Fc receptor binding agents based on invariable region of immunoglobulin

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. There are disclosed versions of a dimer compound for forming a multimer capable to reproduce the effector function of aggregated IgG with identical monomers. Each monomer of the dimer comprises: a monomer of IgG2 link region or a monomer of isoleucine zipper, dimerising each of which forms a multimerising region, and at least Fc-domain monomer containing a link region, CH2 domain and CH3 domain of IgG1. What is described is a multimer compound capable to reproduce the effector function of aggregated IgG and containing two or more dimers. There are disclosed a method for changing the immune response using the dimer or multimer, as well as a multimer-based method of treating an inflammatory disease.

EFFECT: using the invention provides the new compounds capable to bind at least one FcR specified in a group consisting of: FcγRI, FcγRII, FcγRIII, FcγRIV, or their non-human version that can find application in medicine for IVIG substitution for treating a wide range of diseases, including the inflammatory and autoimmune diseases.

7 cl, 25 dwg, 5 tbl, 25 ex

 

The technical field to which the invention relates

[0001] the Present invention generally relates to the field of immunology, inflammatory diseases and oncoimmunology. More specifically, the present invention relates to biologically active biomimetic molecules containing Fc domains of immunoglobulin, to compositions containing such biomimetics, and to methods of using such biomimetics.

[0002] the Invention also relates to the treatment and prevention of disease States mediated by cells derived from monocytes, and, more specifically, to the use of stable functional parts Fc-fragments of IgG during such treatment and prevention.

The level of technology

[0003] immune globulin products from human plasma used since the early 1950s for the treatment of diseases associated with immunodeficiency, and later and more widely for the treatment of autoimmune and inflammatory diseases.

[0004] Initially, the drugs immunoglobulin was administered by intramuscular injection. Later used the intravenous immunoglobulin (IVIG), which was originally shown, was effective in the treatment of autoimmune diseases - idiopathic thrombocytopenic purpura (ITP) (Imbach P, Barandun S, d Apuzzo V, et al: High-dose intravenous gamma globulin for idiopathic thrombocytopenic purpura in childhood. Lancet 181 Jun 6; 1(8232): 1228-31). IVIG (designated in the present application as "hIVIG) is a drug on the basis of sterile, purified immunoglobulin G (IgG) obtained from a mixed human plasma, which typically contains more than 95% unmodified IgG, with a small and variable number of immunoglobulin A (IgA) or immunoglobulin M (IgM) (see, e.g., Rutter A, Luger TA: High-dose intravenous immunoglobulins: an approach to treat severe immune-also been other ideas where and autoimmune diseases of the skin. J. Am. Acad. Dermatol. 2001 Jun; 44(6):1010-24). To date, only the most common clinical application of hIVIG is the treatment of ITP.

[0005] Despite the fact that hIVIG treatment was clinically effective medicines on the basis of hIVIG had a number of shortcomings, which included the probability of lack of sterility, contamination, lack of accessibility, and the dispersion characteristics of drugs from batch to batch. In particular, in the hIVIG preparations may change significantly the content of immunoglobulin A (IgA), which can be dangerous, because IgA cause allergic and anaphylactic reactions in recipients with IgA deficiency.

[0006] in view of the adverse properties of hIVIG there is a need for improved methods of treating autoimmune and inflammatory diseases.

[0007] in addition, cells derived from monocytes that mediate numerous pathologies�their status of various types. Simple therapeutic and/or prophylactic agent designed for use in many, if not all, such States would have been priceless.

Summary of the invention

[0008] Immunoregulatory properties of IVIG is due to the Fc-domain of IgG molecules. For example, in murine models of ITP unmodified IVIG as one Fc-fragment, demonstrates therapeutic efficacy in restoring the number of platelets, whereas wydelennye Fab-fragments of IVIG are not therapeutically effective (Samuelsson, A., Towers, T. L. &Ravetch, J. V. Anti-inflammatory Activity of IVIG also been other ideas where Through the Inhibitory Fc Receptor. Science 291, 484-486 (2001)). Moreover, Fc, but not Fab-fragments of IVIG are also therapeutically effective in the treatment of idiopathic thrombocytopenic purpura in children and adults (Follea, G. et al. Intravenous plasmin-treated gamma globulin therapy in idiopathic thrombocytopenic purpura. Nouv Rev Fr Hematol 27, 5-10 (1985); Solal-Celigny, P., Bernard, J., Herrera, A. & Biovin, P. Treatment of adult autoimmune thrombocytopenic purpura with high-dose intravenous plasmin-cleaved gammaglobulins. Scand J Haematol 31, 39-44 (1983); made significant gains, M. & Bonnet, M.-C. Infusion of Gcgamma fragments for treatment of children with acute immune thrombocytopenic purpura. Lancet 342, 945-49 (1993); Burdach, S. E., Evers, K. & Geurson, R. Treatment of acute idiopathic thrombocytopenic purpura of childhood with intravenous immunoglobulin G: Comparative efficacy of 7S and 5S preparations. J Pediatr 109, 770-775 (1986)).

[0009] therapeutic effect of IVIG initially mediated by Fc-gamma receptor (FcγR) and based on a cross-interaction with dendritic cells(DC) and macrophages, hence, they have long-term tolerogenic effect. FcγRIIIa plays an important role in the initiation phase, and FcγRIIb is involved in the effector phase in models of ITP in mice (Samuelsson, A., Towers, T. L. &Ravetch, J. V. Anti-inflammatory Activity of IVIG also been other ideas where Through the Inhibitory Fc Receptor. Science 291, 484-486 (2001); Siragam, V. et al. Intravenous immunoglobulin ameliorates ITP via activating Fc[gamma] receptors on dendritic cells. Nat Med 12, 688 (2006)). Similarly, human studies have shown that antibodies against Fcγ receptor is effective in the treatment of resistant forms of ITP (Clarkson, S. et al. Treatment of refractory immune thrombocytopenic purpura with an anti-Fc gamma-receptor antibody. N Engl J Med 314, 1236-1239 (1986)). It is important that long-term tolerogenic effects are due to intercellular interactions, and adaptive transfer of IVIG-treated DC is effective in the treatment in models of ITP in mice (Siragam, V. et al. Intravenous immunoglobulin ameliorates ITP via activating Fc[gamma] receptors on dendritic cells. Nat Med 12, 688 (2006)).

[0010] Immunomodulatory effects of IVIG require FcγR aggregation. Aggregation FcγR cause the IgG dimers present in IVIG (5-15% of the total IVIG) (Bleeker, W. K. et al. Vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase. Blood 95, 1856-1861 (2000)). For example, in the model of ITP in mice IVIG treatment with high content of dimers of dimers of full-sized immunoglobulin molecules) led to increased numbers of platelets, whereas "monomers" IVIG (full-size immunoglobulin molecule) were not effective (Teeling, J.. et al. Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia. Blood 98, 1095-1099 (2001)). In addition, although fractionation on ion-exchange resin and the polyethylene glycol is widely used in obtaining IVIG to remove aggregates of IgG, the clinical efficacy of IVIG correlates with the presence of dimers in the blood serum of the patient (Augener, W., Friedman, B. & Brittinger, G. Are aggregates of IgG from the effective part of high-dose immunoglobulin therapy in adult idiopathic thrombocytopenic purpura (ITP)? Blut 50, 249-252 (1985)). Importantly, the percentage of dimers also correlates with vasoactive side effects that are amenable to therapy with the use of acetylhydrolase (Bleeker, W. K. et al. Vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase. Blood 95, 1856-1861 (2000)).

[0011] the Present invention relates to biologically active biomimetic molecules, to compositions containing them and to methods of their use. These biomimetics are widely used for the treatment of immunological and inflammatory diseases including, among others, autoimmune diseases, and also as bioimmunotherapy remedies for the treatment of malignant tumors. In addition, some of these biomimetics also find use as reagents that target, for example, for use in the immunoassay, the OSU�estelauder check the function of immunocytes, as well as in the diagnosis of the disease. In addition, biomimetics and compositions of the present invention have the advantage of which is to overcome the above limitations of hIVIG. The invention also relates to the treatment and prevention of disease States mediated by cells derived from monocytes, and, more specifically, to the use of stable functional parts Fc-fragments of IgG during such treatment and prevention.

[0012] In the first embodiment of the present invention is directed to a dedicated serial strathmere containing two or more monomers of the United strathmere, where each of the monomers of strathmere includes two or more monomer Fc-domain, where the Union of two or more monomers of strathmere leads to the formation of two or more Fc domains, and where consistent streamer specific binds to a first Fcγ receptor through a first of the two or more Fc domains and to a second Fcγ receptor through a second of the two or more Fc domains. In the preferred embodiment, the implementation of two or more monomers of strathmere connected through a covalent bond, a disulfide bond or by chemical cross-linkage.

[0013] In a preferred embodiment of the present invention, a dedicated serial strathmere consist of two with�United monomers of strathmere. In another preferred embodiment, the implementation of a dedicated serial strathmere consist of two monomers of the United strathmere where both monomer strathmere include two monomer Fc-domain, and where the joining of two monomers of strathmere leads to the formation of the two Fc domains. In the first specific example of these embodiments, aimed at dedicated serial strathmere at least one of the two Fc domains comprises the hinge region of IgG and CH2 domain of IgG. In the second specific example, each of the two Fc domains independently comprises a hinge region of IgG and CH2 domain of IgG. In the third specific example, at least one of the two Fc domains comprises the hinge region of the IgG CH2 domain of IgG and the CH3 domain of IgG. In the fourth specific example, each of the two Fc domains independently comprises a hinge region of the IgG CH2 domain of IgG and the CH3 domain of IgG. In the fifth specific example, at least one of the two Fc domains comprises a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 or CH2 IgG3 domain, and CH3 domain of IgG1 or CH3 domain of IgG3. In the sixth specific example, at least one of the two Fc domains comprises a hinge region of IgG1 or hinge region of IgG3 and CH2 domain of IgG1 or CH2 domain of IgG3. In the seventh specific example of each of the two Fc domains independently comprises a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 Ilin domain IgG3, and CH3 domain of IgG1 or CH3 domain of IgG3. In the eighth specific example of each of the two Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG1 domain. In the ninth specific example of each of the two Fc domains independently comprises a hinge region of IgG3, CH2 domain and IgG3 CH3 domain of IgG3. In a tenth specific example of each of the two Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG3 domain.

[0014] Also specified in the first variant of implementation of each of the two or more Fc domains belong to one class of immunoglobulin Fc, and the class Fc of an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. Alternative each of the two or more Fc domains belong to different classes of immunoglobulin Fc, and the specified class Fc immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0015] furthermore, in this first embodiment of the first and second Fc-γ receptors independently represent an Fc-γ receptor I, Fc-γ receptor II, Fc-γ receptor III or Fc-γ receptor IV. Preferably the first and second Fc-γ receptor is an Fc-γ receptor IIIa.

[0016] In the second embodiment of the present invention is directed to a dedicated serial strathmere, including two United monomer strathmere, where each of the monomers of strathmere includes two monomer Fc-domain, where the connection d�wow monomers of strathmere leads to the formation of the two Fc domains where each of the two Fc domains independently comprises a hinge region of the IgG CH2 domain of IgG and the CH3 domain of IgG, and where consistent streamer specific associated with the first Fc-γ receptor through a first of the two Fc domains and the second Fc-γ receptor through a second of the two Fc domains. In the preferred embodiment, the implementation of two or more monomer strathmere is connected through a covalent bond, a disulfide bond or by chemical cross-linkage.

[0017] In the first specific example of the specified second embodiment of each of the two Fc domains refers to one class of immunoglobulin Fc, and the class Fc of an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In the second specific example, each of the two Fc domains refers to different classes of immunoglobulin Fc, and the specified class Fc immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In the third specific example, at least one Fc domain comprises a hinge region of IgG and CH2 domain of IgG. In the fourth specific example each of the Fc domains independently comprises a hinge region of IgG and CH2 domain of IgG. In the fifth specific example, at least one Fc domain comprises a hinge region of the IgG CH2 domain of IgG and the CH3 domain of IgG. In the sixth specific example each of the Fc domains independently comprises a hinge region of the IgG CH2 domain of IgG and the CH3 domain of IG. In the seventh specific example, at least one Fc domain comprises a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 or CH2 IgG3 domain, and CH3 domain of IgG1 or CH3 domain of IgG3. In the eighth specific example each of the Fc domains independently comprises a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 or CH2 IgG3 domain, and CH3 domain of IgG1 or CH3 domain of IgG3. In the ninth specific example each of the Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG1 domain. In the tenth specific example each of the Fc domains independently comprises a hinge region of IgG3, CH2 domain and IgG3 CH3 domain of IgG3. In the eleventh specific example each of the Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG3 domain.

[0018] In the third embodiment of the present invention relates to a dedicated serial strathmere, further comprising Fab-domain, where each of the monomers of strathmere includes a heavy chain Fab fragment and two Monomeric Fc domain of the heavy chain Fab fragment is located on the N-end or C-end of the two monomers of the Fc-domain, where the light chain of the Fab fragment is independently associated with each heavy chain of the Fab fragment, and where the Fab domain has antigen-binding activity. In a preferred embodiment of the implementation of each of the monomers of strathmere additionally includes a monomer hinge region� immunoglobulin, and where the monomer hinge region located between immunoglobulin heavy chain Fab fragment and the two monomers Fc-domain.

[0019] In the fourth embodiment of the present invention is directed to cor-strathmere, including cor-group associated with two or more cor-strdomainname units, where each of two or more cor-strathmere units includes at least one Fc domain, and wherein each of the DAC-strathmere units independently selected from the group consisting of:

(a) Fc-fragment, where the specified Fc-fragment comprises two United monomer Fc-fragment, where each of the monomers of the Fc-fragment comprises an Fc monomer-domain, and where the Union of two monomers of the Fc-fragment leads to the formation of the Fc-domain

(b) incomplete Fc-fragment, where the specified incomplete Fc-fragment comprises two connected incomplete monomer Fc-fragment, where each specified incomplete monomer Fc-fragment comprises an Fc monomer-domain, and where the Union of two incomplete monomer Fc-fragment leads to the formation of the Fc-domain

(c) the Fc-domain, where the specified Fc-domain includes two United Fc monomer-domain, and where the Union of two monomers of the Fc-domain leads to the formation of the Fc-domain

(d) consecutive strathmere where the specified serial streamer includes two or more monomers of the United strathmere, where each of the�azannyh monomers of strathmere includes two or more monomers of the Fc-domain, and where the connection of two or more monomers of strathmere leads to the formation of two or more Fc domains, and

(e) cluster strathmere where the specified cluster streamer includes two or more multimedijalnih cluster strathmere units, where each specified cluster stregoneria unit includes multilaterals region and at least one Fc domain, wherein each specified cluster stregoneria unit includes two United monomer cluster strathmere units, where each specified monomer cluster strathmere unit includes a monomer multimeasure region and at least one monomer Fc-domain, where the connection of the two monomers cluster strathmere units leads to the formation of multimeasure region and at least one Fc domain, and where multimeasure region of two or more cluster strathmere units multimerization with the formation of the cluster strathmere, and

where cor-streamer specific associated with the first Fc-γ receptor through a first of two or more cor-strathmere units and the second Fc-γ receptor through a second of the two or more cor-strathmere units.

[0020] Preferably specified in the fourth embodiment of the KOR group selected from the group consisting of J-chain of an immunoglobulin, albumin, liposomes, particles, peptide and polyethylene glycol.

[0021] In preferred embodiments relating to the cor-page�damerham, each of the two or more cor-strathmere units is independently Fc-fragment. In an alternative embodiment, each of the two or more cor-strathmere units independently is a consistent strathmere.

[0022] In another preferred embodiment of implementation, aimed at cor-strathmere, cor-streamer includes two cor-strathmere units, where each of the two cor-strathmere units independently is a consistent strathmere where consistent streamer includes two United monomer strathmere, where both of the monomers of strathmere include two monomer Fc-domain, and where the joining of two monomers of strathmere leads to the formation of the two Fc domains. In the first specific example, a specified embodiment of at least one of the Fc domains of the two or more cor-strathmere units includes a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 or CH2 IgG3 domain, and CH3 domain of IgG1 or CH3 domain of IgG3. In the second specific example, at least one of the Fc domains of the two or more cor-strathmere units includes a hinge region of IgG1 or hinge region of IgG3 and CH2 domain of IgG1. In the third specific example each of the Fc domains of the two or more cor-strathmere units independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG1 domain. In the fourth specific example for IU�Isha least one of the Fc domains of the two or more cor-strathmere units includes a hinge region of IgG and CH2 domain of IgG. In the fifth specific example each of the Fc domains of the two or more cor-strathmere units independently comprises a hinge region of IgG and CH2 domain of IgG. In the sixth specific example each of the Fc domains of the two or more cor-strathmere units independently comprises a hinge region of IgG3, CH2 domain and IgG3 CH3 domain of IgG3. In the seventh specific example each of the Fc domains of the two or more cor-strathmere units independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG3 domain.

[0023] In a specified embodiment of the first and the second Fc-γ receptor independently represents an Fc-γ receptor I, Fc-γ receptor II, Fc-γ receptor III or Fc-γ receptor IV. Preferably, the first and second Fc-γ receptor is an Fc-γ receptor IIIa.

[0024] In the fifth embodiment of the present invention relates to clustered strathmere comprising two or more multipersoona cluster strathmere units, where each cluster stregoneria unit includes multilaterals region and at least one Fc domain, where each cluster stregoneria unit includes two United monomer cluster strathmere units, where each monomer cluster strathmere unit includes a monomer multimeasure region and at least one monomer Fc-domain, where the connection of the two monomers cluster strathmere units leads to �formirovaniu multimeasure region and at least one Fc domain, where multimeasure region of two or more kesternich strathmere units multimerization with the formation of the cluster strathmere, and where the cluster streamer specific associated with the first Fc-γ receptor through a first Fc-domain, and the second Fc-γ receptor via a second Fc-domain.

[0025] In preferred embodiments, multimeedia region selected from the group consisting of a hinge region of IgG2, CH2 domain of IgE, latinboy, isoleucinol zipper and zinc finger.

[0026] In another preferred embodiment, the implementation of cluster strathmere include two, three, four or five multimedijalnih cluster strathmere units.

