Application of heat-shock proteins for improving effectiveness of antibody therapies

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and aims at treatment of cancer in an individual. It involves introduction to the specified individual of amount of purified heat-shock protein (HSP) preparation. The purified HSP preparation contains purified HSP-peptide complexes containing HSP covalently or non-covalently attached to peptide and where HSP-peptide complexes express antigenicity of said cancer. In addition, an immunogenic reagent is introduced to an individual, where the immunogenic reagent specifically reacts with antigen chosen from the group consisting of VEGF, EGF-R, HER2/NEU, CD25 and CD20, or is being anti-CTLA-4 or anti 41BB antibody.

EFFECT: combined introduction of HSP-peptide complexes and immunogenic reagent allows intensifying immune response in antibody therapy of cancer.

36 cl, 4 ex, 2 dwg

 

Description

This invention claims priority of the provisional application for U.S. patent No. 60/377483, filed may 2, 2002, which is incorporated into this description by reference in its entirety.

1.The scope of the invention

The present invention relates to methods and pharmaceutical compositions useful for the prevention and treatment of any disease where treatment can be made more effective by strengthening the immune response, such as infectious diseases, primary and metastatic neoplastic diseases (i.e. cancer) or neurodegenerative or amyloid disease. In particular, the invention relates to methods, including the introduction of heat shock proteins/stress (HSP or HSP complexes, alone or in combination with each other, in combination with the introduction of the immunoreactive reagent. The invention also provides pharmaceutical compositions containing one or more HSP or HSP complexes in combination with an immunoreactive reagent. In addition, the invention provides for the use of the methods and compositions according to this invention to improve the efficiency or improve passive immunotherapy and functions of effector cells.

2.Background of the invention

2.1Immune response

The immune system of the body that is resulted in two types of reactions to pathogens or other harmful agents humoral response and cell-mediated response (see Alberts, B. et al., 1994, Molecular Biology of the Cell. 1195-96). When resting b cells are activated by antigen proliferation and maturation in antitelomerase cells, they produce and secrete antibodies with unique antigennegative site. This secreting antibody response is known as the humoral response. On the other hand, other reactions T cells collectively referred to as cell-mediated immune reactions. There are two major classes of T cells - cytotoxic T cells and helper T cells. Cytotoxic T cells directly kill cells that are infected by a virus or some other intracellular microorganisms. Helper T-cells, on the contrary, help to stimulate reactions from other cells: they help activate macrophages, dendritic cells and b-cells (see Alberts, B. et al., 1994, Molecular Biology of the Cell, 1228). As cytotoxic T cells and helper T cells recognize antigen in the form of peptide fragments that are generated by the degradation of foreign protein antigens inside the target cells, and both cell types, thus, depend on the molecules of the major histocompatibility complex (MHC), which bind these peptide fragments, carry them to the cell surface and presenterat here T-cells (see Alberts, B. et al. 1994, Molecular Biology of the Cell, 1228). The MHC molecules are usually many on antigen presenting cells (APC).

In addition to acquired immunity, discussed above, innate immunity also plays a role in the immune response of the body. The innate immune system is the first line of immune defense against disease and provides a broad, but relatively nonspecific immune defense of the host, which deprived the properties of antigenic specificity and immunological memory, which characterize the acquired immunity. Effector mechanisms of innate immunity include antimicrobial peptides, granulocytes and phagocytes, natural killer cells, dendritic cells and alternative complementry path, which interact with the adaptive immune responses and control them. Medzhitov and Janeway, 2000, New England J. Med 343: 338-344; floors are only, 2002, Nature Reviews Immunology 2: 957-964.

2.2Presentation of antigen

Antigenpresenting cells (APC)such as macrophages and dendritic cells are key components of innate and acquired immune responses. Antigens usually the T-cells on the surface of other cells, APC. APC can catch migrate to the lymph and blood antigens and after internalization and degradation to presentation of antigenic peptide fragments associated with cellular molecules on top of the spine of the major histocompatibility complex (MHC), T-cells. ARS can then activate T-cells (cell-mediated response) with clonal expansion, and these daughter cells can develop either cytotoxic T cells or helper T cells, which, in turn, activate b-cells (humoral response) the same MHC-associated antigen with clonal expansion and production of specific antibodies (see Alberts, B et al., 1994, Molecular Biology of the Cell. 1238-45).

Found two types antigenprocessing mechanisms. The first type includes the absorption of proteins by endocytosis cells ARS, fragmentation antigens in the vesicles, the Association with molecules of MHC class II expression on the cell surface. This complex is recognized by helper T-cells expressing CD4. The other type is used for proteins, such as viral antigens that are synthesized in the cells and, apparently, includes fragmentation of proteins in the cytoplasm. Peptides produced thus become associated with MHC molecules of class I and recognized by cytotoxic T-cells expressing CD8 (see Alberts, B. et al., 1994, Molecular Biology of the Cell. 1233-34).

Stimulation of T cells includes a number of additional molecules, expressed as T-cells and APC. Co-stimulatory molecules are the molecules that stimulate the growth and activation of T cells. PR is the stimulation of co-stimulatory molecules induce the release of cytokines, such as interleukin 1 (IL-1) or interleukin 2 (IL-2), interferon, etc. that stimulate the growth of T cells and the expression of surface receptors (see Paul, 1989, Fundamental Immunology. 109-10).

APC usually are dormant, and for their operation requires activation. The identity of the signals that activate the APC is a critical and unresolved issue (see Banchereau, et al., 1998, Nature 392: 245-252; Medzhitov, et al., 1998, Curr. ORP. Immunol. 10: 12-15).

2.3Passive immunotherapy

Passive immunotherapy (also called passive immunization) refers to the introduction of immunoreactive reagent, such as molecules containing antigennegative district directed against epitopes on a pathogen, tumor or pathogen protein and the domain Fc-receptornegative district, complimentative district or area, which mediates the action of effector cells, such as antibodies, directly to the patient. Immunoreactive reagent can be injected prophylactically, for example to inhibit infection, or therapeutically to reduce or eliminate the infection, to lower or eliminate cancer cells or for deducing or removing pathogenic proteins, such as protein aggregates or deposits that occur in neurodegenerative and/or amyloidogenic disease. Passive immunotherapy is different from the active IMM is Nataraja, which includes immunization of the patient antigen to induce an in vivo immune response, for example for the production of antibodies or cytotoxic T-lymphocytes. In contrast, passive immunotherapy immunoreactive reagent, for example an antibody, is administered to the patients and this leads to stimulation of effector cells, such as cells with Fc receptors capable of interacting with the Fc-part (i.e. the realm Fc-receptor) is added to the antibody or other immunoreactive reagent that leads to cellular immune functions such as antibody-dependent cellular cytotoxicity (e.g., ADCC) or antiteloobrazovania opsonization and/or phagocytosis, directed against cells of the pathogen or protein having an epitope recognized by the antibody. Efficiency antitelomerase therapy of a tumor, which depends on the effector functions FcR-cells, can be modified through the use of specific cytokines; Keller, et al., 2000, J. Immunol. 164:5746-5752. Therapeutic and/or prophylactic antibodies include, but are not limited to, antibodies that bind to molecules on the cell surface and are opposed to normal functions (e.g., blocking antibodies), antibodies that bind to molecules on the cell surface and mimic the normal function (antibodies-honesty), and requestrefuse antibodies.

2.4CTLA-4

Cytotoxic T-limfotsity antigen-4 (CTLA-4) is a glycoprotein which is expressed on the surface of activated T cells at low levels. CTLA-4 is similar to CD28 and has a higher binding affinity of for family members V7 (e.g., B7-1 and B7-2)than CD28. The binding of CTLA-4 on T cells with B7-ligand mediates a negative signal, which inhibits the secretion of IL-2 and cell proliferation. In the end, CTLA-4-mediated inhibition of activation of T cells leads to "off" adjustable T-cell mediated immune reactions, especially in the early events in the activation of T cells (Brunner, et al., 1999 J. Immunol. 162: 5813-5820).

It was recently described that the blockade of the function of CTLA-4 by treatment with antibodies against CTLA-4 resulted in the strengthening of various immune responses and contributed to the induction of tumor immunity (Leach, et al., 1996, Science 271: 1734-1736; PCT publication WO 00/322231 and WO 01/14424). In mice transplanted with B16 melanoma cells were found regression of tumors and elevated levels of CD8+-T-cells by combination treatment with monoclonal antibodies (mAb) against CTLA-4 and vaccine GM-CSF-producing tumor cells. It was noted that the application of this combinational treatment with prophylactic option (i.e. before the introduction of tumor cells) led to complete protection even in the absence of the influence of CD8 +-T-cells. The data demonstrated that therapeutic self-reactive CD8+T-cells can be induced from having a tumor mice (Van Elsas et al., 2001, J. Exp. Med. 194: 481-489). Co-administration of antibodies against CTLA-4 in combination with a vaccine tumor cells producing GM-CSF, have demonstrated efficacy against established melanoma cells B16-BL6, but little effect was observed when it was applied to only one of therapies (Van Elsas et al., 1999, J. Exp. Med. 190: 355-366). In addition, it was also observed that blockade of CTLA-4 correlates with the increase of helper functions and induced amplification of CD4+-T-cells (Hernandez, et al., 2001, J. Immun. 3908-3914). It was also noted that the blockade by administration of antibodies against CTLA-4 resulted in increased host resistance to the intracellular pathogen, the increase in the number of IFN-g and IL-4-producing cells in the liver and spleen and enhanced, resulting hepatic granulomatous reaction (Murphy, et al., 1998, J. Immun. 4153-4160).

2.5Heat shock proteins

Heat shock proteins (HSP), which are referred to in the present description also interchangeably stress proteins may be selected among any cellular proteins that meet the following criteria. It is a protein, the intracellular concentration increases when a cell is exposed to stress article is the mule, he is capable of binding other proteins or peptides and he is able to release the associated proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH value. In addition, HSP include constitutively expressed conservative cellular homologues of proteins induced by stress. Discovered that a family of Hsp-60, Hsp-70 and Hsp-90 consist of proteins related to protein stress on amino acid sequence, for example, having more than 35% amino acid identity, but their expression levels do not change under the action of stress stimuli. Accordingly, it is assumed that the determination of the stress protein used in the present description, include other proteins, their mutiny, analogs and variants having at least 35-55%, preferably 55-75%, and most preferably 75 to 85% amino acid identity with members of three families, the levels of expression in cells increase in response to stressful stimuli.

The first identified stress proteins HSP were. As indicated by their name, HSP are synthesized by the cell in response to heat shock. Today, on the basis of molecular weight was identified three major HSP family. The family called hsp60, hsp70 and hsp90, where the numbers reflect the approximate molecular weight stress proteins in kilodaltons. It was subsequently identified what about, many members of these families are induced in response to other stressful stimuli, including but not limited to the above, lack of nutrients, metabolic disorder, oxygen radicals and infection by intracellular pathogens (See. Welch, May 1993, Scientific American 56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science 260: 1902-1903; Gething, et al., 1992, Nature 355:33-45; and Lindquist, et al., 1988, Annu. Rev. Genetics 22: 631-677), and the description of these publications is hereby incorporated into this description by reference. It is assumed that hsps/stress proteins belonging to these three families can be used when implementing the present invention.

HSP are intracellular molecules that are represented in large numbers, are soluble and highly conservative. As intracellular "escorts" HSP are involved in many biochemical pathways of maturation proteins and functionally active during stress and normal cellular homeostasis. Many stress can disrupt the three-dimensional structure or folding of the proteins of the cells. The remaining unadjusted, incorrectly folded proteins form aggregates, which can eventually lead to cell death. HSP associated with these damaged proteins, helping them develop in their correct conformation. Under normal (no stress) letocnaolos HSP is required for cellular metabolism. HSP help newly synthesized polypeptides to form and, thus, prevent premature interaction with other proteins. In addition, HSP assist in the transfer of proteins through the various compartments of the cell.

The main HSP can accumulate to very high levels in stressed proteins, but they are at levels from low to moderate in cells that were not subjected to stress. For example, a highly inducible hsp70 protein is difficult to detect at normal temperatures, but becomes one of the most actively synthesized proteins in the cell during heat shock (Welch, et al., 1985, J. Cell. Biol. 101: 1198-1211). In contrast, proteins hsp90 and hsp60 in great numbers at normal temperatures in most, but not all, mammalian cells and additionally induced by heating (Lai, et al., 1984, Mol. Cell. Biol. 4: 2802-2810; van Bergen en Henegouwen, et al., 1987, Genes Dev. 1: 525-531).

Found that HSP have immunological and antigenic properties. Immunization of mice with protein gp96 or R/86, selected from a particular tumor, makes mice immune to this particular tumor, but not to antigenic excellent tumors (Srivastava, P.K., et al., 1988, Immunogenetics 28:205-207; Strivastava, P.K., et al., 1991, Curr. Top. Environ. Immunol. 167: 109-123). In addition, we discovered that protein hsp70 induces immunity to the tumor from which it was isolated, but the e to antigenic excellent tumors. However, found that depleted hsp70 peptides loses its specific immunological activity (Udono, M., and Srivastava, P.K., 1993, J. Exp. Med. 178:1391-1396). These observations suggested that heat shock proteins are not antigenic by themselves, but form non-covalent complexes with antigenic peptides and complexes can induce specific immunity to antigenic peptides (Strivastava, P.K., 1993, Adv. Cancer Res. 62: 153-177; Undono, H., et al., 1994, J. Immunol. 152: 5398-5403; Suto, R., et al, 1995, Science 269: 1585-1588). Recently it was found that hsp60 and hsp70 stimulates the production of proinflammatory cytokines, such as TNFα and IL-6 by monocytes, macrophages or cytotoxic T-cells (Breloer et al., 1999, J. Immunol. 162: 3141-3147; Chen et al., 1999, J. Immunol. 162: 3212-3219; Ohashi et al., 2000, J. Immunol. 164: 558-561; Asea et al., 2000, Nature Medicine, 6: 435-442; Todryk et al., 1999, J. Immunol. 163: 1398-1408). Also found that hsp70 is aimed to act on immature dendritic cells and makes them more capable of capturing antigens (Todryk et al., 1999, J. Immunol. 163: 1398-1408). Postulated that the release or induction of expression of hsp60 and hsp70, e.g., due to cell death may be a signal that the immune response should be increased (Chen et al., 1999, J. Immunol. 162: 3212-3219; Ohashi et al., 2000, J. Immunol. 164: 558-561; Todryk et al., 1999, J. Immunol. 163: 1398-1408; Basu et al., 2000, Intl. Immunol. 12: 1539-1546).

The use of non-covalent complexes of HSP peptide, purified from cancer cells, for the treatment and p is opractice cancer is described in U.S. patent No. 5750119, 5837251 and 6017540.

The complexes of HSP-peptide for sensitization antigen presenting cells in vitro for use in acceptable immunotherapy described in United States patents No. 5985270 and 5830464.

Complexes HPS-peptide can be isolated from infected pathogen cells and used to treat or prevent infections caused by pathogens such as viruses and other intracellular pathogens, including bacteria, protozoa, fungi, and parasites; see patents of the United States No. 5961979 and 6048530.

Immunogenic complexes of HSP-peptide can also be obtained by the formation of complexes of HSP and antigenic peptides in vitro, the use of such complexes for the treatment and prevention of cancer and infectious diseases described in United States patents No. 5935576 and 6030618. The use of heat shock protein in combination with a specific antigen for the treatment of cancer or infectious diseases are described in PCT publication WO 97/06821, dated February 27, 1997.

Purification of complexes of HSP-peptide from the lysate of cells has been described previously; see, for example, the patents of the United States No. 5750119 and 5997873.

3.Summary of the invention

The present invention relates to methods and compositions are applicable for producing or enhancing an immune response comprising the administration of the drug protein th the first shock (HSP) with an immunoreactive reagent. Methods and compositions are applicable to improve the treatment of the subject, which introduced the HSP preparation and/or only immunoreactive reagent. In particular, the invention provides methods and compositions are applicable for producing or enhancing an immune reaction caused immunoreactive reagent, and/or to improve the efficiency of immunoreactive reagent that includes a drug HSP. In accordance with these methods and compositions include the introduction of the drug HSP to improve the efficiency of passive immunotherapy. In specific embodiments, the implementation of such methods and compositions include the introduction of the drug HSP and are applicable to enhance the ability of immunoreactive reagent to stimulate the function of effector cells. The present invention also provides for methods and compositions that can be applied to enhance the immune reaction caused by the drug HSP, including the introduction of immunoreactive reagent. Proposed by the present invention methods and compositions of the invention are useful for the prevention and treatment of diseases and disorders where the effectiveness of treatment or prevention may be improved in the enhanced immune response, such diseases are infectious diseases, primary and metastatic neoplastic diseases the project (i.e. cancer), neurodegenerative or amyloid diseases, or protein deposits or amyloidogenic disease. Thus, the invention includes methods and compositions intended for the treatment or prevention of infectious diseases, primary and metastatic neoplastic diseases (i.e. cancer), neurodegenerative or amyloid diseases or protein deposits or amyloidogenic diseases, including the introduction of one or more immunoreactive reagents in combination with HSP preparation.

In one embodiment, the invention provides a method of inducing or enhancing an immune reaction caused immunoreactive reagent, through the use of the drug HSP, where the HSP preparation to enhance immune response, caused by the number of immunoreactive reagent, which otherwise is suboptimal to induce immune responses when used separately. In some embodiments, implementation, when the HSP preparation is not used in combination with an immunoreactive reagent to induce an immune reaction, the introduction of only the specified drug HSP does not cause or not enhances specified immune response. In alternative embodiments, the implementation of both the HSP preparation and immunoreactive reagent can induce an immune response when introduced on the Telenesti and/or in combination.

In some embodiments, the implementation of the HSP preparation can strengthen action of immunoreactive reagent additive way. In a preferred embodiment, the HSP preparation enhances the actions of immunoreactive reagent synergistic manner. In another embodiment, the immunoreactive reagent enhances the action of the drug HSP additive way. Steps preferably are enhanced synergistic manner. Thus, in some embodiments implementing the invention includes methods of treating or preventing diseases that provide better therapeutic profiles than profiles with the introduction of only drug HSP and/or only immunoreactive reagent. The invention includes combination therapy, which have additive activity or additive therapeutic effect while reducing or excluding unwanted or adverse effects. The invention also includes a synergistic combination whose therapeutic efficacy is higher than additive, while unwanted or adverse effects are reduced or eliminated. In some embodiments, the implementation of the methods of the invention make possible the treatment or prevention of diseases and disorders where treatment becomes more effective by improving the immune response used the eat lower and/or less frequently injected doses of immunoreactive reagents and/or HSP to reduce the incidence of unwanted or adverse effects, caused by the introduction of immunoreactive agents and/or HSP, while maintaining or improving the effectiveness of treatment, preferably increasing the susceptibility of the patient, increasing the effectiveness of therapy and/or reducing unwanted and adverse effects.

The methods and compositions of the present invention are useful not only for nepodvizhnykh treatment of patients, but are also useful in the treatment of patients partially or completely tolerant to the introduction of only HSP or introduction only immunoreactive reagents. In various embodiments implementing the invention provides methods and compositions useful for treating diseases or disorders that were found, are or may be unresponsive or tolerate to therapies, including the introduction of any of the two or both of immunoreactive reagents and HSP, and the effectiveness of treatment is enhanced by increasing the immune response.

Drugs HSP applicable in the methods and compositions of the present invention, may include, but are not limited to, available HSP, not associated with any molecule, and molecular complexes of HSP with another molecule such as a peptide. Complex HSP-peptide contains HSP, covalently or ecovalence attached to the peptide. Complex HSP-peptide may consist of HSP associated with what eptide, originating from the interest type of the tumor, pathogen or cells and/or protein. In one embodiment, the specified peptide is the target that is recognized immunoreactive reagent, or from interest the type of tumor, pathogen or cells and/or protein. Alternatively, the complex HSP-peptide may consist of HSP associated with the endogenous peptide, but not necessarily the peptide from the same source as the target of immunoreactive reagent. Some methods of the invention may not require covalent or non-covalent joining HSP to any specific antigens or antigenic peptides prior to introduction to the subject. Drugs HSP applicable in the methods and compositions of the present invention also include fused proteins HSP. Slit proteins HSP can consist of HSP, merged with any antigenic peptide sequence, where the peptide sequence derived from the interest type of the tumor, pathogen or cells and/or protein. In one embodiment, the specified peptide sequence is the same target recognized immunoreactive reagent, or from interest the type of tumor, pathogen or cells and/or protein.

Immunoreactive reagents that are applicable in the methods and compositions of the present invention, which may include, but not limited to, antibodies, molecules or proteins, constructed by genetic engineering to include antigennegative part of the antibody molecules or proteins, constructed by genetic engineering to include antigennegative domain, which mediates antibody-dependent immune response, peptide or domain, which interacts specifically with interest the antigen or molecule that has any antigennegative domain, which interacts with interest antigen/epitope, and the domain of the constant region of the antibody that mediates antibody-dependent immune reactions, such as reactions or processes of effector cells. Linking antigen domain recognizes a specific target, and the domain of the constant region mediates antibody-dependent immune response of effector cells.

In preferred embodiments, the implementation of the immunoreactive reagent is an antibody, preferably with therapeutic or prophylactic use in vivo, the invention provides methods and compositions that can be applied to improve the effectiveness of such therapeutic or prophylactic antibodies, including the introduction of the drug HSP. In some embodiments, the implementation of the ability of the antibody to stimulate the function effectory the cells increase the drug HSP. In a specific embodiment, antibody-dependent cellular cytotoxicity and/or phagocytosis of tumor cells or pathogens or pathogenic proteins and peptides increase through the use of therapeutic antibodies in combination with HSP preparation. Therapeutic antibody is preferably a cytotoxic and/or opsonizing antibody. In accordance with this invention provides methods and compositions, in which the drug HSP used in combination with an immunoreactive reagent to enhance the function of effector cells (i.e. antibody-dependent cellular cytotoxicity and phagocytosis) for macrophages, natural killer cells (NK-cells) and polymorphing cells. Immunoreactive reagent is preferably an antibody, more preferably a cytotoxic and/or opsonizing antibody. In one embodiment, HSP-mediated amplification of passive immunotherapy is due to stimulation of effector cells, i.e. induction and/or activation of Fc receptors on these cells.

