sarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1", Proc.Natl. Acad. Sci. (U. S. A.), 95, 6343-6348).

Know the antagonists of integrinv3for inhibition angiogenesis, U.S. patent No. 5753230 (Brooks & Cheresh) and U.S. patent No. 5766591 (Brooks & Cheresh), which describe the use ofv3-antagonists, such as synthetic polypeptides, monoclonal antibodies and mimeticsv3that are associated with receptorv3and inhibit angiogenesis.

In addition, the described treatment using fused protein antibody-cytokine, which stimulated mediated immune response inhibition of established tumors, such as metastatic carcinoma. For example, the cytokine interleukin 2 (IL-2) was fused to the heavy chain of the monoclonal antibody, immunoreactive, in two separate fused proteins, associated with tumor antigens, adhesion molecule of epithelial cells (EP ITSELF, KSA, KSl/4-antigen) or disialogangliosides GD2using antibody KS1/4 and ch14.18, respectively, with the formation of the fused proteins chl4.18-IL-2 and KS1/4-IL-2, respectively. See, for example, U.S. patent No. 5650150 (Gillies).

Identification of specific vascular network angiogenesis inhibitors, which are synergistic with therapies, specifiedas characterized by invasion, migration and proliferation of endothelial cells - a process that is dependent on cellular interactions with components of the extracellular matrix. In this context, it was shown that endothelial adhesion receptor integrinv3plays a key role, providing specific for vascular network target for antiangiogenic strategies of treatment (Brooks, P. C., Clark, R. A. & Cheresh, D. A. (1994) "Requirement of vascular integrine alpha v beta 3 for angiogenesis". Science, 264, 569-571; Friedlander, M., et al., (1995) "Definition of two angiogenic pathways by distinct alpha v integrins". Science, 270, 1500-1502). The need for vascular integrinv3angiogenesis has been demonstrated in several models in vivo, where the generation of new blood vessels transplanted human tumors completely inhibited system introduction or peptide antagonists of integrinv3or anti-v3antibodies LM609 (Brooks, P. C., et al., (1994) Science, supra; Brooks, P. C., et al., (1994) "Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels". Cell, 79, 1157-1164). Mouse hybridoma LM609 was japanparts.com contract and she was given the name of ATSS HB 9537 15 September 1987. Such antagonists block the ligation of integrinv3that stimulates the apoptosis of proliferative angiogenic vascular cells and thereby disrupts the maturation of newly formed blood vessels, is essential for the proliferation of tumors.

Vascular endothelial growth factor (VEGF) was identified as a selective angiogenic growth factor that can stimulate mitogenic endothelial cells. Biopsy of human tumors show increased expression of VEGF mRNA in malignant cells and mRNA of VEGF receptor in adjacent endothelial cells. The expression of VEGF, apparently, is the highest in the areas of tumors adjacent to revascularise areas of necrosis (see review by Thomas et al., (1996) "Vascular Endothelial Growth Factor, a Potent and Selective Angiogenic Agent", J. Biol.Chem., 271(2): 603-606). Effective antitumor therapy may be used towards the VEGF receptor monoclonal antibodies for the inhibition of angiogenesis. (L. Witte et al., (1998) "Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an antiangiogenic therapeutic strategy", Cancer Metastasis Rev., 17(2): 155-161.

A major obstacle to effective treatment of disseminirovanne what talasani, which do not have well-developed blood supply for delivery of therapeutic substances. In this regard, a new immunotherapy strategy using specific against tumor compartment monoclonal antibodies were effective for the direction of cytokines in the microenvironment of the tumor. This was achieved by using recombinant fused protein antibody-cytokine generated to preserve the unique ability of tumor-specific targeting of monoclonal antibodies and immunomodulatory functions of cytokines. The use of fused protein antibody-IL-2 for the direction of IL-2 in the tumor compartment induced activation of effector cells, penetrating into the tumor microenvironment, and led to the effective destruction of the registered metastases in three different models of tumors in syngeneic mice (Becker, J. C., et al., (1996) T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy", J. Exp.Med., 183, 2361-2366; Xiang, R., et al., (1997) "Elimination of established murine colon carcinoma metastases by antibody-interleukin 2 fusion protein therapy", Cancer Res., 57, 4948-4955; Lode, H. N., et al., (1998) "Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy", Blood, 91, 1706-1715). Being fully effective in the early stages of metastasis of a tumor, this direction is the tumor, characterized by a fully developed vascular compartment. Here the authors wondered whether there is a complementary advantage in specific vascular and aimed at the compartment of the tumor treatment strategies and whether they are synergistic when used in sequential or simultaneous combinations.

It was investigated on three models of tumors in syngeneic mice colon cancer, melanoma and neuroblastoma, the latter was characterized by spontaneous metastases in the liver. All three models show a close resemblance to these diseases in humans. Model metonomy and neuroblastomas Express disialogangliosides GD2, which, as installed, is associated with tumor antigen in such neuroectodermal malignant lesions (Irie, R. F., Matsuki, T. & Morton, D. L. (1989) "Human monoclonal antibody to ganglioside GM2 for melanoma treatment. Lancet, 1, 786-787; Handgretinger, R., et al., (1995) "A phase I study of human/mouse chimeric antiganglioside GD2 antibody chl4.18 in patients with neuroblastoma", Eur.J.Cancer, 31A, 261-267), and the model for colon cancer characterized by the expression of the adhesion molecule of epithelial cells (EP ITSELF, KSA, KSl/4 antigen), target molecules that have been successfully used for passive immunotherapy in humans (Riethmuller G., et al., (1994) "Randomised trial of monoclonal antidody for a the data models targeting them fused protein antibody-interleukin-2 with chimeric human/mouse anti-GD2-antibody (ch14.18-IL-2) (Gillies, S. D., et al., (1992) "Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells", Proc. Natl. Acad. Sci.(U. S. A.), 892, 1428-1432) and humanized anti-EP-HIMSELF (anti-KSA, anti-KS1/4 antigen) antibody KS1/4-I1-2 (Xiang, R., et al., (1997) supra; Gillies, S., et al., (1998) "Antibody-IL-12 fusion proteins are effective in SCID mouse models of prostate and colon carcinoma metastases", J. Immunol., 160, 6195-6203). Vascular compartment of these models of tumors, as described for several animal models, is determined by the expression of integrinv3in the newly formed blood vessels (Brooks, P. C., et al., (1994) supra). The data presented here demonstrate the synergistic effectiveness of simultaneous and sequential treatments, specifically aimed at the tumor and vascular compartments of primary tumors and distant metastases. The mechanism of this synergism is provided by reducing the formation of blood vessels and increased inflammation only in the animals subjected to this combination therapy. These observations emphasize the beneficial effect of combining antiangiogenic approach with tumor-specific antitumor immunotherapy approach.

The invention

This invention relates to a method of treatment of tumor CL what about the proliferation of tumor cells number inhibiting angiogenesis agent and antitumor immunotherapy agent. The inhibition of proliferation of tumor cells may include inhibition of growth of tumor cells in an existing tumor or tumor metastasis, inhibition of the formation of additional tumor metastasis and death of tumor cells. Inhibiting angiogenesis agent and antitumor immunotherapy agent may be administered essentially simultaneously or sequentially.

In one embodiment, the invention describes a method of treating tumors or tumor metastases in a patient by administration to the patient a combination of at least one inhibiting angiogenesis agent and at least one anticancer immunotherapy agent. Effective inhibition of proliferation of tumor cells in the specified patient can be achieved in this way.

The patient may receive the above-mentioned therapeutic composition before, during or after surgery to remove the entire tumor or part of the tumor. The introduction can be performed by direct immersion; systemic or localized intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.C.), intramuscular (i.m.) injection or direct injection into the tumor weight; and/or by oral administration of PoE, an agent that can inhibit the formation of new blood vessels (revascularization) or the increase of existing capillary network in the tissue near the tumor cells. Appropriate any abscopal angiogenesis agents can be peptides inhibiting angiogenesis activity, such as associated with tumor antigen PSA. Other appropriate any abscopal angiogenesis agents can be antagonists of VEGF-associated angiogenesis, for example, antagonists of VEGF receptor on the cell surface. Preferred inhibiting angiogenesis agent is an antagonist of the binding of integrinv3with cells. Antagonistv3for use in the methods of this invention is the antagonist, which can inhibit angiogenesis associated with tumors or tumor metastases tissue when injected into tissues or target cells. Such antagonists can be unique linear or cyclic polypeptides, linear or cyclo-RGD-containing polypeptides, antibodies or memeticallyv3to migenes.

If the antagonistv3is an antibody, it is assumed that this antibody can be polyclonal, monoclonal antibody or antigennegative fragment having antigennegative specificity in relation tov3or receptorv3. The preferred monoclonal antibody which binds to the integrinv3is a monoclonal antibody identified as LM609 (ATSS HB 9537).

Preferred inhibiting angiogenesis agent is a polypeptide, which is a receptor antagonistv3that can inhibit the integrin receptor on the target cells. The most preferred option antagonistv3is a synthetic RGD-containing peptide cyclo(RGDfN-MeV) (SEQ ID NO:11), etc., Cyclic peptides of this General type is described in U.S. patent No. 5262520 (Plow et al.). He containing RGD peptides described in the patent of Snoho of the invention, is an immunotherapy agent that contains cell effector component, which is connected with a component aimed at associated with tumor antigen. Suitable cell effector components may include cytotoxic chemicals, cytotoxic radioactive isotopes and transfer agents of cellular signals, such as cytokines.

Appropriate aimed at the tumor components are polypeptide chains that are associated with associated with tumor antigens present on the tumor cell or in the surrounding tissue matrix, such as chain proteins, receptors or chains of immunoglobulins.

Associated with tumor antigens that can be used as targets for immunotherapy agents include associated with tumor antigen selected from the group consisting of AFP, CA 125, CEA, CD19, CD20, CD44, CD45, EGF-receptor, GD2, GD3, GM1, GM2, Her-2/Neu, Ep-CAM (KSA), IL-2-receptor, Lewis Y, Lewis-X (CD 15) associated with melanoma of proteoglycan MCSP, PSA and transferrin receptor.