[0027] In the first specific example of the specified fifth embodiment of at least one Fc domain comprises a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 or CH2 IgG3 domain, and CH3 domain of IgG1 or CH3 domain of IgG3. In the second specific example each of the Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG1 domain. In the third specific example, at least one Fc domain comprises a hinge region of IgG and CH2 domain of IgG. In the fourth specific example each of the Fc domains independently comprises a hinge region of IgG and CH2 domain of IgG. In the fifth specific example each of the Fc domains independently comprises a hinge region of IgG3, CH2 domain IgG3 � CH3 domain of IgG3. In the sixth specific example each of the Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG3 domain. In the seventh specific example each of the Fc domains independently comprises a hinge region of the IgG CH2 domain of IgG and the CH3 domain of IgG. In the eighth specific example of at least one cluster strathmere units comprises two or more Fc domain. In the ninth specific example, each cluster stregoneria unit includes two or more Fc domain.

[0028] In a specified embodiment of the first and the second Fc-γ receptor independently represents an Fc-γ receptor I, Fc-γ receptor II, Fc-γ receptor III or Fc-γ receptor IV. Preferably the first and second Fc-γ receptor are Fc-γ receptor IIIa.

[0029] In the sixth embodiment of the present invention is directed to stratatel comprising two or more monomers of the United strathmere and Fab-domain, where each of the monomers of strathmere includes a heavy chain Fab fragment and two or more monomers of the Fc domain of the heavy chain Fab fragment is located on the N-end or C-end of two or more monomers of the Fc-domain, where the Union of two or more monomers of strathmere leads to the formation of two or more Fc domains, where light chain Fab fragment is independently connected to a heavy chain Fab fragment of each monomer strathmere, where the Fab domain has antigen-binding asset�spine and where stratatel specific associated with the first Fc-γ receptor through a first of the two or more Fc domains, and with the second Fc-γ receptor through a second of the two or more Fc domains.

[0030] In preferred embodiments, two or more monomer strathmere connected through a covalent bond, a disulfide bond or by chemical cross-linkage.

[0031] In another preferred embodiment, the implementation of each of these monomers of strathmere in stratatel additionally includes a monomer of the hinge region of immunoglobulin, and where the monomer hinge region located between immunoglobulin heavy chain Fab fragment and the two monomers Fc-domain.

[0032] In a specific embodiment, the implementation of stratatel includes two United monomer strathmere, where each of the monomers of strathmere includes a heavy chain Fab fragment and two monomer Fc-domain, and where the joining of two monomers of strathmere leads to the formation of the two Fc domains. In the first specific example, a specified embodiment of at least one of the two Fc domains comprises the hinge region of the IgG CH2 domain of IgG and the CH3 domain of IgG. In the second specific example, each of the two Fc domains independently comprises a hinge region of the IgG CH2 domain of IgG and the CH3 domain of IgG. In the third specific example, at least one of the two Fc domains comprises the hinge region of IgG and the CH3 domain of IgG. In the fourth specific example, each of the two Fc domains independently comprises a hinge region of IgG and the CH3 domain of IgG. In the fifth specific example �about at least one of the two Fc domains comprises a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 or CH2 IgG3 domain, and CH3 domain of IgG1 or CH3 domain of IgG3. In the sixth specific example, each of the two Fc domains independently comprises a hinge region of IgG1 or hinge region of IgG3, CH2 domain of IgG1 or CH2 IgG3 domain, and CH3 domain of IgG1 or CH3 domain of IgG3. In the seventh specific example of each of the two Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG1 domain. In the eighth specific example of each of the two Fc domains independently comprises a hinge region of IgG3, CH2 domain and IgG3 CH3 domain of IgG3. In the ninth specific example of each of the two Fc domains independently comprises a hinge region of IgG1, CH2 domain and CH3 IgG1 IgG3 domain. In the tenth specific example, at least one of the two Fc domains comprises a hinge region of IgG1 or hinge region of IgG3 and CH2 domain of IgG1 or CH2 domain of IgG3. In the eleventh specific example, at least one of the two Fc domains comprises a hinge region of IgG1 or hinge region of IgG3 and CH2 domain of IgG1.

[0033] In a specified embodiment of the first and the second Fc-γ receptors independently represents an Fc-γ receptor I, Fc-γ receptor II, Fc-γ receptor III or Fc-γ receptor IV. Preferably the first and second Fc-γ receptor are Fc-γ receptor IIIa.

[0034] In the seventh embodiment of the present invention is directed to methods of modifying the immune response in the individual, comprising administering to the individual �if necessary, pharmaceutical compositions containing a therapeutically effective amount of consecutive strathmere, and a carrier or diluent. In a preferred embodiment of the pharmaceutical composition contains a therapeutically effective amount of a heterogeneous mixture of sequential strathmere, and a carrier or diluent.

[0035] In the eighth embodiment of the present invention relates to methods of modifying the immune response in the individual, comprising administering to the individual, if necessary, the pharmaceutical composition comprising a therapeutically effective amount of cor-strathmere, and a carrier or diluent. In a preferred embodiment of the pharmaceutical composition contains a therapeutically effective amount of a heterogeneous mixture of cor-strathmere, and a carrier or diluent.

[0036] In the ninth embodiment of the present invention relates to methods of modifying the immune response in the individual, comprising administering to the individual, if necessary, the pharmaceutical composition comprising a therapeutically effective amount of cluster strathmere, and a carrier or diluent. In a preferred embodiment of the pharmaceutical composition contains a therapeutically effective amount of a heterogeneous mixture to�asternic of strathmere, and a carrier or diluent.

[0037] In the tenth embodiment of the present invention relates to methods of modifying the immune response in the individual, comprising administering to the individual, if necessary, the pharmaceutical composition comprising a therapeutically effective amount Stradale, and a carrier or diluent. In a preferred embodiment of the pharmaceutical composition includes a therapeutically effective amount of a heterogeneous mixture stratatel, and a carrier or diluent.

[0038] In the eleventh embodiment of the present invention relates to methods of screening for antibodies in a subject-specific activity against cells of the immune system, including: (a) the contact of homogeneous populations of immune system cells with an antibody candidate, (b) measuring the activity of a population of cells (a), (c) contact with a homogeneous population of cells of the same type of cells as in (a), consistent with strathmere according to claim 1, (d) measuring the activity of a population of cells (c), and (e) comparing the activity measured in (b)with the activity measured in (d), performing, thus, the antibody screening on a subject-specific activity against cells of the immune system. In a preferred embodiment of the antibody-candidate and consistent streamer selected in accordance with the view�mi and isotype. In another preferred embodiment of the comparison in (e) is the ratio of the activity measured in (d), to the activity measured in (b).

[0039] In a twelfth embodiment of the present invention relates to methods of inhibiting the activity of cells derived from the monocyte (MDC). The method comprises contact of the cell with a composition comprising a substrate, which is associated with the Fc-reactant. Contact can be madein vitro,in vivoorex vivo. The cell can be in your body of an animal, for example, an animal with a disease mediated by cells derived from monocytes (MDCMC), or an animal that is at risk for this disease. The cell can be, for example, dendritic cell, a macrophage, a monocyte, or osteoclast.

[0040] In a thirteenth embodiment of the present invention relates to methods of treatment which include the introduction of the animal a composition comprising a substrate, which is associated with the Fc-reactant, wherein the animal suffers from a disease mediated by cells derived from monocytes (MDCMC), or is at risk of its development.

[0041] Below are embodiments of two of these methods (twelfth and thirteenth version of the implementation).

[0042] the Animal may be, for example, a person.

[0043] the Fc-reactant m�can contain or constitute a functional portion of the Fc-fragment of human rights, for example, the Fc-fragment of human IgG1, Fc fragment of human IgG3, human IgG2 or Fc-fragment of human IgG4. In addition, it may contain, or is a molecule of IgG. Fc-reactant may also be or include a functional part of the Fc-fragment, not a person.

[0044] the Substrate may be or include a synthetic polymer, such as nylon, Teflon, Dacron, PVC, PES (polyester urethane), PTFE (polytetrafluoroethylene) or PMMA (polymethylmethacrylate). The substrate may contain or may consist of metal or metal alloy, such as stainless steel, platinum, iridium, titanium, tantalum, nickeltitanium alloy or cobalt alloy. The substrate may contain or be a tissue of animals or preparation of animal tissue, e.g., tissue or organ transplant, bone (e.g., osteogenic tissue or cartilage. The substrate may contain or be a protein, e.g., collagen or keratin. The substrate may also be or contain a polysaccharide, such as agarose. In addition, the substrate may contain or be a tissue matrix, for example, acellular tissue matrix. The substrate may contain or represent an animal cell (e.g., cell, regenerative tissue, such as fibroblasts or mesenchymal stem cell). Sneaky�GCA may contain or be a salt, for example, calcium sulphate. In addition, the substrate may be or include a gel or cream. Also it may contain or may consist of silicone or Silastic. It can also contain or be a natural fiber such as silk, cotton or wool.

[0045] the Substrate may be a complex form pores in the membrane or implantable medical device, such as a stent (e.g., a vascular stent, such as a coronary stent; a stent placed in the airway such as an endotracheal or nasal stent, gastrointestinal stent, such as a biliary or pancreatic stent; or ureteral stent, such as a urethral stent). It can also be a surgical thread (for example, silk thread, chromed catgut, nylon, polymeric or metallic thread, or a surgical clip (e.g., aneurysm clip)). In addition, the substrate may be artificial hip, an artificial hip, an artificial knee, an artificial knee joint, an artificial shoulder, an artificial shoulder joint, an artificial finger joint (hands or feet), bone plate, bone pin, the implant at bone fractures, intervertebral disc implant, bone cement, or a layer of bone CEM�TA. It may be an arterial-venous shunt, implantable wire, a pacemaker, an artificial heart, a device that supports the heart, cochlear implant, implantable defibrillator, a spinal cord stimulator, a Central nervous system stimulant, implant perifericheskogo nerve, a dental prosthesis or a dental crown. In addition, the substrate may be a device or mesh to protect larger vessels from embolism, subcutaneous device, a skin patch or patches in the submucosa, or implantable device for drug delivery.

[0046] the Substrate may also be a graft of a large blood vessel where the blood vessel is, for example, carotid artery, femoral artery or the aorta. It can also be a subcutaneous implant, corneal implant, intraocular lens or contact lens.

[0047] the Substrate may be in the form of a sheet, pellets, meshes, particles, powder, filaments, granules or fibers. The substrate may contain or be a solid, semi-solid or gel-like substance. Thus, the substrate contains substances which are practically insoluble in aqueous solvents, for example, fat-soluble lipid, such as a liposome.

[0048] MDCMC can be inflammatory �zabolevanie, autoimmune disease, malignancy, impaired bone density, acute infection or chronic infection.

[0049] MDCMC can be hematolymphopoietic process, for example, idiopathic thrombocytopenic purpura, alloimmune/autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, Parvovirus B19-associated red cell aplasia, acquired autoimmunity against factor VIII acquired the disease von Willebrand, multiple myeloma and monoclonal gammopathy of unknown etiology, sepsis, aplastic anemia, pure red cell aplasia, anemia diamond-I am, hemolytic disease of the newborn, immunopositive neutropenia, refractoriness to platelet transfusion, neonatal posttransfusion purpura, hemolytic uremic syndrome, systemic vasculitis, thrombocytopenic thromboembolitic purpura or Evan's syndrome.

[0050] Alternatively, MDCMC can be a neuro-process, for example, Guillain-Barre syndrome, a chronic inflammatory demyelinating polyradiculoneuropathy, paraproteinemic IgM demyelinating polyneuropathy, myasthenic syndrome La�Bert-Eaton, myasthenia gravis, multifocal motor neuropathy, amyotrophic lateral sclerosis, associated with antibodies against GM1, demyelination, multiple sclerosis and optic neuritis, syndrome muscle stiffness, paraneoplastic degeneration of the cerebellum, called anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy caused by anti-Hu antibodies, epilepsy, encephalitis, myelitis, myelopathy especially associated with T-lymphotropic virushumanthe first type, autoimmune diabetic neuropathy, or acute idiopathic autonomic neuropathy.

[0051] MDCMC can be a rheumatic process, for example, Kawasaki disease, rheumatoid arthritis, felty's syndrome, anti-neutrophil cytoplasmic antibodies-positive vasculitis, spontaneous polymyositis, dermatomyositis, antiphospholipid syndromes, recurrent spontaneous abortion, systemic lupus erythematosus, juvenile idiopathic arthritis, Raynaud's syndrome, CREST syndrome, or uveitis.

[0052] in addition, the MDCMC can be dermatovenerologicheskiy process, for example, epidermal necrolysis, gangrene, granuloma, autoimmune skin diseases with the development of abscesses, including vulgar pemphigus, bullous pemphigoid and exfoliative pemphigus, vitiligo, syndrome streptococcal current�demand shock, scleroderma, systemic sclerosis including diffuse and local cutaneous systemic sclerosis, atopic dermatitis or dependent atopic dermatitis.

[0053] in addition, the MDCMC can be musculoskeletal immunological disease such as myositis included with calves, a lot of necrotizing fasciitis, inflammatory myopathies, myositis, anti-decorin (BJ antigen) myopathy, paraneoplastic necrotizing myopathy, aqualicious myopathy coupled to the X-chromosome, penicillamine-induced polymyositis, atherosclerosis, coronary heart disease or cardiomyopathy.

[0054] MDCMC can be written gastrointestinal immunological process, for example, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis, reactive arthritis, Crohn's disease, Whipple's., ulcerative colitis or sclerosing cholangitis.

[0055] MDCMC can be, for example, homologous disease (GVHD), antibody-mediated graft rejection, graft rejection, bone marrow, postinfectious inflammation, lymphoma, leukemia, neoplasia, asthma, diabetes mellitus type I with antibodies against beta cells, Sjogren's syndrome, mixed�th lesions of the connective tissue, Addison disease, syndrome Vogt-Koyanagi-Harada, membranosa-proliferative glomerulonephritis, goodpasture's syndrome, graves ' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropolitical, syndrome Cerca-Strauss, nodular nodosa or multiple organ failure.

[0056] When the MDCMC is a malignant tumor, then MDCMC can be a fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteoblastic bone sarcoma, chordomas, angiosarcoma, endotheliotropic, lymphangiosarcoma, lymphangiosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinoma of the colon, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, carcinoma of the sweat gland, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, počečnokletočnyj carcinoma, hepatoma, carcinoma of the bile duct, choriocarcinoma, seminoma, teratocarcinoma, swelling of Wilma, cervical cancer, tumor of the testis, lung carcinoma, small cell carcinoma of the lung, carcinoma of the bladder, epithelial carcinoma, glioma, asters�cytomas, brain cranyopharyngioma, ependymomas, pinealomas, hemangioblastomas, the auditory nerve neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, waldenstrom's macroglobulinemia, myelodysplastic disease, heavy chain disease, neuroendocrine tumors or Swannanoa.

[0057] If the MDCMC is a disease associated with impaired bone density, MDCMC can be osteoporosis, osteopenia, congenital osteopetrosis, idiopathic hypogonadotropic hypogonadism, anorexia nervosa, non-healing fractures, postmenopausal osteoporosis, deficiency or excess of vitamin D, primary or secondary hyperparathyroidism, thyroid disease or toxicity of the bisphosphonate.

[0058] When the MDCMC is an acute infection, then MDCMC can be: fungal disease, including candidiasis, candidemia or aspergillosis; bacterial disease, including infections caused by Staphylococcus, including methicillin-resistantStaphylococcus aureusstreptococcal infections of skin and oropharyngeal disease or sepsis caused by gram-negative bacteria; Mycobacterium infections, including tuberculosis; viral infection, including mononucleosis, respiratory syncytial virus and�the infection or herpes zoster; parasitic infections, including malaria, schistosomiasis, trypanosomiasis or.

[0059] When the MDCMC is a chronic infection, then MDCMC can be onychomycosis; bacterial disease, including infectionHelicobacter pylori; mycobacterial infection, including tuberculosis; viral infection, including infection caused by the Epstein-Barr infection caused by the human papilloma virus, or infection caused by the herpes simplex virus; or parasitic infections, including malaria or schistosomiasis.

[0060] In the fourteenth embodiment of the present invention relates to a composition that contains or is implantable or attachable medical device, and Fc-reagent associated with it.

[0061] In a fifteenth embodiment of the present invention refers to a kit that contains an implantable or attachable medical device and Fc-reagent. In both of these embodiments, implantable or attachable medical device and the Fc-reactant can be any of those described in the present application. The kit may further contain a suitable container.

[0062] Additional advantages and features of the present invention will be apparent from the following detailed description, figures and examples, which illustrate preferred� embodiments of the invention.

[0063] in the Above General terms quite broadly describes the features and technical advantages of the present invention, subsequent to a detailed description of the invention could be better understood. Additional features and advantages of the invention forming the object of the present invention will be described next. For the person skilled in the art it is obvious that the conception and the disclosed specific embodiments of the invention can be used as a basis for modifying or designing other structures performing the same tasks of the present invention. For the person skilled in the art it is obvious that such equivalent structures do not extend beyond the volume and do not change the essence of the present invention, formulated in the accompanying claims. New features, which are considered to be essential to the invention, its structure and method of implementation, together with other objects and advantages will be more apparent from the following description considered together with the accompanying figures. However, note that each figure is given merely for purposes of illustration and description, and should not be construed as limiting the present invention.

BRIEF DESCRIPTION of FIGURES

[0064] In Fig.1A schematically while�Aza native structure of the monomer Fc fragment of IgG1, the hinge region which is linked to the CH2 domain that is linked to a CH3 domain; Fig.1B shows semiregularly, native Fc fragment of IgG1, formed of two related monomers Fc-fragment.

[0065] In Fig.1C schematically shows the native structure of the monomer Fc-fragment of IgG3 hinge region which is linked to the CH2 domain that is linked to a CH3 domain; Fig.1D shows semiregularly, native Fc-fragment of IgG3, formed of two related monomers Fc-fragment.

[0066] In Fig.2A and 2B shows the aggregates of higher order native structure of the monomer Fc-fragment, shown in Fig.1B. The Fc fragments may be naturally multimerization in dimers of dimers (tetramers) or even in plant-multimer of higher order.