Antibodies used in the methods of the present invention include, but are not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric anti the La, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, fragments F(ab')connected by disulfide bonds Fv (sdFv) and antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the present invention) and epilepsyusa fragments of any of the above. In particular, antibodies used in the methods of the present invention include immunoglobulin molecules and immunologically active portion of immunoglobulin molecules, i.e. molecules that contain antigennegative site that immunospecificity way associated with interest target. The immunoglobulin molecules of the present invention can be a molecule of the immunoglobulin of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2or subclass.

Without intending to be limited by any theory it is believed that the increased concentration of HSP can induce the production of cytokines and surface expression of antigen presenting and co-stimulatory molecules. In accordance with this drug HSP, introduced the subject, can improve the effectiveness of immunoreactive reagent by increasing the effectiveness and efficiency of antigen presentation.

In other preferred embodiments, the implementation of the immunoreactive reagent is lead entity, receiving medication HSP, to improve treatment outcome. In a specific embodiment, the immunoreactive reagent enhances the immune reaction caused by the drug HSP.

In a specific embodiment, the antibody is an antibody against CTLA-4. In another specific embodiment, the antibody is an antibody against the C-erb-2, preferably rhu 4D5 (Herceptin) man, especially useful for the treatment or prevention of cancer, in which is expressed the oncogene Her2/neu. In another specific embodiment, the antibody is anti-tumour Ab (MS11G6), antiidiotypic IgG2a antibody used in cancer therapies, such as, but not limited to, specified disease, resistant to NK-cell lymphoma.

In one embodiment, the HSP preparation is administered in combination with antitumor therapy or anti-cancer antibody directed against cancer. In an alternative embodiment, the HSP preparation is administered in combination therapy with an antibody directed against the pathogen. In another embodiment, the HSP preparation is administered in combination therapy with an antibody directed against pathogenic or undesirable protein or cells affected neurodegenerative or amyloid disease or disorder.

In other embodiments implementing the tvline the methods and compositions of the present invention can be used to generate immune responses against epitopes, associated with neurodegenerative or amyloid diseases, cancer or an agent of infectious disease, or any component, cell, or molecule that has an epitope associated with the above diseases, the introduction to the individual a therapeutic amount of an immunoreactive reagent and drug HSP. When the desired immune response against any type of cancer, use of immunoreactive reagent that specifically binds to the antigen (or "recognizes" an antigen) of this type of cancer, for example, associated with a tumor antigen. In another embodiment, the methods and compositions of the present invention include the introduction of immunoreactive reagent that specifically binds to the antigen specific type of cancer, in combination with HSP preparation for treatment or prevention of this type of cancer. When you need to induce an immune response against the infectious agent of the disease, enter immunoreactive reagent that specifically binds to the antigen or pathogenic protein (e.g., toxin) agent of an infectious disease. In alternative embodiments, the implementation of the methods and compositions of the invention include the introduction of immunoreactive reagent that specifically binds to an agent of an infectious disease in combination with HSP preparation for the treatment or p is opractice specified infectious diseases. In the following other embodiments implement the methods and compositions of the present invention include the introduction of immunoreactive agent that specifically binds to an antigenic molecule associated with a neurodegenerative disease or amyloid disease, in combination with HSP preparation for treatment or prevention of a specified neurodegenerative or amyloid disease. Immunoreactive reagent is preferably an antibody.

The present invention also includes methods and compositions comprising a drug HSP in combination with an immunoreactive reagent patients who have previously received or are currently receiving other forms of drug therapy, including anti-cancer agents, antibiotics and anti-infective agents.

In another embodiment, the present invention provides a method of activating antigen-presenting cells, comprising contacting the APC with the HSP preparation and introduction of such activated APC in combination with the introduction of the immunoreactive reagent. In accordance with this present invention provides methods and compositions for enhancing the immune response induced immunoreactive reagent, including the introduction of activated APC and/or drug HSP. The HSP preparation is preferably not effectively causes an immune response in the OTC is stwie the introduction of the immunoreactive reagent. In some embodiments, the implementation of the HSP preparation does not show immunogenicity of the target recognized immunoreactive reagent. In alternative embodiments, the implementation of the immunogenicity of the drug HSP shows the immunogenicity of the target recognized immunoreactive reagent. Immunogenicity of HSP preparation can be tested in vitro or in vivo method known in this field.

In certain embodiments of the implementation of the methods and compositions of the invention, including the introduction of immunoreactive reagent with the introduction of activated APC and/or drug HSP, are applicable for the treatment of any disease or impairment, where the efficacy of the treatment of such disease may be improved by enhancing the immune response, in particular antitelomerase immune response, and such diseases or disorders are diseases or disorders, but they are not limited to, infectious diseases, cancer or neurodegenerative or amyloid diseases or disorders.

The present invention also includes methods for delivering one or more HSP as adjunctive therapy in combination with immunoreactive reagents; pharmaceutical compositions and preparations for injection containing one or more drugs HSP and one or more of the immunoreactive Reagan is s, kits containing these pharmaceutical compositions, and methods of treatment or prevention of disease, which can be improved by increasing the immune response, and this disease can be infectious diseases, primary and metastatic neoplastic diseases (i.e. cancer), neurodegenerative or amyloid diseases, or the deposition of protein or amyloidogenic disease, with the use of prophylactic or therapeutic pharmaceutical compositions of the present invention. Such methods, kits and compositions may additionally include the introduction of activated APC.

4.Detailed description of the invention

The present invention relates to methods and compositions are applicable for the induction or enhancement of immune response, including the introduction of the drug heat shock protein (HSP) with an immunoreactive reagent. Methods and compositions are applicable to improve the results of treatment of the subject, which introduced the HSP preparation and/or only immunoreactive reagent. In particular, the invention provides methods and compositions for enhancing the preventive and therapeutic effectiveness of immunoreactive agent. The invention also provides methods and compositions suitable for inducing or enhancing an immune reaction caused by immunoreaction the m reagent, including the introduction of the drug HSP. In accordance with these methods and compositions include the introduction of the drug HSP to improve the efficiency of passive immunotherapy. In certain embodiments of the implementation of such methods and compositions include the introduction of the drug HSP and are applicable to enhance the ability of immunoreactive reagent to stimulate the function of effector cells. The present invention also includes methods and compositions that can be applied to enhance the immune reaction caused by the drug HSP, including the introduction of immunoreactive reagent. In this invention, the methods and compositions of the invention are applicable for the prevention and treatment of diseases and disorders where the effectiveness of treatment or prevention may be improved by enhancing the immune response, such as infectious diseases, primary or metastatic neoplastic disease (i.e. cancer), neurodegenerative or amyloid diseases, or deposition of proteins or amyloidogenic disease. Thus, the invention includes methods and compositions intended for the treatment or prevention of infectious diseases, primary or metastatic neoplastic diseases (i.e. cancer), neurodegenerative or amyloid diseases, or deposition of proteins or amyloidogenic diseases, including the General introduction of one or more immunoreactive reagents in combination with HSP preparation.

In one embodiment, the present invention provides a method of inducing or enhancing an immune reaction caused immunoreactive reagent, through the use of the drug HSP, where the HSP preparation to induce an immune reaction so many immunoreactive reagent, which otherwise is insufficient for the induction of an immune response when used alone. In some embodiments, implementation, when the HSP preparation is not used in combination with an immunoreactive reagent for the induction of immune responses, the introduction of only the specified drug HSP does not cause or not enhances specified immune response. In alternative embodiments, the implementation of both the HSP preparation and immunoreactive reagent can induce an immune response individually and/or with the introduction of them in combination.

In some embodiments, the implementation of the HSP preparation can enhance the action of immunoreactive reagent additive way. In a preferred embodiment, the HSP preparation enhances the actions of immunoreactive reagent synergistic manner. In another embodiment, the immunoreactive reagent enhances the action of the drug HSP additive way. Steps preferably are enhanced synergistic manner. Thus, in some embodiments, the implementation of the infusion is her invention includes methods of treatment or prevention of disease, that provide better therapeutic profiles than profiles with the introduction of only drug HSP and/or only immunoreactive reagent. The invention includes combination therapy, which have additive activity or additive therapeutic effect while reducing or excluding unwanted or adverse effects. The invention also includes a synergistic combination that have therapeutic activity was higher than additive, whereas unwanted or adverse effects are reduced or eliminated. In some embodiments, the implementation of the methods of the present invention make possible the treatment or prevention of diseases and disorders where treatment is more effective to increase the immune response, using smaller and/or less frequently injected doses of immunoreactive reagents and/or HSP to reduce the incidence of unwanted or adverse effects caused by the introduction of only one immunoreactive agents or HSP, while maintaining or improving the effectiveness of treatment, preferably increasing the susceptibility of the patient, increasing the effectiveness of therapy and/or reducing unwanted or adverse effects.

The methods and compositions of the present invention are useful not only for non-treated patients, but also are useful is in the treatment of patients partially or completely immune to only introduced HSP or introduced only immunoreactive reagent. In various embodiments, the implementation of the present invention provides methods and compositions useful for treating diseases or disorders in patients, which, as shown, are or may be unresponsive or tolerate to therapies, including the introduction of only any one of the agents, and where the effectiveness of treatment is enhanced by strengthening the immune response.

Drugs HSP applicable in the methods and compositions of the present invention, may include, but are not limited to, available HSP, not associated with any molecule, and molecular complexes of HSP with another molecule such as a peptide. Complex HSP-peptide contains HSP, covalently or ecovalence attached to the peptide. Complex HSP-peptide may consist of HSP associated with peptides derived from interest the type of tumor, pathogen or cells and/or protein, preferably the specified peptide is the same target recognized immunoreactive reagent. In the alternative case, the complex HSP-peptide may consist of HSP associated with the endogenous peptide, but not necessarily the peptide from the same source as the target of immunoreactive reagent. Some of the ways this image is to be placed may not require covalent or non-covalent joining HSP to any specific antigens or antigenic peptides prior to introduction to the subject. The present invention includes the use of complexes of HSP-peptide containing the HSP in the form covalent or non-covalent complexes with exogenous peptides produced in vitro, as well as the use of complexes of endogenous HSP-peptide isolated from cellular sources.

Immunoreactive reagents that are applicable in the methods and compositions of the present invention, may include, but are not limited to, antibodies, molecules or proteins obtained by genetic engineering to enable antigennegative part of the antibody molecules or proteins obtained by genetic engineering to enable antigennegative domain, which mediates antibody-dependent immune response, peptide or domain, which interacts specifically with interest antigen, or any antigennegative domain, which interacts with interest antigen/epitope. Antigennegative domain preferably connected with the domain constant region of the antibody that mediates antibody-dependent immune response, immune reactions or processes of effector cells. Immunoreactive reagent is preferably clear.

In preferred embodiments, the implementation of immunoreactive agent is an antibody, preferably a therapeutic or prophylactic uses, in vivo, and us is Aasee the invention provides methods and compositions applicable to improve the effectiveness of such therapeutic or prophylactic antibodies, including the introduction of the drug HSP. In such scenarios, the implementation of the ability of the antibody to stimulate the function of effector cells is increased by the introduction of the drug HSP. In a specific embodiment, antibody-dependent cellular cytotoxicity and/or phagocytosis of tumor cells or pathogens or pathogenic proteins and peptides increase through the use of therapeutic antibodies in combination with HSP preparation. Therapeutic antibody is preferably a cytotoxic and/or opsonizing antibody. In accordance with this present invention provides methods and compositions, in which the drug HSP used in combination with an immunoreactive reagent to enhance the function of effector cells (i.e. antibody-dependent cellular cytotoxicity and phagocytosis) for macrophages, natural killer cells (NK-cells) and polymorphing cells. Immunoreactive reagent is preferably an antibody, more preferably a cytotoxic and/or opsonizing antibody. In one embodiment, the HSP-mediated increase the effectiveness of passive immunotherapy is through stimulation of effector cells, i.e. induction and/or activation of Fc receptors on these cells.

In other the preferred options for implementation of the immunoreactive reagent is administered to a subject, receiving medication HSP, to improve treatment outcome. In a specific embodiment, the immunoreactive reagent enhances the immune reaction caused by the drug HSP. In other embodiments, implementation of the immunoreactive reagent potentiates the activation of T cells, called HSP.

In a specific embodiment, the antibody is an antibody against CTLA-4. In another specific embodiment, the antibody is an antibody against the C-erb-2, preferably human rhu 4D5 (Herceptin), is particularly useful in the treatment or prevention of cancer, in which is expressed the oncogene Her2/neu. In another specific embodiment, the antibody is anti-tumour MoAb (MS11G6), antiidiotypic antibody IgG2a, useful in the treatment of cancer, such as, but not limited to, resistant to NK-cell lymphoma. In one embodiment, the antibody is an agonist of Toll-like receptor (TLR), such as TLR 2, 7, 8 or 9. In another embodiment, the antibody is an agonist W (see, for example, Miller et al., 2002, J. Immunol. 169: 1792-1800), OX40, ICOS, or CD40. In another embodiment, the antibody is an antagonist of Fas-ligand or PDI. In another embodiment, the antibody is Mab 6B11, which binds to the CDR3 loop of the T-cell receptor (TCR) invariant NKT-cells is to stimulate the reproduction and/or activates these cells. Cm. the patent application U.S. 2002/0164331, published November 7, 2002.

In one embodiment, the HSP preparation is administered in combination with using the antitumor antibody therapy directed against cancer. In an alternative embodiment, the HSP preparation is administered in combination with using antibody therapy directed against the pathogen. In another embodiment, the HSP preparation is administered in combination with using antibody therapy directed against the cells affected neurodegenerative or amyloid disease or disorder.

Without intending to be limited by any theory it is believed that the increased concentration of HSP can induce the production of cytokines and surface expression of antigen presenting and co-stimulating molecules. In accordance with this, it is considered that the drug HSP, introduced the subject, can improve the effectiveness of immunoreactive reagent by increasing the effectiveness and efficiency of antigen presentation. It is believed that in some embodiments, the implementation of the HSP preparation can enhance antitelomerase reactions, such as the function of effector cells.

In other embodiments, implementation of the methods and compositions of the present invention can be used to generate immune responses against epitopes associated with neurodegenerative is passed or amyloid diseases, cancer or an agent of infectious disease, or any component, cell or molecule carrying the epitope associated with the above diseases, the introduction to the individual a therapeutic amount of an immunoreactive reagent and drug HSP. When it is necessary immune response against the cancer type, use the immunoreactive reagent that specifically binds to the antigen (or "recognizes" an antigen) cancer type, i.e. it expresses the immunogenicity of cancer. Examples of such antigens are associated with tumor antigen (i.e. relatively sverhagressivny in tumor cells) or specific for a tumor antigen (i.e. present only in tumor cells). In other embodiments, implementation of the methods and compositions of the present invention include the introduction of immunoreactive reagent that specifically binds to the antigen of the type of cancer, in combination with HSP preparation for treatment or prevention of a specified type of cancer. When necessary is the induction of immune response against the agent of infectious diseases, enter the immunoreactive reagent that specifically binds to the antigen or pathological protein (e.g., toxin) agent of an infectious disease. In alternative embodiments, the implementation of the methods and compositions of the present invention include the maintenance of immunoreactive reagent, which specifically binds to the agent of an infectious disease, in combination with HSP preparation for treatment or prevention of the specified infectious diseases. In the following embodiments, the implementation of the methods and compositions of the present invention include the introduction of immunoreactive reagent that specifically binds to an antigenic molecule or protein epitope associated with a neurodegenerative disease or amyloid disease, in combination with HSP preparation for treatment or prevention of a specified neurodegenerative or amyloid disease. Immunoreactive reagent is preferably an antibody.

The present invention also includes methods and compositions comprising a drug HSP in combination with an immunoreactive reagent patients who have previously received or are currently receiving other forms of drug therapy, including anti-cancer agents, antibiotics and anti-infective agents.

In another embodiment, the present invention provides a method of activating antigen-presenting cells, comprising contacting the APC with the HSP preparation and introduction of such activated APC in combination with the introduction of the immunoreactive reagent. In accordance with this present invention provides methods and compositions for enhancing mannai reaction, caused immunoreactive reagent, including the introduction of activated APC and/or drug HSP. The HSP preparation is preferably not effectively causes an immune response in the absence of the introduction of the immunoreactive reagent. In some embodiments, the implementation of the HSP preparation does not show immunogenicity of the target recognized immunoreactive reagent. In alternative embodiments, the implementation of the immunogenicity of the drug HSP shows the immunogenicity of the target recognized immunoreactive reagent. Immunogenicity of HSP preparation can be tested in vitro or in vivo method known in this field.

In certain embodiments of the implementation of the methods and compositions of the present invention, including the introduction of immunoreactive reagent with the introduction of activated APC and/or drug HSP, are applicable for the treatment of any disease or impairment, where the effectiveness of the treatment of such disease may be improved by enhancing the immune response, and such diseases are, but they are not limited to, infectious diseases, cancer or neurodegenerative or amyloid diseases or disorders.

The invention also includes methods for delivering one or more HSP as adjunctive therapy in combination with immunoreactive reagents; pharmaceutical compo is icii and preparations for the introduction, containing one or more drugs HSP and one or more immunoreactive reagents, kits containing these pharmaceutical compositions, and methods of treatment or prevention of disease, the treatment efficiency can be improved by enhancing the immune response, and such diseases are infectious diseases, primary and metastatic neoplastic diseases (i.e. cancer), neurodegenerative or amyloid disease or deposition of protein or amyloidogenic disease, with the use of prophylactic or therapeutic pharmaceutical compositions of the present invention. Such methods, kits and compositions may additionally include the introduction of activated APC.

4.1Prophylactic/therapeutic methods

The present invention provides methods of inducing or enhancing an immune reaction caused immunoreactive reagent that includes a drug HSP in combination with the introduction of the immunoreactive reagent. The present invention includes methods of treating or preventing diseases and disorders, where the effectiveness of treatment or prevention may be improved by enhancing the immune response. In preferred embodiments, the implementation of enhanced immune response includes the amplification reactions, such as antitilos is independent cellular cytotoxicity (e.g., ADCC) or antiteloobrazovania opsonization and/or phagocytosis, directed against cells of the pathogen or protein having an epitope recognized by the antibody, and mediated complement the cytolysis of cells in the mechanisms of effector cells. In some embodiments, the implementation of the HSP preparation induces the activation of T cells and immunoreactive reagent, for example, the antibody may enhance immune response by potentiating the activation of T cells.

In one embodiment, the term "treatment" refers to reducing the intensity of symptoms of cancer, infectious disease, or a neurodegenerative or amyloid disease, or, at least, one of his apparent symptom. In another embodiment, "treatment" refers to the improvement of at least one measurable physical parameter associated with cancer, infectious disease, neurodegenerative or amyloid disease, but not necessarily visible to the subject. In another embodiment, the term "treatment" refers to inhibiting the development of cancer, infectious diseases, neurodegenerative or amyloid disease or physically, such as stabilization noticeable symptom, physiologically, for example, stabilization of a physical parameter, or both ingibirovaniem. In one embodiment, the implementation of whom tvline "treatment" refers to a slowdown in the beginning of cancer, neurodegenerative or amyloid disease.

In some embodiments, the implementation of the methods and compositions of the present invention are applicable as a preventive measure against cancer, infectious diseases, neurodegenerative or amyloid disease. Used herein, the term "prevention" refers to reducing the risk of acquiring this cancer, infectious diseases, neurodegenerative or amyloid disease. In one variant of implementation, the methods and compositions of the present invention include a drug HSP with the introduction of the immunoreactive reagent as a preventive measure for subject-a person with a genetic predisposition to cancer, infectious disease, neurodegenerative or amyloid disease. In another form of a variant of implementation, the methods and compositions of the invention are applicable as a preventive measure for subject-a person with a genetic predisposition to cancer, or entity subject to the effect of the agent of an infectious disease.

In other embodiments, implementation of the methods and compositions of the present invention are applicable for the treatment or prevention of clinical symptoms or onset of cancer, infectious diseases, or n is rodegenerative, or amyloid diseases.

In some embodiments, the implementation of the present invention provides methods for treatment or prevention of infectious diseases, cancer, neurodegenerative or amyloid diseases or protein deposits or amyloid diseases, including drug HSP in combination with one or more immunoreactive reagents. In some embodiments, the implementation of the HSP preparation is administered to a mammal, preferably a human, concurrently with one or more immunoreactive reagents.

In one embodiment, the HSP preparation and immunoreactive reagent is injected at the same time. In another embodiment, the HSP preparation and immunoreactive reagent is administered to the subject sequentially and that interval of time that the drug HSP could act together with immunoreactive reagent, to provide increased benefits compared to the benefits that would, if entered, only one of them. For example, each (for example, the HSP preparation and immunoreactive reagent) can be entered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they need to enter in the relatively near each other time to provide the desired therapeutic or prophylactic effect. Each can is to be entered separately, in any suitable shape and any suitable means. In one embodiment, the HSP preparation and immunoreactive reagent is injected in the same way the introduction. In another embodiment, the HSP preparation and immunoreactive reagent is injected by different routes of administration. Introduction each can be the same or different places, for example in the arm and leg.