Preferred immunotherapy agent has an effector component, which is a polypeptide cytokine that is connected to the guide the t variable region, which is associated with associated with tumor antigen. Preferably, the chain immunoglobulin when combined with appropriate complementary chain (i.e., a heavy chain complementary light chain) were determined by active site of the antibody, specific associated with tumor antigen.

Aimed at the tumor part of the Ig immunotherapy agent may contain the entire amino acid sequence of the chain of the immunoglobulin or at least for its fragment containing a part of this protein, specific binding of the antigen. Thus, the matching circuit Ig polypeptide will have at least the variable region of the Ig, specific associated with tumor antigen.

The antibody and the polypeptide chain from him, are suitable for use in the methods of the present invention will have an amino acid sequence, which may occur from any mammal. When such protein antibody derived from a mammal other than the intended patient, can be used for protein fragments of this antibody, such as F(ab')2, Fab, Fv, or constructed, containing a single chain Fv protein antibodies. To Telenesti this antibody, to make the protein more similar to the normal components of the antibodies of the patient. For example, the amino acid sequence of mouse monoclonal antibodies can be modified in various ways to humanitarian this antibody, to make it more similar to human antibodies for administration to patients.

Alternatively, the antibody may be of human origin (genetically encoded genes Ig man), but are produced in a transgenic animal transformed with a gene expression Ig man instead of his own native Ig genes. For example, can be obtained by genetic engineering (designed) transgenic mice, which Express DNA of human origin, encoding proteins Ig man. Obtaining monoclonal antibodies from these transgenic mice will result in a murine B-cell hybridomas expressing human DNA encoding antibodies having the amino acid sequence of human origin. This will greatly reduce the immunogenicity of such antibodies for use in the treatment of the person.

For treatment of humans, the preferred antibodies for use in the method of the claimed invention are romantical KS1/4 against associated with tumor antigen, KS1/4 (also known as EP-SDM and KSA).

Component cell effector immunotherapy agent, suitable for use in the methods, compositions and kits of the present invention is a cytokine selected from the group consisting of BDNF, CNTF, EGF, Epo, FGF, Flt3L, G-CSF, GM-CSF, I-309/TCA-3, gamma, IP-10, IFN alpha, IFN beta, IFN gamma, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, LT, MCP-1 - MCP-3, M-CSF, MIF, MIP-lalpha, MIP-1beta, MIP-2, NGF, NT-3, NT-4, OSM, PBP, PBSF, PGFD, PF-4, RANTES, SCF, TGF alpha, TGF beta, TNF alpha, TPO and VEGF. Suitable cytokine, which is a chemokine, can be selected from the group consisting of C10, EMF-1, ENA-78, eotaxin, GCP-2, HCC-1, 1-309, IL-8, IP-10, lymphotactin, MCP-1, MCP-2, MCP-3, MGSA, MIG, MIP-lalpha, MIP-lbeta, MIP-2, NAP-2, PF4, RANTES, SCM-1 and SDF-1. Cytokine part of the above immunotherapy agent can be complete amino acid sequence of the cytokine protein or a fragment of such a fused protein, sufficient for the induction of cytokine-specificheskogo biological response.

In a preferred embodiment, the cytokine portion of the immunotherapy agent has the biological activity of IL-2.

In the immunotherapy agent containing cytokine polypeptide, coupled with Ig polypeptide, a suitable joint between the polypeptide chain cytokines and polypeptide chain Ig includes the direct polypeptide between circuits, includes the use of biotinidase and complex avidin-Biotin. The preferred joint is either direct or separated polypeptide linker polypeptide communication. Direct link makes possible the expression of immunotherapy agent in the form of a single fused protein from a host cell transformed with a suitable expressing vector coding for the immunotherapy agent in the form of a fused protein.

Thus, the preferred immunotherapy agent for use in the methods of the present invention is a bifunctional protein with cytokine component and guide on associated with tumor antigen component, where the guiding component is a polypeptide chain Ig, which has specificity against associated with tumor antigen. Examples of such preferred immunotherapy agents include aimed at GD2 protein chl4.18-IL2 and aimed at associated with tumor antigen, KS1/4 (also known as EP-ITSELF and KSA) protein KS1/4-IL2.

Alternatively, other immunotherapy agents suitable for use in the methods, compositions and kits of this invention may have the CL is m is the agent, which has a direct cytotoxic effect on tumor cells, i.e., immunotoxins, radioactive isotopes, cytotoxic drugs, etc. Like cytokine immunotherapy agents described above, cytotoxic peptides can be connected with guides on associated with tumor antigen is a polypeptide Ig for the formation of a fused protein, either directly or through a linker peptide or chain. Can be used a chemical bond chemical cytotoxins with the guide Ig chains. Can be constructed carrying a radioactive isotope chain antibodies.

Another aspect of this invention relates to therapeutic compositions containing an inhibiting angiogenesis agent and antitumor immunotherapy agent. Preferably, the anticancer immunotherapy agent is directed at the tumor cells or cells of tumor metastases, due to the presence of the component specific associated with tumor antigen, coupled with cellular effector component. In a preferred therapeutic composition of the present invention antitumor immunotherapy agent is a bifunctional b tumor component, which is a polypeptide chain of immunoglobulin (Ig) where the specified Ig-chain contains a variable region that binds to associate with tumor antigen.

Another aspect of the present invention is a kit for treating tumors or tumor metastases. This set includes a package containing an inhibiting angiogenesis agent, such as antagonistv3capable of inhibiting angiogenesis in a given tumor or in these tumor metastasis; bifunctional protein component having cytokine activity and specificity against tumor antigen; and instructions for the treatment of tumor cells in tumors and tumor metastases. This kit can also include a special label indicating the use of the components of the kit for treating tumors or tumor metastases.

Brief description of drawings

Preferred variants of the present invention is described below with reference to the following accompanying chertejei.

The figure 1 presents a graphical representation of the steps combined therapy antiangiogenic integrin antagonistvand primary tumors. Figure 1A shows the results obtained in the primary tumors induced by subcutaneous injection (2106) NXS2 neuroblastoma. The figure 1B shows the results obtained in the primary tumors induced by subcutaneous injection (2106) carcinoma of the colon ST-KSA. Figure 1C shows the results obtained in the primary tumors induced by subcutaneous injection (2106) cells melanoma B78-14 D.

The figure 2 depicts the graphs of the action of the United antivascular and anticancer therapies on the vascularization and antitumor immune response. Figure 2A shows the density of blood vessels in the primary tumor after treatment of vascular and tumor compartment or an antagonist of integrinvor fused protein chl4.18-IL-2, or their combination (*P<0,001, T-student test). Figure 2B shows the degree of infiltration by leukocytes of primary tumors after treatment of vascular and tumor compartments, respectively (*P<0,001, T-student test).

The figure 3 shows the graphs of the action of a sequential combination of anapartment immunotherapy with the merged Belokon antibody-IL-2 on spontaneous neuroblastoma metastases in the liver. The number of spontaneous metastases in the liver was determined by macroscopic counting foci in the liver (n=8) (**P<0,01, criterion rank-sum Wilcoxon signed).

Figure 4 shows in a diagram the effect of essentially simultaneous combination of antiangiogenic integrin antagonistvand specific tumor compartment immunotherapy with fused protein antibody-IL-2 on spontaneous neuroblastoma metastases in the liver. Shows the results of processing the integrin or antagonist (17,5 mg/h) and ofwholesale fused protein chl4.18-IL-2 (5 mg5) initiated before (figure 4A) or after (figure 4B) removal of the primary tumor. Spontaneous metastases in the liver was determined by macroscopic counting foci in the liver (n=8) (**P<0,01, criterion rank-sum Wilcoxon signed).

Detailed description of the invention

The suppression and elimination of primary tumors and distant metastases is the main task of an alternative cancer treatment strategies, such as inhibition of angiogenesis and targeted immunotherapy.

Currently, it was found that there is an unexpected synergy in efficacy in the treatment of tumor cells in the tumor and tumor metastasis by cambiaria inhibition of angiogenesis and (2) anti-tumor immunotherapy. In particular, the combined therapy antagonistvantitumour therapy fused protein antibody against tumor antigen/cytokine.

It was found that there is a synergism between the two unique monoterpene directed against vascular and tumor compartments; accordingly, specific against tumor vasculature angiogenic antagonistvand fused proteins, tumor-specific antibody-interleukin-2.

Simultaneous and sequential combination of these monotherapy effectively eliminated spontaneous metastases in the liver in subimmunogenic syngeneic model of neuroblastoma. In contrast, the controls subjected to monotherapy or antiangiogenic integrin antagonistvor fused protein antibody-IL-2, were only partially effective when applied dose levels.

In addition, the simultaneous processing of an antagonist of integrinvand fused proteins, tumor-specific antibody-IL-2 induced a dramatic regression (reverse development) in three models of tumors in syngeneic mice, i.e., melanoma, carcinoma tree only delay the growth of tumors.

Antitumor response was accompanied by a simultaneous 50% reduction in the density of tumor blood vessels and five-fold increase in inflammatory cells in the microenvironment of tumors. Subsequently, the tumor necrosis was demonstrated only in animals receiving the combination therapy, but not in the case when each agent used in monotherapy. These results show that these synergistic treatments provide a new and effective tool for the future therapy of metastatic cancer.

The present invention describes methods of treating tumors and tumor metastases, therapeutic compositions and therapeutic kits (packaged system), applicable in practice described here synergistic therapeutic effects.

1. Therapeutic compositions

Describes a variety of therapeutic compositions for use in practice of the methods of the present invention. These compositions include (antiangiogenic) inhibiting angiogenesis agents, such as antagonistsv3and antitumor agents, such as fused proteins antibody against tumor antigen/cytokine separately the ilz processed in their tissues, can be used in the compositions and methods of the present invention. A preferred inhibitor of angiogenesis is the antagonistvand, in particular, the antagonistv3. Inhibiting angiogenesis (antiangiogenic) antagonistv3may be a peptide, RGD-containing peptide, anti-v3-a antibody, anti-v3-receptor-antibody orv3-mimetic. Examples of antagonistsv3described in U.S. patent No. 5753230 (Brooks & Cheresh) and U.S. patent No. 5766591 (Brooks & Cheresh), descriptions of which are related to the production and application of antagonistv3specifically incorporated herein by reference.