[0067] In Fig.3A schematically shows the native IgG1 antibody having a native Fab fragment associated with the Fc-fragment in the hinge region of the Fc fragment; Fig.3B shows a similar structure IgG3.

[0068] In Fig.4A shows the monomer streamer consisting of two monomers of the Fc domain of IgG1; Fig.4B shows an alternative structure of the monomer strathmere in which IgG1 Fc - IgG3 Fc - IgE Fc are connected in series.

[0069] In Fig.5A and B shows the monomers of strathmere from Fig.4 A and B, autodemolizioni in serial streamer its ability inherent constituent monomers Fc-home�.

[0070] In Fig.6A shows the monomer strathmere containing IgG1 Fc - IgG1 (hinge region - CH2); Fig.6B shows streamer containing IgG1 (the hinge region - CH2) - IgG3 (hinge region - CH2) - IgE (hinge region - CH2).

[0071] In Fig.7A and B shows the monomers of strathmere 6A and B, autodemolizioni in serial streamer its ability inherent constituent monomers Fc-domain.

[0072] In Fig.7C shows a consistent streamer containing IgE (hinge region)-IgG1 Fc-IgG1 (hinge region-CH2)-IgE (CH3). Fig.7D shows a consistent streamer containing IgG3Fc - IgG1Fc.

[0073] In Fig.8A shows the structure stratatel containing Fab sequential structure of strathmere, with each monomer strathmere monomer contains two IgG1 CH2-CH3 originating from the Fc-domain; Fig.8B shows the structure Stradale, as in 8A, but in the structure of strathmere contains IgG1 Fc associated with IgG3 Fc, associated with IgE Fc.

[0074] In Fig.9A shows stratatel IgG1 Fc - IgG1 (hinge region - CH2); Fig.9B shows stratatel IgG1 (the hinge region - CH2) - IgG3 (hinge region - CH2) - IgE (hinge region - CH2)3.

[0075] In Fig.10A shows the monomer strathmere IgG1 (the hinge region - CH2) - IgG3 CH3 - IgM CH4 and protein J-chain; Fig.10B shows the cor-streamer based pentamere of strathmere Fig.10A, formed as a result of the connection via the CH4 domain of IgM to J-CE�I.

[0076] In Fig.10C shows the monomer strathmere IgG1 Fc - IgG1 Fc - IgM CH4 and protein J-chain; Fig.10D shows the cor-streamer based pentamere of strathmere Fig.10C, formed as a result of the connection via the CH4 domain of IgM to the J-chain.

[0077] In Fig.11A shows the monomer strathmere IgG1 Fc - IgG1 (hinge region - CH2). Fig.11B shows how the monomer strathmere from Fig.11A may autogenerators, forming a consistent streamer. Fig.11C shows how in the same monomer strathmere from Fig.11A Monomeric Fc domains can vyravnivala with the same or similar monomers Fc-domain of another monomer strathmere, but not autotimer, forming, thus, streamer consisting of the same monomer strathmere as autotimer, but with the structure with the effect of lightning.

[0078] In Fig.12A shows the monomer IgG3 Fc - IgG1 Fc strathmere. Fig.12B shows that the addition of a second IgG3 Fc, followed by avtomyerizatsiya, can lead to the formation of branched structured strathmere IgG3 Fc - IgG1 Fc - IgG3 Fc.

[0079] In Fig.13A shows the monomer IgE CH2 - IgG1 Fc - IgG1 (hinge region-CH2) - IgE CH4 strathmere. Fig.13B shows autotimer monomer Fig.13A and selected two formed site FcγR binding.

[0080] In Fig.14A shows streamer consisting of two Fc domains of IgG1) which are attached to the linker. Fig.14B shows streamer consisting of two consecutive streamer�in (namely in each case strathmere 2(IgG1 Fc)), connected by a linker.

[0081] In Fig.15A shows the nucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequence of the Fc fragment of human IgG1. Fig.15B shows the nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequence of Fc fragment of human IgG2. Fig.15C shows the nucleotide (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequences of the Fc-fragment of human IgG3. Fig.15D shows the nucleotide (SEQ ID NO:7) and amino acid (SEQ ID NO:8) sequence of the Fc fragment of human IgG4.

[0082] In Fig.16 shows the nucleotide (SEQ ID NO:17) and amino acid (SEQ ID NO:18) sequence of the design, including (signal sequence IgK - Fc fragment of IgG1 - Fc fragment of IgG1). Amino acid sequence of IgK signal is in bold. Amino acid sequence of the first Fc fragment of IgG1 underlined with a single line. Amino acid sequence of the second Fc fragment of IgG1 accented by a double line. Serine and lysine, marked with an asterisk are amino acids that can be subjected to mutation with the aim of changing the binding to Fc-γ receptor.

[0083] In Fig.17 shows the nucleotide (SEQ ID NO:19) and amino acid (SEQ ID NO:20) sequences of the design, including (restriction sites signal sequence IgK - restriction sites - IgG1(hinge region-CH2-CH3) - restriction sites - epitope tags (V5 and His) - STOP). Am�nochelatina sequence IgK signal is in bold. Amino acid sequence of the Fc fragment of IgG1 underlined with a single line. Amino acid sequence of a V5-tag is underlined with a dotted line. Amino acid sequence His-tag is underlined by a thick line.

[0084] In Fig.18 shows the nucleotide (SEQ ID NO:21) and amino acid (SEQ ID NO:22) sequences of the design, including {the restriction sites - IgK signal - restriction sites-IgG1(hinge region-CH2-CH3)- XbaI site - IgG1(hinge region-CH2-CH3) - STOP}. Amino acid sequence of IgK signal is in bold. Amino acid sequence of the first Fc fragment of IgG1 underlined with a single line. Amino acid sequence of the second Fc fragment of IgG1 accented by a double line.

[0085] In Fig.19 shows the nucleotide (SEQ ID NO:23) and amino acid (SEQ ID NO:24) sequences of the design, including {the restriction sites - IgK signal - restriction sites-IgG1 (hinge region-CH2-CH3) site XbaI - IgG1 (hinge region-CH2-CH3) - epitope tags (V5 and His) - STOP}. Amino acid sequence of IgK signal is in bold. Amino acid sequence of the first Fc fragment of IgG1 underlined with a single line. Amino acid sequence of the second Fc fragment of IgG1 accented by a double line. Amino acid sequence of a V5-tag is underlined with a dotted line. Amino acid posledovatel�sequence His-tag is underlined by a thick line.

[0086] In Fig.20A shows the nucleotide (SEQ ID NO:31) and amino acid (SEQ ID NO:32) sequences N-terminal signal sequence FcRgammaIIIa, in which the polymorphism of phenylalanine (F) are shown in bold and underlined. Variable nucleic acid is also in bold and underlined. Fig.20B shows the nucleotide (SEQ ID NO:33) and amino acid (SEQ ID NO:34) sequences N-terminal signal sequence FcRgammaIIIa in which polymorphism valine (V) are shown in bold and underlined. Variable nucleic acid is also in bold and underlined. Both constructs contain a C-terminal hexa-His-tag for purification.

[0087] In Fig.21 shows the nucleotide (SEQ ID NO:25) and amino acid (SEQ ID NO:26) sequences of the design, including {the restriction sites - IgK signal - EcoRV site - IgG3 (hinge region-CH2-CH3)-IgG1 (hinge region-CH2-CH3) - restriction sites - epitope tags (V5 and His) - STOP}. Amino acid sequence of IgK signal is in bold. Amino acid sequence of the Fc-fragment of IgG3 is underlined with a single line. Amino acid sequence of the Fc fragment of IgG1 accented by a double line. Amino acid sequence of a V5-tag is underlined with a dotted line. Amino acid sequence His-tag is underlined by a thick line.

[0088] In Fig.22 shows the nucleotide(SEQ ID NO:27) and amino acid (SEQ ID NO:28) sequence of construction, including {the restriction sites - IgK signal - EcoRV site - specific IgE (CH2) - IgG1 (hinge region-CH2-CH3)-IgG1 (hinge region-CH2) - IgE (CH4) - STOP}. Amino acid sequence of IgK signal is in bold. Amino acid sequence of IgE(CH2) domain is underlined with a single line. Amino acid sequence of IgG1 (the hinge region-H2-CH3) domain is underlined with a double line. Amino acid sequence of IgG1 (the Hinge region-CH2) domain is underlined with a dotted line. Amino acid sequence of IgE(CH4) domain is underlined with a wavy line.

[0089] In Fig.23A shows the Fc-fragment, and shows that such Fc-fragment is composed of two monomers of the Fc-fragment and optionally includes the Fc-domain (dotted circle) and partial Fc domains (hinge region, CH2 and CH3, as indicated). Fig.23B shows the sequential composition of strathmere, consisting of two monomers of strathmere associated inter-monomer strathmere communication. Serial streamer includes at least two Fc domain (dotted circles) and may optionally include a region linking domains. Fig.23C shows the composition of the cor-strathmere, including cor-the group with which are associated cor-strathmere units, each of which contains at least one Fc domain. Cor-strathmere units can be Fc-fragment, the sequence�individual strathmere or cluster strathmere unit. Fig.23D shows a cluster of strathmere, including multipersoona cluster strathmere units, each of which has multimeasures the region and the region containing at least one Fc domain. Cluster stregoneria unit may be an Fc-fragment or successive strathmere. Multimeedia area when multimerization forms a cluster head strathmere. Legs of cluster strathmere formed areas of the Fc-domain cluster strathmere units that are spatially less compressed than multipersonal head cluster strathmere.

[0090] In Fig.24 shows the amino acid sequence of strathmere described in Table 3.

[0091] In Fig.25 shows the amino acid sequence of monomers partial Fc domain (hinge region, CH2 and CH3) IgG1, IgG2, IgG3 and IgG4 (Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C. 1991. Sequences of proteins of immunological interest 5th Ed. US Public Health Services, NIH, Bethesda).

DETAILED description of the INVENTION

[0092] a rational Approach to the molecular design of compounds described in the present application, which are intended to replace hIVIG, includes recombinant and/or biochemical receipt of immunologically active biomimetic(s). In the preferred ways of connecting substitutes were tested in vitro to evaluate the effectiveness of each with�unity is a good substitute when you bind Fcγ receptor and modulate immune function. Separate connection alternatives selected for further analysis in vivo, as well as optimization of dosage/administration. Connection-substitutes can be used in the treatment of, for example, autoimmune diseases, inflammatory diseases, osteoporosis and malignant tumors. Each phase is described below along with specific examples of the implementation.

[0093] as Used in the original text of the present application, the articles "a" or "an", when used in conjunction with the term "comprising" in the claims and/or description can mean "one", but also does not exclude the meaning of "one or more," "at least one" and "one or more than one."

[0094] Used in the present description, the terms "biomimetic", "biomimetic molecule", "biomimetic compound", and similar terms, refer to the artificial connection that kitfrom a function of another compound, for example, mixed hIVIG, monoclonal antibodies or Fc-fragment of the antibody. "Biologically active" biomimetics are compounds that have biological activities that are identical or essentially identical to the corresponding natural counterparts. "Immunologically active biomimetics are biomimetics, which exhibit immunological activity that is essentially identical to the activity of natural�'s immunologically active molecules, such as antibodies, cytokines, interleukins and other immunological molecules known in the prior art. In preferred embodiments, the biomimetics of the present invention represent strathmere and stratatel defined in the present application.

[0095] the Immunologically active biomimetics of the present invention is designed in such a way that they share one or more immunomodulatory activities Fc-domain of IgG and include at least (i) a first Fc domain capable of binding to FcγR, including FcγRI, FcγRII, FcγRIII and FcγRIV, and (ii) a second Fc domain capable of binding to FcγR, including FcγRI, FcγRII, FcγRIII and FcγRIV.

[0096] In the following paragraphs are defined elementary units of biomimetics of the present invention both structurally and functionally, as well as biomimetics is defined directly. However, it should be noted that, as indicated above, each of the biomimetics of the present invention has at least two Fc-domain. At least the Fc-domain is a dimeric polypeptide (or dimeric region of a larger polypeptide), which consists of two peptide chains or link (monomer), which are connected, forming a functional binding site Fcγ-receptor. Thus, the functional form of individual fragments and domains described in the present application, there is usually �kernou (or multimeric form. The monomers of the individual fragments and domains described in the present application, are single-chain or links, which should be connected with the second circuit or link, in order to form functional dimeric structure.

Fc-fragment

[0097] "Fc-fragment" is a term of art which is used to describe the region of the folded protein or protein structure, which is usually located on the C-end of immunoglobulins (see Fig. 3A-3B). Fc-fragment can be selected from a Fab fragment of monoclonal antibody by papain cleavage that is incomplete and imperfect process (see Mihaesco C and M. Seligmann Papain Digestion Fragments Of Human IgM Globulins. Journal of Experimental Medicine, VoI 127, 431-453 (1968)). With Fab fragment (containing the binding domain of the antibody Fc-fragment is holo-antibody, which in this context means a complete antibody. Fc-fragment consists of the C-terminal parts of the heavy chains of the antibody. Each of the chains in the Fc fragment has a size of approximately 220-265 amino acids, the chains are often linked via a disulfide bond. Fc-fragment often contains one or more independent structural loops and functional domains. In particular, the Fc-fragment comprises an Fc domain that is defined in the present application, as the minimum structure that binds to Fcγ-receptor (see, e.g.�p, Fig. 1B and 1D). Selected Fc-fragment is composed of two monomers of the Fc fragment (for example, two C-terminal parts of the heavy chains of the antibody, also defined in the present application), which demonizovana. Formed by connecting two monomers of the Fc-fragment Fc-fragment has binding activity for Fcγ receptor.

Incomplete Fc-fragment

[0098] "Incomplete Fc-fragment" is a domain that includes non-Fc-fragment of an antibody that still retains a structure that is sufficient to possess the same activity as the Fc-fragment, including Fcγ receptor binding activity. Incomplete Fc-fragment, thus, may be deprived of part or all of a hinge region, a part or all of the CH2 domain of part or all of a CH3 region, and/or part or all of the CH4 region, depending on the isotype of the antibody from which received incomplete Fc-domain. An example of incomplete Fc-fragment comprises a molecule comprising upper, Central and lower hinge region plus the CH2 domain of IgG3 (Tan, LK, Shopes, RJ, Oi, VT and Morrison, SL, Influence of the hinge region on complement activation, CIq binding, and segmental flexibility in chimeric human immunoglobulins, Proc Natl Acad Sci USA. 1990 January; 87(1): 162-166). Thus, in this example, incomplete Fc-fragment does not contain a CH3 domain, which is present in the Fc-fragment of IgG3. Incomplete Fc-fragments consist of two monomers incomplete Fc-fragment. As further defined in N.�standing application when connecting two or more such monomers incomplete Fc-fragment of the resulting incomplete Fc-fragment has Fcγ receptor binding activity.

Fc-domain

[0099] as Used in this application "Fc domain" describes the minimum area (in the context of a larger polypeptide) or least minimized protein structure (in the context of an isolated protein) that may contact or be connected Fcγ receptor. As in the Fc-fragment and incomplete Fc-fragment Fc-domain is the minimal binding region that provides binding molecules with Fcγ-receptor. Although the Fc-domain can be restricted to a single polypeptide, which can bind Fcγ-receptor, it should be understood that the Fc-domain can be part or all of the Fc fragment, as well as part of a whole or partial Fc-fragment. When the term "Fc domain" as used in this invention, a qualified specialist will understand this term more than one Fc-domain. Fc domain comprises two monomers of the Fc-domain. As further defined in this application, the Fc-domain, formed by connecting two or more such monomers Fc domain, has Fcγ receptor binding activity. Thus, the Fc-domain is a dimeric structure, which is functionally able to bind Fcγ-receptor.

Incomplete Fc-domain

[00100] the Use�creating in the present application is "incomplete Fc-domain" describes the portion of the Fc-domain. Partial Fc domains include the individual domains of the constant region of the heavy chain (e.g., CHl, CH2, CH3 and CH4 domains), and hinge region of immunoglobulins of different classes and subclasses. Thus, partial Fc domains of the present invention include the CH1 domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, the CH2 domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, the CH3 domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE CH4 domains of IgM and IgE and a hinge region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE. Incomplete Fc-domain of the present invention may further include combinations of more than one of the specified domains and hinge regions. However, some partial Fc domains of the present invention and their combinations are not able to bind FcγR. Thus, partial Fc domains and combinations thereof comprise less than one Fc-domain. Partial Fc domains can be connected with the formation of a peptide which has an Fcγ receptor-binding activity, forming, thus, the Fc-domain. In the present invention partial Fc domains are used with the Fc-domains as elementary stages to create the biomimetics of the present invention defined in the present application. Each partial Fc domain comprises two monomers incomplete Fc-domain. When two such monomer incomplete Fc-domain are connected, is formed incomplete Fc-domain.

[00101] As described above, each of the Fc-fragments, not�anyh Fc-fragments, Fc domains and partial Fc domains are dimeric proteins or domains. Thus, each of these molecules consists of two monomers, which when connected form a dimeric protein or domain. Although the properties and activity of dimeric forms have been described above, the Monomeric peptides described below.

The monomer Fc-fragment

[00102] as Used in this application monomer Fc-fragment is a single chain protein, which, in conjunction with another monomer Fc-fragment is the Fc-fragment. The monomer Fc-fragment, therefore, is the C-terminal part of one of heavy chain antibodies that comprise Fc-fragment holo-antibody (e.g., the adjacent part of the heavy chain, which includes the hinge region, CH2 domain and CH3 domain of IgG) (see Fig.1A and Fig.1C)). In one embodiment of the monomer Fc-fragment comprises at least one chain hinge region (monomer hinge region), a single chain CH2 domain (monomer CH2 domain) and one chain CH3 region (monomer CH3 domain), connected in series with the formation of the peptide. In another embodiment of the monomer Fc-fragment comprises at least one chain hinge region, a single chain CH2 domain, a single chain CH3 domain and a single chain CH4 domain (monomer CH4 domain) connected in series with the formation of a peptide.

The monomer Fc-domain

[00103] as Used in us�Mr sage request monomer Fc-domain" describes single-chain protein, which, in combination with another monomer Fc-domain that includes the Fc-domain that can bind to Fcγ-receptor. When connecting two monomers of the Fc-domain is formed one Fc-domain. The monomer Fc-domain by itself, including only one side of the Fc-domain can bind Fcγ-receptor.