In various embodiments, the implementation of preventive or therapeutic agents are administered at intervals of time between them, less than 1 hour, 1 hour, from one hour to 2 hours from 2 hours to 3 hours from 3 hours to 4 hours, from 4 hours to 5 hours from 5 hours up to 6 hours over 6 hours up to 7 hours, from 7 hours to 8 hours, from 8 hours to 9 hours, from 9 hours to 10 hours, from 10 hours to 11 hours, 11 hours to 12 hours, but not more than 24 hours or no more than 48 hours. In another embodiment, prophylactic or therapeutic agents are administered at intervals of time between them 1, 2, 4, 8, 12, 24 or 48 hours. In another embodiment, the HSP preparation and immunoreactive reagent is injected in intervals between 2-4 days, 1 week, 1-2 weeks, 2-4 weeks, one month, 1-2 months, or 2 or more months. In preferred embodiments, the implementation of two or more components enter in the same ward patients. The HSP preparation and immunoreactive reagent is administered to a subject, preferably in a period of time is, which allows drug HSP, the immunoreactive reagent, or both can be activated at the same time. The person skilled in the art can determine the distance by determining the half-life from the body of the HSP preparation and immunoreactive reagent.

In a specific embodiment, the HSP preparation is administered before the introduction of the immunoreactive reagent. In an alternative specific embodiment, the HSP preparation is administered after administration of an immunoreactive reagent.

In some embodiments, the implementation of the HSP preparation and/or immunoreactive reagent is administered to a subject repeatedly. Cyclic therapy consists of the administration of the HSP preparation for some period of time, followed by the introduction of immunoreactive reagent and/or any third agent for some period of time and repeating this sequential administration. Cyclic therapy reduces the development of resistance to one or more therapies that eliminates or reduces the side effects of one of therapies and/or increases the effectiveness of the treatment. In such scenarios, the implementation of the present invention provides an alternative drug HSP, followed by the introduction of immunoreactive reagent in 4-6 days, preferably in 2-4 days, more preferably after 2 days, and the subsequent introduction of the drug HSP in 4-6 days, preferably in 2-4 days, more preferably after 1-2 days, etc. these cycles can be repeated as many times as necessary.

In other embodiments, implementation of the HSP preparation is administered to a subject in reasonably the same time as the immunoreactive reagent. Two injection is preferably carried out in the period of time from one injection to another, less than one minute to about five minutes, from about five minutes to about 10 minutes, from about 10 minutes to about 30 minutes, from about 30 minutes to about sixty minutes, for example when the same doctor's visit.

In another embodiment, the immunoreactive reagent and the HSP preparation can be entered simultaneously. In some embodiments, the implementation of the immunoreactive reagent and the HSP preparation is administered in a single pharmaceutical composition. In such scenarios, the implementation of the pharmaceutical composition of the present invention is administered once daily, twice daily or three times a day. In other embodiments, the implementation of the pharmaceutical composition is administered once a week, twice a week, once every two weeks, once a month, once every six months once every two months, twice a year or once a year. Must be the e obviously, the effective dose of immunoreactive reagent used for the treatment may be increased or reduced during the course of a particular treatment.

A different treatment method. In this embodiment, the HSP preparation is administered to a subject, when you need to ARS subject were in an activated state. The HSP preparation can be administered regularly over a period of time, for example daily for up to several weeks 1-2 weeks, 2-4 weeks, 4-6 weeks, up to two months, which may precede, overlap, and/or to follow a regime of treatment immunoreactive reagent. The HSP preparation can improve the results of treatment. Without the intention to communicate with any theory or mechanism, it is believed that the introduction of the drug HSP subject can increase the susceptibility of non-specific immune mechanisms of the subject, for example, by increasing the number of natural killer cells (NK) and/or acceleration of the maturation of dendritic cells. In some embodiments, the implementation of the activation of ARS drug HSP occurs ex vivo and thus activated ARS then injected in accordance with the methods and compositions of the present invention. To activate the APC can be used the drug HSP, which is the same or different from the drug HSP, which must be entered. Each of the drugs the HSP may be or may not show immunogenicity of antigenic molecules, recognized immunoreactive reagent.

In another embodiment, the HSP preparation is administered to a subject over a period of time from one hour to twenty-four hours after administration of an immunoreactive reagent. The time period may be extended, if used immunoreactive reagent slowly or continuously released type. It is believed that this method helps to activate effector cells, such as APC, which are present at the site or near the site of injection and which cannot be activated by the presence of immunoreactive reagent.

In another embodiment, the HSP preparation is administered to the subject in the period of time from one to 12 hours 12-24 hours 24-48 hours before the introduction of the immunoreactive reagent. It is believed that this method of pre-activates the APC of the subject before the collision with the immunoreactive reagent.

In other embodiments, implementation of the treatment introduction carried out simultaneously, i.e. individual dose HSP and immunoreactive agent(s) administered separately, but in this time interval that the drug HSP could act together with immunoreactive reagent(s). For example, the HSP preparation can be injected once a week in combination with an immunoreactive reagent(s)that can be injected once every two weeks is or once every three weeks. In other words, regimens for drug HSP and immunoreactive reagent(s) provide for the simultaneous introduction, even if they are administered simultaneously or not in the same patient visit.

In one embodiment, the HSP preparation is administered concurrently with one or more immunoreactive reagents in the same pharmaceutical composition. In another embodiment, the HSP preparation is administered concurrently with one or more immunoreactive reagents in different pharmaceutical compositions. In another embodiment, the HSP preparation is administered before administration or after administration of one or more immunoreactive reagents. The invention provides a drug HSP in combination with one or more immunoreactive reagents the same or different routes of administration. In a preferred embodiment, the HSP preparation is administered intradermally. In another preferred embodiment, the immunoreactive agent is injected. In a particularly preferred embodiment, the HSP preparation is administered intradermally and immunoreactive agent is injected. In some embodiments, implementation, when the HSP preparation is administered simultaneously with an immunoreactive reagent, which may cause adverse or unwanted side de is affected, including, but not limited to, specified, toxicity, specified immunoreactive reagent can mainly be entered at a dose that falls below the threshold at which apparent adverse side effects.

In another embodiment, the invention provides a method of inducing an immune reaction to a suboptimal number of immunoreactive reagent, where the drug HSP contributes to the induction of immune responses by the number of immunoreactive reagent, which otherwise is insufficient for the induction of an immune response when used alone. In some embodiments, the implementation of suboptimal number is a number, otherwise, which is insufficient for efficient induction of immune responses or achieve the desired prophylactic or therapeutic effect. In particular, the method includes the stage of (a) the introduction of the subject of the preparation of heat-shock proteins and (b) introducing the subject of the immunoreactive reagent, against which must be induced the desired immune response, in an amount that is suboptimal in the absence of stage (a), whereby the subject is induced immune response. The drug HSP may be or may not show the immunogenicity of the antigen molecule recognized immunoreactive re is Ghent.

In another embodiment, the present invention provides a method of inducing an immune reaction subimmunogenic number of drug HSP, where immunoreactive reagent to induce immune responses number of drug HSP, less than sufficient to induce an immune response when used alone. The drug HSP may be or may not show the immunogenicity of the antigen molecule recognized immunoreactive reagent.

The present invention provides methods for treatment, prevention or reduction of the intensity of one or more symptoms associated with a disease, disorder or infection, the introduction of the subject pharmaceutical compositions containing the immunoreactive reagent and HSP. In a preferred aspect of the immunoreactive reagent and HSP are essentially treated (i.e. essentially free from substances that limit its effect or produce undesirable side effects). In accordance with the present invention the composition of the invention containing immunoreactive reagent and HSP, is administered to a subject, the person with cancer, infectious disease or neurodegenerative or amyloid diseases as treatment.

The present invention relates also to methods of use of compositions of this is subramania for the treatment of infectious diseases, primary and metastatic neoplastic diseases (i.e. cancer), neurodegenerative or amyloid diseases, deposition of proteins/amyloidogenic diseases or any other disease treatment, symptoms of which can be improved by increasing the immune response.

4.2Patient population

The entity to which the drug is administered HSP and immunoreactive reagent is preferably a mammal such as non-Primate (for example, cattle, pigs, horses, cats, dogs, rats etc) and a Primate (e.g., a monkey, such as cynomolgus monkey, and human). In a preferred embodiment, the subject is the man.

In other various ways of implementing the methods and compositions of the invention are used for treatment or prevention of any diseases or disorders for which therapeutic or prophylactic immunoreactive reagent is useful for treatment or prevention. Preferably the disease or disorder is amenable to treatment or prevention by improving the immune response, more preferably infectious disease, cancer or neurodegenerative or amyloid disorder.

The composition can be used to prevent various types of cancer, for example, individuals who Pedras is juxtaposed to this in the family history, or individuals with an increased risk of cancer due to environmental factors, for the prevention of infectious diseases, for example, individuals with elevated risk of exposure to the effects of agents of infectious diseases and for the prevention of neurodegenerative or amyloid diseases, for example, individuals with genetic predispositions to neurodegenerative or amyloid diseases.

The methods and compositions of the present invention can be used for patients who have not previously received such treatment, patients who have previously received or are currently receiving treatment with HSP, patients who have previously received or are currently receiving treatment immunoreactive reagent, or patients who have previously received or are currently receiving treatment with other pharmaceutical agents or combinations, including but not limited to the above, anticancer agents, antibiotics, antibacterial agents, antifungal agents and antiviral agents. In a specific embodiment of the present invention, the HSP preparation is administered to a patient who has previously received or is currently receiving immunotherapy treatment reagents. In another embodiment of the invention immunotherapy reagent is injected, patsie the one who has previously received or is currently receiving treatment with HSP. In another embodiment of the present invention, the HSP preparation is administered to a patient who has previously received or is currently receiving treatment, which includes the application, not limited to the above, anticancer agents, antibiotics, antibacterial agents, antifungal agents or antiviral agents, optionally with an immunoreactive reagent. In another embodiment, the immunotherapy agent is administered to a patient who has previously received or is currently receiving treatment, which includes the application, not limited to the above, anticancer agents, antibiotics, antibacterial agents, antifungal agents or antiviral agents, optionally with an HSP preparation.

In a preferred embodiment, the pharmaceutical composition of the present invention, consisting of immunotherapy reagent and drug HSP, is administered to a patient who has previously received or is currently receiving treatment, which includes the application, not limited to the above, anticancer agents, antibiotics, antibacterial agents, antifungal agents or antiviral agents.

The methods and compounds of the present invention can also be used for l the treatment of patients who have previously received treatment with HSP or immunoreactive reagents and are currently inefficient treat only the HSP preparation or an immunoreactive reagent.

In one embodiment, the composition of the present invention, consisting of HSP preparation and immunoreactive reagent is administered to the patient, not responsive to treatment with one agent only drug HSP. In another embodiment, the composition of the present invention, consisting of HSP preparation and immunoreactive reagent is administered to a patient who is unresponsive to treatment with one agent only immunoreactive reagent. In another embodiment, the composition of the present invention, consisting of HSP preparation and immunoreactive reagent is administered to a patient who is unresponsive to treatment as soon as the HSP preparation or an immunoreactive reagent, but not to both treatments. In another embodiment, the composition of the present invention, consisting of HSP preparation and immunoreactive reagent is administered to a patient who is not receiving any form of medical treatment.

4.3Treatment and prevention of cancer

The invention includes methods of treatment or prevention of cancer or metastasis in a subject, comprising in any order the introduction phase the subject is the immunoreactive reagent, contains a component that recognizes the antigen or epitope of a cancer cell (i.e. immunogenic amount of an antigen of a cancer, including but not limited to the above specific to the tumor antigen associated with a tumor antigen or molecule, showing its antigenicity), and introducing the subject of many drug HSP, effective to induce or enhance an immune response in the subject against the component of the detected immunoreactive reagent.

In some embodiments, the implementation of the compositions and methods of the present invention can be used for preventing, inhibiting or reducing the growth of cancer cells or reduce metastasis of cancer cells. In a specific embodiment, the drug HSP in combination with an immunoreactive reagent inhibits or reduces the growth of cancer cells or reduces the metastasis of cancerous tumors, at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20% or at least 10%, relative to cell growth or metastasis in absence of the introduction of yazanarabiat HSP in combination with the specified immunoreactive reagent.

Cancers that can be treated in accordance with the methods of the invention include, but are not limited to, leukemia (e.g., acute leukemia such as acute lymphocytic leukemia and acute military leukemia), neoplasma, tumors (e.g., non-Hodgkins lymphoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordomas, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangiosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, carcinoma of the sebaceous glands, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic cancer, kidney cancer, hepatoma, carcinoma of the bile ducts, horiokartsinoma, seminoma, embryonal carcinoma, Wilms ' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell carcinoma of the lung, carcinoma of the bladder, epithelial carcinoma, glioma, glioblastoma polymorphic, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuromas, oligodendroglioma, meningioma, melanoma, for neuroblastoma and retinoblastoma), disease heavy chain (b-cell lymphoma, metastases, or any disease or disorder characterized by unregulated cell growth.

Tumor antigens or associated with the tumor antigens include antigens of the cancer germ cells (CG) (MAGE, NY-ESO-1), mutational antigens (MUM-1, p53, CDK-4), sverhagressivny autoantigens (p53, HER2/NEU), viral antigens (virus, papilloma virus, Epstein-Barr), tumor proteins derived from mRNA sequences non-primary open reading frame (NY-ESO1, LAGE1), Melan F, MART-1, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-1, tyrosinase, gp100, gp75, HER-2/neu, c-erb-B2, CEA, PSA, MUC-1, CA-125, Stn, TAG-72, KSA (17-1A), PSMA, p53 (having point mutations and/or sverhagressivny), RAS (with point mutations), EGF-R, VEGF, GD2, GM2, GD3, anti-Id, CD20, CD19, CD22, CD36, aberrant class II, B1, CD25 (the IL-2R), (anti-TAC) or HPV.

In a preferred embodiment, the method or composition of the present invention used for the treatment or prevention of cancer or metastasis in a subject by administration of the drug HSP and immunoreactive reagent, where the immunoreactive reagent is an antibody against CTLA-4, or an antibody against VW. In another preferred embodiment, the method or composition of the present invention used for the treatment or prevention of cancer or metastasis in a subject by administration of the drug HSP and immunoreactive reagent, where monoreactive reagent is an anti-cancer monoclonal antibody. In another preferred embodiment, the method or composition of the present invention used for the treatment or prevention of cancer or metastasis in a subject by administration of the drug HSP and immunoreactive reagent, where the immunoreactive reagent is Herceptin.

4.4The treatment of infectious diseases

The present invention also includes methods of treating or preventing an infectious disease in a subject, comprising conducted in any sequence stage of introducing the subject of the immunoreactive reagent and the introduction of the subject of the preparation of heat-shock proteins that are effective in combination with an immunoreactive reagent for inducing or enhancing an immune response against the component in the subject.

Infectious diseases that can be treated or prevented through the use of immunoreactive reagent in combination with the methods of the present invention are caused by infectious agents, including but not limited to the above, viruses, bacteria, fungi protozoa and parasites. Some of the commonly used immunoreactive reagents against infectious diseases and their appropriate dosages and applications known in the field and described in the literature, such as Physician''s Desk Reference (56thed., 2002).

Infectious agents, which is you can fight in accordance with the present invention, include, but are not limited to, viruses, bacteria, fungi and protozoan agents of diseases.

Viral diseases that can be treated or prevented through the use of immunoreactive reagent in combination with the methods of the present invention include, but are not limited to, diseases caused by a pathogen hepatitis type a, hepatitis type b, hepatitis type C, influenza, varicella, adenovirus, the causative agent of herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, Echovirus, rotavirus, respiratory syncytial virus, human papilloma virus, papovavirus, cytomegalovirus, ehirovipuka, arboviruses, Hantavirus, Coxsackie virus, virus Partita, measles virus, measles virus rubella, polio, smallpox virus, virus Epstein-Barr, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and agents of viral diseases such as viral meningitis, encephalitis, Dengue fever, or smallpox.

Bacterial diseases that can be treated or prevented through the use of immunoreactive reagent in combination with the methods of the present invention, caused by bacteria, including but not limited to the above, the tubercle bacilli is ' series, rickettsiae, Mycoplasma, Neisseria, S. pneumoniae, Borellia burgdorferi (the causative agent of Lyme disease), Bacillus nthracis (the causative agent of anthrax), the causative agent of tetanus, Streptococcus, Staphylococcus, Mycobacterium, the causative agent of tetanus, the causative agent of pertussis, the causative agent of cholera, the causative agent of plaque, the causative agent of diphtheria, chlamydia, S. aureus and Legionella.

Protozoal diseases and/or parasitic diseases that can be treated or prevented through the use of immunoreactive reagent in combination with the methods of the present invention, are caused by protozoa and/or parasites, including but not limited to the above, Leishmania, coccidia, Triangoli, the causative agent of malaria, chlamydia, Rickettsia, the causative agent of Chagas disease, filaree, the causative agent of toxoplasmosis, the causative agent of schistosomiasis and tapeworms, which cause diseases.

4.5Treatment of neurodegenerative diseases

Can also be used immunoreactive reagent that specifically binds an antigenic molecule in the cell or on the cell or structure, such as extracellular deposits or plaques containing peptide and/or protein fibrils, which are hallmarks of neurodegenerative or amyloid disease. When you want to treat or prevent neurodegener is positive or amyloid disease preferably use immunoreactive reagents that specifically bind to molecules containing epitopes of antigenic molecules associated with neurodegenerative diseases, or epitopes of antigenic molecules associated with melodrami diseases, including but not limited to the above, the peptides or proteins fibrillin. Such related neurogenerative disease antigenic molecules may be molecules that are associated with Alzheimer's disease, age-related loss of cognitive function, hydrocyanic dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's disease, cortical palsy, progressive supranuclear palsy, a disease of GUAM, dementia associated with the disease Taurus Levi, diseases caused by prion, spongiform encephalopathy, disease of Creutzfeldt-Jakob disease, polyglutamine disease, Huntington's disease, motonishi dystrophy, hereditary ataxia, ataxia syndrome Gilles de La Tourette's, seizure disorders, epilepsy, chronic epileptic disorder, stroke, trauma brain, spinal trauma, dementia associated with AIDS, alcoholism, autism, retinal ischemia, glaucoma, vegetative functional disorders, hypertension, neuropsyche the electrical violation, schizophrenia or schizoaffective disorder.

Examples of such antigenic molecules described in WO 01/52890, published on July 26, 2001, which is incorporated into this description by reference in its entirety, and include, but are not limited to, β-amyloid or its fragment, oligomeric β complex or its fragment, complex Area-β, Tau-protein or its fragment, the precursor protein of amyloid or its fragment, the precursor protein mutant amyloid or its fragment, presenilin or a fragment, mutant presenilin or its fragment, α-synuclein or its fragment or protein or prion his fragment and antigenic derivatives of any of the above proteins or their fragments. Associated with amyloid disease antigenic molecules may be molecules associated with diseases characterized by the extracellular deposition of protein and/or peptide fibrils, which form amyloid deposits or plaques, and including but not limited to the above, type II diabetes and amyloidosis associated with chronic inflammatory or infectious diseases and malignancies such as myeloma. Some amyloid diseases, such as, but not limited to, Alzheimer's disease and diseases caused by prions, such as disease Creutzfeldt-Jacob, also Vlada neurodegenerative diseases.

4.6Drugs HSP

Any HSP or HSP preparation, are known in this field can be used in the compositions and methods of the present invention. For the purposes of this invention, the HSP preparations may include, without limitation, free HSP that is not associated with any molecule, molecular complexes of HSP with another molecule such as a peptide and fused proteins HSP. Complex HSP-peptide contains HSP, covalently or ecovalence attached to the peptide. Complex HSP-peptide may consist of HSP associated with peptides derived from the interest of the tumor, pathogen or cell type and/or protein (e.g., the same target, which is recognized by the antibody). Alternatively, the complex HSP-peptide may consist of HSP associated with the endogenous peptide, but not with peptide from the same source as the target of therapeutic antibodies. The methods of the present invention do not require covalent or non-covalent joining some specific antigens or antigenic peptides prior to introduction to the subject. The HSP preparation can be or can not be obtained from a subject who is administered the drug. HSP complex HSP-peptide or protein HSP preferably clear. The HSP preparation can include crude cell lysate containing the HSP, and the amount of lysate soo the same 100-10 8equivalents cells. When the peptide is attached to the HSP, the peptide can be any peptide that can be ecovalence, covalently linked or merged with HSP. HSP can be an accepted way cleared of the majority of cellular sources, because the population of complexes of different peptides ecovalence associated with HSP. HSP can be separated from ecovalence related peptide processing environment with a low pH value and/or triphosphate, or other methods known in this field. The HSP preparation is usually administered separately from the immunoreactive reagent. The peptide(s) may not be specific immunoreactive reagent or infectious disease or disorder. For the comfort and convenience of the recipient, the HSP preparation can be mixed with an immunoreactive reagent immediately before the introduction.

In various embodiments, the implementation of source HSP is preferably eukaryotic, more preferably a mammal and most preferably a human. In accordance with this drug HSP used by the methods of the invention include eukaryotic HSP, HSP mammal and HSP man. Eukaryotic source from which to obtain the drug HSP, and the subject receiving the drug HSP, preferably are in the same mind.

In various embodiments, the implementation of the HSP preparation can in order to keep HSP, including but not limited to the above, hsp60, hsp70, hsp90, hsp110, gp96, grp170 or calreticulin, separately or in combination with each other. HSP preferably represents hsp60, hsp70, hsp90, hsp110, gp96, grp170 or calreticulin. The present invention also includes complexes of HSP-peptide, such as complexes of hsp60-peptide complexes of hsp70-peptide complexes of hsp90-peptide complexes hsp110-peptide complexes gp96-peptide complexes grp170-peptide or complexes calreticulin-peptide. The present invention also includes fused proteins HSP, such as fused proteins hsp60, fused proteins hsp70 fused proteins hsp90, fused proteins, hsp110, fused proteins gp96, fused protein grp170 or fused proteins calreticulin.