Preferred antagonists are RGD-containing peptides, such as cyclic peptide cyclo(RGDfN-MeV) (SEQ ID NO:11). Additional antagonists have been described in the literature, including organic mimet the3. See, for example. Brooks et al. International Publication No. WO 97/45137, (PCT/US97/09158).

Tests for identification of antagonistv3suitable for use as an antagonist, as described in the cited U.S. patents, and therefore, it is assumed that alternative antagonists can be easily identified for use in the practice of this invention.

Suitable monoclonal anti-v3the antibody may be modified to include antigenspecific fragments, including F(ab')2, Fab and designed Fv or single-chain antibodies (SCA). Methods of obtaining such fragments are known in the art (see, for example, Hermanson, G. T., Bioconjugate Techniques/ Academic Press, 1996). The methods of the present invention, describes the use of antibodies also include a suitable modification of whole antibodies before it antigenspecific fragments. One suitable monoclonal antibody identified as LM609 (ATSS HB 9537).

It has been shown that other antagonistsv3are effective in the inhibition angiokeratoma, 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), was tested in various model systems of mammals (see, for example, Keenan RM, et al., (1998) Bioorg.Med.Chem.Lett., 8(22): 3171-3176; Ward KW, et al., (1999) Drug Metab.Dispos., 27(11): 1232-1241).

Vascular endothelial growth factor (VEGF) was identified as a selective angiogenic growth factor. Thus, alternative or additional inhibiting angiogenesis agent may be an inhibitor of VEGF activity, sufficient for inhibition of angiogenic growth. Such an inhibitor could be competitive inhibitor or VEGF-binding/inactivating the molecule, or a VEGF receptor antagonist. For example, possible inhibitors could be neoangiogenesis chemical mimetic of VEGF to competition for binding to the VEGF complex-target/receptor, modified neoangiogenesis derived VEGF antibody that specifically binds VEGF is sufficient for inhibiting angiogenic activity, or perhaps another specific protein which binds to VEGF (such as a dedicated receptor protein VEGF), or an antibody that binds to the VEGF receptor and blocks the interaction with VEGF.

Other compounds were iden is th with tumor antigen PSA.

Century Antitumor immunotherapy agent

Methods and compositions for use in such methods of this invention involve the joint introduction of at least one (antiangiogenic) inhibiting angiogenesis therapeutic agent with at least one anticancer immunotherapy agent. Preferred anticancer immunotherapy agent combines aimed at the tumor component with the component cells-effector, such as a cytokine, immunotoxin, a radioactive agent, or etc., Aimed at the tumor component preferably contains at least antigennegative part of antibodies directed against associated with tumor antigen. In one preferred embodiment, the effector component is a cytokine.

a) Associated with tumor antigens

Associated with the tumor antigen is a target, by which immunotherapy agent used in the methods, compositions and kit of the present invention, in the final account is sent to a tumor cell. Associated with tumor antigens are considered in this area correlated with certain types of cancer. In the vast majority stakim way what is not generally found in normal cells, associated with the source of the cancer. Other associated with tumor antigens are sekretiruemyi molecules or related extracellular matrix components that are not normally found in Association with Mature normal tissue.

Some associated with tumor antigens are proteins expressed in earlier stages of development, tissue, and is usually associated with Mature tissues (sometimes called oncofetal proteins). Other associated with tumor antigens expressed in normal Mature cells, but is expressed in large amounts by cancer cells. Associated with tumor antigens can be mutated forms normally expressed cell markers or extracellular components. Other associated with tumor antigens are modified or unusual forms of glycosylation of proteins, lipids or extracellular components, or new carbohydrate parts of the molecules.

Thus, associated with tumor antigens can vary widely and define, at least partially, the type of tumor to be treated using this sobremesa well studied in the areas of cancer research.

Many additional markers of tumors are in the process of research and after identification and characteristics as associated with tumor antigen they are also a suitable target antigen for immunotherapy targeting agent to the desired site of the tumor or metastasis.

It was reported about the direction vectors in gene therapy of cancer cells using monoclonal antibodies against antigen Lewis Y (Kurane S. et al., Jpn.J.Cancer Res., 89(11): 1212-1219 (1998)). Similarly, it was reported about the direction of liposomes using anti-ganglioside-GM3 antibodies or antibodies against the antigen Lewis X (Nam, S. M., et al., Oncol.Res., 11(1): 9-16 (1999).

As discussed below, in this area known methods of using the identified antigen to generate appropriate antibodies, which will connect specifically with that antigen. Thus, tumor antigens can be identified, and can be produced monoclonal antibodies that are applicable in this invention. In the review of the receipt of tumor antigens, see the descriptions in Human Cancer Markers, The Humans Press Inc., eds. Sell and Wahren, 1982. Description standard biochemical and molecular biological techniques that can be applied to obtain antigenaemia surface antigens can be specific for a class of tumors or for individual tumor type. Many nucleic acid sequences and/or amino acid sequences relating to associated with tumor antigens and coding associated with tumor antigens, available from a public computer databases, which can be found on the Internet using various services, i.e., the NCBI (national center for biotechnology information; www3.ncbi.nlm.nih.gov), and are usually identified by name and access number (a few non-limiting examples of the types of available sequence information cited by the comment "see the access number").

The preferred associated with tumor antigens target include, but are not limited to, AFP (see access number NH 001125), SA (see access number NP 005890), CEA (see access number AAA), CD19 (see access number R), CD20 (see access number R), CD44 (see access number R), CD45 (see access number R), EGF-receptor (see access number R), GD2, GD3, GM1, GM2, Her-2/Neu (see access number AAA), Ep-CAM (KSA) (see the access number R, AAA 36151), IL-2-receptor (see access number R, NP000197, R), Lewis Y, Lewis-X (CD 15) associated with melanoma proteoglycan MCSP (see access number NP001888), PSA (see access number R), PMSA, the transferrin receptor (see access number NP 003218, NP 003225, AAAF04564), Mar), L6 (see access number R) and CO-029 (see access number A, R).

Especially preferred associated with tumor antigens target for immunotherapy targeting agents are GD2and KSA (Ep-CAM; KS1/4 antigen), as described here.

Associated with tumor antigen protein is commercially available or can be obtained using standard methods of recombinant DNA and cell culture, methods of protein expression, known in this area. For example, lists Sigma (St.Louis, MO) for sale disialo-ganglioside GD 2 (G 0776), disialogangliosides GD3 (G 5904), monosialoganglioside GM1 (G 7641), monosialoganglioside GM2 (G 4651), tumor antigen gastrointestinal tract CA 19-9 (G 8660, G 8535), carcinoembryonic antigen CEA (S), Lewis-X-trisaccharide (L 5152) and Lewis-Y (L 7401).

Antibody specific for many of these associated with tumor antigens are commercially available (Eq. USB, RDI, Accurate Chemical and Scientific Corp., Zymed Lab). For Example, Sigma (St.Louis, MO) sell many monoclonal antibodies, such as anti-AFP (8452), anti-CEA (S), anti-EGF-receptor (S 3138), anti-IL-2 soluble receptor, (I 5652, I 5777, I 5902), anti-D19 (F 3899), anti-D20 (S), anti-D44 (S) and anti-D45 (S).

Even if antibodies are not commercially available is aligned with tumor antigens, can be generated using the antigen as an immunogen. For example, were described by the variable domains of the heavy and light chains of murine antibodies Lewis-Y (see access numbers AAV and AAV). Similar methods were described variable domains of the heavy and light chains of murine antibodies against asialo-CM1-ganglioside (see access number AAD09194 and AAD09195). Was elucidated crystal structure of the heavy and light chain of monoclonal antibodies against ganglioside GD2 (see access number 2554841, 2554842). It was reported on antibodies that bind to the folate receptor (binding protein), identified as a marker of ovarian cancer (see access number NP 000793). The nucleic acid sequence of the heavy and light chain variable regions of monoclonal antibodies against SA was also published and used for inclusion in the cassette vector (see access number AAV, 33455).

Particularly preferred antibodies are antibodies described here ch14.18 and KS1/4.

b) Monoclonal antibody and its antigennegative parts and etc.

Since the advent of methods of generating monoclonal antibodies, first published Kohler and Milstein, became well-known advanced techniques in this area (subel, F. M. et al. Short Protocols in Molecular Biology, 2nded. (Current Protocols, John Wiley & Sons, NY, NY, 1992) chapter 11). Currently obtaining monoclonal antibodies that bind to a specific antigen, is a common practice. The screening protocols were also developed to selection, if necessary, high-affinity binding of antibodies.

Usually the owner to obtain hybridomas are mice or other rodents.

One of the barriers to reparse human mouse monoclonal antibodies are HAMA (human antimurine antibodies) generated by the patient in response to this treatment. Ways to overcome this barrier include humanization of murine proteins antibodies replacement antigenic amino acid mouse protein protein sequences of human, who are presumably less antigenic. Other ways include transplant determining the binding specificity of amino acid residues or regions in the frames of the human protein.

The ability to Express the protein antibodies in phage systems view allows you to select antibodies which have been subjected to mutagenesis or improve the binding, or to reduce immunogenicity (see, the and in transgenic mouse was allowed to use mice hosts to generate antibodies and, therefore, monoclonal antibodies that occur from the nucleic acid sequence of a human.

Antibodies can also be reduced in size by fragmentation to obtain a reduced antigenicity or to create a smaller therapeutic molecules. For example, the full protein antibodies can be reduced either by cleavage with appropriate enzymes, or by getting the smaller protein by methods of recombinant DNA. Suitable fragments will comprise at least antigennegative part of the full molecule and may contain design Fab, F(ab)2F(AB)3, Fv or Fv, single chain antibody with one chain; SCA).

It seems that all the components of therapeutic agents based on monoclonal antibodies for use in the methods of this invention for the treatment of a person can benefit from modifications to reduce immunogenicity and potential KAMA, as described above.