The monomer incomplete Fc-domain

[00104] Used in the present application is "incomplete monomer Fc-domain" describes single-chain protein, which, in conjunction with other incomplete monomer Fc-domain is incomplete Fc-domain. The amino acid sequence of the hinge region partial Fc domain monomers CH2 and CH3 for IgG1, IgG2, IgG3 and IgG4 are shown in Fig.25. The incomplete connection of two monomers of the Fc-domain leads to the formation of one incomplete Fc-domain.

Strathmere

[00105] In specific embodiments, the biomimetics of the present invention include strathmere. Strathmere represent a biomimetic compounds, capable of binding two or more Fcγ-receptors (see, e.g., Fig.13B). In a preferred embodiment of the implementation of strathmere of the present invention are used to bind Fcγ receptors on effector cells such as NK cells, immature dendritic cells and other cells derived from monocytes. In one embodiment of the Fcγ-receptor is an Fcγ-receptors low affinity. Stradone� can have four different physical conformation: serial, cluster, cor - or Fc-fragment, each of which is described in the following paragraphs. As will be obvious, Fc-fragments, incomplete Fc-fragments, Fc domains and partial Fc domains described above are used in the design of various conformations of strathmere. In addition, individual monomers Fc domain monomers and partial Fc domain, as described above, after receiving coassociation, forming dimeric structures, which are strathmere of the present invention.

Serial streamer

[00106] "Serial streamer" is a dimeric polypeptide consisting of two linear monomers of strathmere that when the compound forms two or more Fc domain. Fc-domains of strathmere are functional only in the case when the two peptide chains (monomers of strathmere) are associated (that is non-functional in a Monomeric state). Thus, consistent streamer is a biomimetic compound capable of binding two or more Fcγ receptors. In various embodiments, the serial streamer may include two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more Fc domains and partial Fc domains. Fc domains and partial Fc domains in sequential strathmere can be related�Ana domain links as defined below.

[00107] as Used in this application "dimer of strathmere" is a particular form of strathmere consisting only of two strathmere. In one embodiment, the implementation of the dimers of strathmere are molecules formed as a result of samegreloshi of the respective monomers of strathmere. In another embodiment of the monomers of strathmere in dimers of strathmere physically linked through inter-monomer strathmere communication, as defined in the present description. "The multimeric streamer" consists of three or more strathmere formed as a result of samegreloshi monomers of strathmere or the formation of inter-monomer strathmere ties, as defined in the present description.

Monomer strathmere

[00108] as Used in this application, the term "monomer strathmere" refers to single, whole peptide molecule, which when connected at least with the second monomer strathmere forms a polypeptide comprising at least two Fc domain (see, e.g., Fig.6A-6B, Fig.12A). Although in preferred embodiments, the serial strathmere consist of two monomers of the United strathmere (see, e.g., Fig.5A, 5B, 7A, 7B, 1C, 7D), consistent streamer may also contain three (see Fig.11C) or more of the monomers of strathmere. The monomers of strathmere can �be connected with the formation of strathmere through the formation of inter-monomer strathmere relations, or they can form strathmere through samegreloshi.

[00109] the Monomer strathmere can have such an amino acid sequence that, when connecting with another monomer strathmere formed one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more Fc domains, and is formed streamer. Monomer strathmere may also have amino acid sequence that, when connecting with another monomer strathmere formed one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more incomplete Fc-domain, and is formed streamer.

[00110] the Field of the monomers of strathmere that form the Fc domains and partial Fc domains in the context of strathmere can be located just at the end to N-end in the form of successive regions of a molecule of monomer strathmere (see, e.g., Fig.4A-4B). In the alternative, a sequential region of the monomers of strathmere can be linked through the peptide sequence, which in this application is called the "domain" link. Mutual arrangement of the individual monomers Fc domain monomers and partial Fc domain comprising the monomer strathmere, is not critical. However, the specified location must allow the formation of two functions�functional Fc domains when connecting two monomers of strathmere.

[00111] In one embodiment, the implementation of strathmere of the present invention receive the monomers of strathmere that on the N-end peptide contain a monomer Fc-domain monomer or incomplete Fc-domain, such as one or two end of the monomer CH2 domain of IgE monomer or partial hinge region of IgG3, which are strongly associated with themselves, forming the Fc-domain or incomplete Fc-domain, respectively. Each of these monomers of strathmere is a necessary addition to the monomer Fc-domain and/or monomers incomplete Fc-domain, so that after the formation of streamer could link two Fc-gamma receptor. Strathmere, which are formed by a combination of such monomers of strathmere represent biomimetics, capable of binding two or more Fc-gamma receptors. In a preferred embodiment of the N-terminal Fc-domain or incomplete Fc-domain containing additional glycosylation site, for example, such as is contained in the CH2 domain of IgE.

[00112] as an explanatory example, a qualified specialist will understand that the molecules of strathmere of the present invention can be constructed by obtaining a polynucleotide molecule that encodes various combinations of monomers Fc domain monomers and incomplete Fc-domain, but the combination should be such that gives at least two monomer Fc-domain. Such p�dinucleotide molecule may be embedded in the expression vector, which can be used to transform the bacteria population. Then the monomers of strathmere can be obtained by growing the transformed bacteria under appropriate conditions. Then the monomers of strathmere can form functional strathmere either through samegreloshi monomers of strathmere, either by joining monomers of strathmere using inter-monomer strathmere ties. The present invention covers both strathmere formed by linking monomers of strathmere having the identical amino acid sequence of monomers of strathmere having essentially the same amino acid sequence, or monomers of strathmere having different sequences. In the latter embodiment of the amino acid sequence of monomers of strathmere comprising streamer shall bear only such similarity, to form two or more functional Fcγ-receptornegative site.

[00113] As described above, the Fc-domain can functionally defined by their ability to bind Fcγ-receptor. As a result of specific amino acid sequence of the Fc-domain change-based incomplete Fc-domains, which constitute the Fc-domain. However, in one embodiment of the present invention, the Fc domain comprises a hinge�th region and CH2 domain of the immunoglobulin molecule. In another embodiment, the implementation of the Fc domain comprises a hinge region, CH2 domain and CH3 domain of the immunoglobulin molecule. In another embodiment, the implementation of the Fc domain comprises a hinge region, CH2 domain, CH3 domain and CH4 domain of an immunoglobulin molecule. In yet another embodiment of the Fc domain comprises a hinge region, a CH2 domain and a CH4 domain of an immunoglobulin molecule.

Domain relationship

[00114] As mentioned above, "domain relationship" is a peptide bond between the monomers Fc-domain and/or monomers incomplete Fc-domain that make up each of the individual monomers of strathmere serial strathmere or stratatel of the present invention. Domain relationship may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Domain relationship is not present between the monomers incomplete Fc-domain that are in their natural sequence. Thus, when used parts of monomers Fc-domain associated in the natural order, for example, a hinge region, CH2 domain and CH3 domain of IgG, these monomers incomplete Fc-domain include contiguous sequence, so the domain relationship between these elements is not needed. In contrast, for example, when two or more monomers of the Fc-domain or monomers incomplete Fc-domain are connected in this order, in which usually are not, and form in d�altace separate monomer strathmere, can be used in the domain of communication. An example might be the relationship between the two peptide of the hinge region, CH2/CH3 a more isolated monomer strathmere constituting streamer, which includes: a hinge region, CH2/CH3/L/hinge region, CH2/CH3, where "L" denotes a domain relationship (see, eg., Fig.4A, where the domain relationship (not shown) is formed between the CH3 domain of IgG1 and hinge region of IgG1). In various described cases domain relationship can be one of the natural parts of the heavy chain, which is attached to a hinge region of the CH domains in the monomer Fc-domain of an antibody. In the alternative, domain relationship may be any other amino acid sequence that provides the necessary spacing and flexibility between the monomers Fc domain monomers and incomplete Fc-domain of the individual monomer strathmere, and which enables the individual monomers of strathmere to connect with each other, forming strathmere of the present invention.

[00115] a Qualified technician will be able to understand that the identity of the domain context is not particularly important, provided that it allows two or more individual monomers of strathmere to form the biomimetic compounds of the present invention, and that the resulting compounds have the ability transversely to associate more than one FcγR. It is assumed that each �immunologicheski active biomimetic compound preferably contains at least one domain relationship in each monomer strathmere serial strathmere or Stradale, which operates so that supports Fc-domains of immunologically active biomimetics within a limited spatial area, which increases the activity when activating FcγR, e.g., through aggregation Fcγ receptors by binding to the Fc-domains in the immunological active biomimetic. Preferably, domain relationships provide the same or higher degree of conformational variability, which provides a hinge region of IgG molecules. All of the above relation is known in the prior art.

Inter-monomer stregoneria link

[00116] a single bond, present in the biomimetic compounds of the present invention is the inter-monomer stregoneria communication", which is located between two or more distinct monomers of strathmere that make up strathmere and stratatel of the present invention. Whereas the domain of communication are short amino acid sequences that serve to link together monomers Fc domain monomers and incomplete Fc-domain, which are individual monomers of strathmere biomimetic connections, inter-monomer strathmere links are used to connect two or more distinct monomers of strathmere that are biomimetic compounds. Inter-monomer strathmann�I connection may be any connection, able to stable connection of the individual monomers of strathmere. In some embodiments, the implementation of the inter-monomer stregoneria bond may be a covalent bond between the monomers of strathmere. In the alternative, inter-monomer stregoneria the relationship between monomers of strathmere can be a direct chemical crosslinking. In preferred embodiments, the structures of the monomers of strathmere have the advantage of allowing natural samegreloshi monomer Fc-domain that allows you to create samearearule strathmere. In such embodiments, between the individual monomers of strathmere disulfide bonds are formed, resulting in the formation of strathmere (see, e.g., Fig.5A, where the inter-monomer strathmere connection (not shown) are used to connect two separate monomers of strathmere comprising streamer). Disulfide bonds formed between cysteine residues of the monomer Fc-domain that are biomimetic molecule using cysteine residue present in the natural sequence of the monomer Fc-domain or the cysteine residue introduced into the monomer Fc-domain by using site-directed mutagenesis. Such natural properties of samegreloshi can also be used for the purpose of forming inter-monomer strathmere connection between individual monomer�mi strathmere in multimer of strathmere. Alternative implementation options include inter-monomer strathmere communication, where disulfide bonds are formed between cysteine residues introduced by site-directed mutagenesis in amino acid sequence constituting a separate monomers of strathmere.

[00117] As described above, in a preferred embodiment of the inter-monomer stregoneria connection, which is formed streamer, is a bond formed as a result of samegreloshi monomers of strathmere. In one embodiment, the implementation of the two monomer strathmere that make up streamer, peptides are identical, that is, two separate monomer strathmere that make up streamer, have identical sequences. However, a qualified technician will be able to understand what other options for implementation include strathmere in which the monomers of strathmere differ from each other in amino acid sequences.

[00118] the Two monomer strathmere can form streamer, for example, there is a scene in parallel, thus, the connection occurs between identical monomers incomplete Fc-domain monomers of strathmere (see, e.g., Fig.5A-B). However, the present invention also includes variants of implementation, in which the connection is Mezhdunarodnye monomers incomplete Fc-domain, and implementation options (see Fig.11C), in which coupling occurs between identical monomers incomplete Fc-domain monomers of strathmere, but in which the alignment of two monomers of strathmere occurs with offset.

[00119] For the regulation of products and samovlastii monomer strathmere can be used "kiperousa region". For example, the sequence of monomer strathmere may include the following partial Fc domains: CH2 IgE/hinge region of IgG1/IgG1 CH2/CH3 IgG1/hinge region of IgG1/IgG1 CH2/CH4 of IgE (see Fig.13A), where the domains of IgE serve as a cap which prevents the binding effect". The effect of buckling may occur when the monomer strathmere (see Fig.11A) may autogenerators (see Fig.11B) or may Orient itself not in the form of autotimer, and in the form of parallel alternating monomers (see Fig.11C). The average person skilled in the art is aware that in order to call automerization of strathmere and prevent the binding effect, if required, can be used in various partial Fc domains, such as the hinge region of any immunoglobulin or CH4 domain of IgM or IgE, separately or in combination. Other inconsistent patterns may contain molecules with branched chain (see Fig.12B), two or more strathmere arranged in parallel and connected by linkers, such as through a simple covalent bond, peptide linkers or the non-peptide linkers (see Fig.14A and 14B).

Cor-streamer

[00120] the Cor-streamer" consists of cor-group, with which are connected two or more cor-strathmere units, where each cor-stregoneria unit includes at least one Fc domain, forming, thus, biomimetic compound capable of binding two or more Fcγ receptors. Fc-fragment, incomplete Fc-fragment, consistent streamer or cluster stregoneria unit can independently serve as one or both (if they include two Fc-domain) cor-strathmere units in cor-strathmere, since each of these molecules contains at least one Fc domain. Thus, cor-streamer may include cor-the group with which is associated at least one serial streamer.

[00121] as Used in this application cor-group cor-strathmere is any physical structure, which can be connected or covalently bound cor-strathmere units. Preferred polypeptides that can serve as a cor group, include keyhole lymph snails, bovine serum albumin and ovalbumin. Chemical cross-linking between such cor-groups and cor-strdomainname units (for example, Fc-fragment, incomplete Fc-fragment Fc-domain, consistent strathmere and�esternay strathmere unit) can be implemented by a variety of chemical reagents using known methods. Examples of chemicals that were routinely used in cross-stitching, include glutaraldehyde, carbodiimide, esters succinimides (e.g. MBS, SMCC), benzidine, periodate, isothiocyanate; PEO (polyethylene oxide)/PEG (polyethylene glycol) spacers, such as bis(NHS)PEO5, DFDNB (1,5-debtor-2,4-dinitrobenzene); and aminonicotinate homobifunctional cross-linking reagents, including aldehyde-activated dextran, bis(sulfosuccinimidyl)suberate, bis[2-(succinimidylester-oxy)ethyl]sulfone, dimethylpiperidin·2 HCl, dimethylpyrimidin·2 HCl, dimethylsuberimidate·2 HCl, disuccinimidyl glutarate, dithiobis(Succinimidyl)propionate, disuccinimidyl, disuccinimidyl, dimethyl-3,3'-databasprojekt·2 HCl, 3,3'-dithiobis(sulfosuccinimidyl), ethylene glycol bis[Succinimidyl], ethylene glycol bis[sulfosuccinimidyl-succinate], β-[Tris(gidroximetil)phosphino]propionic acid and Tris-succinimidylester. The person skilled in the art will be able to choose the appropriate cross-linking reagent and conditions based on specific selected cor-group and sequence Fc-domain-containing polypeptides, connected with the formation of immunologically active biomimetics. See, for example, Wong, Shan S. Chemistry of protein conjugation and cross-linking. Boca Raton: CRC Press, c1991 (ISBN 0849358868).

[00122] In another predpochtitel�nom embodiment, the implementation as cor-group can be used the connective polypeptide (J) chain. When the cor-group use the J-chain cysteine bridges can be used to connect the individual cor-strathmere units with the formation of cor-strathmere (Cm. Fig.10A-10D). In one embodiment, the implementation of the cor-streamer, serial strathmere (serving as cor-strathmere units) containing the end of the CH4 domain of IgM, are connected by a J-chain with the formation of cor-strathmere. The inclusion of the CH4 domain of IgM leads to samegreloshi of strathmere, this includes thecomplete Fc-domain with the J-chain, with the formation of biomimetic, is able to associate multiple Fc-gamma receptors. Another example of cor-strathmere is cor-streamer comprising Fc domains (serving as cor-strathmere units), where Fc domains have the following structure: hinge region of IgG3/IgG3 CH2/CH3 IgG3/IgM CH4. Fc-domains constituting the molecule, cannot individually to associate more than one Fc-gamma receptor, and the complete structure can bind five Fc-gamma receptors, when the components of the Fc-domains are connected by a J-chain.

[00123] In another embodiment, the implementation of CDF-group can be polipeptide molecule. With cor-strdomainname units can be physically connected by various suitable compositions, ensuring production of immunologically active biomimetics. Can be used with non-toxic pellets, hyperazotemia floor�measures and dendrimers, nanoparticles, as well as various compounds, which are classified by the FDA as completely safe (for example, propylene glycol, sorbitol, liposomes and calcium silicate). See, for example, Nanoparticulates as Drug Carriers (Vladimir P. Torchilin (Editor)), Imperial College Press (Sept. 2006) ISBN: 1860946305/ISBN-13: 9781860946301.

[00124] the Preferred cor-group of the present invention include granules, albumin, liposome, peptide and polyethylene glycol.

Cluster streamer

[00125] "Cluster streamer" is biomimetic that has osmanagaoglu shape with a Central team, "head" and two or more "legs", each leg includes one or more Fc domains, which can bind at least one Fc-gamma receptor, forming, thus, biomimetic capable of binding two or more Fc-gamma receptor. Each of the cluster streamer consists of more than one dimeric protein, which is called "cluster strathmere unit". Each cluster stregoneria unit consists of the area that multimeasures, and region "legs", which includes at least one functional Fc domain. Multimeedia region forms the "head" of the cluster strathmere if multimerization with multimeasure area of another cluster strathmere units. The area of the leg is capable of communicating so much Fcγ-receptors, how many Fc-home�new contains in each area of the leg. Thus, cluster streamer is a biomimetic compound capable of binding two or more Fcγ-receptors.

[00126] Multimeedia region may be a peptide sequence that induces further multimerization dimeric proteins, or, in the alternative, multimeedia region may be glycosylation, increases multimerization dimeric proteins. Examples of peptide multiperiodic areas include the hinge region of IgG2, CH2 domain of IgE, isoleucinol the zipper and the zinc fingers. The influence of glycosylation on multimerization peptides are well described in the prior art (e.g., Role of Carbohydrate in Multimeric Structure of Factor VIII/V on Willebrand Factor Protein. Harvey R. Gralnick, Sybil B. Williams and Margaret E. Rick. Proceedings of the National Academy of Sciences of the United States of America, Vol. 80, No. 9, [Part 1: Biological Sciences] (May 1, 1983), pp. 2771-2774; Multimerization and collagen binding of vitronectin is modulated by its glycosylation. Kimie Asanuma, Fumio Arisaka and Haruko Ogawa. International Congress Series Volume 1223, December 2001, Pages 97-101).