In a preferred embodiment, the HSP preparation contains a single (free) HSP, HSP complex or protein HSP. In another embodiment of the present invention, the HSP preparation contains a mixture of HSP, HSP complexes or fused proteins HSP. Preferably the mixture of HSP, HSP complexes and/or fused proteins HSP contains two or more essentially pure HSP, HSP complexes and/or fused proteins HSP. Used herein, the term "essentially pure" means essentially free from compounds normally associated with HSP or HSP complex in its natural state, and exhibiting a constant and reproducible chromatographic characteristics, the elution profiles and biologicallyactive. Essentially pure HSP complexes do not produce peptides, which form with HSP complex covalent or non-covalent manner, or peptides, which form an endogenous complex with HSP. The term "essentially pure" does not mean that it does not include artificial or synthetic mixtures HSP, HSP or fused proteins HSP with other compounds. A large number of non-limiting examples of HSP, HSP complexes and merged HSP proteins and methods for their preparation are presented below.

In one embodiment, when the drug HSP do not use in combination with an immunoreactive reagent for the induction of specific immune response, the introduction of only one drug does not induce HSP antigen-specific immune response, which could be induced immunoreactive reagent. In another embodiment, the HSP preparation is really induces antigen-specific immune response, which could be induced immunoreactive reagent.

Assumes that all HSP, belonging to the family hsp60, hsp70 and hsp90, including fragments of such HSP, can be used to practice the present invention.

In the present invention use purified unrelated HSP, HSP, covalently or ecovalence associated with specific peptides or non-specific peptides (collectively called herein described and complexes of HSP-peptide), merged HSP proteins, and combinations thereof. Purification of HSP in the form of complexes or in free form is described in the following subsections. In addition, the person skilled in the art can synthesize HSP and fused proteins recombinant HSP expression or peptide synthesis, which are also described below.

In another embodiment assumes that proteins HSP may be other proteins, mutiny, analogs, and variants having at least 35-55%, preferably 55-75%, and most preferably 75 to 85% amino acid identity with members of the three main families of stress proteins, the levels of expression in the cell increases in response to a stressful stimulus. Production, isolation and purification of stress proteins belonging to the class of HSP known in this field and described in the literature. For example, the production and purification of calreticulin described in the publication Basu and Srivastava, 1999 J. Expt. Med. 189: 797-802, incorporated herein by reference in its entirety. The invention also includes methods of preparation and purification of HSP and complexes of HSP-peptide, they are described below and are presented by way of example, but not by way of limitation.

4.6.1Production and purification of Hsp70 or complexes of Hsp70-peptide

Purification of complexes of hsp70-peptide has been described previously, see, for example, Udono et al., 1993, J. Exp. Med. 178: 1391-1396. The procedure, which can be used and presented by way of example, and without whom ograniczenia, is the following.

First, cells of a human or mammal suspended in 3 volumes of buffer to 1X lysis, consisting of a buffer, 5 mm of sodium phosphate (pH 7), 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2and 1 mm phenylmethylsulfonyl (PMSF). Then the precipitate is subjected to the action of ultrasound with ice cooling up until >99% of the cells will be lysed, and the % lysis determine microscopic examination. Alternatively, ultrasonic treatment, the cells can be lysed by mechanical fragmentation and in this approach, cells are usually resuspended in 30 mm sodium bicarbonate (pH 7.5), 1 mm PMSF, incubated on ice for 20 minutes and then homogenized in the homogenizer of downs until, until lysed >95% of the cells.

Then the lysate centrifuged at 1000 × g for 10 min to remove intact cells, nuclei and other cellular debris. The resulting supernatant is again centrifuged at 100000 × g for 90 minutes, the supernatant harvested and then mixed with separateTMCon And balanced phosphate buffered saline (PBS)containing 2 mm CA2+and 2 mm Mg2+. When cells are lysed by mechanical fragmentation, the supernatant is diluted with equal volumes of buffer for lysis 2 before mixing with separateTMCon A. the Supernatant is then leased the claim to associate with separate TMCon And for 2-3 hours at 4°C. the Material is not contacted, collect and cialiswhat within 36 hours (three times, 100 volumes each time) against 10 mm Tris-acetate (pH 7.5), 0.1 mm EDTA, 10 mm NaCl, 1 mm PMSF. Then dialysed centrifuged at 17000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and injected into the ion-exchange chromatographic column Mono Q FPLCTM(Pharmacia), equilibrated to 20 mm Tris-acetate (pH 7.5), 20 mm NaCl, 0.1 mm EDTA, 15 mm 2-mercaptoethanol. To the column and then apply the gradient of 20 to 500 mm NaCl and then erwerbende faction fractionary gel electrophoresis in the system sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and characterized by Western blot turns with suitable antibodies against hsp70 (for example, from clone N27F3-4, StressGen).

Faction, strongly immunodiagnosis with antibody against hsp70, unite and complexes of hsp70-peptide precipitated with ammonium sulfate; especially 50-70% ammonium sulfate. The resulting precipitate is then collected by centrifugation at 17000 rpm (Sorvall SS34 rotor) and washed with 70% ammonium sulfate. The washed precipitate is then solubilizers and any residual ammonium sulfate removed by gel-filtration on a column of SephadexRG25 (Pharmacia). If necessary, the thus obtained preparation hsp70 can be re-purified by passing through ion exchange chromatographic column ono Q FPLC TM(Pharmacia), as described above.

Using this method, the complex of hsp70-peptide can be purified to apparent homogeneity. Typically, 1 mg of the complex of hsp70-peptide can be cleaned from 1 g of cells/tissue.

Improved method of purification of complexes of hsp70-peptide includes contacting the cell proteins with ADP or digitalisieren analogue of ATP associated with the solid substrate, so that hsp70 in the lysate can contact ADP or digitalisieren analogue of ATP, and elution of the bound hsp70. In a preferred method using column chromatography with ADP, associated with a solid substrate (for example, ADP-agarose). The resulting hsp70 preparations are very clean and do not contain contaminating proteins that are not related endogenous peptides associated with HSP in the complex HSP-peptide. The outputs of the complex of hsp70 significantly increase, approximately more than 10 times. Alternatively, purification of complexes of hsp70-peptide can be used chromatography with neytrolizatsii analogues of ATP instead of ADP. As an example, but without limitation of the invention, the purification of complexes of hsp70-peptide by chromatography on ATP-agarose can be performed as follows.

Cell sarcoma Meth F (500 million cells) homogenized in hypotonic buffer and the lysate centrifuged at 100000 × g for 90 minutes at 4°C. Supernatant is injected into the column ADP-agarose. The column is washed with buffer and elute with 5 column volumes of 3 mm ADP. Complexes of hsp70-peptide suiryudan in fractions 2-10 from the elution just 15 fractions. Erwerbende fractions analyzed by SDS-PAGE. Using this procedure, the complexes of hsp70-peptide can be purified to apparent homogeneity.

The selection of HSP complex of hsp70-peptide can be carried out in the presence of ATP or low pH value. For elution of the peptide from the complex of hsp70-peptide can be used two ways. The first approach involves the incubation of the drug complex of hsp70-peptide in the presence of ATP. The second approach involves the incubation of the drug complex of hsp70-peptide in a buffer with low pH value. These methods and any other methods known in this field can be used to highlight HSP peptide from the complex hsp-peptide.

4.6.2Production and purification of Hsp90 or complexes of Hsp90-peptide

The procedure, which can be used and which is represented by example and without limitation, is as follows.

First, cells of a human or mammal suspended in 3 volumes of buffer to 1X lysis, consisting of a buffer, 5 mm of sodium phosphate (pH 7), 150 mm NaCl, 2 mm CaCl2, 2 mm MgCl2and 1 mm phenylmethylsulfonyl (PMSF). Then the precipitate is subjected to the action of ultrasound with ice cooling up until >99% of the notches will not be lysed, and % lizirovania determine microscopic examination. Alternatively, ultrasonic treatment, the cells can be lysed by mechanical fragmentation, and in this approach, cells are usually again suspended in 30 mm sodium bicarbonate (pH 7.5), 1 mm PMSF, incubated on ice for 20 minutes and then homogenized in the homogenizer of downs until, until lysed >95% of the cells.

Then the lysate centrifuged at 1000 × g for 10 min to remove intact cells, nuclei and other cellular debris. The resulting supernatant is again centrifuged at 100000 × g for 90 minutes, the supernatant harvested and then mixed with separateTMCon And balanced PBS containing 2 mm CA2+and 2 mm Mg2+. When cells are lysed by mechanical fragmentation, the supernatant is diluted with equal volumes of buffer for lysis 2 before mixing with separateTMCon A. the Supernatant is then incubated for binding to separateTMCon And for 2-3 hours at 4°C. the Material which is not bound, collect and cialiswhat within 36 hours (three times, 100 volumes each time) against 10 mm Tris-acetate (pH 7.5), 0.1 mm EDTA, 10 mm NaCl, 1 mm PMSF. Then dialysed centrifuged at 17000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and injected into the ion-exchange khromatograficheskoe the column Mono Q FPLC TM(Pharmacia), equilibrated with buffer for lysis. Proteins then elute with a gradient of salt from 200 to 600 mm NaCl.

Erwerbende faction fractionary using SDS-PAGE and fractions containing complexes hsp00-peptide, identified by Western blot turns using antibodies against hsp90, such as 3G3 (Affinity Bioreagents). Using this procedure, the complexes of hsp90-peptide can be purified to apparent homogeneity. Usually 150-200 μg of hsp90-peptide can be purified from 1 g of cells/tissue.

The selection of HSP complex hsp90-peptide can be carried out in the presence of ATP or low pH. For elution of the peptide of hsp90-peptide can be used two ways. The first approach involves the incubation of the drug complex of hsp90-peptide in the presence of ATP. The second approach involves the incubation of the drug complex of hsp90-peptide in a buffer with low pH value. These methods and any other methods known in this field can be used to highlight HSP peptide from the complex hsp-peptide.

4.6.3Obtaining and purification of Gp96 and complexes Gp96-peptide

The procedure, which can be used and which is represented by example and without limitation, is as follows.

Sediment cells of a human or mammal resuspended in 3 volumes of buffer containing buffer consisting of 30 mm sodium bicarbonate (pH 7.5) and 1 mm PMF, and cells allow to swell for 20 minutes on ice. The precipitated cells are then homogenized in a homogenizer of the downs (suitable clearance homogenizer will vary in accordance with each cell type) with ice cooling to lizirovania >95% of the cells.

The lysate centrifuged at 1000 × g for 10 min to remove intact cells, nuclei and other debris. The supernatant from this stage centrifugation then again centrifuged at 100000 × g for 90 minutes. Complex gp96-peptide can be purified either from the precipitate in the test tube, or from the supernatant after centrifugation at 100000 × g.

When cleaning of the supernatant, the supernatant is diluted with equal volumes of buffer for lysis 2 and the supernatant is stirred for 2-3 hours at 4°C With separateTMSOP A balanced PBS containing 2 mm CA2+and 2 mm Mg2+. Then, the suspension is injected into a column and washed with buffer to 1X lysis up until OD280drops back down to baseline. Then the column was washed with 10% α-methylmagnesium (α-MM)dissolved in PBS containing 2 mm CA2+and 2 mm Mg2+applied to the amount of 1/3 of the volume of the layer of the column, the column sealed with a piece of paraffin and incubated at 37°C for 15 minutes. Then the column is cooled to room temperature and the paraffin is removed from the bottom of the column. Enter in column five column volumes of α-MM-Boo the EPA and the eluate analyzed by SDS-PAGE. The resulting material typically has a purity of about 60-95%, but it depends on the type of cells and the relationship of tissue to buffer for lysis. The sample was then injected into the ion-exchange chromatographic column, Mono Q FPLCTM(Pharmacia), equilibrated with buffer containing 5 mm sodium phosphate (pH 7). Proteins then elute from the column with a gradient of 0-1M NaCl and the gp96 fraction elute between 400 mm and 550 mm NaCl.

This procedure, however, can be modified by two additional stages, used separately or in combination, in order to constantly obtain explicitly homogeneous complex gp96-peptide. One optional step includes the deposition of ammonium sulfate before the stage of purification on Con A, and another optional step includes cleaning on DEAE-sepharoseTMafter cleansing stage at A Con, but before the stage of purification on a Mono Q FPLC.

In the first optional stages described by way of example, as described below, the supernatant, which was formed after the stage of centrifugation at 100000 × g, adjusted to a final concentration of 50% ammonium sulfate by the addition of ammonium sulfate. Ammonium sulfate is added slowly with gentle stirring of the solution in the beaker, placed in a pan of ice water. The solution is stirred for from about 1/2 to 12 hours at 4°C and the resulting solution centrifuged the ri 6000 rpm (Sorvall SS34 rotor). The supernatant formed at this stage, remove, saturate up to 70% ammonium sulfate by addition of a solution of ammonium sulfate and centrifuged at 6000 rpm (Sorvall SS34 rotor). Formed in vitro sludge of this stage is collected and suspended in PBS containing 70% ammonium sulphate to wash the precipitate. This mixture is centrifuged at 6000 rpm (Sorvall SS34 rotor) and the precipitate in the test tube and dissolved in PBS containing 2 mm CA2+and Mg2+. Undissolved material is removed brief centrifugation at 15,000 rpm (Sorvall SS34 rotor). Then the solution is mixed with separateTMCon a and treated by a procedure which was applied to it.

In the optional second stage described by way of example, as described below, containing the gp96 fraction, erwerbende with speakers with Con A, unite and carry out the replacement of the buffer to a buffer of 5 mm sodium phosphate (pH 7), 300 mm NaCl by dialysis or, preferably, buffer replacement on a column of Sephadex G25. After replacing the buffer solution is mixed with DEAE-separateTMpre-equilibrated with buffer 5 mm phosphate (pH 7), 300 mm NaCl. The protein solution and balls gently stirred for 1 hour and poured into the column. Then the column was washed with buffer 5 mm solution of sodium phosphate (pH 7), 300 mm NaCl, up until the absorption at 280 nm has dropped to baseline. Then associated protein elute from the column is the first volumes of buffer 5 mm phosphate (pH 7), 700 mm NaCl. Containing protein fractions are combined and diluted with buffer 5 mm phosphate (pH 7) to reduce the salt concentration to 175 mm. The resulting material is then injected into the ion-exchange chromatographic column Mono Q FPLCTM(Pharmacia), equilibrated with buffer 5 mm phosphate (pH 7), and a protein that binds to the ion exchange chromatographic column Mono Q FPLCTM(Pharmacia), elute, as described above.

However, it is obvious that the person skilled in the art can appreciate the usual experimentation the advantage of including the optional second stage in the purification Protocol. In addition, it is also clear that the benefits from adding each of the optional stages will depend on the source material.

When the gp96 fraction isolated from the sediment in the tube after centrifugation at 100000 × g, the residue is suspended in 5 volumes of PBS containing either 1% of deoxycholate sodium, or 1% of octylglucoside (but without Mg2+and CA2+), and the suspension is incubated on ice for 1 hour. The suspension is centrifuged at 20000 × g for 30 minutes and the resulting supernatant cialiswhat against several substitutions PBS (without Mg2+and CA2+to remove the detergent. Dialysate centrifuged at 100000 × g for 90 minutes, the supernatant is collected and the supernatant add calcium and magnesium to produce final conc the Nations 2 mm, respectively. The sample was then purified either unmodified or modified method for separation of complex gP96-peptide from the supernatant of centrifugation at 100000 × g, see above.

Using this procedure, the complex Dr-peptide can be purified to apparent homogeneity. Approximately 10-20 μg gp96 can be isolated from 1 g of cells/tissue.

The selection of HSP complex gp96-peptide can be carried out in the presence of ATP or low pH. For elution of peptides from complex gp96-peptide can be used two ways. The first approach involves the incubation of the drug complex of the gp96-peptide in the presence of ATP. The second approach involves the incubation of the drug complex of the gp96-peptide in a buffer with low pH value. These methods and any other methods known in this field can be used to highlight HSP peptide from the complex gp96-peptide.

4.6.4Production and purification of complexes Hsp110-peptide

The procedure described by Wang et al., 2001, J. Immunol. 166(1): 490-7, can be used and presented by example without limitation, is as follows.

The precipitate (40-60 ml) of cells or tissue, for example tissue of tumor cells, homogenized in 5 volumes of hypotonic buffer (30 mn sodium bicarbonate, pH of 7.2, and protease inhibitors) using a homogenizer of the downs. The lysate centrifuged at 4500 × g and then 100,000 × g in ECENA 2 hours. If the cells or tissue are of hepatic origin, the resulting supernatant is first injected into the column blue sepharose (Pharmacia) to remove albumin. Otherwise, the resulting supernatant is injected into the column with separate Con-A (Pharmacia Biotech, Piscataway, NJ)pre-equilibrated with buffer for binding (20 mm Tris-HCl, pH 7.5; 100 mm NaCl; 1 mm MgCl2; 1 mm Cl2; 1 mm MnCl2and 15 mm 2-ME). Associated proteins elute buffer binding, containing 15% of α-D-o-methyladenosine (Sigma, St. Louis, MO).

Non-separate Con-A first material cialiswhat against a solution of 20 mm Tris-HCl, pH 7.5; 100 mm NaCl and 15 mm 2-ME, and then injected into a column of DEAE-sepharose and elute the salt gradient from 100 to 500 mm NaCl. The fractions containing hsp110, collect, dialist and loaded into a column of Mono Q (Pharmacia) 10/10, balanced 20 mm Tris-HCl, pH 7.5; 200 mm NaCl and 15 mm 2-ME. Associated proteins elute with a gradient 200-500 mm NaCl. Fractions analyzed by SDS-PAGE with subsequent Western blot turns with Ab for hsp110, as described by Wang et al., 1999, J. Immunol. 162: 3378. Combined fractions containing hsp110, concentrating device Centriplus (Amicon, Beverly, MA) and injected into the column superose 12 (Pharmacia). Proteins elute 40 mm Tris-HCl, pH 8.0; 150 mm NaCl and 15 mm 2-ME at a flow rate of 0.2 ml/min

4.6.5Production and purification of biogas produced complexes Grp170-peptide

The procedure described by Wang et al., 2001, J. Immunol. 166(1): 490-7, may be the used and presented by means of example, without limitation, is the following.

The precipitate (40-60 ml) of cells or tissue, for example tissue of tumor cells, homogenized in 5 volumes of hypotonic buffer (30 mn sodium bicarbonate, pH of 7.2, and protease inhibitors) using a homogenizer of the downs. The lysate centrifuged at 4500 × g and then 100,000 × g for 2 hours. If the cells or tissue are of hepatic origin, the resulting supernatant is first injected into the column blue sepharose (Pharmacia) to remove albumin. Otherwise, the resulting supernatant is injected into the column with separate Con-A (Pharmacia Biotech, Piscataway, NJ0, pre-equilibrated with buffer for binding (20 mm Tris-HCl, pH 7.5; 100 mm NaCl; 1 mm MgCl2; 1 mm Cl2; 1 mm MnCl2and 15 mm 2-ME). Associated proteins elute buffer binding, containing 15% of α-D-o-methyladenosine (Sigma, St. Louis, MO).

Associated with separate Con-A first material cialiswhat against a solution of 20 mm Tris-HCl, pH 7.5, and 150 mm NaCl and then injected into the column Mono Q and elute with a gradient of salt 150-400 mm NaCl. Combined fractions are concentrated and injected into the column superose 12 (Pharmacia). Collect the fractions containing homogeneous grp170.

4.6.6.Recombinant expression of HSP

Methods known in this field can be used for the recombinant production of HSP. The sequence of a nucleic acid encoding a HSP, can be inserted into the expression vector d is I the propagation and expression in host cells.

Used here the expression design refers to a nucleotide sequence encoding HSP, functionally associated with one or more regulatory regions that make possible the expression of HSP in a suitable cell host. "Functionally linked" refers to a linkage in which the regulatory region and the HSP sequence to be expressed are joined and positioned in such a way as to make possible the transcription and, ultimately, translation.

Regulatory region required for transcription of HSP, can be provided by expressing vector. Can also be provided with the codon of translation initiation (ATG), if must be expressed sequence HSP gene devoid of its own initiation codon. In a compatible system design host-cell transcription factors, such as RNA polymerase, will contact the regulatory regions on the expression constructs for transcription of the modified HSP sequence in the body of the host. The exact nature of the regulatory regions needed for gene expression may vary from one host cell to another cell-master. Typically requires a promoter that is able to bind RNA polymerase to start transcription of the functionality is Ino associated nucleic acid sequence. Such a regulatory region can include 5'-non-coding sequences involved with initiation of transcription and translation, such as TATA-box, kiperousa sequence, SAAT-sequence, and the like. Non-coding region 3' (right) relative to the coding sequence may contain regulatory sequences termination of transcription, such as the terminator and polyadenylation sites.

To attach DNA sequences with regulatory functions, such as promoters, to the HSP gene sequence, or embed HSP gene sequence into the cloning site of a vector, linkers or adapters providing the appropriate compatible restriction sites may be legirovanyh to the ends of the cDNA by methods well known in the art (Wu et al., 1987, Methods in Enzymol. 152: 343-349). For cleavage by the restriction enzyme can be followed by modification to create blunt ends, reverse the removal or completing single-stranded DNA before legirovaniem. Alternatively, the desired restriction site of the enzyme can be introduced into the DNA fragment amplification of this DNA by using PCR with primers containing the restriction site of the enzyme.

Expression construct that contains the HSP sequence functionally linked to a regulatory region is s, can be directly entered in the appropriate cell hosts for the expression and production of complexes of HSP-peptide without further cloning. See, for example, U.S. patent No. 5580859. The expression construct can also contain DNA sequences that facilitate integration HSP sequences in the genome of the host cell, for example, by homologous recombination. In this case there is no need to use expressing a vector containing the origin of replication suitable for appropriate host cells in order to reproduce and the expression of HSP in cells of the host.

Can be used in a variety of expression vectors, including but not limited to the above, plasmids, Comedy, phage, family or modified viruses. Typically, these expressing vectors contain a functional origin of replication for propagation of the vector in a suitable cell host, one or more sites of restriction endonucleases to embed sequences of HSP gene and one or more breeding markers. The expression vector should be used with a compatible cell host, which can be obtained from prokaryotic or eukaryotic organism, including but not limited to the above, bacteria, yeast, insects, mammals and humans.