You can also use specific receptor protein that can specifically bind to a particular associated with a tumor antigen, in the form of a guide component immunotherapy agents described above. Functionally, a specific receptor may be that the OPA in respect associated with tumor target antigen. Be careful to minimize the impact of cross-reactive binding with similar proteins that are not necessarily associated with tumor proteins.

(C) cytokines

In one embodiment, the anticancer immunotherapy design of the present invention include cellular effector part, which preferably is a cytokine.

The effector component of anticancer therapeutic agents of this invention may include any of a variety of cytokines for the induction of cytokine-specific biological response of a cell bearing a receptor for this cytokine. Cytokines are well characterized, and therefore, the invention should not be construed as limited thereby. Cytokines include as a subclass of molecules called chemokines. In the context of this invention, the chemokine is considered as a member of the superfamily of cytokines. Thus, the term "cytokine" as applied here refers, in General, as cytokines and chemokines. In relation to descriptions of the types of cytokines, see Callard. and Gearing, The Cytokine Facts Bool, Academic Press, Inc., 1994. In the description of the types of chemokines see Vaddi et al. The Chemokine Facts Book, Academic Press, Inc., 1997. Many of which may be available from public databases, available online through various services, i.e., the NCBI (national center for biotechnology information; www3.ncbi.nlm.nih.gov) and are usually identified by name and access number (link to several non-limiting examples of access to cited here sequence information given by the note "see the access number" with reference to the identifying number).

Cytokines mammals can be species-specific and may also vary within a species due to mutation and/or allelic variation. Suitable cytokine may be selected for use in accordance with the type of mammal to be treated. In the case of the existence of multiple alleles may be made in accordance with the activity of a cytokine or can be used a mixture of allelic variants in the quality of the selected cytokine. Thus, the use of veterinary methods of this invention can be adapted to the species to be treated animal or selection of the cytokine from more closely related species, if one of the types of targets is not available. For treatment of humans, preferably using human homologue, if known.

Cytokines, suitable for use as 4758020), EGF (see access number R), EPO (see access number 4503589), FGF (see access number SAV), Flt3L, G-CSF (see access number SAA), GM-CSF (see access number 4503077), I-309/TCA-3, gamma, IP-10 (see the access number R), IFN alpha (see access number R), IFN beta (see the access number R), IFN gamma (see access number R), IL-1 - IL-18 (see access number R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, R, Q14005, NP002181, Q14116), LIF (see access number AAS), LT (see access number 4504031), MCP-1 - MCP-3, M-CSF (see access number 4503075), MIF (see access number 4505185), MIP-lalpha, MIP-lbeta, MIP-2, NGF (see access number 4505391), NT-3 (see access number R), NT-4 (see access number R), OSM (see access number R), PBP (see access number 4505621), PBSF, PGFD, PF-4, RANTES, SCF (see access number R), TGF alpha (see access number R), TGF beta (see the access number R), TNF alpha (see access number R), Tro (see access number R) or VEGF (see access number AAD03710).

Chemokines, suitable for use in this invention include, but are not limited to, C10 (see access number R), EMF-1 (see access number R), ENA-78 (see access number A), eotaxin (see access number VIA), GCP-2 (see access number R), NSS-1 (see access number g1004267), I-309 (see access number g4506833), IL-8 (see the access number AAA), IP-9 (see the access number SAA), IP-10 (but see the MCP-3 (see the access number SAUL), MCP-4 (see access number Q99616), MGSA (see access number R), MIG (see access number R), MIP-lalpha (see access number R), MIP-lbeta (see access number R), MIP-2, NAP-2 (see access number R), PF4 (see access number 4505733), RANTES (see access number 4506847), SCM-1 (see access number R) or SDF-1 (see access number R). Preferred cytokines for use in this invention include IL-2 and IL-12 or their biologically active fragments that retain at least part of the effector activity full of intact molecules.

A preferred cytokine for use in this invention is IL-2.

Suitable cytokines for use in the methods of the present invention can be obtained from the corresponding nucleic acid sequence using standard molecular biological methods. Methods the expression of genes known in the art (for example, see Goeddel, D. V., editor, Methods in Enzymology, Vol.185: Gene Expression Technology (Academic Press, Inc., NY NY, 1991)). Cytokines are also available from commercial sources (i.e. Sigma, St.Louis MO).

d) Immunotherapy agent in the form of a complex of antibody against tumor/cytokine

These antitumor immunotherapy agents suitable for use in practice of this tion with aimed at the tumor component. Binding a tumor-binding component of the effector component can be done in different ways.

1) Merged with antibodies to the proteins

Were described anticancer immunotherapy agents that direct the function of cytokines on cells or tissues, which are tumor antigens, and the use of these cytokines provide recruiting immune response against cells bearing tumor associated antigen or tumor antigen. These immunotherapy agents called fused protein antibody against tumor antigen/cytokine, since this protein contains a merge of the cytokine with the polypeptide chain recombinant immunoglobulin (Ig), which is immune reacts with the pre-selected associated with tumor antigen.

In the application here immunotherapy agent, which is fused protein antibody against tumor antigen/cytokine that includes the slit structure between protein fragments of antibodies, which contain at least antigennegative part, and cytokines, which contain at least the effector part of the cytokine sufficient to maintain the functions of the biological signal transmission. Slit proteins dannemora peptide or peptides.

Slit proteins antibody against tumor antigen/cytokine known in this field and, in particular, are described, for example, in U.S. patent No. 5650150 (Gillies), the disclosure of which relating to the receipt and application of fused proteins, specifically incorporated here by reference.

Protein can be directed to any of a variety of tumor antigens, which are cell surface antigens, and therefore, the invention should not be construed as limited thereby. For example, we know getting fused protein using cytokine and heavy chain Ig, and the preparation of recombinant heavy chain Ig derived from monoclonal antibodies. In addition, obtaining monoclonal antibodies is developed field, and know how to obtain such antibodies against tumor antigens. Obtaining monoclonal antibodies described in "Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., eds, Reisfeld and Sell, 1985.

Usually polypeptide chain Ig is a polypeptide heavy or light chain Ig, which contains the N-terminal variable region that is specific for cells bearing a tumor antigen on the cell surface. Protein usually has a specified Ig polypeptide, linked on the carboxy end of the peptide is a rule contains additional domains CN and CH2 and may optionally additionally contain domain snz. However, if desirable, such domains of the constant region may be removed to reduce immunogenicity, size, or nonspecific binding of the final design of the fused protein. To facilitate the Association of the domains of the heavy and light chain may be desirable to design molecules Fv, where a heavy chain Fv linked to a light chain Fv. This relationship is between the carboxy end of one chain and the amino end of the other and is long enough not to cause significant steric changes in antigennegative "pocket" after re-stacking/domain Association.

Preferred protein contains the cytokine IL-2 and a heavy chain Ig, which immunoreagent with associated with tumor antigen GD2. In another embodiment, the preferred protein contains the cytokine IL-2 and a heavy chain Ig, which is immune reacts with associated with tumor antigen EP-HIMSELF (also known as KSA antigen, KS1/4). Examples of fused proteins of these options is described below as ch14.18-IL-2 and KS1/4-IL-2, respectively.

2) the Conjugates of antibodies

Alternative immunotherapy designs can be used in the methods and compositions of the present invention. For example, using high-affinity system Eotyrannus with a tumor antigen, associated with Biotin (or Avidya), and cytokine associated with the appropriate conjugate (Avidya or Biotin), can be constructed using a suitable connection. When using protein biotinylated antibodies can be combined with protein cytokine in vitro or in vivo before or after administration to create bifunctional immunotherapy agent that has a component that binds a tumor antigen and effector cytokine component associated complex of Biotin-avidin.

Biotinylated antibodies and proteins are known in this field and can be obtained using commercially available reagents. One indication of use of the biotinylated targeting tumor antigen antibodies is that the Biotin molecule has multiple binding sites avidin that will make possible a greater potentiation of the effector component, such as associated with Avidya cytokine, for each linked antibodies or combined use of more than one effector component. Other direct chemical methods of conjugation and reagents well known in this area and was used for the conjugation of antibodies with effector molecules, cal Protocols, 2nded. (Pound, J. D. editor, Humana Press, Inc., Totowa NJ, 1998) chapter 17; and Hermanson, G. T., Bioconjugate Techniques (Academic Press, NY NY, 1996). Thus, immunotherapy agents of this invention also include biotinylation design, described above, between the protein fragments of the antibody that contains at least antigennegative part, and cytokines, containing at least the effector part of the cytokine, sufficient to preserve the biological function of the transmission signal of the cytokine.

e) Other immunotherapy agents

Antitumor immunotherapy agents suitable for use in the methods, compositions and kits embodying the invention, may include effector part, which is not a cytokine. It is assumed that the cellular effector component can also be a toxin or otherwise damaging the tumor cytotoxic agent. Thus, other applicable anticancer therapeutic agents may include design between protein fragments of antibodies, which contain at least antigennegative part, and radioactive isotopes, immunotoxin, cytotoxic peptide or cytotoxic drugs.

1) is Italo and radioactive isotope-label, used as anticancer agents. Antigennegative specificity of monoclonal antibodies provides the opportunity for directed localization at tumor sites, while radioactive label provides cytotoxic properties (see Hermanson, G. T., Bioconjugate Techniques (Academic Press, NY NY, 1996) chapter 8). These radioimmunoconjugates may be suitable for use as anticancer therapeutic agents in the methods of the present invention.

2) Immunotoxin-antitelomerase monoclonal antibodies and protein toxins from various sources, also tested for anti-tumor treatment (See. Hermanson, G. T., Bioconjugate Techniques (Academic Press, NY NY, 1996) Chapter 8). Such conjugates may be suitable for use in the methods of the present invention.

3) cytotoxin Drug-antibody

Conjugates of monoclonal antibodies and drug toxins, chemically synthesized and/or purified from a variety of sources, can also be tested for anticancer treatments (See. Hermanson, G. T., Bioconjugate Techniques (Academic Press, NY NY, 1996)). Such conjugates may be suitable for use in the methods of the present invention.