[00127] an Experienced specialist is aware that the cluster stregoneria unit itself may include a serial streamer (containing two or more Fc domains) along with multimeasure area. Thus, the "legs" of the cluster strathmere may consist of consecutive strathmere any of the types described in the present application, and/or one or more Fc fragments of IgG1, �/or Fc-fragments IgG3, and/or an Fc-domain. An experienced specialist is aware that each of the Fc fragments of IgG1 and each Fc-fragment of IgG3 in such biomimetics can be modified and will include incomplete Fc-fragments from any immunoglobulin. The monomers that make up the cluster strathmere unit (which, as stated above, exists as a dimeric complex of two peptides), are monomers cluster strathmere chart. An example of the constructed cluster strathmere, cluster stregoneria unit which connects not more than one Fc-gamma receptor low affinity before multimerization is CH2 IgE/hinge region of IgG1/IgG1 CH2/CH3 IgG1.

[00128] an Experienced specialist is aware that when the serial streamer used as "legs" cluster strathmere, each "leg" is able to bind more than one Fc-gamma receptor (as in sequential strathmere present at least two Fc-domain), then the result is biomimetic capable of binding more than one Fc-gamma receptor. Partial Fc domains, other sequences of immunoglobulins and nimmanahaeminda sequences can be placed on the ends of the individual monomers cluster strathmere units that made up the legs, to obtain cluster strathmere in which each leg p�Solorina at the required distance, which increases their accessibility upon binding with one or more than one Fc-gamma receptor.

[00129] Multimeedia region may be a peptide sequence that causes dimerization or multimerization peptides and includes the hinge region of IgG2, CH2 domain of IgE, isoleucinol zipper and zinc finger. As known in the art, the hinge region of human IgG2 can form covalently linked dimers (Yoo, E. M. et al. J. Immunol. 170, 3134-3138 (2003); Salfeld, Nature Biotech. 25, 1369-1372 (2007)). The formation of dimers IgG2 potentially mediated by the structure of the hinge region of IgG2 in the formation of C-C bond (Yoo et al, 2003), suggesting that the structure of only one hinge region may cause the formation of dimers. Thus, consistent strathmere containing the hinge region of IgG2 (and, thus, serve as cluster strathmere units), will form a cluster streamer, which may include two serial strathmere or even three consecutive strathmere.

[00130] the Amino acid sequence of monomer a hinge region of human IgG2 is as follows: ERKCCVECPPCP (SEQ ID NO:36). Key structure of the hinge region is part of the C-X-X-C monomer hinge region. Thus, the monomers of strathmere of the present invention can include a full posledovatelnostei hinge region of IgG2 12 amino acids, or four main amino acids, along with the monomer Fc-domain. Although X-X in the key structure may denote any amino acid, in a preferred embodiment of the sequence X-X is a V-E or P-P. For qualified specialist it is obvious that the monomer of the hinge region of IgG2 may consist of any part of the sequence of the hinge region in addition to the basic structure of the four amino acids, including the full sequence of the hinge region of IgG2, as well as part or complete the sequence of monomers CH2 and CH3 domain of IgG2. Specific examples of possible structures consistent strathmere based on the hinge region of IgG2-Fc-domain of IgG1 are as follows:

Table 1
N-endHCH2CH3HCH2CH3HCH2CH3C-end
SHHS111��
SHHS111111
222111
222111111
2111
2111111
222111
222111111
22211111 1Sharn.IgE
2111
Notation: H=hinge region, CH2=constant heavy domain 2, CH3=constant heavy domain 3, 1=IgG1, 2=IgG2, X=any amino acid; 2x=two pivot region in sequential order

[00131] these are just a few of the many examples. Any of the Fc domains of IgG1 can be replaced, for example, the Fc-domain of IgG3. Additional proteins with dimerization domains include IgG2 chimeric proteins IgG2-IgG1 with an additional N - and/or C-terminal sequences, including sequences of monomer domains of IgM or IgE. These N - and C-terminal sequences can be articulated by the regions, constant regions, or both.

[00132] As described above, lacinova and isoleucine lightning can also be used as multimeasure area. Lacinova and isoleucine zipper (coiled domains) are known to contribute to the formation of protein dimers, trimers and tetramers (Harbury et al.Science 262:1401-1407 (1993); O'shea et al. Science 243:538 (1989)). Cluster strathmere with the advantage of natural tendencies isoleucinol zipper to form a trimer, can be obtained using successive strathmere, including isoleucinol lightning. Association of three or more consecutive strathmere (as clustered strathmere units) containing isoleucine lightning, leads to the formation of cluster strathmere with at least six areas, connecting the Fc-gamma receptors.

[00133] Although for qualified specialist it is obvious that can be used by different types latinovich and isolating lightning, in the preferred embodiment, the implementation uses isolationa lightning from the modified regulator of GCN4 transcription (described in the article by Morris et al., MoI. Immunol. 44:3112-3121 (2007); Harbury et al. Science 262:1401-1407 (1993)): YTQKSLSLSPGKELLGGGSIKQIEDKIEEILSKIYHIENEIARIKKLIGE RGHGGGSNSQVSHRYPRFQSIKVQFTEYKKEKGFILTS (SEQ ID NO:37). The sequence isoleucinol lightning is only one of several possible sequences that can be used to multimerization monomer Fc-domain. Although it can be used the entire sequence shown in SEQ ID NO:37, the underlined portion of the sequence represents a key sequence isoleucinol zipper that can be used in a clustered Stra�the baths of the present invention. Thus, the monomers of strathmere of the present invention may include either the full sequence of 88 amino acids isoleucinol zipper (ILZ), or 28 key amino acids, along with one or more monomers Fc-domain. A qualified specialist is also clear that isolationa lightning may consist of any part of the zipper in addition to the basic structure of 28 amino acids and, thus, may consist of more than 28 amino acids, but less than 88 amino acids isoleucinol lightning. Specific examples of possible structures based on the ILZ-Fc-domain of IgG1 below.

Table 2
HCH2CH3HCH2CH3
ILZ111
ILZ11111
ILZ111111
ILZ111333
Notation: H=hinge region, CH2=constant heavy domain 2, CH3=constant heavy domain 3, 1=IgG1, 3=IgG3, ILZ=domain isoleucinol lightning

[00134] these are just a few of the many examples. Any of the domains of IgG1 can be replaced, for example, domains IgG3. Additional proteins with ILZ domains include chimeric IgG1 proteins with additional N - and/or C-terminal sequences from other Ig molecules such as IgM or IgE. These N - and C-terminal sequences can be articulated by the regions, constant regions, or both.

Fc-fragment-streamer

[00135] "Fc-fragment-streamer" consists of more than one Fc-fragment. In some circumstances related to posttranslational modification of Fc-fragment Fc-fragment with sufficient force associated with a different Fc-fragment, providing the formation of molecules that binds more than one Fcγ-recepto�ohms. Post-translational modification, which provides such linkage includes glycosylation and methylation. The identity of cell lines, in which the recombinant Fc-fragments, as well as the conditions under which they are obtained, determine whether the Fc-fragments of form Fc-fragment-strathmere. For example, recombinant Fc-fragment obtained in transient transfected CHO cells FreestyleMax forms of multimer that visually recorded on a Western blot, is associated according to a bivalent standard in the analysis of binding method plasmon resonance, and demonstrates biological activity in the analysis on dendritic cells, comparable with the activity of IVIG. On the contrary, the same recombinant Fc-fragment, obtained in a stable CHO cell line does not form multimeric Fc-fragment, detected on a Western blot, is associated according to a monovalent standard in the analysis of binding method plasmon resonance, and does not show comparable biological activity. Thus, the Fc-fragment-streamer is biomimetic compound capable of binding two or more Fcγ-receptors.

[00136] it is Also used in the present application, the term "Fc-dimer" denotes a dimer Fc-fragments (see Fig.2A), the term "Fc-trimer" refers to a trimer Fc-fragments and the term "Fc-multimer" oboznachaet� of multimer Fc-fragments (see Fig.2B).

Stratatel

[00137] the Present invention also encompasses Stradale. Used in this application "stratatel" refers to a molecule comprising two or more Fc domains, preferably in the context of strathmere (including serial strathmere, cor-strathmere, cluster strathmere and Fc-fragment-strathmere) to which is attached one or more Fab domains (see, e.g., Fig.8A-B and 9 A-B). Thus, by the presence of the Fab domains stratatel possess antigen-binding ability, and strathmere Fcγ receptor-binding activity. In some embodiments, the implementation of the Fcγ receptor-binding activity may be due to the ability to bind and cross-link in FcγR is equal to or greater extent compared to the Fc-part of the native structure holo-antibody. Preferably Fab-part Stradale and includes heavy and light chains. Variable heavy chain and light chain can independently be from any compatible immunoglobulin, e.g., IgA1, IgA2, IgM, IgD, IgE, IgG1, IgG2, IgG3 or IgG4, and may refer to the same or a different Ig isotype, but preferably they belong to the same Ig isotype. Light chains Kappa or lambda can also occur from various Ig isotypes. Stratatel as strathmere, can link two or more Fc?-receptors and modulate th�structural function.

[00138] In one embodiment, the implementation of strathmere may contain Fab immunoglobulin associated with Fc hinge (H) domain of strathmere forming stratatel (e.g., Fig.8A and B). In another embodiment, the implementation of stratatel may consist of IgG1 Fc - (hinge region - CH2) IgG1 (e.g., Fig.9A). In other embodiments, stratatel may consist of a domain and hinge region of IgG1, domain and hinge region of IgG3 and domain and hinge region IgGE (e.g., Fig.9B). Fab and includes heavy and light chain present in the native structure of immunoglobulins (Fig.3A-B).

[00139] Stratatel have antigen-binding properties of the Fab part and the above-described properties of strathmere. This combination is used for binding, cross-linking and activating Fcγ receptors on effector cells with higher speed than can be achieved Fc-basis holo-antibodies, especially in an environment with low expression of epitopes (for example, 90% of patients with breast cancer whose tumors are not classified as expressing her/2-neu high) induces ADCC in patients with a higher percentage. As indicated above, one or more antigen-binding Fab fragments can be introduced into strathmere with the formation stratatel. Preferably, the polypeptides (except for relationships described in the present application), entered� in strathmere, are not all nimmanahaeminda polypeptides or parts thereof.

[00140] Fab may be a chimeric structure consisting of constant regions that are related to human and non-human variable regions, such as the variable region derived from mouse antibody, rat, rabbit, monkey, or goat. The average person skilled in the art can obtain various Fab-chimeric structures with the aim of incorporating them in stratatel using techniques currently available and described in the scientific literature in relation to such structures. Thus, the "humanized" Stradale can be similarly created "humanized" monoclonal antibodies.

Variants and homologues

[00141] a Qualified specialist it is obvious that can be developed strathmere and other biomimetics of the present invention, which includes a specific Fc domains of immunoglobulins, such as the two Fc domain of IgG1 (the hinge region of IgG1/IgG1 CH2/CH3 IgG1/hinge region of IgG1/IgG1 CH2/CH3 IgG1). This streamer can be constructed by obtaining at the beginning of the polynucleotide encoding two Monomeric Fc domain of IgG1 (i.e., monomer a hinge region of IgG1/monomer IgG1 CH2/monomer CH3 IgG1/monomer hinge region of IgG1/monomer IgG1 CH2/monomer CH3 IgG1), and expression of a monomer of strathmere. �ATEM when associating two or more such monomers of strathmere formed consistent streamer, including two Fc domain of IgG1.

[00142] Strathmere and other biomimetics of the present invention can also be created based on the identity of certain incomplete Fc-domain of an immunoglobulin that comprise Fc domains. For example, can be obtained consistent streamer containing two Fc domain, wherein the first Fc domain comprises a hinge region of IgG1/IgG3 CH2/IgG1 CH3, and the second Fc domain comprises a hinge region of IgG3/IgG1 CH2/CH3 IgG3.

[00143] it Should be understood that strathmere and other biomimetic molecules disclosed in the present application, can be derived from any organisms of different species. Actually Fc domains or partial Fc domains in any biomimetic molecules of the present invention can be derived from immunoglobulin occurring more than one (e.g. two, three, four, five or more) species. However, usually they come from the same species. In addition, it should be understood that any of the methods disclosed in the present application (e.g., treatments), can be applied to any kind. Basically, all components of biomimetics applied to the target species, originate from the same species. However, it can also be applied biomimetics, where all the components come from different species or from more than one species (including or not including the species to which the subject method is used).

p> [00144] in Certain CH1, CH2, CH3 and CH4 domains and a hinge region, which include Fc domains and partial Fc domains of strathmere and other biomimetics of the present invention, can be independently selected on the basis of a subclass of immunoglobulins, and on the basis of the organism from which they are derived. Thus, strathmere and other biomimetics disclosed in the present application may include Fc domains and partial Fc domains, which occur independently of immunoglobulins of various types, for example, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. Similarly, each Fc-domain and incomplete Fc-domain can be derived from different species, preferably mammals, including all primates except humans (e.g., monkeys, baboons and chimpanzees), humans, mice, rats, cattle, horses, felines, dog, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, Guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys and ducks), fish and reptiles used to obtain species-specific or chimeric molecules of strathmere.

[00145] a Separate Fc domains and partial Fc domains can also be humanized. The person skilled in the art it is obvious that different Fc domains and partial Fc domains provide functional properties of various types. For example, Fcγ-specific receptors bind to immunoglobulins IgG and no other�their classes of immunoglobulins. Thus, the person skilled in the art, intending to create streamer that has the ability to bind multiple Fcγ-receptors, will create the Fc-region of strathmere that include at least a well-described sequence of IgG, Fcγ binding receptor, including the corresponding sequence of the hinge region of IgG and CH2 and CH3 domains of IgG. The person skilled in the art it is also obvious that with the use of specific Ig domains can be linked to various unwanted effects, for example, anaphylaxis associated with injections IgA. Biomimetics disclosed in the present application, in General, need to be developed to avoid such effects, although in certain circumstances, such effects may be desirable.

[00146] the Present invention also covers strathmere comprising Fc domains and partial Fc domains, which contain amino acids that differ from the natural amino acid sequence of the Fc-domain or incomplete Fc-domain. Preferred Fc-domains included in the biomimetic compounds of the present invention, have apparently detected specific binding affinity or in relation to the holo-Fcγ-receptor, or a soluble extracellular domain part of FcγR. The primary amino acid sequence and are generated by roentgenogram�holographie patterns of different Fc domains and Fc monomers domain known in the prior art. See, for example, Woof JM, Burton DR. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol. 2004 Feb;4(2):89-99. Typical Fc domains with Fcγ-receptornegative ability include Fc domains of the human IgG isotypes 1-4 (hIgG1-4) (SEQ ID NOS: 1, 3, 5 and 7 respectively; see also Fig.15A-D). (Cm. Fig.2 the article Robert L. Shields, et al. High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR. J. Biol. Chem., Feb 2001; 276:6591-6604). These native sequences have been subjected to extensive structural and functional analysis, including the mapping of site-directed mutagenesis of the functional sequences 14. On the basis of these previous structural and functional studies and available data of crystallography specialist in this field can create a functional sequence variants Fc-domain (e.g., SEQ ID NOS: 1, 3, 5 and 7) with preservation of Fcγ-receptornegative ability of the Fc domain.

[00147] Amino acid substitutions can be detected throughout the sequence by the Fc-domain or localized to a particular incomplete Fc-domains, which constitute the Fc-domain. Functional variants of the Fc domain used in trademark and other biomimetics of the present invention have at least approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to native� Fc-domain. Similarly, functional variants incomplete Fc-domains used in trademark and other biomimetics of the present invention have at least approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a native Fc incomplete-domain.

[00148] a Qualified specialist it is obvious that the present invention also encompasses the use of functional variants of monomers Fc-domain in the construction of monomers Fc fragment monomers incomplete Fc fragment monomers of strathmere and other monomers of the present invention. Functional versions of the monomer Fc-domain to have at least approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the native sequence of the monomer Fc-domain.

[00149] Similarly, the present invention also encompasses the use of functional variants of monomers incomplete Fc-domain in the construction of monomers Fc fragment monomers incomplete Fc fragment monomers of the Fc domains of the monomers of strathmere and other monomers of the present invention. Functional versions of the monomers incomplete Fc-domain to have at least approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the native sequence of the monomer incomplete Fc-domain.

[00150] Amino acid substitutions can decrease, increase or not change the binding affinity of strathmere with Fcγ-receptor. Preferably, such amino acid substitutions are conservative substitutions of amino acids, however, such changes include deletions, additions and other replacement. The conservative replacement of amino acids typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

[00151] the Term "functional variant" as used in this application, refers to sequences that are related by homology with the original sequence, which is able to mediate the same biological effects as the original sequence (in the case of a polypeptide), or which encodes a polypeptide that is able to mediate the same biological effects as the polypeptide encoded by the original sequence (in the case of the polynucleotide). For example, a functional variant of any of the biomimetics described in the present application, has given homology or identity and is able to immune modulation of dendritic cells. Functional sequence variants include polynucleotides and polypeptides. Identity last�dovalidate usually evaluated using BLAST 2.0 (Basic Local Alignment Search Tool), working with default parameters: Filter - On, Scoring Matrix BLOSUM62, Word Size is 3, E value is 10, Gap Costs - 11.1 and Alignments - 50.

[00152] based On the foregoing, it should be understood that strathmere of the present invention include strathmere containing: (a) Fc only natural domains; (b) a mixture of natural Fc domains and Fc domains with altered amino acid sequences; and (c) Fc domains with altered amino acid sequences. It is only necessary that strathmere containing the modified amino acid sequence that possess at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% or even greater ability than strathmere comprising Fc domains with natural sequences, contacting two or more Fcγ receptors.