DL the long-term production with high yield properly processioning HSP or HSP complexes-peptide is preferred stable expression in mammalian cells. Cell lines that stably Express HSP or HSP complexes-peptide, can be constructed with the use of a vector that contains breeding marker. By way of example, and without limitation, after the introduction of expression constructs engineered cells may be given the opportunity to grow for 1-2 days in an enriched medium, and then transferred to selective medium. Breeding marker in the expression of a design confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and grow in culture and proliferate in cell lines. These cells can be cultivated over a long period of time with continuous expression of HSP.

Recombinant cells can be grown under standard conditions of temperature, time of incubation, the optical field and the composition of the medium. However, conditions for growth of recombinant cells may be different from the conditions of HSP expression and antigenic proteins. To improve the production of HSP may also be used modified culturing conditions and environment. For example, recombinant cells containing the HSP with their own promoters, may be subjected to heat or other environmental stress and chemical stress. Any method known in this field, can be used to create optimal conditions for the production of HSP or HSP complexes-peptide.

4.6.6.1.Recombinant expression of the fused protein HSP

Methods known in this field can be used for producing recombinant image fused protein consisting of the sequence of heat shock protein and the sequence of the antigenic peptide. For producing such recombinant fused protein expressing vector design with nucleic acids encoding heat shock protein, fused to sequences encoding the antigenic peptide using recombinant methods known in this field, such as the methods described in the above section 4.6.6. Slit proteins HSP-antigenic peptide then Express and secrete. Such fused proteins can be used for the induction of immune response; Suzue et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 13146-51. Through specific design antigenic peptide part of the molecule such fused proteins can be used for the induction of immune response and immunotherapy against a subject to treatment of diseases or disorders.

4.6.7Peptide synthesis

An alternative way of obtaining recombinant HSP ways is the synthesis of peptides. For example, a HSP or peptide, sootvetstvujushij part of the HSP, can be synthesized using a peptide synthesizer. Can be used in the conventional synthesis of peptides or other synthetic protocols known in this field.

Peptides having the amino acid sequence of HSP or part thereof, can be synthesized by solid-phase peptide synthesis using procedures similar to the procedures described by Merrifield, 1963, J. Am. Chem. Soc. 85: 2149. During the synthesis of N-α-protected amino acids having protected side chains, attached step by step to growing polypeptide chains linked to its C-terminal part with an insoluble polymer carrier, i.e. granules of polystyrene. The peptides are synthesized by linking an amino group of the N-α-unprotected amino acid with the α-carboxyl group of N-α-protected amino acids, which was activated by its interaction with this reagent, as dicyclohexylcarbodiimide. Join free amino group to an activated carboxyl leads to the formation of the peptide bond. The most commonly used N-α-protective groups include BOC, which is unstable under the action of acid, and Fmoc, which is unstable under the action of bases. Details of suitable chemical analyses, resins, protective groups, protected amino acids and reagents are well known in this field and therefore detail in the data is m description not discussed (see Atherton, et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nded., Springer-Verlag).

Purification of the resulting HSP is carried out using conventional procedures, such as preparative HPLC, gel permeation, distribution and/or ion exchange chromatography. The selection of appropriate matrices and buffers is well known in this area and therefore, this description is not described in detail.

4.7Activated antigenpresenting cells (APC)

In a different implementation, as described above, instead of the HSP preparation to a subject to achieve a similar result can be entered activated APC. The present invention includes a method of activating antigen-presenting cell, comprising contacting the APC with the HSP preparation. Before processing the HSP preparation for the activation of APC cells can be optionally enriched or purified and/or reproduced ex vivo methods, well known in this field. APC can be obtained from the body of the subject, preferably of the same subject, which again enter treated APC (i.e. use of autologous APC), can also be used neutogena ARS. Neutogena APC can be syngeneic (i.e. taken from an identical twin of an individual, which will be introduced activated APC) or allogeneic (i.e. taken from in the of epiderma, which has at least one common MHC allele with the individual activated APC will be entered).

Monitoring activation of ARS can be carried out by methods well known in the field, such as the methods described in section 6 to test CD11b+cells, but not limited to such methods. In a particular embodiment, the activated APCS can be used in vivo to induce or enhance the immune response induced immunoreactive reagent that is administered to a subject in reasonably the same time. Activated APCS can be an alternative introduced in different time intervals, as described above, but these intervals are not limited to these intervals, from one to twelve hours before or after administration of an immunoreactive reagent, or periodically over several days or more after using immunoreactive reagent slowly or continuously releasing type. Processed ARS is preferably introduced at a location at or near the introduction of the immunoreactive reagent. Introduction of activated APC may be performed by any means known in this field.

4.8Immunoreactive reagents

Immunoreactive reagents include antibodies, molecules or proteins, in which the genetic engineering enabled the anti-Christ. envasada part of the antibody, molecules or proteins, antibodies, molecules or proteins, in which the genetic engineering enabled antigennegative domain that recognizes interest antigen target, and the domain of the constant region, which mediates antibody-dependent immune response, peptide or domain, which interacts specifically with interest antigen, or any antigennegative domain, which interacts with interest antigen/epitope, and the domain of the constant region of the antibody that mediates antibody-dependent immune reactions or processes of effector cells. Examples of such domains or areas in the constant region Ab, which can be used in the present invention include domains and areas described in the publications Reddy et al., 2000, J. Immunol. 164(4): 1925-33; Coloma et al., 1997, Nat. Biotechnol. 15(2): 159-63; Carayannopoulos et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91(18): 8348-52; Morrison, 1992, Annu. Recombinant Expression Vector Immunol. 10:239-65; Traunecker et al., 1992, Int. J. Cancer Suppl. 7: 51-2; Gillies et al., 1990, Hum. Antibodies Hybridomas 1(1): 47-54; each of which is incorporated in this description by reference in its entirety.

Immunoreactive reagents of the present invention preferably contain 1) antigennegative region 2) region, which mediates one or more antibody-dependent immunological processes. Antigennegative area may contain or SOS is Oyat from antigennegative region of the antibody. Antigennegative region can contain any peptide or domain that specifically interacts with interest antigen. For example, antigennegative area can be a ligand or other specific binding partner of interest antigen, or may be a fragment of such a ligand or binding partner, or can be obtained from such a ligand or binding partner.

Region, which mediates one or more antibody-dependent immunological processes, may contain or consist of the area that are capable of binding Fc receptor, for example the part of the antibody that binds Fc-receptor, or the region that binds the complement, for example complimentative region of the antibody. This area can be antigenspecific domain antibody that binds to Fc receptors or complement.

Such antibody-dependent processes include, but are not limited to, antibody-dependent cellular cytotoxicity, activation of complement, opsonization and phagocytosis. Effector cells that mediate some of antibody-dependent processes include monocytes, macrophages, natural killer cells and polymorphically cells. Without communication with a specific mechanism believe that HSP is able to increase the number of receptors on the effector what's cells, responsible for mediating antibody-dependent reactions. These receptors include Fc-alpha - and Fc-gamma receptors and their isoforms or any combination of them. Thus, in a specific embodiment, the area of immunoreactive reagent, which mediates one or more antibody-dependent immunological processes, contains or consists of a region, which is a ligand for Fc-receptors, preferably Fc-α receptor or Fc-gamma receptor or both. In another embodiment, the area of immunoreactive reagent, which mediates one or more antibody-dependent immunological processes, contains or consists of a region that stimulates the function of immune effector cells, preferably monocytes, macrophages, natural killer cells, polymorphing cells, or any combination of two or more types of such cells, to achieve a prophylactic and/or therapeutic effect. In some embodiments, implementation, when a drug induces HSP immune response, such as cytokine and/or activation of T cells, immunoreactive reagent potentiates or costimulate such immune response.

In a preferred embodiment, the immunoreactive reagent is an antibody or a composition comprising the antibody or antibodies is, such as serum. In a specific embodiment, the immunoreactive reagent is an antibody IgA, IgG or IgM, or it contains a fragment. In a particularly preferred embodiment, the immunoreactive reagent is a monoclonal antibody, or he includes fragments of monoclonal antibodies. Immunoreactive reagent may also contain or consist of immunoglobulin for treatment of hepatitis b; respigam for the treatment of RSV; sandoglobulin or immune globulin IV (IGIV). In another embodiment, the immunoreactive reagent is not directed to any one epitope, but instead is a mixture of one or more molecules, which is associated with the population epitopes. An example of such an immunoreactive reagent is serum or antibodies, concentrated from serum or plasma. Such serum or plasma can be taken from a subject immunized against a particular antigen, or a subject who has not been immunized.

Antibodies that can be used in the methods of the present invention include, but are not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, polyspecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, Advocaat cnie Fv (scFv), single-chain antibodies, Fab fragments, F(ab')fragments linked by disulfide bonds Fv (sdFv), and antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the present invention) and epilepsyusa fragments of any of the above. In particular, antibodies used in the methods of the present invention include immunoglobulin molecules and immunologically active portion of immunoglobulin molecules, i.e. molecules that contain antigennegative site that immunospecificity way associated with interest target. The immunoglobulin molecules of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecules.

In another embodiment, the immunoreactive reagent is bespecifically molecule having two antigennegative of different specificity, i.e. one recognizes an epitope on the target cell or protein, and another that recognizes the epitope effector cells, such as epitope FcR. In another embodiment, the immunoreactive reagent is bespecifically molecule having two antigenspecific domain for different epitopes on the targeted cell/protein and domain, which mediates antitilos the independent immune response. Such bespecifically molecules that targeting cancer cells or pathogens, and their therapeutic effects were studied both in vivo and in vitro (for example, in the publications by Wallace et al., 2001, J. Immunol. Methods 248(1-2): 167-82; Sundarapandiyan et al., 2001, J. Immunol. Methods 248(1-2): 113-23; Honeychurch et al., 2000, Blood 96(10): 3544-52; Negri et al., 1995, Br J Cancer 72(4):928-33; Wang et al., 1994, Zhonghua Zhong Liu Za Zhi 16(2): 83-7, Chinese) (each of the publications included in this description by reference in its entirety).

In a preferred embodiment, the immunoreactive reagent cleanse. The term "purified"as used here to describe some of the peptides, antibodies, molecules, proteins, antigens, HSP, HSP-peptide and the like, refers to a state outside the state in which molecules, proteins, antigens and the like is separated from the more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of proteins, polysaccharides, and/or lipids, which peptides antibodies, molecules, proteins, antigens, HSP, complexes of HSP-peptide and the like normally associated in nature. If the selected molecules, proteins, antigens, HSP, complexes of HSP-peptide and the like are synthesized, they are polluted less than 50%, 40%, 30%, 20%, 10%, 5%, 1% or 0.1% chemical precursors or synthesis reagents used in the synthesis of molecules, proteins, antigens, HSP, HSP-peptide and the like. In preferred embodiments, the implementation of the peptides, anti the ate, molecules, proteins, antigens, HSP, complexes of HSP-peptide and the like have at least 1% pure, 5% purity, 10% purity, 20% purity, 30% purity, 40% purity, 50% purity, 60% purity, 70% purity, 80% purity, 90% purity, 95% purity, 99% pure or 100% pure. Used in the present description, the term "% purity indicates the mass percentage of the overall composition, which makes the molecule "interesting". Thus, the composition with a mass of 100 grams, containing 50 grams of interest molecules, has a 50% purity in respect of interest of the molecule.

Monoclonal antibodies can be obtained using a wide variety of methods known in this field, including the use of technology hybridoma, recombinant, and phage presentation (display) or their combination. For example, monoclonal antibodies can be obtained using methods hybridoma, well known in this field, and are described, for example, in the publications Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nded. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas, pp 563-681 (Elsevier, N.Y., 1981) (both publications included in the present description by reference in their entirety). Used herein, the term "monoclonal antibody" is not limited to antibodies produced through technology hybridoma. The term "monoclonal antibody which" refers to the antibody, which is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the way in which it is received.

4.8.1Getting immunoreactive reagents

Immunoreactive reagents of the present invention can be obtained by any method known in the field of synthesis of antibodies, especially by chemical synthesis or preferably, by means of recombinant expression. Such methods are described below with reference to the immunoreactive reagent is an antibody, but they are easily suitable for other immunoreactive reagent.

Methods of producing and screening for specific antibodies using technology hybridoma, are conventional and well known in this field. In a non-limiting example, mice can be immunized interest antigen or a cell expressing such antigen. After detection of the immune response, for example, antibodies specific for this antigen to be detected in the mouse serum, the mouse spleen is separated and isolated splenocytes. The splenocytes are then subjected to fusion of well-known methods with any suitable myeloma cells. Hybridoma select and clone by limiting dilution. Clones of hybridoma then analyzed by methods known in this field, on prisutstvie the e cells, which secrete antibodies capable of binding the antigen. Ascitic fluid, which typically contains high levels of antibodies, can be generated by inoculation of mice intraperitoneally positive gibridnye clones.

Fragments of antibodies that recognize specific epitopes may be generated by known methods. For example, Fab fragments and F(ab')2can be obtained by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2-fragments. F(ab')2the fragments contain a complete light chain and the variable region SN and hinge region of the heavy chain.

Antibodies can also be generated, for example, using different methods phage views known in this field. In the methods of phage representation of the functional domains of antibodies are located on the surface of phage particles which carry the coding them polynucleotide sequence. In a specific embodiment, such phage can be used to represent antigenspecific domains, such as Fab and Fv or stabilized by a disulfide bond Fv, downregulation of a set or library of chimeric antibodies (e.g., human or murine). Phage expressing ant is enswathe domain, which binds a domain of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound to a solid surface or bead or captured such surface or granule. Phage used in these methods is typically filamentous phage including fd and M13. Antigennegative domains Express in the form of recombinant image fused proteins or gene III or gene VIII phage. Examples of methods of phage views that can be used to produce immunoglobulin or its fragments of the present invention include the methods described in the publications Brinkman et al., 1995, J. Immunol Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al, 1994, Advances in Immunology 57:191-280; PCT application no PCT/GB91/01134; PCT publication WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401 and U.S. patents№№ 5698426; 5223409; 5403484; 5580717; 5427908; 5750753; 5821047; 5571698; 5427908; 5516637; 5780225; 5658727; 5733743 and 5969108; and each of the publications, applications and patents included in this description by reference in its entirety.

As described in the above references, after selection of phage encoding the antibody region of the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired f is Ahmetov and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, you can use the methods for recombinant obtain Fab fragments, Fab' and F(ab')2using methods known in this field, such as the methods described in PCT publication WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; and Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043 (each of these publications is incorporated by reference in its entirety). Examples of methods that can be used to produce single-chain FV antibodies, include the methods described in U.S. patent No. 4946778 and 5258498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu et al., 1993, PNAS 90:7995-7999; and Skerra et al., 1988, Science 240:1038-1040.

For some applications, including the use of antibodies in vivo in humans, can be preferably used chimeric, humanized antibodies or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are obtained from different animal species, such as antibodies having a variable region derived from murine monoclonal antibody and a constant region derived from human immunoglobulin. Methods of obtaining chimeric antibodies are known in the literature. See, for example, the publication Mrrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gilles et al., 1989, J. Immunol. Methods 125:191-202; U.S. patent No. 5807715; 4816567 and 4816397, which is incorporated into this description by reference in their entirety. Humanitarianism antibodies are antibody molecules from view, is not a man that bind to the desired antigen, have one or more complementarity determining regions (CDR), which is not a person, and a frame region of the molecules of the immunoglobulin. Often the frame remains in the frame areas of a person can be replaced by the corresponding residue CDR-donor antibody to change, preferably enhance the binding of the antigen. These frame replacement identify ways well known in this field, for example by modeling the interactions of the CDR and framework residues to identify the frame of residues important for antigen binding, and comparing the sequences to identify unusual frame residues in certain positions. See, for example, Queen et al., U.S. patent No. 5585089; Reichmann et al., 1988, Nature 332:323, which are included in this description by reference in their entirety. Antibodies can be humaniterian using various methods known in this field, including, for example, CDR-transfer (EP 239400; PCT publication WO 91/09967; U.S. patent No. 5225539; 5530101 and 5585089), wrapping (wrapping) or images of what the other surface (EP 592106; EP 519596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al, 1994, Proc Natl Acad. Sci. USA, 91:969-973), and the permutation circuit (U.S. patent No. 5565332), all of these publications and patents are included in this description by reference in their entirety).

Prolonged use of therapeutic and/or prophylactic antibodies may be limited by the immunogenicity of the antibody that causes an immune reaction in the host, which limit their functional efficiency and application. Strategies have been developed, including technology TolerMabTM(TolerRx, Cambridge, MA), for modification of monoclonal antibodies to reduce immunogenicity of the antibody, thereby to enable the ability to perform prolonged and/or intermittent introduction of antibodies for therapy and at the same time to avoid neutralization by the immune system of the host. In line with this, "taurisano" a monoclonal antibody, which was given the ability to induce tolerance to itself while maintaining the ability to have a target antigen and to carry out its function in viv may be necessary for therapeutic or prophylactic treatment of patients in accordance with the present invention. Gilliland et al., 1999, J. Immunol. 162:3663-71.

Fully humanized antibodies are especially necessary for therapeutic or prevention is the definition of the treatment of patients-people. Human antibodies can be obtained by various methods known in this field, including the above-described methods phage representation using libraries of antibodies obtained from sequences of human immunoglobulin. Cm. U.S. patent No. 4444887 and 4716111 and PCT publication WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735 and WO 91/10741, each of which is incorporated into this description by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are unable to Express a functional endogenous immunoglobulins, but which can Express human immunoglobulin genes. An overview of this technology for obtaining human antibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol 13:65-93. A detailed discussion of this technology for obtaining human antibodies and monoclonal human antibodies and protocols for producing such antibodies, see, for example, in PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European patent No. 0598877; U.S. patents№№ 5413923; 5625126; 5633425; 5569825; 5661016; 5545806; 5814318; 5885793; 5916771 and 5939598, and all publications and patents are included in this description as a reference in their entirety. In addition, to provide human antibodies directed against a selected antigen, can be specified companies such as Abgenix®, Inc. (Freemont, CA), Medarex® NJ) and Genpharm® (San Jose, CA), using technology similar to the technology described above.

Completely human antibodies which recognize a selected epitope can be generated using a method called "guided selection". In this approach, selected nonhuman monoclonal antibody, such as a mouse antibody, is used to control the selection of a completely human antibody recognizing the same epitope (Jespers et al., 1988, Bio/technology 12:899-903).

In a preferred embodiment, antibodies have in vivo therapeutic and/or prophylactic use. Examples of therapeutic and prophylactic antibodies include, but are not limited to, MDX-010 (Medarex®, NJ)which is a humanized antibody against CTLA-4, currently used in the clinic for the treatment of prostate cancer; synagis® (MedImmune®, MD)which is a humanized monoclonal antibody against respiratory Intellinova virus (RSV), used for the treatment of patients with RSV infection; Herceptin® (Trastuzumab) (Genentech®, CA)which is a humanized monoclonal antibody against HER2, used for the treatment of patients with metastatic cancer breast cancer; Remicade® (infliximab) (Centocor®, PA), which is a chimeric monoclonal antibody against TNFα, used to treat patients with bliznukova; reopro® (abciximab) (Centocor®), which is an antibody against glycoprotein (IIb/IIIa) receptor on platelets, used to prevent the formation of a clot; zenapax® (daclizumab) (Roche Pharmaceuticals®, Switzerland)which is an immunosuppressive, humanized monoclonal antibody against CD25 used to prevent acute rejection of renal allograft. Other examples are humanitarianly F(ab')2against CD18 (Genentech®); CDP860, which is a humanized F(ab')2against D18 (Celltech®, UK); PRO542, which is an antibody against gp 120 of HIV fusion with CD4 (Progenics®/Genzyme Transgenics®); Octaver, which is an antibody against the hepatitis b virus human (Protein Design Lab®/Novartis®); protivin™which is a humanized IgGl antibody against CMV (Protein Design Lab®/Novartis®); MAK-195 (Secord®)which is a murine F(ab')2against TNF-α (Knoll Pharma®/BASF®); IC14, which is an antibody against D14 (ICOS Pharm®); humanitariannet IgG1 antibody against VEGF (Genentech®); ovarex™which is a murine antibody against CA 125 (Altarex®); panorex™which is a murine IgG2a antibody against a cell surface antigen 17-IA (Glaxo Wellcome®/Centocor®); BEC2 which is a murine antiidiotypic antibody IgG (GD3 epitope) (ImClone System®); IMC-C225 which is a chimeric the IgG antibody against EGFR (ImClone System®); vitaxin™, which is GU is Anisimovna antibody against αVβ3-integrin (Applied Molecular Evolution®/MedImmune®); campath 1H/LDP-03 which is a humanized IgGl antibody against CD52 (laconic®); smart Ml95, which is a humanized IgG antibody against CD33 (Protein Design Lab®/Kanebo®); Rituxan™which is a chimeric antibody against the CD20 IgGl (ID Pharm®/Genentech®, Roche®/Zettyaku®); lymphocide™which is a humanized IgG against CD22 (Immunomedics®); smart ID10, which is a humanized antibody against HLA (Protein Design Lab®); ancoli™ (Lym-1), which is a radiolabelled murine antibody against HLA used as a diagnostic reagent (Techniclone®); ABX-IL8 is a human antibody against IL8 (Abgenix®); anti-CDl1a, which is a humanized IgGl antibody against CD11 (Genentech®/Xoma®); ICM3, which is a humanized antibody against ICAM3 (IOS Pharm®); Idec-114, which is primaryservername antibody against CD80 (ID Pharm®/Mitsubishi®); zevalin™is a radiolabelled murine antibody against CD20 (IDEC®/Schering AG®); Idec-131, which is a humanized antibody against CD40L (IDEC®/Eisai®); Idec-151, which is primaryservername antibody against CD4 (ID); Idec-152, which is primaryservername antibody against CD23 (IDEC®/Seikagaku®); smart anti-CD3, which is a humanized IgG antibody against CD3 (Protein Design Lab®); 5G1.1, which is a humanized antibody against sets the elemental factor 5 (C5) (Alexion Pharm®); D2E7, which is a humanized antibody against TNF-α (CAT®/BASF®); CDP870, which is a humanized Fab fragment against TNF-α (Celltech®); Idec-151, which is primaryservername IgGl antibody against CD4 (ID Pharm®/SmithKline Beecham®); MDX-CD4, which is a human antibody IgG against CD4 (Medarex®/Eisai®/Genmab®); CDP571, which is a humanized IgG4 antibody against TNF-α (Celltech®); LDP-02, which is humanized antibody against α4β7 (LeukoSite®/Genentech®); orthoclone OKT4A, which is a humanized IgG antibody against CD4 (Ortho Biotech®); antova™which is a humanized IgG antibody against CD40L (Biogen®); antegren™which is a humanized IgG antibody against VLA-4 (Elan®); MDX-33, which is an antibody (FcγR) man against CD64 (Medarex®/Centeon®); SCH55700, which is a humanized IgG4 antibody against IL-5 (Celltech®/Schering®); SB-240563 and SB-240683, which are humanitarianism antibodies against IL-5 and IL-4 respectively (SmithKline Beecham®); rhuMab-E25, which is a humanized IgGl antibody against IgE (Genentech®/Norvartis®/Tanox Biosystems®); ABX-CBL, which is a murine IgM antibody against CD-147 (Abgenix®); BTI-322, which is a mouse-IgG antibody against CD2 (Medimmune®/Bio Transplant®); orthoclone/ACTS, which is a murine IgG2a antibody against CD3 (ortho Biotech®); simulect™which is a chimeric IgGl antibody against CD25 (Novartis Pharm®); LDP-01, which are the two which is a humanized IgG antibody against β 2-integrin (LeukoSite); anti-LFA-1, which is a murine F(ab')2against CD18 (Pasteur-Merieux®/Immunotech®); CAT-152, which is a human antibody against TGF-β2(Cambridge Ab Tech®), and Karsavin M, which is a chimeric antibody against factor VII (Centocor®). The above immunoreactive reagents, and any other immunoreactive reagents can be introduced in accordance with any scheme taking medicines known to specialists in this field, including the schemes recommended by those prescribing immunoreactive reagents.