4) Multispecific antibody

Manipulation of fragments of antibodies generated farmacevticheskie antibodies (BsAbs), which are hybrid molecules combining two antibodies with different specificnosti binding. Thus the specific binding to the tumor target can be combined with binding to cellular effector pharmaceutical agent (i.e., cytokine, drug, or toxin) to localize the treatment of tumor cells (See, for example, French, R. R., in Methods in Molecular Biology Vol. 80: Immunochemical Protocols, 2nded. (Pound, J. D. editor, Humana Press, Inc., Totowa NJ, 1998) chapter 12). Such bi - or in another case, multifunctional antibodies may be suitable for use in the methods of the present invention.

2. Therapeutic methods

therapeutic methods of this invention for the treatment of tumor cells in tumors and tumor metastases based on the combined use of inhibiting angiogenesis (antiangiogenic therapy and anti-tumor immunotherapy. More than one type of inhibiting angiogenesis agent may be used in combination with more than one type of antitumor immunotherapy agent. The combined use can be performed simultaneously, sequentially, or with the time interval between treatments. Any specific terapevticheskii the use of inhibiting angiogenesis therapeutic agents and antitumor immunotherapy agents, that can lead to synergistic potenzirovania inhibitory effect on the proliferation of tumor cells of each of the individual therapeutic agents, leading to more effective treatment than the treatment that can be detected by the introduction of only one individual component. Thus, in one aspect, the method of this invention involves the administration to a patient in combination quantities of inhibiting angiogenesis agent, anti-cancer and immuno-therapeutic agent, which may not lead to effective inhibition of angiogenesis or activity against tumor cells when they are separate injection in the same quantities.

The method of this invention provides many ways of impacts in relation to the stages for the application of the invention in practice. For example, the antagonist and anti-tumor immunotherapy agent (such as protein antibody against tumor antigen/cytokine in the preferred embodiment) can be introduced after mixing, i.e., simultaneously, or can be introduced sequentially, i.e., separately. In addition, the antagonist and protein can be entered separately in the time interval of about 3 weeks between injections, i.e. essentially aqueous agent. In addition, it is assumed that the order may vary, i.e., that the antagonistv3can be entered before the introduction of the fused protein, or that the introduction can be carried out in reverse order.

In one embodiment, the method of this invention involves the administration to a patient in need of treatment of a tumor or metastasis, inhibiting amount of inhibiting angiogenesis agent, such as antagonistv3and the number of antitumor immunotherapy agent, sufficient for the induction of biological responses. For example, sufficient for the induction of cytokine-specific biological response where, for example, a bifunctional protein is a cytokine and the polypeptide chain recombinant immunoglobulin (Ig), where this Ig-chain contains a variable region specific for tumor cells, the carrier associated with a tumor antigen on the cell surface, and where Ig chain is associated peptide bond with this cytokine. And when the immunotherapy agent contains a cytotoxic agent, this number may be such that it induces to cytotoxicitekristin in combination with surgical procedures where were removed part of the tumor mass or the entire mass of the tumor. In this case, this method can be applied after the surgical procedure. Alternatively, a surgical procedure may be performed in the interval between introduction of the first active agent and a second active agent. An example of this method is the combination of this method with the following surgical removal of the tumor.

Treatment in accordance with this method will usually provide for the introduction of therapeutic compositions in one or more cycles of administration. For example, if practiced simultaneous introduction of a therapeutic composition comprising the antagonistv3and antitumor immunotherapy agent is administered for a period of time from about 2 days to 3 weeks in a single cycle. After this, the loop can be optionally repeated in accordance with the decision of the practitioner. Similarly, if it is assumed sequential introduction time introduction for each individual therapeutic agent is adapted so that in a typical case, to cover the introduction can be performed using periodic single dose, continuous infusion, etc., Methods of administration may include intravenous, subcutaneous, intramuscular, orthotopic injection, orthotopic infusion, oral administration, etc.,

therapeutic composition used in the method of the present invention contains an active agent in a pharmaceutically acceptable carrier, as is well known, and, therefore, the invention should not be construed as limited in respect of this song up until the concentration of the active agent (active agents) in the composition is sufficient to deliver (introduction) of the indicated active agent described herein.

The usual dose of antitumor immunotherapy agent, such as a fusion protein directed from the antigen/cytokine 0.01-10 mg, preferably approximately 0.1-1 mg, and more preferably about 0.6 mg per kg of body weight per day.

Dose of antitumor immunotherapy agent, such as directed to the antigen cytotoxic agent may be usually 0.01-10 mg, preferably approximately 0.1-1 mg, and more preferably about 0.6 mg per kg of body weight per day, and the dose may be correspond to the I radiation therapy, include 57 cobalt; 67-copper; 99m-technetium, 123-iodide; 131-iodide and 111-indium, etc., Radioactive pharmaceutical agents and doses will vary depending on the radioactive isotope and the target tissue. Immunologia therapy of malignant neoplasm (neoplasms) may use higher doses than conventional radiation therapy because of the immunotherapy agent is directed to the site (the site) of the tumor and/or tumor cell. For example, it was found that treatment of b-cell non-Hodgkin lymphoma radioimmunotherapeutic agent, which is directed at b cells, has the maximum tolerated dose of 0.4 MCI/kg of body weight (1 MCI=37 MBq) (Witzig, THOSE et A1., (1999) "Phase I/II trial of IDEC-Y2B8 radioimmunotherapy for treatment of relapsed or refractory CD20(+) B-cell non-Hodgkin e lymphome", J. Clin.Oncol., 17(12): 3793-3803. However, focusing on a specific tumor or specific tumor metastases radioimmunotherapy can afford higher-tolerated dose than for treatment of lymphoma. Naked muscles tolerated the introduction of two doses of 18.5 MBq of 111-indium-labeled antibodies (Saga T, et A1., (1999) "Radioimmunotherapy of human glioma xenografts in nude mice by indium-111 labelled internalising monoclonal antibody", Eur.J.Cancer, 35(8): 1281-1285).

The typical dose of antagonistv3costela in the day.

It is clear that the cancer is found throughout the animal Kingdom, and that the principles described here apply to all animals, which angiogenesis can be inhibited by the antagonistv3and cytokines in the immune system. Thus, it is expected that this invention can be applied to all animals and, in particular, to the man.

In addition, it is known that a large variety of tumors, which require vascularization for growth and, therefore, are candidates for the mode of action of combination therapy of these methods. Tumors that can lead to growth and inducing angiogenesis, include tumors arising from neuroectodermal, epithelial, etc. fabrics. Examples of tumors and tumor metastases include adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hamartoma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma, etc. of the tumor.

3. therapeutic system

In one embodiment, the present invention races is placed on the practice of the methods of the present invention. Set for treatment of tumor cells in tumors or tumor metastases contains packaging

a) inhibiting angiogenesis agent, such as antagonistv3able to inhibit angiogenesis in tumors or tumor metastases;

b) antitumor immunotherapy agent, such as a bifunctional protein-reagent having cytokine polypeptide and chain recombinant immunoglobulin (Ig), where this Ig-chain contains a variable region specific for tumor cells, the carrier associated with a tumor antigen on the cell surface, where Ig is the chain of linked peptide bond with a cytokine; and

c) instructions for use of these agents in the treatment of tumors and tumor metastases.

The reagent kit of the present invention is usually prepared in the form of a therapeutic composition described herein and, therefore, may be represented in any of many forms, suitable for placing in the set. Such forms may include a composition in the form of liquid, powder, tablets, suspensions, etc. to ensure antagonist and/or a fused protein of the present invention. These reagents may be provided in otive can be provided combined in the composition in a single container in this package.

Similarly, such a package can contain, instead of the above components, or in addition to the above components, any other antitumor immunotherapy agents, such as described above.

The package may contain a quantity sufficient for one or more doses of reagents as described here, the methods of treatment. Usually the package will contain a quantity sufficient for one treatment cycle, as described here. The label may indicate the combined or sequential use of prisoners of reagents for therapeutic treatment of tumors and/or tumor metastases in accordance with the methods of the present invention. This package label can be attached to each bottle with reagents and/or all the packaging materials.

The kit of this invention also contains "instructions for use" of the materials contained in the package. For instructions, refer to the way the joint application of the antagonist and the slit protein for treating tumors or tumor metastases in accordance with the methods of the present invention. Because these methods can vary widely depending on the tumor, the patient and condition of sabreena should not be construed as limiting, depending on the nature of the instructions, other than the specifics regarding the joint application of the antagonist and the slit protein in accordance with the methods of the present invention.

Similarly, the reagents may include antitumor immunotherapy cytotoxic agents, such as binding tumor antigen antibody associated with radioactively labeled isotope or cytotoxic agent such as a cytotoxic peptide or cytotoxic drugs, etc.,

4. Obtaining synthetic peptide

A. The procedure of synthesis

Linear and cyclic polypeptides listed in the table below, was synthesized using standard solid phase synthesis methods, such as described by Merrifield, RB, (1969) "Solid-Phase Peptide Synthesis", Adv. Enzymol.Relat.Areas Mol.Biol., 32:221-296; Merrifield, RB, (1969) "The synthesis of biologically active peptides and proteins", JAMA, 210(7): 1247-1254; and Fields, G. B. and Noble, R. L., (1990) "Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids". Int.J.Peptide Protein Res., 35(3): 161-214.

Two grams (g) of BOC-Gly-DArg-Gly-Asp-Phe-Val-OMe (SEQ ID NO:1) was first dissolved in 60 milliliters (ml) of methanol, to which was added 1.5 ml of 2n sodium hydroxide solution to obtain a mixture. Then this mixture was stirred for 3 h at 20C. After evaporation the residue was placed in water, acidified to pH 3 RA the config BOC-Gly-DArg-Gly-Asp-Phe-Val-OH (SEQ ID NO:2) was stirred at 20C for 2 h with 20 ml of 2n model HC1 in dioxane. The resulting mixture was evaporated to obtaining H-Gly-DArg-Gly-Asp-Phe-Val-OH (SEQ ID NO:3), which is then dissolved in a mixture of 1800 ml of dichloromethane and 200 ml of dimethylformamide (DMF) followed by cooling to 0C. was then added sequentially 0.5 g dicyclohexylcarbodiimide (DCCI), 0.3 g of 1-hydroxybenzotriazole (HOBt) and 0.23 ml of N-methylmorpholine under stirring.