[00153] the Sequence above Fcγ-receptornegative sites present in strathmere and stratatel of the present invention, can be altered through genetic engineering in predicted order of receipt of binding sites with altered binding capacity and affinity compared to the native sequence. For example, certain residues may be changed, which reduces the binding of the Fc-domain of biomimetic compounds with FcγRII, increasing the binding to FcγRIIIa. An example of the deployed structure-function analysis on the basis of the mutagen�and Fcγ-receptornegative sequences hIgG can be found in the article by Robert L. Shields, et al. High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR. J. Biol. Chem., Feb 2001; 276: 6591-6604. Similar studies were performed on Fc mouse IgG (mIgG Fc). Based on the homology of the structure and primary sequence of a native Fc domain of IgG of different types of specialist in this field can transfer a wealth of information on structural and functional properties of the hIgG Fc and mIgG Fc in rational mutagenesis of all sequences of a native Fcγ-receptornegative sites in the biomimetic compounds of the present invention, creating binding sites with a given specificity and affinity of binding to Fcγ-receptor.

[00154] In addition to the composition of the amino acid sequence of the native fields of the Fc-domain of the carbohydrate in the Fc-domain, as it is known, has a great influence on the structure of the Fc domain and the interaction with FcγR. See, for example, Robert L. Shields, et al. Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human Fc RIII and Antibody-dependent Cellular Toxicity. J. Biol. Chem., JuI 2002; 277: 26733 - 26740 (doi:10.1074/jbc.M202069200); Ann Wright and Sherie L. Morrison. Effect of C2 - Associated Carbohydrate Structure on Ig Effector Function: Studies with Chimeric Mouse-Human IgG1 Antibodies in Glycosylation Mutants of Chinese Hamster Ovary Cells. J. Immunol, Apr 1998; 160: 3393-3402. The carb can be adjusted using, for example, specific protein expression systems, including the specific cell line or enzymatic modification ofin vitro. Still� way the present invention includes strathmere and stratatel comprising Fc domains with native carb holo-antibody, which were obtained from these domains, as well as biomimetic compounds in which changed the carb.

[00155] the Introduction of additions to the incomplete polypeptide chain Fc domain, multimerization region or changes in glycosylation may cause a conformational change in the Fc-domain, leading to enhanced binding of the Fc domain with a Fcγ receptor. Thus, seemingly minor modifications of the polypeptide may also result in strathmere able to associate multiple Fcγ-receptors.

Incomplete domains and incomplete fragments

[00156] a Qualified specialist, it is also evident that the Fc domains and partial Fc domains used in embodiments of the present invention, may not be the full-sized versions. Thus, the present invention encompasses the use of monomers Fc domain monomers and incomplete Fc-domain containing N-terminal, C-terminal amino acid or amino acids from the Central part of specific monomers Fc domain monomers and incomplete Fc-domain that constitute strathmere and other biomimetics of the present invention.

[00157] for Example, was described binding site Fcγ-receptors on immunol�Bulino of human IgG (e.g., Radaev, S., Sun, P., 2001. Recognition of Immunoglobulins by Fcγ Receptors. Molecular Immunology 38, 1073-1083; Shields, R. L. et. al., 2001. High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR. J. Biol. Chem. 276 (9), 6591-6604). Based on this information it is possible to remove amino acids from the Fc-domain of these antibodies and to determine the influence exerted on the interaction between the Fc domain and the receptor. Thus, the present invention encompasses Fc domains of IgG containing at least about 90% amino acids, including the provisions 233-338 the lower hinge region and CH2, as defined in article Radaev, S., Sun, P., 2001.

[00158] Partial Fc domains of immunoglobulins IgG according to the present invention includes all of the hinge region or a portion of, the entire CH2 domain or portion thereof, as well as the entire CH3 domain or a portion thereof.

[00159] Partial Fc domains of IgG containing only a portion of the hinge region, the portion of the CH2 domain or a portion of a CH3 domain, constructed from monomers incomplete Fc-domain. Thus, the present invention includes monomers of the hinge region of IgG, obtained from the N-terminal part of the hinge region or the C-terminal part of the hinge region. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62 (up to 15 for IgG1, up to 12 for IgG2, to 62 for IgG3, up to 12 for IgG4) amino�of islote hinge region.

[00160] the Present invention also includes monomers CH2 domain of IgG, obtained from the N-terminal portion of the CH2 domain or the C-terminal portion of the CH2 domain. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 (up to 110 for IgG1 and IgG3, to 109 for IgG2 and IgG4) amino acids of the CH2 domain.

[00161] the Present invention further includes monomers CH3 domain of IgG, obtained from the N-terminal part of the CH3 domain or the C-terminal part of the CH3 domain. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 or 107 (up to 106 for IgG1 and IgG3, and 107 for IgG2 and IgG4) amino acids CH3 domain.

[00162] Partial Fc domains of immunoglobulin IgA1, and IgA2 IgD of the present invention includes all of the hinge region or a portion of, the entire CH2 domain or portion thereof, as well as the entire CH3 domain or a part of it. In addition, as partial Fc domains can be used the entire CH1 domain of an immunoglobulin IgA1, IgA2, or IgD, or a portion thereof.

[00163] Incomplete domains IgA1, and IgA2 IgD containing roofing�on the part of the hinge region, part of the CH1 domain, the portion of the CH2 domain or a portion of a CH3 domain, constructed from monomers incomplete Fc-domain. Thus, the present invention includes monomers hinge region derived from the N-terminal part of the hinge region or the C-terminal part of the hinge region of IgA1, IgA2 or IgD. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 (up to 26 for IgA1, to 13 for IgA2, to 64 for IgD amino acids of the hinge region.

[00164] the Present invention includes monomers CH2 domain derived from the N-terminal portion of the CH2 domain or the C-terminal portion of the CH2 domain IgA1, IgA2 or IgD. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 or 107 (102) for IgA1, up to 96 for IgA2, to 107 for IgD) amino acids of the CH2 domain.

[00165] the Present invention includes CH3 domains derived from the N-terminal part of the CH3 domain or the C-terminal part of the CH3 domain IgA1, IgA2 or IgD. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 or 131 (to 113 for IgA1, to 131 for IgA2, to 110 for IgD) the amino acid CH3 domain.

[00166] Partial Fc domains of immunoglobulins IgM and IgE present invention includes all of the hinge region, CH2 domain or portion thereof, the entire CH3 domain or portion thereof, and the CH4 domain, or a portion, of these molecules. In addition, as partial Fc domains can be used the entire CH1 domain, or a portion, of the immunoglobulins IgM and IgE.

[00167] a Partial domain of IgM and IgE containing only a portion of the hinge region, CH2 domain, a part of the CH3 domain, or a portion of the CH4 domain constructed from monomers incomplete Fc-domain. Thus, the present invention includes monomers of the hinge region, CH2 domain derived from the N-terminal part of the hinge region, CH2 domain or the C-terminal part of the hinge region, CH2 domain of IgM or IgE. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 or 112 (to 112 for IgM, to 109 for IgE) amino acids of the hinge region, CH2 domain.

[00168] the Present invention includes� monomers CH3 domain of IgM and IgE, obtained from the N-terminal part of the CH3 domain or the C-terminal part of the CH3 domain of IgM or IgE. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106 (up to 106 for IgM, to 105 for IgE) amino acids CH3 domain.

[00169] the Present invention includes monomers CH4 domain of IgM and IgE derived from the N-terminal part of the CH4 domain or C-terminal part of the CH4 domain of IgM or IgE. They, therefore, may contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 (130 for IgM, to 105 for IgE) amino acids CH4 domain. However, the size of the parts of the CH4 domain of IgM or IgE, which include the C-end of the CH4 domain, preferably more than 18 amino acids and more preferably more than 30 amino acids, and most preferably greater than 50 amino acids.

[00170] From the above it is obvious that various embodiments of the present invention include strathmere containing: (a) full-size Fc-domains; (b) Kombi�ation full-size Fc domains and partial Fc domains; and (c) partial Fc domains. In each of these embodiments of strathmere can optionally enable CH1 domains. As described in the present application, in each variant of implementation of strathmere present invention, strathmere have the ability to link two or more Fcγ-receptors.

Preferred embodiments of strathmere and monomers of strathmere

[00171] examples of monomers of strathmere of the present invention:

1. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1

2. The hinge region of IgG1 - IgG3 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1

3. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG3 - hinge region of IgG1 - IgG1 CH2 - CH3 IgG1

4. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG3 - IgG1 CH2 - CH3 IgG1

5. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG3 CH2 - CH3 IgG1

6. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG3

7. The hinge region of IgG1 - IgG3 CH2 - CH3 IgG1 hinge region of IgG3 - IgG1 CH2 - CH3 IgG1

8. The hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1

9. The hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 hinge region of IgG3 - IgG1 CH2 - CH3 IgG1

10. The hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG3 - hinge region of IgG1 - IgG3 CH2 - IgG3 CH3

11. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG3 - IgG3 CH2 - IgG3 CH3 - �arnima region IgG1 - IgG1 CH2 - CH3 IgG1

12. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG3 - hinge region of IgG1 - IgG3 CH2 - CH3 IgG3 - hinge region of IgG1 - IgG1 CH2 - CH3 IgG1

13. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG3 - IgG3 CH2 - CH3 IgG3 - hinge region of IgG1 - IgG2 CH2 - IgG3 CH3.

14. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG4 - IgG4 CH2 - CH3 of IgG4 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1

[00172] it Should be understood that in each of these embodiments and in other embodiments, the implementation presented in this application domain relationships can be used to link the individual monomers incomplete Fc-domains, which form a separate monomers incomplete Fc-domains, which form the monomers of strathmere. In one of the embodiments of the monomers incomplete Fc-domain for each of the monomers of strathmere listed above, monomers are incomplete Fc-domain of human rights.

[00173] the Present invention includes strathmere comprising two or more monomers of strathmere listed above. In preferred embodiments, the present invention includes a serial strathmere, consisting of two identical monomer strathmere above.

[00174] As stated above, the functional properties of strathmere regarding the binding of more than one Fcγ receptor can also be �the conduct of J chain as cor-group cor-streamer, similar or the molecule of natural IgM, or IgA. In native immunoglobulins IgA and IgM connective (J) chain is a 15 kDa peptide that connects the heavy and light chains of IgA and IgM with 18 amino acids "secretory end pieces" Fc portions of antibodies via disulfide bridges. Braathen, R., et al., The Carboxyl-terminal Domains of IgA and IgM Direct Isotype-specifϊc Polymerization and Interaction with the Polymeric Immunoglobulin Receptor, J. Bio. Chem. 277(45), 42755- 42762 (2002).

[00175] Such cor-strathmere can consist of monomers of strathmere containing natural CH4 Fc-domains, preferably from IgM immunoglobulins, thus ensuring that the Association of such monomers of strathmere with J chain (see Fig.10A-10D). The following are examples of monomers of strathmere that can santimonious forming streamer, and then connect with J chain, forming a cor-streamer consisting of a plurality (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, fifteen, eighteen, twenty or more) strathmere:

1. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 - IgM CH4 (see Fig.10C-10D)

2. The hinge region of IgG1 - IgG3 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 - IgM CH4

3. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG3 - hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 - IgM CH4 (see Fig.10A-10B)

4. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 - IgM CH4

5. SARN�RNA region IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG3 CH2 - CH3 IgG1 - IgM CH4

6. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 - IgM CH4

7. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - CH2 IgG1 - IgG3 CH3 - CH4 IgM

8. The hinge region of IgG1 - IgG3 CH2 - CH3 IgG1 hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 - IgM CH4

9. The hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 - IgM CH4

10. The hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 - IgM CH4

11. The hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 hinge region of IgG1 - IgG1 CH2 - hinge region of IgG1 - IgG3 CH2 - IgG3 CH3 - CH4 IgM

[00176] it Should be understood that in each of these embodiments and in other embodiments, the implementation presented in this application domain relationships can be used to link the individual monomers incomplete Fc-domains, which form a separate monomers incomplete Fc-domains, which form the monomers of strathmere. In one of the embodiments of the monomers incomplete Fc-domain for each of the monomers of strathmere listed above, monomers are incomplete Fc-domain of human rights.

[00177] Cor-strathmere on the basis of J chain may also consist of Fc-fragments, incomplete Fc-fragments and/or Fc domains which contain CH4 Fc-domain. In this example, each of the Fc-fragments, incomplete Fc-fragments and/or Fc domains, CH4 Fc-domain of which�azan with KOR group can contain only one Fcγ-receptornegative website, however in the context of such cor-strathmere, forms a biologically active biomimetic containing more than one Fcγ-receptornegative site. A qualified specialist it is obvious that partial Fc domains from different native immunoglobulins can be used to obtain functional Fc-fragments, incomplete Fc fragments and Fc-domain of such cor-strathmere. The following are examples of monomers Fc-fragments, incomplete Fc-fragments and the Fc-domain that can santimonious, and then be associated with the J chain, forming a cor-streamer:

1. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 - IgM CH4

2. The hinge region of IgG3 - IgG1 CH2 - CH3 IgG1 - IgM CH4

3. The hinge region of IgG1 - IgG3 CH2 - CH3 IgG1 - IgM CH4

4. The hinge region of IgG1 - CH2 IgG1 - IgG3 CH3 - CH4 IgM

5. The hinge region of IgG1 - IgG3 CH2 - IgG3 CH3 - CH4 IgM

6. The hinge region of IgG3 - IgG3 CH2 - CH3 IgG1 - IgM CH4

7. The hinge region of IgG3 - IgG3 CH2 - CH3 IgG1 - IgM CH4

8. The hinge region of IgG1 - IgG3 CH2 - IgG2 CH3 - CH4 IgM

9. The hinge region of the IgG1 hinge region of IgG3 - IgG3 CH2 - IgG2 CH3 - CH4 IgM

10. The hinge region of IgG1 - IgG1 CH2 - CH3 IgG1 - and IgE CH4 - CH4 IgM

[00178] it Should be understood that in each of these embodiments and in other embodiments, the implementation presented in this application domain relationships can be used to link the individual monomers incomplete� Fc-domains, which form of individual monomers incomplete Fc-domains, which form the monomers of strathmere. In one of the embodiments of the monomers incomplete Fc-domain for each of the monomers of strathmere listed above, monomers are incomplete Fc-domain of human rights.

[00179] As can be seen from the foregoing examples, the monomers of strathmere can have different length and composition, in accordance with the purpose of forming a cor-streamer containing more than one Fcγ-receptornegative website when connected through samegreloshi or inter-monomer strathmere ties with a second monomer strathmere and when you connect with J chain. These examples in no way limit the invention, the person skilled in the art it is obvious that strathmere can have many other configurations.

Fcγ-receptors

[00180] the Terms "FcγR" and "Fcγ-receptor used in the present application, include each representative of a family of Fc gamma receptor proteins, expressed on the surface of immunopositive, as described in Nimmerjahn F and Ravetch JV. Fcgamma receptors: old friends and new family members. Immunity. 2006 Jan; 24(1): 19-28, or which may be described later. It is assumed that the term "FcγR" described in the present application covers all the representatives of Fc gamma families of RI, RII and RIII. Fcγ-receptor includes Fcγ-receptors low affinity and high affinity,�Lucie, in addition, FcγRI (CD64); FcγRII (CD32) and its isotypes, and allotype FcγRIIa LR, FcγRIIa HR, FcγRIIb and FcγRIIc; FcγRIII (CD16) and its isotypes FcγRIIIa and FcγRIIIb. A qualified specialist it is obvious that the present invention, which includes compounds that bind FcγR, relates to future FcγR receptors and corresponding isotypes and allotypes that are still unknown to date.

[00181] it has Been described that IVIG binds and fully saturates the neonatal Fc-receptor ("FcRn") and what is konkurente inhibition of FcRn may play an important role in the biological activity of IVIG (e.g., Mechanisms of Intravenous Immunoglobulin Action in Immune Thrombocytopenic Purpura. F. Jin, J. Balthasar. Human Immunology, 2005, Volume 66, Issue 4, Pages 403-410.) Because immunoglobulins that bind to Fcγ-receptors, have also been associated, at least to some extent, with FcRn, then a qualified specialist it is obvious that strathmere who is able to communicate with more than one Fcγ receptor will also bind and can fully saturate FcRn.

[00182] "Immunological activity of native aggregated IgG" refers to the properties multisensornogo IgG that affect the functioning of the immune system when exposed to the immune system IgG aggregates. Specific properties of native multisensornogo IgG include altered specific binding to FcγR receptors, cross the hol�ivanie with FcγR receptors on the surface of immune cells or effector functional properties multisensornogo IgG, such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis (ADCP), or associate a compliment (see, e.g., Nimmerjahn F, Ravetch JV. The anti-inflammatory activity of IgG: the intravenous IgG paradox. J Exp Med. 2007; 204:11-15; Augener W, Friedman B, Brittinger G. Are aggregates of IgG from the effective part of high-dose immunoglobulin therapy in adult idiopathic thrombocytopenic purpura (ITP)? Blut. 1985;50:249-252; Arase N, Arase H, Park SY, Ohno H, Ra C, Saito T. Association with FcRgamma is essential for activation signal through NKR-Pl (CD 161) in natural killer (NK) cells and NKl.1+ T cells. J Exp Med. 1997;186:1957-1963; Teeling JL, Jansen - Hendriks T, Kuijpers TW, et al. Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia. Blood. 2001;98: 1095-1099; Anderson CF, Mosser DM. Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors. J Immunol. 2002; 168:3697-3701; Jefferis R, Lund J. Interaction sites on human IgG-Fc for Fc[gamma]R: current models. Immunology Letters. 2002;82:57; Banki Z, Kacani L, Mullauer B, et al. Cross-Linking of CD32 Induces Maturation of Human Monocyte - Derived Dendritic Cells Via NF-{kappa} B Signaling Pathway. J Immunol. 2003;170:3963-3970; Siragam V, Brine D, Crow AR, Song S, Freedman J, Lazarus AH. Can antibodies with specificity for soluble antigens mimic therapeutic effects of intravenous IgG in the treatment of autoimmune disease? J Clin Invest. 2005;115:155-160). Usually these features are assessed by comparing with the properties of Monomeric IgG.

[00183] "Comparable or superior to cross-linking of Fcγ receptor or effector functional properties of many natural, aggregated immunoglobulins IgG" as used in this application, means that stromer shows the analysis result of the order of 70% or more p� comparison with the result, get to IVIG. In other embodiments, the result of the analysis is at least within the standard root mean square error of the analysis results obtained for IVIG. In other embodiments, the result of the analysis is 110% or higher compared to the result obtained for IVIG. Tests for cross-linking of FcγR-known medium-sized specialists in this field (see for example, Falk Nimmerjahn and Jeffrey Ravetch. Fcγ receptors as regulators of immune responses.Nature Reviews Immunologypublished online 7 December 2007).