The nucleotide sequence encoding the antibody or other immunoreactive reagent may be obtained from any information available to the person skilled in the art (i.e., from Genbank, the literature, or by cloning). If a clone containing a nucleic acid sequence that encodes a specific antibody or epitopespecific fragment, or other immunoreactive reagent is not available, but the sequence of the molecule antibodies or epilepsyusa fragment or other immunoreactive reagent is known, a nucleic acid encoding the immunoglobulin or other immunoreactive reagent, can be chemically synthesized or obtained from a suitable source (e.g., cDNA library of antibodies Il the cDNA library, generated from any tissue or cells expressing the antibody, such as hybridoma cells selected to Express an antibody or nucleic acid, preferably poly-A+-RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected for expression of the antibody) by PCR amplification using synthetic primers, hybridisierung with 3'- and 5'-ends of the sequence or by cloning using oligonucleotide probe specific for the sequence of a particular gene, to identify, for example, a cDNA clone from a cDNA library that encodes this antibody. Then amplificatoare nucleic acid generated using PCR can be cloned into replicable cloning vectors using any method well known in this field. In the case of immunoreactive reagents that do not exist in nature, nucleic acids encoding different areas of immunoreactive reagent, can be obtained from existing libraries or genes known or can be synthesized.

After determining the nucleotide sequence of the antibody or other immunoreactive reagent manipulation with the nucleotide sequence of the antibody or the other what about the immunoreactive reagent can be carried out using, well known in this area for the manipulation of nucleotide sequences, for example, methods of recombinant DNA, site-directed mutagenesis, PCR, etc. (see, for example, the methods described in the publications Sambrook et al., 1990, Moleular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley and Sons, NY, both of which are included in the present description by reference in their entirety), to generate an antibody or other immunoreactive reagent with a different amino acid sequence, for example, by the introduction of amino acid substitutions, deletions and/or insertions in the field epilepsyusa domain antibody or other immunoreactive reagent or constant (Fc) region of an antibody or other immunoreactive reagent that participate in the interaction with immune effector cells.

For recombinant expression of the antibody or other immunoreactive reagent required design expressing vector containing a nucleotide sequence that encodes the antibody or other immunoreactive reagent. After the obtained nucleotide sequence encoding the antibody molecule or a heavy or light chain of the antibody or part thereof (preferably, but not necessarily, containing the variable region in the heavy or light chain), or other immunoreactive reagent, a vector for the production of molecules of antibody or other immunoreactive reagent can be obtained by recombinant DNA technology using methods well known in the field. So, here it describes how to obtain the protein by expression of polynucleotide containing the nucleotide sequence encoding the antibody or other immunoreactive reagent. To construct expressing vectors containing sequences encoding the antibody or other immunoreactive reagent and the appropriate signals to the regulation of transcription and translation, can be used in ways that are well known to the person skilled in the art. These methods include, for example, methods of recombinant DNA in vitro, synthetic methods and genetic recombination in vivo. The nucleotide sequence encoding the variable or constant region of the heavy chain variable or constant region of light chain, the variable region of both heavy chain and light chain, epitopespecific fragment of the variable region of the heavy and/or light chain, or one or more complementarity determining regions (CDR) of the antibody or other immunoreactive reagent, can be cloned into such a vector for expression. Expressing the vector Perrin is placed in a cage-the owner accepted methods and transfetsirovannyh cells are then cultivated in a common manner.

For expression of antibody molecules or other immunoreactive reagent of the present invention can be used in a variety of host system-expressing vector. Such systems owner-expressing vector are the media through which can be obtained and then cleared of interest coding sequences, but are also cells that can transform or transfetsirovannyh appropriate nucleotide coding sequences to Express the molecule, antibody or other immunoreactive reagent of the present invention in situ. They include, but are not limited to, microorganisms such as bacteria (e.g. E. coli and B. subtilis)transformed with recombinant expressing vector DNA bacteriophage, plasmid DNA or kosmidou containing DNA sequences encoding the antibody or other immunoreactive reagent; yeast (for example, Saccharomyces and Pichia)transformed with recombinant yeast expressing vectors containing sequences encoding the antibody or other immunoreactive reagent systems; insect cells infected with recombinant expressing the vectors of the virus (e.g. baculovirus)containing the sequence encoding antic the lo or other immunoreactive reagent; system of plant cells infected with recombinant expressing the vectors of the virus (e.g., cauliflower mosaic virus, CaMV; and tobacco mosaic virus, TMV) or transformed with recombinant plasmid expressing vectors (e.g., Ti plasmid)containing the sequence encoding the antibody or other immunoreactive reagent; and systems of mammalian cells (such as COS cells, Cho, BHK, 293, 3T3 and NSO)carrying the recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the promoter metallothionein) or from mammalian viruses (e.g., the late promoter of adenovirus; the promoter 7,5K cowpox virus). For expression of the recombinant molecule of the antibody or other immunoreactive reagent is preferably used bacterial cells such as Escherichia coli, more preferably, eukaryotic cells, especially for the expression of a molecule of recombinant antibody or other immunoreactive reagent. For example, mammalian cells such as cells of the Chinese hamster ovary (Cho), in conjunction with a vector such as an element activator primary intermediate early gene of human cytomegalovirus is an effective expression for antibodies (Foecking et al., 1986, Gene 45: 101 and Cockett et al., 1990, Bio/Technology8:2).

In the bacterial system, the number expressing vectors can be advantageously selected depending on the intended use of the expressed molecule antibody or other immunoreactive reagent. For example, when it received a large quantity of such a protein to obtain pharmaceutical compositions of antibody molecules may be desired vectors, which are aimed at the expression of high levels of products in the form of a fused protein, which are easy to clean. Such vectors include, but are not limited to expressing vector pUR278 E. coli (Ruther et al., 1983, EMBO 12:1791), in which the sequence encoding a molecule of the antibody or other immunoreactive reagent can be Legerova individually into the vector in frame with the lacZ coding region so that the protein is produced, and pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13: 3101-3109 and Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509).

In the system of the insect as a vector for expression of foreign genes using the nuclear polyhedrosis virus of Autographa californica NPV (AcNPV). The virus grows in the cells of Spodoptera frugiperda. The sequence encoding the antibody or other immunoreactive reagent, can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

p> In the cells of the host mammal can be used a number of systems in the expression of viral-based expression of the molecule antibody or other immunoreactive reagent of the present invention. In cases where as a vector expression using adenovirus of interest sequence encoding the antibody or other immunoreactive reagent, can be Legerova with adenovirus regulatory complex of transcription/translation, e.g., the late promoter and consists of three parts leader sequence. This chimeric gene may then be inserted in the adenovirus genome by recombination in vitro or in vivo. Insertion in a nonessential region of the viral genome (e.g., region El or E3) can lead to the formation of a recombinant virus that is viable and capable to Express the molecule, antibody or other immunoreactive reagent in infected hosts (e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 355-359). For efficient translation of inserted sequences encoding the antibody or other immunoreactive reagent may also need to be specific initiation signals. These signals include the initiation codon ATG and neighboring sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence is lnasty to ensure translation of the entire insert. These exogenous regulatory signals broadcast and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate elements of the enhancer of transcription, transcription terminators, etc. (see, e.g., Bitter et al., 1987, Methods in Enzymol. 153:516-544).

In addition, can be selected strain host cell that modulates the expression of the sequences of the antibody or other immunoreactive reagent, or modifies and processes the antibody or other immunoreactive reagent is needed in a specific way. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the antibody or other immunoreactive reagent. Different cell owners have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. To ensure the correct modification and processing of the expressed antibody or other immunoreactive reagent can be selected from appropriate cell lines or system owners. This purpose can be used eukaryotic cells-owners who have a cellular mechanism for the proper processing of the primary transcript, glycosylation, and phosphorylation grams of the spent product. These cells are the owners of the mammal include, but are not limited to, Cho, VERY, KSS, HeLa, COS, MDCK, 293, 3T3, W138, and particularly myeloma cells such as NS0 cells and related cell lines, see, for example, Morrison et al., U.S. patent No. 5807715, which is included, therefore, by reference in its entirety.

For continuous production with high yield of recombinant antibody or other immunoreactive reagent is preferred stable expression. For example, cell lines which stably Express molecule antibody or other immunoreactive reagent, can be obtained by genetic engineering.

Instead of using expressing vectors, which contain the beginning of the replication of viral origin, cells of the hosts can be transformed with DNA controlled by appropriate elements of the regulation of expression (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, and so on), and controlled marker. After the introduction of foreign DNA obtained by the genetic engineering of cells may be provided the ability to grow for 1-2 days in an enriched environment with subsequent growth in the selected environment. Breeding marker in the recombinant plasmid confers resistance to the selection and allows the cell is m stable integerate plasmid into their chromosomes and grow to form focus which, in turn, can be cloned and expanded into cell lines. This method can be favorably used for the obtained genetic engineering of cell lines that Express molecule antibody or other immunoreactive reagent. Obtained such genetic engineering of cell lines may be particularly applicable in screening and evaluation of compositions that interact directly or indirectly with one molecule of the antibody or other immunoreactive reagent.

Can be used a number of breeding systems, including but not limited to those listed genes timedancing of herpes simplex virus (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine (Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adrinfo.standortstr (Lowy et al, 1980, Cell 22:8-17), which can be used in cells tk-, hgprt-or aort-respectively. Also, antimetabolite resistance can be used on the basis of selection for the following genes: dhfr, which gives resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357, and O'hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which gives resistance to mycophenolate acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which gives resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol Toxicol. 3:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; and May, 1993, TIB TECH 11(5):155-215); and hygro, which gives resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the field of recombinant DNA technology, it is possible in the usual way to apply for selection of the desired recombinant clone, and such methods are described, for example, in the publications of Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley and Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al (eds.), 1994, Current Protocols in Human Genetics, John Wilew and Sons, NY; and Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which is included in this description by reference in their entirety.

The levels of expression of molecules of antibody or other immunoreactive reagent can be increased by amplification of the vector (for a review, see Bebbington and Hentschel, 1987, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DN cloning, Vol. 3. Academic Press, New York). When a marker in the vector system expressing antibody or other immunoreactive reagent is amplificare, raising the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Because amplificatory region associated with the gene of the antibody or other immunoreactive reagent, producing the antibody or other immunoreactive reagent will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

p> A host cell can be cotransfection two expressing the vectors of the present invention, the first vector encoding a polypeptide derived from the heavy chain and the second vector encoding a polypeptide derived from a light chain. Two vectors can contain identical breeding markers that are able to equal expression of the polypeptides of the heavy and light chains, or different breeding markers in order to guarantee the maintenance of both plasmids. In an alternative embodiment, can be used one vector that encodes and is capable to Express the polypeptides of the heavy and light chains. In such cases, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). Coding sequences for the heavy and light chains may contain cDNA or genomic DNA.

After the molecule, antibody or other immunoreactive reagent of the present invention obtained by recombinant expression, it can be cleaned by any method known in the field of purification of immunoglobulin molecules or other immunoreactive reagent, for example by chromatography (e.g. ion exchange, affinity, particularly by affinity chromatography for the specific antigen after protein purification and column chromatograph is it to divide by the size of molecules), by centrifugation, based on the different solubility, or by any other standard methods for protein purification. In addition, antibody or other immunoreactive reagent of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or known in this area for easier cleaning.

The present invention also includes the use of antibodies or their fragments, recombinant way fused or chemically conjugated (including both covalent and non-covalent conjugation) with a heterologous polypeptide (or any part thereof, preferably a polypeptide containing at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate the fused proteins. The merger does not necessarily need to conduct directly, it can be done via a linker sequence. For example, antibodies can be used for "targeting" heterologous polypeptides to specific types of cells, either in vitro or in vivo, the fusion or conjugation of antibodies with antibodies specific for particular cell surface receptors. Antibodies fused or conjugated heterological polypeptides, can also be used in immunological assays in vitro and purification methods using methods known in this field. See, for example, PCT publication WO 93/21232; EP 439095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. patent 5474981; Gillies et al., PNAS 89:1428-1432 (1992); and Fell et al., J. Immunol. 146:2446-2452 (1991), all of these publications and patents are incorporated by reference in their entirety.

The present invention further includes a composition comprising heterologous polypeptides fused or conjugated with fragments of antibodies. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, an Fc fragment, a Fv fragment, a fragment F(ab)2or part of it. Methods of fusion or conjugation of polypeptides with parts of the antibodies known in the field. See, for example, U.S. patents№№ 5336603, 5622929, 5359046, 5349053, 5447851 and 5112946; EP 307434; EP 367166; PCT publication WO 96/043388 and WO 91/06570; Ashkenazi et al., Proc. Natl. Acd. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154: 5590-5600 (1995) and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (these publications and patents are incorporated by reference in their entirety).

The present invention further includes the use of antibodies or their fragments, conjugated to a therapeutic agent.

The antibody or its fragment can be anywhereman with therapeutic part, such as a cytotoxin, such as cytotoxic or citied the first agent, therapeutic agent or an ion of a radioactive metal, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-tioguanin, cytarabine, 5-perurail, dacarbazine), alkylating agents (e.g., mechlorethamine, tiapa chlorambucil, melphalan, carmustine (BCNU) and lomustin (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and condicionamento(II) (DDP)cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and astromicin (AMC)), and antimitoticescoe agents (e.g. vincristine and vinblastine).

In addition, the antibody or its fragment can be anywhereman with a portion of therapeutic agent or drug is tion means, which modifies a given biological response. Part of therapeutic agents or drugs should not be construed as limited to classical chemical therapeutic agents. For example, part of a medicinal product may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, exotoxin seudomonas, cholera toxin, diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, a growth factor derived from platelets, tissue activator of plasminogen, apoptotic agents, such as TNF-α, TNF-β, AIM I (see, international patent application WO97/33899), AIM II (see international patent application WO 97/34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6:1567-1574), and VEGI (see international patent application WO 99/23105), a thrombotic agent or an antiangiogenic agent, e.g. angiostatin or endostatin, or modifier of biological reactions, such as, for example, a lymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), - stimulating factor granulocyte-macrophage colonies (“GM-CSF”), and the stimulating factor granulocyte colony (“G-CSF”)), or growth factor (e.g., growth hormone (“GH”)).

In addition, the antibody can is to be conjugated to a therapeutic parts, such as an ion of a radioactive metal, such as alpha-emitters, such as213Bi or macrocyclic chelating agents suitable for conjugation to polypeptides ions of radioactive metals, including but not limited to the above,131In131Lu,131Y131Ho,131Sm. In some embodiments, the implementation of the macrocyclic helatoobrazouatelem is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraoxane acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in this field and are described in publications Denardo et al., 1998, Clin. Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50, each of which is incorporated by reference in its entirety.

Methods of conjugation of therapeutic parts with antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs in Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (eds.), pp. 243-56 (Alan R Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd ed.), Robinson et al (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, 1982, Immimol Rev. 62:119-58.

Alternatively, the antibody may be conjugated with a second antibody with education heteroconjugate antibodies, as described by Segal in U.S. patent No. 4676980, which is included in this description by reference in its entirety.

4.9Therapeutic/prophylactic use

Determination of immunogenicity of immunoreactive agents after treatment HSP

In an optional procedure of inducing or enhancing immunogenicity of immunoreactive reagent, which is used with the HSP preparation of the present invention, may be analyzed using various methods, well known in this field and are given as examples in section 5.

Other ways to identify antigen-specific T cells can be used to analyze the tetramer staining" (Altman et al., 1996, Science 274:94-96). For example, in one embodiment, the MHC molecule containing a specific peptide antigen, such as a specific for tumor antigen, multimeric to obtain soluble peptide tetramers and mark, for example, by complexation with streptavidin. The complex of the MHC-peptide antigen is then mixed with a population of T cells obtained from a patient who had been treated immunoreactive reagent and drug HSP. Then use Biotin for staining the cells, who Express interest are tumor-specific antigen.

In addition, using the analysis of mixed linfocitos culture-target cytotoxicity of T cells can be tested 4-hour analysis51Cr-release (see Palladino et al., 1987, Cancer Res. 47:5074-5079). In this analysis a mixed limfocitna culture is added to a suspension of target cells to produce different relations effector:target (E:T) (usually 1:1 - 40:1). Target cells previously have been labelled by incubating 1×106target cells in culture medium containing 500 µci51Cr ml, for one hour at 37°C. Cells are washed three times after tagging. Each point of analysis (the ratio E:T) are obtained in triplicate and suitable controls include to measure spontaneous release51Cr (analysis not add lymphocytes) and 100% release (the cells are lysed with detergent). After incubation mixtures of cells within 4 hours, the precipitated cells in vitro by centrifugation at 200 × g for 5 minutes. The number of51Cr released into the supernatant, measure gamma counter. The percentage of cytotoxicity was measured as the number of pulses per minute in the sample to be tested minus spontaneously released the number of pulses per minute, divided by the total number of pulses per minute after treatment with detergent minus spontaneously in the exempted number of pulses per minute. To block the cascade of MHC class I concentrated supernatant of hybridoma obtained from cells hybridoma To-44 (anti-MHC-hybridoma class I), is added to the test samples to a final concentration of 12.5%.

In the alternative case, the ELISPOT analysis can be used to measure cytokine release in vitro cytotoxic T-cells after stimulation of immunoreactive reagent and drug HSP. Cytokine determined by antibodies that are specific for a particular cytokine, such as interleukin-2, factor α tumor necrosis or interferon-γ (see, for example, Scheibenbogen et al., 1997, Int. J. Cancer 71:932-936). The analysis is performed in the title microplates, which were pre-coated with an antibody specific for the interest of the cytokine, which capture cytokine secreted by T-cells. After incubation of T cells within 24-48 hours in covered holes cytotoxic T-cells are removed and replaced with a second labeled antibody that recognizes a different epitope on the cytokine. After extensive washing the product to remove unbound antibodies tablet add enzyme substrate that produces a colored reaction product. The number of cytokine producing cells are counted under a microscope. This method has advantages in the short time of the Lisa and sensitivity without the need of a large number of cytotoxic T cells.

4.10The pharmaceutical composition

The present invention also provides pharmaceutical compositions. Such prophylactically or therapeutically effective compositions contain immunoreactive reagent and HSP and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means a term adopted by "Regulatory agence" of the Federal government or the state government or listed in the U.S. Pharmacopoeia or other generally recognized Pharmacopoeia for use in the treatment of animals and, more specifically, people. The term "carrier" refers to a diluent, excipient or carrier with which the injected therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils, petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. As liquid carriers, particularly for injectable solutions can be also used saline solutions and aqueous dextrose and glycerol. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, is Latin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if necessary, can also contain minor amounts of wetting or emulsifying agents or buffering agents to establish the pH. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, preparations with protracted release and the like. Oral drug can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium salt of saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are described in “Remington''s Pharmaceutical Sciences” by E.W. Martin. Such compositions will contain a prophylactically or therapeutically effective amount of immunoreactive reagent and HSP preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration patient. The drug must correspond to the method of administration.

Immunoreactive reagents and HSP of the present invention can also be advantageously used in combination with one or more drugs used for the treatment of diseases, disorders the treatment or infection, such as, for example, anticancer agents, anti-inflammatory agents or anti-bacterial/fungal or anti-viral agents. Examples of anticancer agents include, but are not limited to, cisplatin, carboplatin, cyclophosphamide, doxorubicin, etoposide, ifosfamide, paclitaxel, taxanes, CPT-11, topotecan, gemcitabine, oncovin, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, levamisole, BCNU, vinorelbine, Temodal, vincristine and Taxol.