The resulting mixture was stirred additionally for 24 h with 0And then when 20Even within 48 hours. The solution was concentrated and treated mixed ion exchange layer to free it from salts. After removal of the obtained resin clarified by filtration, the solution was evaporated and the residue was purified by chromatography to obtain cyclo(Gly-DArg-Gly-Asp-Phe-Val) (SEQ ID NO:4).

The following peptides listed in table using abbreviations amino acid residues using single-letter code and identified by the symbol number of the peptide was obtained in a similar way: cyclo(Arg-Gly-Asp-DPhe-Val) (SEQ ID NO:5); cyclo(Arg-Ala-Asp-DPhe-Val) (SEQ ID NO:6); cyclo(Arg-DAla-Asp-Phe-Val) (SEQ ID NO:8); cyclo(AGD-Gly-Asp-Phe-to dval) (SEQ ID NO:7) and cyclo(Arg-Gly-Asp-DPhe-NmeVal) (where methylation is an alpha-amino-nitrogen s is ptib 62184, differed from the latter only in salt model HC1, and not TFU-salt present in 62184 (SEQ ID NO:5). The same is true for peptide 69601 and 62185 (SEQ ID NO:6) and for 85189 and 121974 (SEQ ID NO:11).

b. Alternative synthesis procedure

i. Synthesis TFU-salt of cyclo(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11)

Fmoc-Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-ONa (SEQ ID NO:14) are synthesized using solid-phase procedures, the type synthesis of Merrifield sequential addition of NMeVal, DPhe, Asp(OBut), Gly and Fmoc-Arg(Mtr) stepwise manner to 4-hydroxyethylnitrosamine resin (resin type Wang) (apply the usual methods of peptide synthesis type synthesis of Merrifield). Polystyrene resin and precursor amino acid residues are commercially available from chemical companies Aldrich, Sigma or Fluka. After successive addition of amino acid residues resin is then separated from the peptide chain using a mixture of 1:1 TFU/dichloromethane, which provides the product Fmoc-Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH (SEQ ID NO:15). Then Fmoc group is removed with a mixture of 1:1 piperidine/DMF, which provides a crude predecessor Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH (SEQ ID NO:16), which is then purified using HPLC accepted way.

For cyclization solution of 0.6 g Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH (synthesized above) (SEQ ID NO:16) in 15 ml DMF ice/acetone add 40 ál diphenylphosphinite (Aid-rich). After standing at room temperature for 16 h, the solution concentrate. The concentrate is subjected to gel filtration (column Sephadex G10 in a mixture of isopropanol/water 8:1) and then purified using HPLC accepted way. Processing TFU (triperoxonane acid/N2Oh (98:2) gives cyclo(Arg-Gly-Asp-DPhe-NMeVal) (also referred to here as the "cyclo(RGDfN-MeV)"; SEQ ID NO:11) x TFU, which is then purified using HPLC accepted way; RT=19,5; FAB-MS (M+H):589.

ii. Synthesis of "inner salts"

TFU-salt is removed from the obtained as described above cyclic peptide by suspendirovanie cyclo(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11) x TFU in water, followed by evaporation under vacuum to remove TFU. Educated cyclic peptide called "inner salt", and he identified as cyclo(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11). Use the term "internal salt", as this cyclic peptide contains two oppositely charged residue, which vnutrielitnogo protivorechivaya each other with education in General uncharged molecules. One of the charged residues is the acid portion of the molecule, and the other charged residue contains amino-part. When the acid portion of the molecule and amino-part are in close proximity to each other, the acid portion of the molecule may be the charge.

iii. HCl-processing with the formation of cyclo(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11)HCl

80 mg cyclo (Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11) was dissolved in 0.01 M HCl five or six times and lyophilized after each dilution. Subsequent purification using HPLC gives cyclo(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11)HCl; FAB MS (M+H):589.

iv. Processing methanesulfonic acid with the formation of cyclo(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11)SO3N

80 mg cyclo(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11) was dissolved in 0.01 M MeSOsH (methanesulfonate acid) five or six times and lyophilized after each dilution. Subsequent purification using HPLC gives cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (SEQ ID NO:11)MeSO3H; RT=17,8; FAB MS (M+H):589.

Alternative ways of cyclization include the derivatization of the side chains groups acyclic peptide precursor sulfhydryl parts of the molecule which, when exposed to pH conditions, slightly higher than the normal physiological pH (pH 7.5), form an intramolecular disulfide bond with other sulfhydryl groups present in the molecule, with the formation of the cyclic peptide. In addition, the C-terminal carboxylate portion of the acyclic peptide precursors is of containing the thioester bond cyklinowanie peptides.

5. Generation and characterization of fused protein tumor-specific antibody-cytokine

A. Proteins and specific for vascular network integrin antagonistv

Design and characterization of fused proteins antibody-cytokine ch14.18-IL-2 and huKS1/4-IL-2 were described previously (Xiang, R., et al., (1997); Gillies, S., et al., (1992) supra). Antigennegative characteristics of both structures were identical to the characteristics of their respective specific antibodies and EL-2 activity was equivalent to the activity of commercially available rhIL-2. Were synthesized and characterized antagonistic cyclic peptide integrinv3121974 (cyclo(RGDfN-MeV)) (SEQ ID NO:11) and the control peptide 135981 (cyclo(RADfN-MeV) (SEQ ID NO:13).

6. Cell lines and animal models

All cell lines and corresponding animal models were established essentially as described previously (Becker, J. C., et al., (1995); Xiang, R., et al., (1996); Lode, H. N., et al., (1996) supra). The absence of integrinv3on the NXS2 cells and CT26-KSA demonstrated using antibodies against mouse CD61 (chain integrin3) (Pharmingen, La Jolla, the falsity of the integrinv3-positive cells in murine melanoma B16FG3 and B78-D14 used as positive controls. In addition, NXS2 cells were unable to attach to plastic coated with mAB against mouse CD61 (10 μg/ml, 4C, 24 h), in contrast to antibody against GD2ch14.18 (10 μg/ml, 4C, 24 h), used as positive control. However, all tumor cells Express integrinvaccording to FACS, and are attached to vitronectin that indicates the presence of integrinv3.

For all surgical procedures mice were anestesiologi injection of ketamine (100 mg/kg i.p.) and while inhalation of metofane (Pitman-Moore, Munlelein, IL).

Osmotic pumps (Alzet, model 2001, Palo Alto, CA) for introducing an antagonist of integrinvand the control peptide used to speed delivery of 17.5 µg/H. These pumps are manipulated in accordance with the manufacturer's instructions and implanted in the subcutaneous tissue of the back, under sterile conditions. All pumps replaced at day 7 after implantation and were removed at day 10 the care and use of laboratory animals of the National Institute of Health, USA (NIH).

7. Histology and immunohistochemistry

Fixed with acetone, frozen sections of primary tumors were incubated with 4% goat serum to block nonspecific binding. Incubation with mAb against mouse CD31 and anti mouse CD45 (Pharmingen, La Jolla, CA) (1:100) and subsequent staining labeled with rhodamine goat Anticriminal antibody (1:300) was performed in a humidified chamber at room temperature. For each incubation was followed by washing SFR (3). The number of vessels and leukocytes in the field of view of the microscope with high magnification (HPF) was determined microscopically at a magnification 200x (Brooks, P. C., et al.,(1995) "Anti-integrin alpha v beta 3 blocks human breast cancer growth and angiogenes in human skin", J. Clin.Inves., 96, 1815-1822). Representative areas were photographed at 200x (vessels) and only 800 meters (leukocytes), respectively.

8. The reduction of primary tumors only in mice treated with an antagonist of integrinvcombined with the merged protein antibody-IL-2

The synergistic action of inhibiting angiogenesis therapy (integrin antagonistv) and immunotherapy (slit proteins antibody-IL-2) was determined in mice with established subcutaneous tumors (110-130 mm3in all three syngeneic models, sootvetstvennovand antitumor immunotherapy compartment-specific immunotherapy with fused protein antibody-IL-2 in the primary tumors. Figure 1A shows the results obtained in the primary tumors induced by subcutaneous injection (2106) NXS2 neuroblastoma. The figure 1B shows the results on the primary tumors induced by subcutaneous injection (2106) carcinoma of the colon ST-KSA. Figure 1C shows the results obtained from primary tumors induced by subcutaneous injection (2106) cells melanoma B78-D 14. Treatment of established tumors (110-130 mm3) began intravenous injection once a day fused proteins tumor-specific antibody-IL-2 huKSI/4-IL-2 (10 μg, carcinoma of the colon) and ch.l4.18-IL-2 (5 ág, neuroblastoma, 10 μg, melanoma) (5) and continuous subcutaneous infusion of the antagonist specific for vascular network integrinvor control peptide osmotic pump within 7 days of 17.5 µg/h (upper graph). The start time of the processing is indicated by a black arrow. The size of the primary tumors in the mouse(Averagethe standard error). The reduction in size of the primary tumor mice receiving the combined treatment, in comparison with the size of established tumors at the time of starting treatment was statistically significant in three different syngeneic models of tumors (P<0,001, criterion rank-sum Wilkinson) in contrast with all the controls (P>0,05).

First set of suboptimal quantities for each therapeutic method of treatment and began their subsequent use in combination. Only mice treated with an antagonist of integrinvand fused proteins with IL-2, was found to reduce tumors in all three models in the range from 59 to 90% (P<0,001). In fact, half of the animals inoculated with neuroblastoma cells and carcinoma of the colon, completely rejected the primary tumor, data not shown. In contrast, each strategy used in monotherapy, in the best case, delayed growth compared with the control group, respectively. Then analyzed the effect of each processing method and their combination on vascular and tumor compartment.

Performed the histology after combined antiangiogenic and tumor is tan 20 days after inoculation of tumor cells. Briefly, formalin fixed primary tumor was subjected to fill in paraffin and subsequent staining with hematoxylin/eosin. Identified necrotic areas and infiltrates of leukocytes.