[00184] "Immune activity", "modulating the immune response", "modulation of the immune system" and "immunomodulation" means impacts on your immune system modifying activities, functional properties and relative amounts of one or more of immunocytes, including the maturation of cells of any type within its type cells or by transformation into other types of cells. For example, immunomodulation immature monocytes may lead to increase in population more Mature monocytes, dendritic cells, macrophages or osteoclasts, which are formed from immature monocytes. For example, the immune cell receptors can be bound immunologically active biomimetics and can activate intracellular signaling, induces various changes of immunocytes, se�till then referred to as "activation modulation". Blocking of receptors of immune cells to prevent activation of receptors is also included in the scope of "immunomodulation" and may separately be referred to as "inhibitory immunomodulation".

[00185] the Modulation of the maturation of monocyte relates to monocyte differentiation into Mature DC, macrophage or osteoclast. Differentiation can be modulated in order to accelerate the maturation and/or uvelichenie number of monocytes that undergo differentiation. In the alternative, the differentiation can be reduced in terms of speed differentiation and/or the number of cells undergoing differentiation.

[00186] as Used in this application, the term "isolated" polypeptide or peptide refers to the polypeptide or peptide, which has no natural counterpart, or has been isolated or obtained by purification from components that typically surround it, for example, in tissues such as pancreas, liver, spleen, ovary, testis, muscle, joint tissue, neural tissue, gastrointestinal tissue, breast tissue or tumor tissue (e.g., tissue breast cancer), or in fiziologicheskii fluids such as blood, serum or urine. Typically, the polypeptide or peptide are considered to be "highlighted" when the degree of purity of proteins and other naturally occurring organic molecules with which they are usually associated with�provide at least 70% on a dry weight basis. Preferably, the preparation of the polypeptide (or peptide) of the invention contains at least 80%, more preferably at least 90% and most preferably at least 99% by dry weight of the polypeptide (peptide) of the invention, respectively. Since the polypeptide or peptide is synthesized chemically by nature separated from components that accompany it in nature, synthetic polypeptide or peptide are "selected".

[00187] the Selected polypeptide (or peptide) of the invention may be obtained, for example, by extraction from a natural source (e.g., from tissues or bodily fluids); by expression of a recombinant nucleic acid that encodes a polypeptide or peptide; or by chemical synthesis. The polypeptide or peptide that is produced in a cellular system different from the source from which it occurs in nature, are "selected" because it is not necessarily will contain components that accompany it in nature. The degree of purification or purity may be determined using any suitable method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The pharmaceutical composition

[00188] the Introduction of immunologically active biomimetic compositions described in us�Mr sage application shall be in any usual way - orally, parenterally or topically. Examples of routes of administration include, among others, oral, nasal, buccal, rectal, vaginal, ocular, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intratumoral, spinal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, sublingual, oral mucosa, bronchial, lymphatic, intrauterine, subcutaneous, intratumoral, integrable on an implantable device, intradural, intracardially or skin. Such compositions are generally administered in the form of pharmaceutically acceptable compositions, as described in the present application. In a preferred embodiment of the implementation of selected immunological active biomimetic injected.

[00189] the Term "pharmaceutically acceptable carrier", ispolzuemyi in the present application, includes any and all solvents, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, absorption inhibitors, etc. the Use of such media and agents in combination with pharmaceutically active substances is known in the prior art. Except in those cases where any conventional media or agent is incompatible with the vectors or cells us�Mr sage of the invention, their use in therapeutic compositions is contemplated. Also in the composition may include additional active ingredients.

[00190] the Immunologically active biomimetic compositions of the present invention can be prepared in a neutral form or in salt form. Pharmaceutically acceptable salts include salts of joining acids (formed with the free amino groups of the protein), and salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, almond, etc. Salts formed free carboxyl groups can also be derived from inorganic bases such as, for example, hydroxides of sodium, potassium, ammonium, calcium or a hydrate of oxide of iron, and such organic bases as Isopropylamine, trimethylamine, histidine, procaine, etc.

[00191] Sterile injectable solutions are obtained by introducing the necessary amount of immunologically active biomimetics in a suitable solvent, optionally together with other various components listed above, followed by sterilization by filtration. Usually the dispersion is prepared by introducing the various sterilized active ingredients into a sterile environment, which� contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile solutions for injection, the preferred methods of cooking methods include vacuum drying and freeze drying to obtain the powder of the active ingredient, also contains any additional desired component from a corresponding previously presterilization by filtration of the solution.

[00192] in addition, one of the embodiments relates to immunologically active biomimetic compositions suitable for oral administration, which is prepared in a pharmaceutically acceptable carrier with or without an inert diluent. The media must be digestible or edible, and includes liquid, semi-solid, i.e., pasty, or solid carriers. Except in those cases where any conventional media, agent, diluent or carriers are undesirable regarding the recipient or therapeutic efficacy of immunologically active biomimetic drug contained in them, their use in orally administered immunologically active biomimetic composition used in implementing the methods of the present invention, is provided. Examples of carriers or diluents include fats, oils, water, salt solutions, lipids, liposomes, resins, binders, N.�politely, etc. or combination of these. The term "oral administration" as used in this application, includes oral, buccal, enteral or intragastric administration.

[00193] In one embodiment of the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulation, microencapsulation, absorption, etc., these operations are standard for specialists in this field.

[00194] In a particular embodiment of the immunologically active biomimetic composition in powder form unite or mixed thoroughly with a semi-solid or solid carrier. Mixing can be accomplished by any convenient method, for example, by grinding. In the process of mixing can also be added stabilizers to protect the composition from loss of therapeutic activity in the application, i.e., denaturation in the stomach. Examples of stabilizers used in orally administered compositions include buffers, antagonists of the secretion of gastric acids, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc., inhibitors of proteolytic enzymes, etc. More preferably, stabilizat�R for orally administered compositions may also include antagonists of the secretion of gastric acid.

[00195] in addition, immunologically active biomimetic composition for oral administration, combined with semi-solid or solid carrier, can also be prepared in the form of gelatin capsules, tablets or pills with a hard or soft shell. More preferably, gelatin capsules, tablets or pills are enteric coated. Enteric coatings prevent denaturation of the composition in the stomach or upper intestine, where the pH is acidic. See, for example, U.S. patent 5,629,001. When injected into the small intestine with a basic pH, the coating dissolves, and the composition is released, interacting with the cells of the intestine, for example, M-cells of Peyer plaques.

[00196] In another embodiment of the immunologically active biomimetic composition in powder form is combined or mixed with materials forming the nanoparticles encapsulating immunologically active biomimetic, or to which is attached immunologically active biomimetic. Each nanoparticle has a size less than or equal to 100 microns. The nanoparticles may have mucoadhesive properties that provide the absorption of immunologically active biomimetics of the gastrointestinal tract, which otherwise would not be bioavailable by oral administration.

[00197] In another variation�the implementation of the powder composition is combined with a liquid carrier, such as, for example, water or saline, with or without the tail.

[00198] a Specific dosage form of immunologically active biomimetic that can be used is a solution of the immunologically active biomimetic protein in hypotonic buffer on the basis of phosphate, which does not contain potassium, where the composition of the buffer is as follows: 6 mm monobasic monohydrate sodium phosphate, 9 mm heptahydrate dibasic sodium phosphate, 50 mm sodium chloride, pH 7.0+/-0,1. The concentration of immunologically active biomimetic protein in hypotonic buffer can vary from 10 micrograms/ml to 100 mg/ml of the Indicated dosage form can introduce any by introducing, for example, among other things, intravenously.

[00199] in addition, immunologically active biomimetic composition for topical application, combined with semi-solid carrier, can also be prepared in the form of cream or gel. The preferred carrier for the preparation of the gel is the gel polymer. The preferred polymers used to prepare the compositions of the present invention in gel form, include, among others, carbopol, carboxymethylcellulose and plurality polymers. In particular, a powder composition containing a Fc-multimer, combined with a water-based gel, �terrasim curing agent, such as Carbopol 980, in a concentration of from 0.5% to 5% W/V, for application to the skin to treat diseases or under the skin. The term "topical application" as used in this application, comprises applying to the skin surface, epidermis, subcutaneous layer, or mucosa.

[00200] In the process of preparation of dosage form solutions are administered in a manner consistent with the preparation of metered-dose dosage forms, and in such quantity which is therapeutically effective and leads to the relief or elimination of symptoms. Formulations are generally administered in various dosage forms such as solutions for internal use, medicinal capsules, etc., Some variation in dosage may occur depending on the condition of the subject to treatment of the individual. The person responsible for the introduction, can, in any case, to determine the appropriate dose for the individual. In addition, for the introduction of human drugs must comply with the standards of sterility, safety and cleanliness as required by Department standards of biologics of the FDA.

[00201] the route of administration is changed, as one would expect, in accordance with the localization and the nature of the disease being treated, and may include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, VNU�rinally, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, rinsing, direct injection and oral administration.

[00202] the Term "parenteral administration" as used in this application, includes any form of administration in which the compound is absorbed in the body of the individual, excluding the absorption through the intestinal tract. Examples of methods of parenteral administration, as used in the present invention include, among others, intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, or intra-articular injection.

[00203] the Following are examples of different categories of pharmaceutical forms and the preferred routes of administration, as indicated, for specific examples of diseases:

[00204] for Buccal or sublingual dissolvable tablet: angina, polyarteritis nodosa.

[00205] Intravenous: idiopathic thrombocytopenic purpura, myositis with included cells, paraproteinemic IgM demyelinating polyneuropathy, necrotizing fasciitis, vulgar pemphigus, gangrene, dermatomyositis, granuloma, lymphoma, sepsis, aplastic anemia, multiple organ failure, multiple myeloma and monoclonal gammopathy of unknown etiology, chronic inflammatory demyelinating polyradiculoneuropathy, inflammatory myo�Atia, termicheskaya thrombocytopenic purpura, myositis, anemia, neoplasia, hemolytic anemia, encephalitis, myelitis, myelopathy especially associated with T-lymphotropic virus human first type, leukemia, multiple sclerosis and optic neuritis, asthma, epidermal necrolysis, myasthenic syndrome of Eaton-Lambert, myasthenia gravis, neuropathy, uveitis, Guillain-Barre syndrome, homologous disease, a syndrome of muscle stiffness, paraneoplastic degeneration of the cerebellum, called anti-Yo antibodies, paraneoplastic encephalomyelitis and sensory neuropathy caused by anti-Hu antibodies, systemic vasculitis, systemic lupus erythematosus, autoimmune diabetic neuropathy, acute idiopathic autonomic neuropathy, syndrome Vogt-Koyanagi-Harada, multifocal motor neuropathy, amyotrophic lateral sclerosis, associated with antibodies against GM1, demyelination, membranosa-proliferative glomerulonephritis, cardiomyopathy, Kawasaki disease, rheumatoid arthritis and syndrome Evan IM-ITP, CIDP, MS, dermatomyositis, myasthenia gravis, muscular dystrophy. The term "intravenous administration", as used in this application, includes all methods of delivery of compounds or compositions of the present invention into the bloodstream through intravenous injection or infusion.

[00206] Skin gel, l�Sion, cream or patch: vitiligo, herpes zoster, acne, cheilitis.

[00207] Rectal suppository, gel or infusion: ulcerative colitis, the inflammation of hemorrhoids.

[00208] Oral form such as a pill, lozenge, or encapsulated with an enteric coating: Crohn's disease, celiac disease, irritable bowel syndrome, inflammatory liver disease, Barrett's esophagus.

[00209] Intracardially: epilepsy, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's chorea.

[00210] Intraperitoneal infusion or implant: endometriosis.

[00211] the Intravaginal gel or suppository: bacterial, Trichomonas or yeast vaginitis.

[00212] Medical devices: introduced on coronary artery stent, prosthetic connection.

[00213] the Immunologically active biomimetics described in the present application can be administered in dosages from about 0.01 mg per kg to about 300 mg per kg of body weight and especially from 0.01 mg per kg of body weight to about 300 mg per kg of body weight, and can type at least daily, weekly, fortnightly or monthly. Can be used two-phase scheme of introduction, when the first phase of implementation includes from about 0.1% to about 10% of the second phase of the introduction.

Therapeutic applications of strathmere and stratatel

[00214] On the basis of rational design and testsin vitroandin vivoimmunologically active biomimetics of the present invention can serve as an important biological products for the treatment of autoimmune diseases and to modulate immune function in various other contexts, for example, in bioimmunotherapy cancer and inflammatory diseases. Pathological conditions suitable for the treatment of immunologically active biomimetics described in the present application, include diseases, which today is usually subjected to treatment with hIVIG, or diseases in which hIVIG, as installed, were clinically effective, such as autoimmune cytopenia, Guillain-Barre syndrome, myasthenia gravis, autoimmune disease with autoimmunity against factor VIII, dermatomyositis, vasculitis, and uveitis (See, F. G. van der Meche, P. I. Schmitz, N. Engl. J. Med. 326, 1123 (1992); P. Gajdos et al, Lancet, i, 406 (1984); Y. Sultan, M. D. Kazatchkine, P. Maisonneuve, U. E. Nydegger, Lancet, ii, 765 (1984); M. C. Dalakas et al., N. Engl. J. Med. 329, 1993 (1993); D. R. Jayne, M. J. Davies, C. J. Fox, C. M. Black, C. M. Lockwood, Lancet 337, 1137 (1991); P. LeHoang, N. Cassoux, George F., N. Kullmann, M. D. Kazatchkine, Ocul. Immunol. Inflamm. 8, 49 (2000)), as well as the types of cancer or inflammatory diseases in which can be used or is already in clinical use of a monoclonal antibody. Diseases that can� effectively treated by the compounds, which are the object of the present invention, include inflammatory disease with impaired balance ways of cytokines, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or acute or chronic phase of chronic relapsing autoimmune, inflammatory or infectious disease or process.

[00215] in addition, treatment of immunologically active biomimetics can be successfully subjected to other pathological conditions with an inflammatory factor, such as amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, Parkinson's disease, myocardial infarction, stroke, hepatitis B, hepatitis C, inflammation associated with the virus immunodeficita human adrenoleukodystrophy and epileptic diseases, especially those that are considered to be associated with posterunek encephalitis, including Rasmussen syndrome, West syndrome and Lennox-Gastaut syndrome.

[00216] the General approach to therapy using selected immunologically active biomimetics described in the present application, includes introduction to the individual with the disease or pathological condition a therapeutically effective amount of the selected immunologically active biomimetics for the implementation of treatment. In some embodiments, zābol�ing or pathological condition can be roughly classified as inflammatory diseases with impaired balance ways of cytokines, autoimmune disorders mediated by pathogenic autoantibodies or autoaggressive T cells, or acute or chronic phase of chronic relapsing disease or process.

[00217] the Term "treatment" as used in this application, refers to the introduction to the individual a therapeutically effective amount of biomimetics of the present invention, resulting in the individual improvement of the disease or pathological condition, or symptom of the disease or pathological condition. Improvement is any improvement or elimination of a disease or condition, or symptom of the disease or pathological condition. The improvement is noticeable or measurable improvement or could be improved overall well-being of the individual. Thus, the person skilled in the art it is obvious that the treatment may improve the disease, but it may not lead to full recovery. In particular, improvements in individuals can include one or more of the following: a reduction of inflammation; the analytical reduction of the inflammatory markers such as C-reactive protein; reducing autoimmune reactions, as evidenced by one or more of the following: improvement of the picture autoimmune markers, for example, autoantibodies or Chi�La platelets, the number of leukocytes or red blood cells, reducing rash or purpura, a decrease of weakness, numbness or tingling, increased glucose levels in patients with hyperglycemia, decrease joint pain, inflammation, swelling or degradation, reduction in the frequency and intensity of cramps and diarrhea, reduction of angina, reduction of tissue inflammation or reducing the frequency of paroxysms; reducing the severity of the cancer, slow progression of the tumor, reducing pain in cancer, increase survival or improve quality of life; or delay of progression or improvement in the course of osteoporosis.

[00218] the Term "therapeutically effective amount" as used in this application, refers to the amount that leads to improvement or elimination of symptoms of the disease or pathological condition.

[00219] Used in the present application "prevention" can mean complete prevention of disease symptoms, delay the development of symptoms or decrease in severity of symptoms of the disease developing later.

[00220] the Term "individual" used in the present application, is used to refer to any patient is a mammal that is administered biomimetics of the present invention according to the methods described in this application. In a specific embodiment of the methods of the present�General description used for the treatment of humans. The methods of the present description can also be applied to processing all primates except humans (e.g., monkeys, baboons and chimpanzees), mice, rats, cattle, horses, felines, dog, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, Guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys and ducks), fish and reptiles, for the purpose of obtaining species-specific or chimeric molecules of strathmere.

[00221] In particular, the biomimetics of the present invention can be used for the treatment of pathological conditions, including, among others, coronary heart disease (CHD), vasculitis, rosacea, acne, eczema, myocarditis and other lesions of the myocardium, systemic lupus erythematosus, diabetes, spondylopathies, synovial fibroblasts and bone marrow stroma; bone loss; Paget's disease of osteoclastoma; multiple myeloma; breast cancer; dysfunctional osteopenia; malnutrition, periodontal syndrome, Gaucher's disease, histiocytosis of Langerhans cells, the lesion of the spinal cord, acute septic arthritis, osteomalacia, the syndrome Itsenko-Kushinga, manasalu fibrous dysplasia, pelissolo fibrous dysplasia, reconstruction of the periodontium and bone fractures; sarcoidosis; osteolytic cancer, lung cancer, kidney cancer and rectal cancer; bone metastases, pain in regulation to�ti, and humoral malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathies; graft rejection, viral infections, hematologic neoplasia and diseases like neoplastic, for example, Hodgkin's lymphoma; non-Hodgkin's lymphoma (Burkitt's lymphoma, small cell lymphocytic lymphoma/chronic lymphocytic leukemia, mushroom granuloma, lymphoma cells, mantle zone, follicular lymphoma, diffuse large b-cell lymphoma, lymphoma of the boundary zone, histiocytic and reticuloendotheliosis lymphoplasmacytic leukemia), tumors of precursor cells of lymphocytes, including B-cell acute lymphoblastic leukemia/lymphoma and T-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of the Mature T - and NK-cells, including nodal T-cell leukemia, T-cell leukemia/T-cell lymphoma adult T-cell leukemia of large granular lymphocytes, Langerhans cell histiocytosis (X), myeloid neoplasias such as acute myelogenous leukemia, including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute mielomonocitarnyi leukemia and acute monocytic leukemia, myelodysplastic syndromes and chronic myeloproliferative diseases, including chronic myelogenous leukemia, tumors of the Central nervous system�status, for example, brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancer, ovarian cancer, primary liver cancer or endometrial cancer, tumors of the vascular system (angiosarcoma and hemangiopericytoma)) or other forms of cancer.