Various delivery systems are known and can be used for the introduction of therapeutic and preventive agents covered by the present invention, i.e. immunoreactive reagents and HSP, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of Express immunoreactive reagent, the HSP preparation, antibody, or antibody fragment, receptorpositive endocytosis (see, e.g., Wu and Wu, 1997, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, and the like. Methods of introduction of immunoreactive reagent or HSP preparation or pharmaceutical compositions containing the same include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), the disease is real and through the mucous membrane (for example, intranasal and oral routes). In a specific embodiment, the immunoreactive reagents, such as antibodies, injected intramuscular, intravenous or subcutaneous means. The administration can be systemic or local. In addition, can also be used pulmonary introduction, for example, through the use of an inhaler or nebulizer and the drug with aerosolizing agent. See, for example, U.S. patents№№ 6019968; 5985320; 5985309; 5934272; 5874064; 5855913; 5290540 and 4880078 and PCT publication WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346 and WO 99/66903, each patent and publication are included in this description by reference in its entirety. In one embodiment, therapeutic or prophylactic agent is injected with the use of technology delivery pulmonary drug Alkermes AIRTM(Alkermes, Inc., Cambridge, MA).

Solubility and injection are factors that must be considered when selecting the route of administration. The method of administration can vary and includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intradermal route or path through the mucous membrane. Route of administration through the mucosa may further include a form for oral, rectal and nasal administration. Taking into account the above factors, it is preferable to introduce the first of those is piticescu or preventive agent in place, which is the same or proximal to the site of administration of the second agent. In the method of treatment of a tumor, the HSP preparation is injected near the tumor, most preferably inside the tumor injection.

In one embodiment, the HSP preparations and immunoreactive reagents administered using any desired route of administration. Advantages intradermal include the use of lower doses and rapid absorption, respectively. The advantages of subcutaneous or intramuscular injection include its suitability for some insoluble suspensions or oil suspensions, respectively. Route of administration through the mucosa include, but are not limited to, oral, rectal and nasal administration. Preparations for the introduction through the mucous membrane are appropriate in various forms, as described below.

In another embodiment, therapeutic or prophylactic agents of the present invention is injected intramuscular, intravenous or subcutaneous means. The composition can be introduced by any suitable means, for example by infusion or bolus injection, by absorption through epithelial or skin-mucous lining (e.g., mucosa of the oral cavity, the mucous membrane of the rectum and intestine and the like), and can be introduced into the natural with other biologically active agents.

In a specific embodiment, it may be desirable to introduce the pharmaceutical compositions of the present invention locally to the area in need of treatment or prevention of a disease. In one embodiment, the treatment or prevention can be achieved, for example, but not limiting way, local infusion, by injection, or by means of an implant, with the specified implant is porous, non-porous, or gelatinous material, including membranes, such as calascione membranes, or fibers. Preferred is the use of the materials on which the agent is not absorbed.

In another embodiment, the composition may be delivered in the vesicles, especially the liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in Thepapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In another embodiment, the composition may be delivered to the system with controlled release or extended release. Any method known to the person skilled in the art, may be used to obtain preparations with protracted release, containing one or more antibodies or one or more fused proteins. See, for example, U.S. patent No. 4526938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al, 199, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel, "Radiotherapy &Oncology 39:179-189; Song et al, 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science &Technology 50:372-397; Cleek et al, 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Intl. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l Symp. Control Rel. Bioact Mater. 24:759-760, 1997, and each of the publications included in this description by reference in its entirety. In one embodiment, the controlled release can be used a pump (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, to achieve controlled release of immunoreactive reagents or drugs HSP can be used polymeric materials (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol Sci. Rev. Macromol Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al, 1989, Ann. Neurol. 25:351; Howard et al, 1989, J. Neurosurg. 71:105); U.S. patent No. 5679377; U.S. patent No. 5916597; U.S. patent No. 5912015; U.S. patent No. 5989463; patent No. 5128326; PCT publication WO 99/15154 and PCT publication WO 99/20253). In another embodiment, the controlled release may be placed about therapeutic target (e.g., lungs), the button, thus, the need was only part of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other systems with controlled release are discussed in the review Langer, 1990, Science 249:1527-1533.

In one preferred embodiment, the HSP preparation is administered in conjunction with the introduction of the immunoreactive reagent. Co-administration of the HSP preparation and immunoreactive reagent means that the HSP or HSP complex-peptide is administered together in the form of a mixture with an immunoreactive reagent or injected separately, but in close enough in time that the immunoreactive reagent. This method requires that two injections are conducted within a time range from less than one minute to about five minutes, or up to about sixty minutes from each other, for example, when the same doctor.

In a preferred embodiment, the present invention provides a method of injection HSP, including but not limited to the above, hsp60, hsp70, hsp90, hsp110, gp96, grp170 or calreticulin, separately or in combination with each other, the subject together with the introduction of the immunoreactive reagent in the same place or in close proximity to this place.

If a candidate therapeutic or prophylactic agent is a water-soluble, it is may be prepared in a suitable buffer, for example, phosphate buffered saline or other physiologically compatible solutions, preferably sterile. Alternatively, if the resulting complex has a low solubility in aqueous solutions, it can be made with nonionic surface-active agent, such as twin or polyethylene glycol. Thus, the compounds and their physiologically acceptable solvate can be made for inhalation or insufflation (either through the mouth or the nose) or oral, transbukkalno, parenteral or rectal administration, or, in the case of tumors, they are directly injected into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquid form, for example in the form of solutions, syrups or suspensions, or may be presented in the form of a medicinal product for transformation of water or other suitable filler prior to use. This liquid product can be obtained by conventional means with pharmaceutically acceptable additives such as suspendresume agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or Arabian gum); non-aqueous vehicles (e.g. almond oil, oily esters or akcionirovanie vegetable oils), preservatives (for example, methyl - or propyl-p-hydroxybenzoate or sorbic acid). The pharmaceutical preparation may be in the form of, for example, tablets or capsules, obtained by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pre-klasterizovannykh maize starch, polyvinylpyrrolidone or hypromellose); fillers (e.g. lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrators (e.g., potato starch or sodium salt glycolate, starch) or wetting agents (e.g. sodium lauryl sulphate). Tablets can be coated by methods well known in the field.

Composition for oral administration may be suitably formulated to provide controlled release of the active connection.

For transbukkalno the introduction of the composition may be in the form of tablets or pellets, made the conventional way.

Agents can be made for parenteral administration by injection, for example bolus injection or continuous infusion. Preparations for injection can be presented in unit dosage form, for example in ampoules or containers for many doses, added to what servants. Drugs can be in such forms as suspensions, solutions or emulsions in oily or aqueous fillers, and may include forming the shape of agents, such as suspendida, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for the preparation of the drug with a suitable filler, such as sterile, pyrogen-free water, before use.

The composition can also be made in the form of rectal medication, such as a suppository or retention enema, for example, containing conventional for suppository bases such as cocoa butter or other glycerides.

In addition to the drugs described earlier, the agents of the present invention can be also manufactured in the form of the drug depot. These drugs for long periods can be introduced by implantation (for example, subcutaneous or intramuscular) or by intramuscular injection. For example, the drug can be manufactured with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or in the form of slightly soluble derivatives, for example in the form of a slightly soluble salt. Liposomes and emulsions are well known examples of fillers or carriers for the delivery of hydrophilic drugs.

DL is the introduction of inhalation drugs for use in accordance with the present invention is suitable manner delivered in the form of an aerosol spray from pressurized packs or nebulizer with the use of a suitable propellant, e.g. DICHLORODIFLUOROMETHANE, trichloromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol single dose may be provided with a valve for delivering measured quantities. Can be made capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

Finished formulations of pharmaceutical compositions containing the HSP, and procedures for their manufacture can be found in the literature and in the U.S. patents incorporated by reference in this document.

The invention also provides that the immunoreactive reagent, for example an antibody, or an HSP preparation is Packed in hermetically insulated container such as an ampoule or sachet, which shows the number of immunoreactive reagent. In one embodiment, the immunoreactive reagent and HSP put together or separately in the form of dry sterilized liofilizovannyh powders or concentrates without water in one or more hermetically sealed containers, they can be recreated, for example, water or saline to the appropriate concentration for administration to a subject. Efficiency is the usual dose of each immunoreactive reagent can be initially estimated from the data analysis in vitro. It also depends on the nature of the target antigen, the antigen density in tumors, tumor type, method of administration, which can be optimized by the person skilled in the art without undue experimentation. The usual effective dose for injection is approximately from 0.1 to 5 mg/kg/day, preferably from about 1 to 4 mg/kg/day and more preferably to 2 to 4 mg/kg/week. Immunoreactive reagent is preferably supplied as a dry sterile liofilizirovannogo powder in a hermetically insulated container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg

In a specific embodiment, the immunoreactive reagents introduced animal, have the origin of species or the reactivity of the species, which is the same as the species of animal. Thus, in a preferred embodiment, human antibodies or humanized antibodies are administered to the patient is a person for therapy or prevention.

Depending on the route of administration and type of HSP the HSP preparation number of HSP the HSP preparation can be, for example, from 0.1 to 1000 μg per administration. The preferred amount of gp96 or hsp70 are in the range is from 10 to 600 micrograms on the introduction and 0.1-50 μg, preferably 10-25 µg, if the HSP preparation is administered intradermally. For hsp90 preferred amount of approximately 50-1000 µg introduction and approximately 5-50 µg for intradermal injection.

In other embodiments, implementation of the heat shock protein is hsp60, hsp70, hsp90, gp96 or calreticulin. The dose of injected drug HSP depends largely on the condition and size of the exposed treatment of the patient, as well as the amount of typing immunoreactive reagent, frequency of treatment and route of administration. Modes of continuous therapy, including the place, the dose and frequency of injection may be determined by the initial response and clinical evaluation.

The optimal number of specific HSP for use with a specific composition of the invention can vary. Optimization of a certain number of HSP for this composition is, as demonstrated above examples, it is within the competence of the specialist in this field.

Due to the introduction of the drug HSP fewer immunoreactive reagent may be required for the induction of immune response in the subject. The number of immunoreactive reagent, which is used with the drug, HSP, including the number in the suboptimal range, can be determined in a dose-dependent experiments conducted in animal models is the means, known in this field.

In a preferred embodiment, the heat shock protein is hsp70. The amount of hsp70 in the pharmaceutical compositions is preferably in the range of 10 to 600 micrograms on the introduction and 0.1-50 μg, preferably 10-25 µg, if the HSP preparation is administered intradermally.

In a particularly preferred embodiment, the heat shock protein is gp96. The number hsp96 in the pharmaceutical compositions preferably is in the range of 10 to 600 micrograms on the introduction and 0.1-50 μg, preferably 10-25 µg, if the HSP preparation is administered intradermally.

In an alternative embodiment, the immunoreactive reagent and HSP supplied in liquid form in a hermetically sealed container, which is indicated by the number and concentration of HSP and immunoreactive reagent. The liquid form of immunoreactive reagent is preferably supplied in sealed container containing at least 1 mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml or at least 25 mg/ml Liquid HSP preferably put in sealed container containing at least 0.1 mg/ml, more preferably, at least 1.0 mg/ml, at least 5 mg/ml, at least 1 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, or at least 250 mg/ml

In a preferred embodiment, the composition is manufactured in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to humans. Compositions for intravenous administration usually are solutions in sterile isotonic aqueous buffer. When necessary, the composition may also include solubilizers agent and a local anesthetic, such as lignocaine to relieve pain at the injection site.

The ingredients of the compositions of the present invention is usually supplied in the form of a set or separately, or mixed together in a dosage form, for example, in the form of dry liofilizirovannogo powder or not containing water concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition should be introduced by infusion, it can be dosed vessel for infusion containing sterile pharmaceutical water purity or saline. When the composition is administered by injection, may be provided ampoule of sterile water for injection or physiological saline so that the ingredients can be mixed prior to introduction. In another variant implementation, the value set of the present invention further comprises a needle or syringe, preferably packaged in sterile form, for injection of the composition, and/or a packaged alcohol pad. Optionally include instructions for administration of the compositions of the invention the Clinician or patient.

Compositions of the present invention can be prepared in the form of the free form or in salt form. Pharmaceutically acceptable salts include salts formed with anions such as anions formed from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and salts formed with cations such as the cations derived from sodium, potassium, ammonium, calcium, hydroxide iron(III), Isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The number of compositions of the present invention, which may be effective in the treatment, prevention or attenuation of one or more symptoms associated with a disease, disorder, or infection, can be determined by standard clinical techniques. The exact dose you need to apply in the product will depend on the route of administration, age of the subject and the severity of the diseases, disorders or infection and should be determined in accordance with the opinion of the practitioner and of all circumstances relevant to the patient. Effective doses may be extrapolated from the curves dose-response obtained is s in vitro or used as a test system model-animal (for example, cotton hamster or Javanese Macaca). Models and methods of estimation of activity of the HSP and an antibody or other immunoreactive reagents are known in the art (publication Wooldridge et al., 1997, Blood 89(8):2994-2998 included in this description by reference in its entirety).

For antibodies therapeutically or prophylactically effective dose, administered to a subject, typically 0.1 - 200 mg/kg body weight of the subject. Dose, administered to a subject, preferably is between 0.1 mg/kg and 20 mg/kg body weight of the subject, and more preferably the dose, administered to a subject is between 1 mg/kg and 10 mg/kg of body weight of the subject. The dose, however, will depend on the extent to which increased half-life of the molecules from the serum. Human antibodies usually have a longer half-life from the human body than antibodies from other species due to the immune response against the foreign polypeptides. Thus, it is often possible to lower doses of human antibodies and less frequent administration. In addition, the dose and frequency of injection of immunoreactive reagents can also be reduced by enhancing uptake and tissue penetration (e.g., light) for immunoreactive reagents, such as, for example, lipidation. Information about a specific dose of antibodies can be found in the liner unit is Cai manufacturer for the specified antibodies or Physician''s Desk Reference (56 thel., 2002).

The treatment of the subject a therapeutically or prophylactically effective amount of an immunoreactive reagent and HSP can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject treated immunoreactive reagent in the range between approximately 0.1 and 30 mg/kg of body weight once a week for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably for about 4, 5 or 6 weeks. Immunoreactive reagents and their dosages, routes of administration and recommended usage are known in this field and have been discussed in the literature, as Physician''s Desk Reference (56thed., 2002). In a preferred example, a subject treated HSP in the range between from about 0.1 to 1000 mg, more preferably 1-500 mg, most preferably 2-250 mg once a week for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably at least 4, 5 or 6 weeks. Specialist in this field should be obvious dependence of a suitable dose of HSP on the condition of the subject being treated, the input immunoreactive reagent, as well as from the subject.

In some embodiments, the implementation nastojasih the invention compositions of the invention contain HSP in combination with fillers. The heat shock protein is preferably hsp60, hsp70, hsp90, gp96 or calreticulin, and fillers selected from nonionic surfactants, polyvinylpyrrolidone, bovine serum albumin human serum and various unmodified and derivatizing cyclodextrins. More preferably in these embodiments, the implementation of the nonionic surfactant is selected from Polysorbate-20, Polysorbate-40, Polysorbate 60 and Polysorbate-80. The polyvinylpyrrolidone may be preferably plasdone C15, polyvinylpyrrolidone pharmaceutical grade. The preferred cyclodextrin is hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin and methyl-β-cyclodextrin. The cyclodextrins are preferably β-cyclodextrin. Compositions of the present invention preferably contain a prophylactically or therapeutically effective amount of HSP preparation or an immunoreactive reagent and a pharmaceutically acceptable carrier.

In a specific embodiment, the term "pharmaceutically acceptable" means permitted "Regulatory agency" of the Federal government or the state government or listed in the U.S. Pharmacopoeia or other generally recognized Pharmacopoeia for use in the treatment of animals and, more specifically, in the treatment of people. The term "carrier" relative who are getting ready for the diluent, adjuvant (for example, adjuvant's adjuvant (complete or incomplete), excipient or carrier with which the injected therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils, petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A preferred carrier is water, when the pharmaceutical composition is administered intravenously. As liquid carriers may also be used saline solutions and aqueous dextrose and glycerol, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. If necessary, the composition may also contain minor amounts of wetting or emulsifying agents or buffering agents to control pH. These compositions can be in the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, drugs with long release, and the like.

The drug can be, if necessary, provided in the package or the metering device, which may contain one or more unit dosage forms containing the HSP preparation. Packaging can represent, for example, metal foil or plastic film and can be "bubble" packaging. Packaging or dispensing device may be accompanied by instructions for administration.

Compositions of the present invention can be introduced to an animal, preferably a mammal and most preferably a human, to treat, prevent or ameliorate one or more symptoms associated with a disease, disorder or infection. In a preferred embodiment of the present invention the composition is placed on the outer part of the body. It is established that the immunoreactive reagent preferably has a therapeutic advantage in the absence of heat shock protein and recognizes an epitope on the cell or molecule associated with the cause or symptoms of diseases, disorders or infections.

The compositions contain immunoreactive reagent (i.e. antigennegative protein containing antigennegative area and region, which mediates one or more antibody-dependent immunological processes, such as Fc-receptor-binding region) and HSP.

Each composition of the present invention must contain at least one immunore the active agent (as defined here for example, antibody) and HSP, and compositions of the present invention may also be used in combination with other forms of therapy for a particular disease. One or more immunoreactive reagents that immunospecificity way associated with one or more antigens on target, can be used locally or systemically in the body as a prophylactic or therapeutic agent.

4.11Sets

The proposed well as kits for performing methods of the present invention. In a specific embodiment, the kit contains a first container containing the product heat shock protein in a quantity effective to enhance the immune response induced immunoreactive reagent against the target of immunoreactive reagent, against which the required immune response, and a second container containing immunoreactive reagent in a quantity that, when introduced prior to, simultaneously with or after the injection of heat-shock proteins of the first container, is effective in the induction of immune responses against the target.

The proposed sets of the present invention, which contain in the container immunoreactive reagent in a quantity effective to treat or prevent diseases or disorders, and in another container preparation of heat-shock proteins in Koli is este, effective to enhance or maintain the immune response induced immunoreactive reagent. In the embodiment, the number of immunoreactive reagent that is present in the container, is suboptimal for the induction of immune response in the subject with the introduction regardless of the preparation of heat-shock proteins in another container. The kit can, optionally, be accompanied by instructions.

The present invention also provides kits containing one or more containers with one or more ingredients of the pharmaceutical compositions of the present invention. Such set(s) may not necessarily be accompanied by a notice in the form prescribed by the governmental Agency regulating the manufacture, use or sale of pharmaceuticals or biological products, and this notice reflects approval by this Agency of manufacture, use and sale of the compositions of the container for introducing a person. In one embodiment, the kits may optionally further comprise a predetermined number of immunoreactive reagent (i.e. antigennegative protein containing antigennegative district and the district, which mediates one or more antibody-dependent immunological processes, such as Fc-receptornegative district) and HSP In a preferred embodiment, the kit contains immunoreactive reagent and HSP in different containers.

5.EXAMPLES

5.1Amplification-mediated antibody lysis in vitro

Murine splenocytes (effector cells) obtained from spleens processed 6-8 weeks mice. These effectiye cells incubated with the appropriate number of HSP preparation and a suitable number of monoclonal antibodies in 24-72 hours. At the end of the period of incubation of the target cells (E.G7-OVA or M04;) burden51Cr. Effector cells and labeled target cells incubated in certain respects, the effector:target in the presence or in the absence of antibodies against SIINFEKL/class I MHC (1-10 μg/ml) at 4°C for 30-60 minutes. Lysis in the presence of HSP and antibodies compared with controls without HSP, without antibodies or without both.

5.2Increased protection in the model, inoculated with tumor

Mice C57B1/6 inoculant subcutaneous (SC) by the side of the tumor M04 (1×105) or tumor EG7-OVA. 24-48 hours after inoculation of mice injected intraperitoneal (IP) by the appropriate number of immunoreactive reagent (e.g. antibody) to SIINFEKL/MHC class I or local by SC in the presence or in the absence of a suitable quantity of the drug HSP. Antitumor effect of combination therapy compared with only antibody for monitoring of tumor growth over a period of 30-60 days (measured with calipers). Set the Ute survival, and the influence of time on the death of the mice analyze regression analysis of Sokh. Mice are examined daily to signals of toxicity, including activity level, uneven coating on the tongue, diarrhea, and General appearance. Models and methods of evaluation of the actions of an antibody or other immunoreactive reagents are known in the art (publication Wooldridge et al., 1997, Blood 89(8):2994-2998 included in this description by reference in its entirety).

5.3Increased opsonization of bacteria

Increased opsonization of bacteria by the addition of HSP preparation for therapeutic treatment of the antibody is demonstrated in vitro by incubation of effector cells for analysis of opsonophagocytosis (HL-60) with an HSP preparation. Cells are evaluated on whether they are more effective in opsonization of S. pneumoniae or S. aureus at this titer antibodies (for example, a sample of human serum with opsonizing activity specific to S. pneumoniae or S. aureus, respectively).

5.4Positive regulation of Fc-receptors

Monocytes, natural killer cells or polymorphically cells incubated in the presence or in the absence of a suitable quantity of the drug HSP. Trypsinization cells incubated at 4°C for 60 min with monoclonal antibodies specific for the Fc αR, Fc gamma R1, Fc gamma RII or Fc gamma RIII. Cells are then incubated with FITC-probe to Antim is Shinobu IgG, washed, fixed in paraformaldehyde and analyzed FACScan. Monitor positive regulation of Fc-receptors on these cells.

In addition, conduct monitoring positive regulation of TNF-alpha, IL-6 and MIP-1-alpha drug HSP in cells-macrophages.

Experimental examples

The experiments were performed in the mouse model. Was carried out by the introduction of a complex of purified gp96-peptide and anti-L-4 antibody and complex purified gp96-peptide and anti-D25 antibody. Compared antitumor activity in joint injection or separate administration. On day 0 the mice were injected tumor cells SM1, as specified in figures 1 and 2 days were injected antibodies or preparation of purified gp96. Figure 2 shows the mean tumor size (for combination with anti-D25 antibody), figure 1 - average tumor diameter (for combination with anti-L-4 antibody) in the period after the introduction of tumor cells.