Figure 2 graphically depicts the action of the United antivascular and antitumor therapy on the vascularization and antitumor immune response. Mice (n=6) with the established primary neuroblastoma tumors received combination treatment specific for vascular network integrin her antagonist, non-specific control peptide and tumor-specific fused protein ch14.18-IL-2 as described for figure 1, including the controls, which received each treatment separately. At the end of the treatment's.with.-the tumor was removed surgically. Frozen sections of each tumor were analyzed immunohistochemically using antibodies specific against endothelial cells of blood vessels (CD-31) and infiltration of leukocytes (CD45), respectively. The latter is a well-established marker for specific tumor compartment immune response induced fused protein ch14.18-IL-2 (Becker, J. C., et al., (1996), supra; Xiang, R, et al., (1996), supra; Lode, H. N., et al., (1998), supra).

In figure 2A the image is tion and tumor compartment or an antagonist of integrinvor fused protein chl4.18-IL-2, or their combination (*P<0,001, T-student test). Figure 2B shows the results of the infiltration of leukocytes primary tumors after treatment of vascular and tumor compartments, respectively (*P<0,001, T-student test).

In mice receiving the integrin antagonistvidentified 50% reduction in vascularization (figure 2), coinciding with the stunted growth of primary tumors, demonstrating effective focus on vascular compartment. In this case, the tumor compartment was not direct action (figure 1). In contrast, in mice treated only fused protein antibody against GD2-IL-2, showed an obvious leukocyte infiltration, a well-established characteristic of this directed against tumor compartment therapy (Becker, J. C., et al., (1996), supra; Xiang, R., (1996), supra; Lode, H. N., et al., (1998), supra), resulting in a significant decrease in growth s.with.-the tumor (figure 1).

However, only mice treated with the combination of an antagonist of integrinvand fused protein anti-GD2-IL-2, found a five-fold increase in the infiltration of leukocytes into the tumor, in comparison with mice treated only what was shown histologically and immunohistochemically and was attributed to the influx of macrophages, picture, often observed in necrotic tissues during removal of cellular debris. In fact, these necrotic areas were present only in tumors after combinatorial treatment, in contrast to controls, treated each component separately.

9. Sequential and simultaneous vascular and tumor directed induces the destruction of spontaneous liver metastases

In addition to the successful treatment of primary tumors, a more pertinent question is the question, are distant metastases impact of this strategy combined antivascular and specific antitumor therapy. This was explained on the model of neuroblastoma, which is characterized by spontaneous liver metastases. For this purpose the treatment of the primary tumor angiogenic integrin antagonistvconsistently combined with antitumor immunotherapy slit protein antibody-IL-2.

Figure 3 graphically depicts the effect of sequential combinations of antiangiogenic integrin antagonistvand specific tumor compartment immunotherapy with fused protein antibody-IL-2 on spontaneous met what ol, as described for figure 1, for 10 days in General. After surgical removal of primary tumors, mice received specific tumor compartment immunotherapy by injection once a day 5 mg fused protein ch14.18-IL-2 (5). The number of spontaneous metastases in the liver was determined by macroscopic score foci in the liver (n=8) (**P<0,01, criterion rank-sum Wilcoxon signed).

Only in mice treated consistently by both agents, found 1.5 to 2 log reduction of metastases in the liver, in contrast with all the controls, where the processing of each agent used in monotherapy was ineffective (P<0,01) (figure 3). In fact, 4/8 mice subjected to combined therapy, revealed the complete absence of liver metastases, whereas the remaining animals found only 1-5 small metastatic lesions. Essentially similar results were obtained by simultaneous combinations of integrin antagonistvwith the merged protein chl4.18-IL-2 (upper part of figure 4).

The figure 4 shows the effect of simultaneous combinations of antiangiogenic integrin antagonistvand specific tumors of the Yeni. Spontaneous metastases induced after induction of primary tumors's.c. injection 2106cells NXS2 neuroblastoma. Treatment with integrin or antagonist (17,5 mg/h) and opukholespetsificheskaya fused protein ch14.18-IL-2 (5 mg5), was started before (figure 4A) or after (figure 4B) removal of the primary tumor. Spontaneous metastases in the liver was determined by macroscopic score foci in the liver (n=8) (**P<0,01, criterion rank-sum Wilcoxon signed).

Only in mice treated with both agents showed either complete absence (figure 4A) or >a 1.5 log reduction (figure 4B) liver metastasis, depending on their introduction before or after removal of primary tumors. In contrast, all controls when each agent used in monotherapy, the treatment was ineffective.

10. The synergistic combination and effective therapy

Destruction of blood vessels in the vascular compartment of malignant tumors is a powerful strategy for cancer control. The direction of the endothelial cells of the vascular network of tumors can be treated successfully. Peptide antagonist that affects vascular network through interaction with integrinsvvfirst and foremost was sent tov3it communicates also with the closely related integrinv5. Tested tumors carcinoma of the colon and neuroblastoma explicitly deniedv3but, probably, Express a certain amount ofv5. The model of melanoma also expressesv5. However, the effect of this antagonist of integrin was clearly limited tumor vasculature in all three animal models, as shown for the model of neuroblastoma (figure 2). It is important that the antitumor effect of targeting tumor vascular network is enhanced by the simultaneous attack on the compartment of the tumor that is effective against primary tumors and against spontaneous metastases. This is especially appropriate, as the eve ENT, well documented on other models of tumors, due to a decrease in circulating blood levels of inhibitors of angiogenesis after excision of the primary tumor (Holmgren, L., et al., (1995), supra; Folkman, J., (1995), supra). Simultaneous targeting vascular and tumor compartments have proven to be very effective because it combines the reducing power of tumor cells with active destruction of tumor cells, leading to regression of primary tumors and the elimination of remote Metastasio. In contrast, the approach only on vascular compartment, which uses two different strategies antiangiogenic treatments led to suppression of growth s.with.-tumors in syngeneic model (Mauceri, H. J., et al., (1998) "Combined effects of angiostatin and ionizing radiation in antitumor therapy", Nature, 394, 287-291).

In this strategy, specific tumor compartment response is mediated by inflammatory cells, which are activated and sent to the microenvironment of the tumor fused proteins tumor-specific antibody-IL-2. It is important that antiangiogenic strategy, although it is effective in suppressing the growth of primary tumors with well-developed blood supply, has a similar efficacy against from the enough disease with low tumor load, characterized by poor vascularization, branch processing antitumor compartment used in combination therapy is quite effective when used as monotherapy (Xiang, R., et al., (1997), supra; Lode, H. N., et al., (1998), supra). In this situation the role of antiangiogenic treatment is the suppression induced by micrometastasis revascularization and subsequent increase of metastatic foci (Volpert, O. V., et al., (1998), supra). This, in turn, facilitates the elimination of such metastases aimed at the tumor compartment therapy, which is optimally effective in conditions of minimal residual disease (Becker, J. C., et al., (1996), supra).

Effective treatment of primary tumors and disseminated metastasis remains a major problem in clinical Oncology. The results of this message show that the combination of specific antiangiogenic and immunotherapy act synergistically on the primary tumor, causing them to regress and the elimination of metastases. Since both ways of processing, i.e., antagonists of integrinvand fused proteins antibody-interleukin-2, are currently undergoing clinical evaluation as mo the e examples describe some variants of this invention are illustrative and, of course, should not be construed as specifically limiting the invention. In addition, such variations of the present invention, now known or later developed, which are in the competence of the person skilled in the art should be considered within the scope of the present invention, shown attached claims.

Claims

1. A method for the treatment of neuroectodermal or epithelial tumor in a patient, comprising the introduction of a specified patient inhibiting the proliferation of neuroectodermal or epithelial tumor cell number (a) agent that inhibits angiogenesis and which is the antagonistv3; and b) antitumor immunotherapy agent address associated with tumor antigen and containing the variable region, which is associated with associated with tumor target antigen.

2. The method according to p. 1, where the specified agent, inhibiting angiogenesis, and the anticancer immunotherapy agent is administered essentially simultaneously.

3. The method according to p. 1, where the specified agent, inhibiting angiogenesis, and the anticancer immunotherapy agent is administered sequentially in a time interval of about 3 weeks.

4. The method according to p. 3, where the specified agent, inhibiting angiogenesis, injected before a specific antitumor immunotherapy agent.

5. The method according to p. 3, where specified antitumor immunotherapy agent is administered before the specified agent, inhibiting angiogenesis.

6. The method according to p. 3, where the aforementioned introduction is performed in a time when the tumor or tumor metastases surgically remove the specified patient during a specified time interval.

7. The method according to p. 1, where the aforementioned introduction perform after the tumor or tumor metastases were surgically removed in the specified patient.

8. The method according to p. 1, where the specified agent, inhibiting angiogenesis, is administered at a dose of 10-1000 mg per kilogram of body weight per day.

9. The method according to p. 1, where the specified antitumor https://img.russianpatents.com/chr/945.gif">v3selected from the group consisting of a peptide, RGD-containing peptide, monoclonal antibodies againstv3monoclonal antibodies against receptorv3and mimeticav3.

11. The method according to p. 10, where specified RGD-containing peptide has the sequence of amino acid residues cyclo(RGDfN-V) (SEQ ID NO:11).

12. The method according to p. 1, where the indicated cytokine selected from the group consisting of BDNF, CNTF, EGF, Epo, FGF, Flt3L, G-CSF, GM-CSF, I-309/TCA-3, gamma IP-10, IFN-alpha, IFN-beta, IFN - gamma, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, LT, MCP-1, MCP-2, MCP-3, M-CSF, MIF, MIP-1-alpha, MIP-1-beta, MIP-2, NGF, NT-3, NT-4, OSM, PBP, PBSF, PGFD, PF-4, RANTES, SCF, TGF-alpha, TGF-beta, TNF-alpha, TPO and VEGF.

13. The method according to p. 1, where the indicated cytokine is a chemokine selected from the group consisting of 10, EMF-1, ENA-78, eotaxin, GCP-2, HCC-1, 1-309, IL-8, IP-10, lymphotactin, MCP-1, MCP-2, MCP-3, MGSA, MIG, MIP-1-alpha, MIP-1-beta, MIP-2, NAP-2, PF4, RANTES, SCM-1 and SDF-1.