The combination of purified gp96 and anti-L-4 antibodies provided 86% of the tumor suppression in mice. With the introduction of only antibodies was noted 14% suppression, and the introduction of only cleared Dr - 17% tumor suppression. The combination of the drug cleared Dr and anti-D25 antibody gave a reduction in the size swelled by 89% compared with the group treated with the buffer, while a decrease was not observed in mice that received only the drug is cleared Dr, and in mice, recip is our only anti-D25 antibody noted a decrease in size at 72%.

The combination of purified Dr and anti-L-4 antibodies provides 86% of the tumor suppression in mice, while mice treated with some antibodies, noted 14% suppression, and of mice treated with one drug cleaned Dr, noted 17% tumor suppression. The number 86% of the above total by 31% and is synergistic.

The combination of the drug cleared Dr and anti-D25 antibodies provides a reduction of tumor size by 89% compared with the group treated with the buffer, it was not marked reduction of the tumor in mice treated with a single drug is cleared Dr and mice treated with a single anti-D25 antibody, the tumor was reduced by 72%. Thus, the tumor 89% is not total, more than 72% and is synergistic.

All cited in this description reference is included as a reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated, as incorporated by reference in its entirety for all purposes.

Can be done many modifications and variations of the present invention, is not beyond the scope and essence of the invention, as should be obvious to a person skilled in this field. Specific options for the implementation described here, PR is dstanley only as examples, and the invention should be limited only by the attached claims along with the full scope of equivalents that are included in this invention.

1. A method of treating cancer in an individual, comprising the stage of:
(a) introducing a specified individual of the preparation of purified heat shock protein (HSP), where the product purified HSP complexes contain purified HSP - peptide containing HSP, covalently or ecovalence attached to the peptide, and where the complexes of HSP - peptide display the antigenicity of a specified cancer; and
(b) the introduction of a specified individual number immunogenic reagent, where immunoreactive reagent which specifically interacts with the antigen selected from the group consisting of VEGF, EGF-R, HER2/NEU, CD25 and CD20, or anti-L-4 or anti - VV antibody
where these quantities are synergies at the specified treatment, providing treatment specified cancer in the specified individual.

2. The method according to claim 1, where the specified immunoreactive reagent is a preventive or therapeutic antibody.

3. The method according to claim 1, where the specified immunoreactive reagent is a monoclonal antibody.

4. The method according to claim 1, where the specified drug purified HSP HSP contains selected from the group consisting of hsp60, hsp70, hsp90, gp96, hsp110, grp170 and calreticulin alone or in combination with each other.

5. The method according to claim 1, where the specified drug purified HSP complexes contain purified HSP-peptide selected from the group consisting of complexes of hsp60-peptide complexes of hsp70-peptide complexes of hsp90-peptide complexes gp96-peptide complexes hsp110-peptide complexes grp170-peptide complexes calreticulin-peptide alone or in combination with each other.

6. The method according to claim 1, where the individual is the person and the product purified HSP HSP contains a mammal.

7. The method according to claim 1, where the preparation of purified HSP is injected before the introduction of the immunoreactive reagent.

8. The method according to claim 1, where the preparation of purified HSP is injected simultaneously with the introduction of the immunoreactive reagent.

9. The method according to claim 1, where the preparation of purified HSP is administered after administration of an immunoreactive reagent.

10. The method according to claim 1 where the cancer is selected from the group consisting of carcinoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, bronchogenic carcinoma, renal cell carcinoma, lung carcinoma, small cell carcinoma of the lung, leukemia, chronic malacitana leukemia, melanoma and lymphoma.

11. The method according to claim 1, where the complexes purified HSP - peptide include peptides with a peptide sequence which is derived from the sequence of the same target recognized immunoreactive reagent, and where the immunoreactive Reagan which specifically interacts with the antigen, selected from the group consisting of VEGF, EGF-R, HER2/NEU, CD25 and CD20.

12. The method according to claim 1, where these complexes are purified HSP - peptide include peptides derived from the tumor.

13. The method according to claim 1, where these complexes are purified HSP - peptide include peptides that contain the epitope of the tumor-specific antigen or an antigen associated with a tumor.

14. The method according to claim 1, where the complexes purified HSP - peptide obtained from the individual who had introduced these complexes.

15. The method according to claim 1, where the immunoreactive reagent is purified.

16. The method according to claim 1, where the specified number of immunoreactive reagent is administered to a specified individual in a dose that is below the threshold of side effects.

17. The method according to claim 1, where the specified individual partially or completely unresponsive to either stage (a)or stage (b).

18. The method according to claim 1, where these complexes are purified HSP - peptide purified from cells of the indicated cancer.

19. The method according to claim 1, where the specified amount of purified HSP preparation is administered to a specified individual in an amount effective for the induction of the immune response in the absence of stage (b).

20. The method according to claim 1, where the specified number of immunoreactive reagent is administered to a specified individual in number, are not effective for the induction of immune responses in the absence of stage (a).

21. The method according to claim 1, where the specified immunoreacted wny reagent which specifically interacts with CD25.

22. The method according to claim 1, where the specified immunoreactive reagent is an anti - CTLA-4 antibody.

23. Method of strengthening the immune response to the first antigen, caused by the preparation of purified HSP from the individual, to whom the drug is administered purified HSP, including the introduction of individual immunoreactive reagent, where immunoreactive reagent which specifically interacts with the second antigen is selected from the group consisting of CD25 and CD20, or anti-CTLA-4 or anti - VV antibody, where the drug is purified HSP complexes contain purified HSP-peptide containing HSP, covalently or ecovalence attached to the peptide, and where the complexes of HSP - peptide display the antigenicity of the specified first antigen, and where these quantities are synergies in relation to the specified gain, thereby exacerbating the specified immune response.

24. The method according to item 23, where the specified drug purified HSP HSP contains selected from the group consisting of hsp60, hsp70, hsp90, gp96, hsp110, grp170 and calreticulin.

25. The method according to paragraph 24, where HSP is a gp96.

26. The method according to paragraph 24, where HSP is a hsp70.

27. The method according to item 23, where the specified immunoreactive reagent is a monoclonal antibody.

28. The method according to item 23, where the specified drug purified HSP complexes contain purified HSP - peptide selected from the group consisting of complexes hsp6-peptide, complexes of hsp70-peptide complexes of hsp90-peptide complexes gp96-peptide complexes hsp110-peptide complexes grp170-peptide complexes calreticulin-peptide, alone or in combination with each other.

29. The method according to item 23, where the individual is the person and the product purified HSP HSP contains a mammal.

30. The method according to item 23, where said first antigen is a cancer antigen.

31. The method according to item 30, where these complexes are purified HSP - peptide include peptides derived from tumor specified cancer.

32. The method according to item 30, where said first antigen contains an epitope of a tumor-specific antigen or an antigen associated with a tumor.

33. The method according to item 23 or 30, where these complexes are purified HSP - peptide obtained from the individual who had introduced these complexes.

34. The method according to item 23, where the specified immunoreactive reagent is purified.

35. The method according to item 23, where the specified immunoreactive reagent which specifically interacts with CD25.

36. The method according to item 23, where the specified immunoreactive reagent is an anti-L-4 antibody.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to virology, immunology and medicine. The composition contains antigen bound or fused with virus-like particle (VLP), completed with immunostimulating nucleic acid containing at least one unmathylated CpG-sequence and ligand of toll-like receptor (TLR). There are also disclosed vaccine composition and method for potentiating immune response in an animal.

EFFECT: invention can be used for inducing strong antibody and T-cell response and, particularly it is effective in treatment of allergy, tumours and virus chronic diseases, and also other chronic diseases.

25 cl, 12 dwg, 1 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to immunomodulatory drugs. Application Helepin D or Helepin as an immunomodulatory drug in secondary immunodeficiency states.

EFFECT: Helepin D or Helepin possess effective immunomodulatory action in immunodeficiency states.

1 dwg, 4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely pulmonology, and can be used for treating community-acquired pneumonia in the patients with mid-severe and severe clinical course involving immunogram abnormalities. That is ensured by antibacterial therapy. In addition, for 3-4 day of treatment, the treatment regimen is supplemented with Polyoxidonium in a dose 6 mg daily intramuscularly within 5 days.

EFFECT: early positive dynamics of the main clinical symptoms of disease and normalisation of immunological indicators which allow reducing treatment time and preventing complications.

1 tbl, 1 ex, 1 dwg

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary medicine, particularly to anthelmintic preparations for treating small cattle and can be used for treatment and prevention of helminthic invasions accompanied by reduced immunobiological properties. The anthelmintic preparation for treating small cattle contains albendazole, 40% Echinacea tincture, 4% methylcellulose - 100 and sorbic acid and distilled water in the ratio as follows, wt %: albendazole 2.5-5.0; 40% Echinacea tincture 9-12; 4% methylcellulose-100 20-25; sorbic acid 0.5-1.0; distilled water - the rest.

EFFECT: higher effectiveness and lower toxicity of the preparation.

2 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula (I)

, where R is selected from ethyl, n-propyl, iso-propyl, n-butyl and allyl; R' is selected from hydrogen, straight, branched or cyclic C1-C4alkyl; straight, branched or cyclic C1-C3alkoxy; fluorine, chlorine, bromine, trifluoromethyl and OCHxFy, where x=0, 1, 2, y=1, 2, 3 under the condition that, x+y=3; R" is selected from hydrogen, fluorine and chlorine, with the condition that, R" is selected from fluorine and chlorine only when R' is selected from fluorine and chlorine; R3 is selected from hydrogen and straight, branched or cyclic C1-C5alkyl; R4 is selected from hydrogen, CH2OCOC(CH3)3, pharmaceutically acceptable inorganic or organic cations, and COR4', where R' is straight, branched or cyclic C1-C5alkyl, phenyl, benzyl or phenethyl; R7 is selected from methyl and ethyl; one of A and B is sulphur, and the other is C-R2; when A is S, R2 is selected from hydrogen and methyl, with the condition that R2 is methyl only when R3 is not hydrogen; and when B is S, R2 is hydrogen; and to any tautomer thereof, as well as to a pharmaceutical composition which contains formula (I) compound, to a method of producing said compounds and to a method of treating diseases which are a result of autoimmune response or pathologic inflammation.

EFFECT: new compounds are disclosed, which can be used in treating diseases which are a result of autoimmune response or pathologic inflammation.

35 cl, 2 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a new derivative of sulfonamide substituted imidazoquinolines, and specifically to N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo-[4,5-c]-qunolin-1-yl]-1,1-dimethylethyl}-methanesulfonamide and its pharmaceutically acceptable salts, as well as to a pharmaceutical composition based on this compound.

EFFECT: invention also relates to a method of inducing biosynthesis of cytokines in an animal organism and methods of treating viral and oncological diseases in animals using said compound and pharmaceutical composition based on said compound.

5 cl, 1 tbl, 39 ex

FIELD: chemistry.

SUBSTANCE: invention relates to organic chemistry, particularly to the technology of producing selenoxanthenes and can be used in producing food additives, medicinal agents and cosmetic agents, which exhibit broad biological activity. An agent is described, which is an α-crystalline form of 9-phenyl-sym-octahydroselenoxanthene, which has antixodant, detoxication, immunomodulating, antiatherogenic, antisclerotic, anabolic, hypolipid action, and the corresponding structural formula with powder X-ray pattern obtained on Cu-K radiation sources with characteristic reflection indices expressed in degrees of the diffraction angle 2θ: 6.0 12.0 15.0 17.0 19.0 20.0 21.5, 21.7, 20.9 25.0 27.0 28.0 29.0 37.0 and melting temperature 96.8°C, as well as to a method of producing said agent, involving crystallisation of the corresponding 9-R-sym-hydroselenoxanthene from low-polar or non-polar solvent, preferably hexane, chloroform or isopropyl alcohol.

EFFECT: design of an efficient method of producing selenoxanthenes.

3 cl, 1 dwg, 1 tbl

FIELD: food industry.

SUBSTANCE: preparation for usage as feeding contains Bifidobacterium breve in quantity 1×104 -1×1010 cfu/g and a mixture of indigestible carbonhydrates. Carbohydrates are chosen from indigestible monosaccharides up to hexasaccharides of the same carbohydrate structure and from indigestible heptasaccharides and top polysaccharides of the same structure including inuline. Addition to children feeding and infant food containing mentioned preparation are proposed as its usage for healing or prevention of children immune disturbances as for usage for children obtaining artificial of partly breast-feeding.

EFFECT: usage of the preparation for producing of composition for prevention and healing of insufficient energy consumption, also for preparation of composition for inhibiting of eosinophils infiltration when having allergy.

16 cl, 7 tbl, 29 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and design of agents with immunomodulating properties. A new fungal strain Penicillium verrucosum VKPM F-984 and a new immunomodulating agent based on the strain are proposed. The strain is extracted from microflora of ginseng roots and is kept in a medium which contains mineral salts, glucose and asparagine. Fungus mycelium is extracted with a water-alcohol solution (70% ethanol solution). The advantage of this agent is its natural occurrence and effecient stimulation of adaptive capabilities of the body.

EFFECT: obtaining an extract which has stimulating action on cell and humoral immunity, improves immune status of the body.

3 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to new derivatives of imidazo[1,2-c]pyrimidinyl acetic acid of formula (I) or to its salts: , where R1 is ,, in which n is an integer ranging from 0 to 6; Y is aryl, where the said aryl is optionally substituted at a substitutable position with one or more substitutes selected from a group which consists of halogen or C1-6alkyl, optionally substituted with mono-, di- or trihalogen; R2 is hydrogen; R3 is hydrogen or halogen; and R4 is hydrogen. The invention also relates to derivatives of imidazo[1,2-c]pyrimidinyl acetic acid of formula (I-i) or to its salts, to a drug, to use of compounds in paragraph 1, as well as to a drug in form of a standard single dosage.

EFFECT: obtaining new biologically active compounds, which are active towards CRTH2.

23 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely oncology, and can be used for integrated therapy of stomach cancer. The therapy involves surgical procedure and pharmacotherapy. For 3-7th postoperative day, removal of drainages is immediately followed with endolymphatic infusion with single-stage introduction of 5-fluorouracil 750 mg, etoposide 100 mg and ceftriaxon 3.0 g in total amount up to 20 ml. The procedure is performed every 7 days.

EFFECT: application of the invention provides prevention of local recurrence of stomach cancer in the early postoperative period ensured by action on subclinical micrometastases in lymphatic channels, reduces risk of postoperative pancreatitis due to inhibitory action of 5-fluorouracil on pancreatic secretion and rate of postoperative intra-abdominal inflammatory processes owing to ensured great concentration of antibacterial preparations in abdominal cavity.

2 ex

FIELD: medicine.

SUBSTANCE: present invention concerns medical products, particularly pharmaceutical combination for development inhibition or treatment of proliferative disease which contains in therapeutically effective amounts (a) compound PTK787 and (b) epothilone derivative of formula , where A means O or NRN, where RN means hydrogen or lower alkyl, R means hydrogen or lower alkyl, and Z means oxygen or chain, for simultaneous, separate or consecutive application. Besides the invention concerns a pharmaceutical composition, application and commercial package thereof.

EFFECT: specified combination of active components ensures synergetic effect in treatment of proliferative diseases.

7 cl, 6 ex

FIELD: medicine.

SUBSTANCE: invention concerns pharmacology, veterinary science and medicine and covers a cytostatic composition containing coordination compounds of platinum, ascorbic acid and phenanthroline derivatives where as coordination compound of platinum, it contains cisplatin, as ascorbic acid and phenanthroline derivatives - ascorbate phenanthrolinate complex of platinum of formula [PtPhen2]Asc and ascorbate phenanthrolinate complex of europium of formula EuAscNO3Phen·4·H2O at the following mass ratio: cisplatin - 1; ascorbate phenanthrolinate complex of platinum - 2; ascorbate phenanthrolinate complex of europium - 3.

EFFECT: composition with antioxidant activity.

1 cl, 1 dwg, 2 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to compounds of formula (I) and their pharmaceutically acceptable salts. The disclosed compounds have inhibitory effect on HsEg5. In formula (I) A is C=O or CH2; B is optionally substituted C1-6alkyl, D is O or N, where O is substituted with one R8, and where N is substituted with one or more R8, R1 and R2 together with the carbon atoms with which they are bonded form optionally substituted isothiazole or isoxazole, condensed with a pyrimidine ring, optionally substituted with a substitute which is C1-6 alkyl. Values of the rest of the radicals are given in the formula of invention.

EFFECT: invention relates to use of disclosed compounds in making medicinal agents with inhibitory effect on HsEg5, to a method of obtaining inhibitory effect on HsEg5, to a pharmaceutical composition which contains the disclosed compound as an active ingredient.

22 cl, 31 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a new derivative of sulfonamide substituted imidazoquinolines, and specifically to N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo-[4,5-c]-qunolin-1-yl]-1,1-dimethylethyl}-methanesulfonamide and its pharmaceutically acceptable salts, as well as to a pharmaceutical composition based on this compound.

EFFECT: invention also relates to a method of inducing biosynthesis of cytokines in an animal organism and methods of treating viral and oncological diseases in animals using said compound and pharmaceutical composition based on said compound.

5 cl, 1 tbl, 39 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to compounds of formula (I), their R and S isomers; or a mixture of R and S isomers; or pharmaceutically acceptable salts. Disclosed compounds can be used as a medicinal agent with agonist properties towards PPAR. In formula (I) and L represents (II) or (III); R1, R2, R3, Ya, R4a, R", Yb, R4b are hydrogen; R and R' are independently hydrogen, C1-C4alkoxy; n equals 0, 1 or 2; m equals 0, 1 or 2; X1 is a -Z-(CH2)P-Q-W group; X2 is -CH2-, -C(CH3)2-, -O- or -S-.

EFFECT: invention relates to a pharmaceutical composition, which contains the disclosed compound, to use of the pharmaceutical composition as a medicinal agent, to use of the disclosed compound in making the pharmaceutical composition.

13 cl, 35 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a compound of formula: which is N-{2-tert-butyl-1-[(4,4-diflurocyclohexyl)methyl]-1H-benzimidazol-5-yl}ethanesulfonamide and its pharmaceutically acceptable salt, their diastereomers, enantiomers or their mixture. The invention also relates to use of this compound in making a medicinal agent with modulator activity of CB1 receptors; to a pharmaceutical composition based on this compound; to a method of modulating CB1 receptors, based on use of effective quantities of this compound, as well as to a method of producing the compound described above.

EFFECT: obtaining a new derivative of benzimidazole with useful biological activity.

8 cl, 1 tbl, 28 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely oncology and can be used for treating prostate intraepithelial neoplasia. The methods according to the invention involve introduction of a composition containing therapeutically effective amounts of supercritical extracts of rosemary, turmeric, origanum and ginger; and therapeutically effective amounts of water-alcohol extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, Polygonum cuspidatum, Coptis chinensis and barberry.

EFFECT: invention allows inhibiting growth of intraepithelial neoplasia cells due to antineoplastic activity of herbal extracts of the composition, inhibition of cyclooxygenase 2 and induction of apoptosis.

43 cl, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to oncology, and can be used for optimisation of treatment tactics in case of Hodgkin's lymphoma in children and teenagers. Method is realised in the following way. Such unfavourable prognostic factors as: age over 10 years, IV stage of disease, conglomerate dimensions more than 5 cm and/or value of mediastinal-thoracic index greater than 0.33, number of affected zones more than 4, symptoms of intoxication and biological activity of process. After that three risk groups are determined by the sum of detected unfavourable factors. In the first group, if 0-2 factors are detected, 2 cycles of polychemotherapy in accordance with regimen VBVP are carried out, in the second group, if 3-4 risk factors are detected - 4 cycles in alternating regimen VBVP-ABVD-VBVP-ABVD, in the third group, if 5-6 factors are detected - 6 cycles in alternating regimen VBVP-ABVD-VBVP-ABVD-VBVP-ABVD. Two weeks after carrying out polychemotherapy in all groups irradiation of all earlier affected zones is performed.

EFFECT: method allows to reduce risk of post-cytostatic and radiation complications development due to reduction of treatment loading in patients.

1 tbl, 1 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: new pyrrolotriazine derivatives of general formula (I) are described, where R1 is possibly substituted piperidinyl or piperazinyl; R2 is possibly substituted phenyl; R3 is hydrogen; X is -NH-; Y is -CH2-; as well as their pharmaceutically acceptable salts or steroisomers, and pharmaceutical compositions containing said compounds.

EFFECT: given compounds are kinase inhibitors and can be used in medicine, for example as anticancer agents.

9 cl, 267 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to virus inactivation in production of immunoglobulins, particularly of G fraction. The method virus inactivation for production of G fraction immunoglobulin includes purification of dissolved immunoglobulin recovered by Kohn's spirit fractionation. Purified dissolved immunoglobulin is prepared with a solvent detergent mixture which is acetate buffer solution 0.05 M at pH 5.5, containing 1 wt % tri-n-butylphosphate and 1 wt % polysorbate 80. Preparation represents stirring of the mixture within 12-16 hours followed with dilution with acetate buffer solution 0.05 M at pH 5.5 containing 1 wt % sodium octanoate, sodium chloride 0.15 M and propylene glycol concentrated 0.2 g/l. Prepared immunoglobulin is immobilised on sulphoprolylcation sorbent with grain size 50 mcm and sorption capacity 55 mg/cm3, and two-stage washed by column chromatography followed with elution. The first stage of washing implies acetate buffer solution 0.05 M at pH 5.5 containing 1 wt % sodium octanoate, sodium chloride 0.15 M and propylene glycol concentrated 0.2 g/l. The second stage of washing applies acetate buffer solution 0.05 M at pH 5.5. At the first stage the product is washed with volume related to 5 volumes of sorbent, while at the second stage washing is performed with volume related to three volumes of sorbent.

EFFECT: invention provides high-effective virus inactivation that improves viral safety of immunoglobulin preparations.

3 cl, 7 ex

Up!