14. The method according to p. 1, where the specified associated with tumor antigen target selected from the group consisting of AFP, CA 125, CEA, CD 19, CD20, CD44, CD45, EGF-receptor, GD2, GD3, GM1 is the transferrin receptor.

15. The method according to p. 1, wherein the specified antitumor immunotherapy agent contains the cytokine IL-2 and a heavy chain Ig, which is immune reacts with associated with tumor antigen GD2.

16. The method according to p. 1, where the specified antitumor immunotherapy agent contains the cytokine IL-2 and a heavy chain Ig, which is immune reacts with associated with tumor antigen KSA (EP-HIMSELF; KS1/4 antigen).

17. A therapeutic composition comprising at least one agent which inhibits angiogenesis and which is the antagonistv3and at least one anticancer immunotherapy agent containing cytokine component cells-effector component and immunoglobulin (Ig) polypeptide, aimed at associated with tumor antigen and containing a variable region that binds to associate with tumor target antigen, and the anticancer immunotherapy agent is the slit protein which contains the cytokine IL-2 and a heavy chain Ig, which is immune reacts with associated with tumor antigen GD2or KSA antigen (EP-HIMSELF; KS1/4 antigen); and where specified, associated with the CCCs.

18. Therapeutic composition for p. 17, containing (a) an amount of at least one inhibiting angiogenesis agent, sufficient for inhibition of angiogenesis in tumors or tumor metastases; and (b) the amount of at least one anticancer immunotherapy agent, sufficient for the induction of biological responses.

19. The composition according to p. 17, where the specified antagonistv3selected from the group consisting of a peptide, RGD-containing peptide, monoclonal antibodies againstv3monoclonal antibodies against receptorv3and mimeticav3.

20. The composition according to p. 19, where specified, the antagonist is an RGD-containing peptide having the sequence of amino acid residues cyclo(RGDfN-MeV) (SEQ ID NO:11).

21. Set for the treatment of neuroectodermal or epithelial tumors or tumor metastases containing package comprising (a) an agent which inhibits angiogenesis and which is the antagonistv2or KSA antigen (EP-HIMSELF; KS1/4 antigen); and where the specified associated with tumor antigen target is from the tumor cells with neuroectodermal or epithelial nature.

22. Set on p. 21, where the specified antagonistv3selected from the group consisting of a peptide, RGD-containing peptide, monoclonal antibodies againstv3monoclonal antibodies against receptorv3and mimeticav3.

23. Set on p. 22, where the specified RGD-containing peptide is a peptide, and the inhibiting angiogenesis, and the specified antitumor immunotherapy agent placed in separate containers in the specified package.

25. Set on p. 21, where the specified agent, inhibiting angiogenesis, and the anticancer immunotherapy agent are combined in one container at the specified package.

26. Set on p. 21, where the specified agent, inhibiting angiogenesis, and the anticancer immunotherapy agent are combined in one container at the specified packaging and have a label indication for combined use.

 

Same patents:

The invention relates to new peptides having the amino acid sequence of the 9-55 amino acid residues, comprising the amino acid sequence FTLASAETT (SEQ ID NO:l), pharmaceutical compositions for treatment of autoimmune diseases, containing one or more of these peptides and a pharmaceutically acceptable carrier

The invention relates to a LHRH antagonists - compounds of General formula Iin which a represents acetyl or 3-(4-forfinal)propionyloxy group Xxx1mean D-Nal(1) or D-Nal(2), Xxx2-Xxx3mean D-Cpa-D-Pal(3) or a simple link, Xxx4means Ser, Xxx5means N-Me-Tyr, Xxx6mean D-Hci or a residue of D-amino acids of General formula (II)

where n means the number 3 or 4, a R1means a group of the General formula IIIwhere R denotes an integer from 1 to 4, R2means hydrogen or alkyl group, and R3means unsubstituted or substituted aryl group or heteroaryl group, or R1mean 3-amino-1,2,4-triazole-5-carbonyl group,Xxx7means Leu or Nle, Xxx8means Arg or Lys(iPr), Xxx9means Pro and Xxx10means A1A or Sar, and their salts with pharmaceutically acceptable acids: process for the preparation of these compounds, pharmaceutical compositions having the properties of an LHRH antagonist, comprising as an active narushenie compounds according to the invention has a high solubility in water

The invention relates to biotechnology and can be used to obtain a polypeptide with IL-10 similar properties

The invention relates to a method of producing tridecapeptide formula I: H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH and aims to simplify the process and increase the yield of the target product, as well as Pentapeptide formula II: X-His(X)-Gly-Val-Ser(Y)-Gly-OH, which is an intermediate compound in the synthesis

The invention relates to oligopeptides comprising the amino acid sequence of the B-X-X-X-B-X-X-X-J-Tyr, which is Lys or Arg; X represents any amino acid, other than charged aliphatic amino acid or its D-isomer; and J is Gly, Lys or Arg; these amino acids are found in nature L-isomer, D-isomer and norleucine, and where specified Oligopeptide includes other than (a) naturally occurring sequencedomain antigen In human leukocyte (HLA-B) 75-84, (b) a naturally occurring sequence of the transmembrane sequence-chain T-cell receptor of human rights and (in) consistency, which is the mutant with respect to either (a) or (b) having no more than two mutations as an active part of the above oligopeptides; the method of suppressing the activation limfozitah cells, the method of transplantation of donor organs or mammalian cells, methods of inhibiting protein synthesis of inflammatory cytokines by cells capable of producing the specified protein inflammatory cytokine, the method of suppressing an inflammatory response in a mammal, the method of modulation of the activity hemade the
The invention relates to a group of new protected linear peptides containing the amino acid sequence, which can be used as starting compounds to obtain RGD-containing cyclopeptides, the General formula

R3-Arg-Gly-Asp(OR1)-OR2,

where R1is benzyl or tert-butyl; R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl; R3is a hydrogen atom or a urethane protective group of the B1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues; and the peptides, where R3- peptidyl structure E-Z-Y-X-, in which E is a hydrogen atom or a urethane protective group IN2O-CO-, where2not equal to R1and can take values tert-butyl; benzyl; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophen is IN3O-CO-, in which3= R1attached to the omega-amino group; Z can take values Phe or D-Phe

The invention relates to a new compound - peptide of General formula Thr-Gly-Glu-Asn-His-Arg, possessing the biological activity of inducing differentiation and inhibiting cell proliferation in tumors and activity of the tread and the normalizing action on the vital processes of mammalian cells obtained by the method of solid-phase peptide synthesis by sequential growth of the peptide chain and having the following properties: mol

The invention relates to peptides of General formula a-B-C-D-Pro-Gly-Pro-X, where A - O-Met(O), -Met, -Thr, -Ala, -Lys-Gly, -Glu, -Arg, -His, -Phe -,- Tyr, -Trp, B - O, -Glu, -Lys, -Tyr, -Gly, -Arg-Val-Pro; - O, -His, -Pro-Asp-Gly, -Arg, -Tyr, -Val-Phe; D - O-Phe, -His, -Arg, -Lys, -Ala, -Tyr, -Thr, -Pro-Ile; X - O-Ilе, highly neurotropic activity (refers to the lack of amino acids), provided that excluded peptides: Glu-His-Phe-Pro-Gly-Pro, Thr-Lys-Pro-Arg-Pro-Gly-Pro, Tyr-Pro-Phe-Pro-Gly-Pro-Ile and Met-Glu-His-Phe-Pro-Gly-Pro

The invention relates to peptides-immunoregulators and their therapeutic use

The invention relates to the medical industry and refers to polypeptides that are specific against CD19 and CD3, and their application

Improved vaccines // 2214279
The invention relates to medicine and is vaccine conjugates, immunogenic polypeptides, encoding immunogenic polypeptides molecules of nucleic acids, cell host containing encoding immunogenic polypeptides molecules of nucleic acids, and methods of obtaining the vaccine conjugates and immunogenic polypeptides as well as encoding immunogenic polypeptides molecules of nucleic acids

The invention relates to medicine

The invention relates to the field of medicine and relates to a composition for inhibition of angiogenesis, monoclonal antibodies, polypeptide and method of inhibiting tumor growth

The invention relates to biotechnology and can be used to create a fused protein fragments of antibodies with bi - or multifunctional binding sites

The invention relates to film-coated tablets comprising as active compounds cyclophosphamide and containing core tablets cyclophosphamide, one or more fillers, one or more dehumidifiers-binding except for the swollen starch, regulator fluidity and sizing

The invention relates to the field of medicine and relates to a stable form of anti-cancer drug containing paclitaxel, and how it is received by dissolving crystalline paclitaxel in a neutral organic solvent selected from the group consisting of acetonitrile, dioxane, ethanol or mixtures thereof, provided that the contents of the individual solvents in the mixture is in the range from 5 to 95%, whereas the water content is in the range from 0 to 60%, optionally filtering the resulting solution, freezing and removal of the solvent by sublimation under reduced pressure at low temperature, and optional separation of dose in terms providing sterility

The invention relates to compositions for oral administration of paclitaxel and related taxan patients and methods of treatment using such compositions

The invention relates to a polypeptide (I), a mutant protein of IL-2 people with numbering according to IL-2 wild-type human IL-2 is substituted by at least one position, 20, 88 or 126, allowing the specified mutant protein activates T-cells, preferably before natural killer (NK) cells; pharmaceutical composition having immunostimulatory activity, comprising the polypeptide of I; polynucleotide, representing a DNA sequence encoding a mutant protein of IL-2 persons; vector pBC1IL 2SA for expression of mutant protein IL-2 persons; line ovary cells of Chinese hamsters; cell line of African green monkeys, the E. coli strain, cell line Spodoptera fugiperda, transformirovannykh vector pBC1IL 2SA that produce mutant protein IL-2; the method of treating mammals suffering from oncological diseases, as well as to method of selection of mutant proteins IL-2 assessment in studies using IL-2Rin comparison with IL-2Rwhere the activity of mutant b is
Up!