[00222] In the present application is "a malignant tumor" refers to or describes the physiological condition in mammals that is typically characterized by unregulated growth of cells. Examples of malignant tumors include carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteoblastic bone sarcoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, myxosarcoma, chondrosarcoma), neuroendocrine tumor, mesothelioma, chordoma, synovioma, sandamu, meningioma, adenocarcinoma, melanoma, and leukemia or lymphocytic leukemia. More specific examples of such forms of malignant tumors include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma �egcogi and squamous cell carcinoma of the lung, small cell carcinoma of the lung, cancer of the abdominal cavity, hepatocellular cancer, gastric cancer, including cancer of the gastro-intestinal tract, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial carcinoma or uterine carcinoma of the salivary gland, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, carcinoma of the penis, testicular cancer, esophageal cancer, tumors of the biliary tract, the Ewing's tumor, basal cell carcinoma, adenocarcinoma, carcinoma of the sweat glands, carcinoma of the sebaceous glands, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, počečnokletočnyj carcinoma, hepatoma, carcinoma of the bile duct, choriocarcinoma, seminoma, teratocarcinoma, Wilms tumor, a tumor of the testis, carcinoma of the lung, carcinoma of the bladder, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, cranyopharyngioma, ependymoma, pinealoma, hemangioblastoma, the auditory nerve neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, waldenstrom's macroglobulinemia, myelodysplastic disease, bolezn� heavy chains, neuroendocrine tumors, Sandamu and other carcinomas, and head and neck cancer.

[00223] the Biomimetics of the present invention can be used for the treatment of autoimmune diseases. The term "autoimmune disease" as used in this application, refers to a large group of more than 80 different diseases and pathological conditions. The main problem with such diseases and pathological conditions is that the immune system attacks your own body. Autoimmune diseases affect all major systems of the body, including connective tissue, nerves, muscles, endocrine system, skin, blood, respiratory and gastrointestinal systems. Autoimmune diseases include, for example, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and type I diabetes.

[00224] the Disease or pathological state that can be treated using compositions and methods of the present invention may be hematolymphopoietic process, including, but not limited to, idiopathic thrombocytopenic purple, alloimmune/autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, Parvovirus B19-associated red cell aplasia, acquired autoimm�nice against factor VIII, acquired disease von Willebrand, multiple myeloma, and a monoclonal gammopathy of unknown etiology, sepsis, aplastic anemia, pure red cell aplasia, anemia diamond-I am, hemolytic disease of the newborn, immunopositive neutropenia, refractoriness to platelet transfusion, neonatal posttransfusion purple, hemolytic uremic syndrome, systemic vasculitis, Trombitsky thrombocytopenic purple, or Evan's syndrome.

[00225] the Disease or pathological condition can be written neuro process, including but not limited to, Guillain-Barre syndrome, chronic inflammatory demyelinizing the polyradiculoneuropathy, paraproteinemic IgM demyelinizing polyneuropathy, myasthenic syndrome of Eaton-Lambert, myasthenia gravis, multifocal motor neuropathy, amyotrophic lateral sclerosis, associated with antibodies against GM1, demyelination, multiple sclerosis and optic neuritis, a syndrome of muscle stiffness, paraneoplastic degeneration of the cerebellum, called anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy caused by anti-Hu antibodies, epilepsy, encephalitis, myelitis, myelopathy, especially associated with T-lymphotropic virus human PE�cost type, autoimmune diabetic neuropathy, or acute idiopathic autonomic neuropathy.

[00226] the Disease or pathological condition can be written rheumatic process, including, but not limited, Kawasaki disease, rheumatoid arthritis, felty's syndrome, anti-neutrophil cytoplasmic antibodies-positive vasculitis, spontaneous polymyositis, dermatomyositis, antiphospholipid syndrome, recurrent spontaneous abortions, systemic lupus erythematosus, juvenile idiopathic arthritis, Raynaud's syndrome, CREST syndrome, or uveitis.

[00227] the Disease or pathological condition can be written dermatovenerologicheskiy process, including, among other things, toxic epidermal necrolysis, gangrene, granuloma, autoimmune skin disease with the development of abscesses, including vulgar pemphigus, bullous pemphigoid and exfoliative pemphigus, vitiligo, syndrome streptococcal toxic shock, scleroderma, systemic sclerosis including diffuse and local cutaneous systemic sclerosis and atopic dermatitis (especially dependent atopic dermatitis).

[00228] the Disease or pathological condition can be written musculoskeletal immunological disease, including, but not limited to, myositis with included cells, necrotizing fasciitis, inflammatory myopathies, myositis, anti-decorin (BJ of antig�n) myopathy, paraneoplastic necrotizing myopathy, aqualicious myopathy coupled to the X-chromosome, penicillamine-induced polymyositis, atherosclerosis, coronary heart disease or cardiomyopathy.

[00229] the Disease or pathological condition may also be gastrointestinal immunological process involving, among other things, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, celiac enteropathy, dermatitis herpetiformis, cryptogenic cirrhosis, reactive arthritis, Crohn's disease, Whipple's., ulcerative colitis or sclerosing cholangitis.

[00230] the Disease or pathological condition may also be homologous disease (GVHD), antibody-mediated graft rejection, graft rejection, bone marrow, postinfectious inflammation, lymphoma, leukemia, neoplasia, asthma, diabetes mellitus type I with antibodies against beta cells, Sjogren's syndrome, mixed lesions of the connective tissue, Addison disease, syndrome Vogt-Koyanagi-Harada, membranosa-proliferative glomerulonephritis, goodpasture's syndrome, graves ' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropolarization, syndrome Cerca-Strauss, nodular nodosa or multiple organ failure.

[00231] In another embodiment, the implementation of strathmere described in the present application, can be used in Primerose system in which the blood taken from the patient and subjected to a short contact with strathmere (strathmere) during the period of time of approximately half an hour to approximately three hours, after which the blood is injected back to the patient. In this form of cell therapy own effector cells of the patient are exposed to contact with strathmere, which is immobilized on the matrix ex vivo, with the purpose of modulation of effector cells through contact of effector cells with strathmere. Then blood containing modulated effector cells are administered back to the patient. This primirea the system can find the different clinical and therapeutic applications.

Therapeutic applications stratatel in Oncology

[00232] In addition to the presence of clinical use for the treatment of immunological diseases stratatel find therapeutic use in the treatment of malignant tumors and inflammatory diseases. Stradale can be used essentially according to known protocols for any relevant therapeutic antibodies. Stratatel are usually created with the purpose of strengthening effect displayed effector cells under the influence of� monoclonal antibodies, for example, ADCC when a malignant tumor or decrease of monocytes and maturation of DC with a reduced secretion of cytokines in autoimmune disease and, thus, potentiation of the immune response against cancer, which developed, for example, as a result of the use of monoclonal antibodies, which served as the source of the Fab-part Stradale.

[00233] Examples of the Fab domains of monoclonal antibodies, which can be created stratatel include cetuximab, rituximab, muromonab-CD3, abtsiksimab impact, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, tiuxetan of ibritumomab, adalimumab, omalizumab, tositumomab, 1-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101, volociximab, anti-CD80 mAb, anti-CD23 mAb, CAT-3888, CDP-791, epratuzumab, MDX-010, MDX-060, MDX-070, matuzumab, CP-675,206, CAL, SGN-30, zanolimumab, adecatumumab, oregovomab, nimotuzumab, ABT-874, denosumab, AM 108, AMC 714, fontolizumab impact, golimumab, CNTO 1275, to have a clear significant HuMax-CD20, belimumab, epratuzumab, MLN 1202, visilizumab, tocilizumab, have a clear significant certolizumab pegol, eculizumab, pexelizumab, abtsiksimab, ranibizumab, mepolizumab and TNX-355, MYO-029.

[00234] Strathmere and stratatel, in the aggregate immunologically active biomimetics disclosed in the present application, have many other applications.

<> Alteration of immune responses

[00235] the Immunologically active biomimetics disclosed in the present application, can also be successfully used to modify the responses of the immune system in various situations for specific changes of the profiles of the immune response. Change or modulation of the immune response in the individual are increase, decrease or change in ratio or components of the immune response. For example, the levels of production or secretion of cytokines, if necessary, can be increased or decreased by a directed impact on the appropriate combination of Fc-receptor strathmere created to interact with these receptors. Autoantibody production may also be increased or decreased. can be changed the ratio of two or more cytokines or immune cell receptors; or it may be induced by the development of additional types of cytokines or antibodies. The immune response can be written effector function immunocyte expressing FcγR, including increased or decreased the phagocytic capacity of the monocyte macrophage derived cells, increase or decrease in the function of osteoclasts, increased or decreased antigen presentation antigen-presenting cells (e.g. dendritic cells), increased or reduced function of NK cells, increased or �originou the function of B-cells, compared with immune response, which is not subjected to modulation with the use of immunologically active biomimetics disclosed in the present application.

[00236] In a preferred embodiment of the immune response of an individual with a malignant tumor, or an autoimmune or inflammatory disease changed, which includes the stage of introduction to the individual a therapeutically effective amount of immunologically active biomimetics described in the present application, where a therapeutically effective amount of immunologically active biomimetics alters the immune response in the individual. Ideally, such intervention is the treatment of diseases and pathological conditions of the individual. The altered immune response may be increased or decreased response and may include altered levels of cytokines, including levels of any of IL-6, IL-10, IL-8, IL-23, IL-7, IL-4, IL-12, IL-13, IL-17, TNF-alpha and IFN-alpha. However, the invention is not limited to any specific mechanism of action of biomimetics described. The altered immune response may be altered by the level of antibodies in the individual. The altered immune response may be altered by the level of autoaggressive T-cells in the individual.

[00237] for Example, a decrease in production of TNF-alpha in autoimmune diseases can have tera�eticheskoe impact. Practical application of this method is therapy with anti-TNF-alpha antibodies (e.g. REMICADE®), which has been shown in clinical trials, is effective in the treatment pied psoriasis, rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis and ankylosing spondylitis. Listed autoimmune diseases have different etiologies, but include common key immunological components of disease processes associated with inflammation and activity of immune cells. Streamer designed to reduce production of TNF-alpha, similarly, will be effective at these and possibly other autoimmune diseases. The changed profile of the immune response may also be direct or indirect modulation that causes decreased production of antibodies, such as autoantibodies that attack its own tissue of the individual, or the change in the level of autoaggressive T-cells in the individual. For example, multiple sclerosis is an autoimmune disorder involving autoreactive T-cells that can be treated with therapy of beta-interferon. See, for example, Zafranskaya M, et al., Interferon-beta therapy reduces CD4+ and CD8+ T-cell reactivity in multiple sclerosis, Immunology 2007 May;121(l):29-39-Epub 18 December 2006 Design strathmere aimed at reducing the level autoreactive T-cells analogic�therefore be effective in multiple sclerosis and perhaps, in other autoimmune diseases involving autoreactive T-cells.

Use in immunological analysis

[00238] the Immunologically active biomimetics disclosed in the present application may be applied when performing immunological analysis to test the function of immune cells, to modulate which created immunologically active biomimetics.

[00239] the Transmission of signals over the path of Fcγ-receptors low affinity requires aggregation of the receptors and cross-linking on the cell surface. These aggregation and cross-linking, as suggested, are performed with Fab-antigen specific binding of the target with the subsequent interaction between the Fc region and Fcγ-receptors low affinity on the surface of reacted cells. In this case, the antibody is able to induce cell responses in two different ways: 1. Fab-interaction/lock epitopespecific target and 2. Fc-Fc interactions with Fc receptors. Despite the above mechanisms, the existing means of control for the majority of therapeutic studies using monoclonal antibodies usedin vivothe potential interactions Fc:Fcγ receptor as participants observed functional effects use not up to par. Many policies nowadays�tense, aimed at addressing the interactions Fc:FcR as unwanted variables. For example, some studies used Scv (single chain variable region) or Fab fragments, which retain the epitope-specificity, but it does not have a Fc-domain. These approaches are limited by short half-life of these reagents and their limited capacity in the induction of signaling. In other studies using fusion proteins consisting of a receptor or ligand, fused with the Fc-fragment. Although these methods help to differentiate Fab-specific effects from effects associated with ligand-receptor interactions, they have not effectively regulate Fc-mediated effects. In the evaluation of therapeutics based on antibodies in animal models can also be used antibody isotype control with any other extraneous binding site Fab. The justification for this choice is based on an assumed functional similarity between antibodies of the same isotype, regardless of their Fab specificity or affinity. However, the use of foreign control isotypes has several significant drawbacks:

1. If Fab fragments of these antibodies can not bind ligand or antigenic epitope, it is likely that the Fc-fragments will not stimulate signaling through interaction FcR low affinity for OTS�lack of cross-linking Fcγ-receptor. Thus, the observed functional differences between test and control antibodies cannot be accurately attributed to Fab-interaction with the epitope-specific target, no ability transversely to bind FcγR.

2. If these isotypes obtained in cells that produce a variety glycoform or other percentage of individual glycoforms compared to the original antibody, binding to Fc - receptors of both low and high affinity, will be changed, even if Fab-affinity will be identical.

[00240] While there is no precise method of control to solve this problem, there are, one possibility is the use of an isotype-specific strathmere obtained in the same cells as the original antibody, and taken at a dose proportional to the levels of expression of the epitope to which specific experimental antibody. For example, appropriate control for epitope-specific antibodies obtained in the rat, may be a rat isotype-specific streamer able to bind Fcγ-receptor on the surface of effector cells.

[00241] Usually immunocyt subjected to contact with an effective amount of immunologically active biomimetics, modulating the activity of immunocyte known manner, with the specified immunomodulation compare� with the test compound or molecule, to determine whether the test compound similar immunomodulatory activity.

[00242] In another embodiment of termoaktivirovannye strathmere and aggregated immunoglobulins can be used as reagents for the laboratory control in various immunological assays described in this application and known medium-sized specialists in this field.

[00243] immunoassays can be the testsin vitroorin vivoand can include human or non-human immunocytes using species-specific or species-specific immunologically active biomimetics. In one embodiment of the immunoassay is carried out using an effective amount of immunologically active biomimetics, modulating the activity of immunocyte, and then carried out a comparison of this modulation with modulation of immunocyte under the action of the tested compounds. Streamer or stratatel can perform the function of the reagent positive control in the tests, including testing other compounds on the subject of immunological actions. The analysis can compare the effect of the tested monoclonal antibody in comparison with strathmere when binding to Fcγ-receptors on effector cells as well as functional response, �smilemy changes in the expression level of the receptor, cytokine secretion, and functional properties, for example, using the reaction of the mixed culture of lymphocytes. In this case, if streamer (which does not contain Fab) gives the answer, which is partly similar to the response of monoclonal antibodies, the effect of monoclonal antibodies, to some extent, due to the specificity of its Fab, but is a consequence of the General effect of binding and cross-linking of more than one Fcγ receptor on the effector cell. Stratatel, which contains the same streamer, and Fab from monoclonal antibodies, can also help to distinguish the specificity of monoclonal Fab antibodies from the overall effect of cross-linking and binding of more than one Fcγ receptor on the effector cell.

[00244] If the biological activity is species-specific and isotype-specific antibodies partially or fully reproduced species-specific and isotype-specific strathmere, then it is obvious that Fc-Fcγ-receptor activity is part of the observed biological activity related to species-specific and isotype-specific strathmere. Thus, species-specific and isotype-specific strathmere can be applied to the evaluation of potential therapeutic antibodies to determine whether and to what extent the observed biological activity to Fab-h�STI test antibody or a nonspecific effect of the Fc-part of the molecular binding and cross-linking of more than one Fcγ receptor.

[00245] In one embodiment, the implementation of selected immunological active biomimetics of the present invention includes at least one streamer, which includes at least two Fc-domain or the corresponding partial domains of the same class of immunoglobulin Fc, where Fc immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations thereof. Such biomimetics are also capable of specific contact with the first FcγRx1where x1represents the I, II, III, or IV, and with the second FcγRx2where x2represents the I, II, III or IV. These biomimetics can additionally be characterized by the presence of immunological activity, including cross-linking of Fcγ-receptor or effector functional properties comparable or superior to cross-linking of Fcγ-receptor or effector functional properties of many natural, aggregated immunoglobulins IgG.

[00246] In another embodiment of the present invention includes selected immunologically active biomimetic, which includes at least one streamer comprising at least two Fc domains from different classes of immunoglobulin, or the corresponding incomplete domains, where biomimetic is bound to the first specific FcγRx1where x1represents the I, II, III, or IV, and with the second FcγRx2where x represents the I, II, III or IV. The specified biomimetic may further be characterized by the presence of immunological activity, including cross-linking of Fcγ-receptor or effector functional properties comparable or superior to cross-linking of Fcγ-receptor or effector functional properties of many natural, aggregated immunoglobulins IgG against Fcγ-receptors.

[00247] In the following embodiment of the present invention includes selected immunologically active biomimetic that includes one or more strathmere, each of which independently comprises three or more Fc-domain, where three or more Fc domain include: (a) a first Fc domain, wherein the first Fc-domain includes the Fc-hinge region (H) of the first immunoglobulin, (b) a second Fc domain, wherein the second Fc domain comprises a constant region 2 (CH2) of the second immunoglobulin, wherein the second Fc domain capable of specific contact FcγRx1where x1represents the I, II, III, or IV; (c) the third Fc-domain, where the third Fc domain comprises a constant region 3 (CH3) of the third immunoglobulin, where the third Fc domain capable of specific contact FcγRx2where x2represents the I, II, III or IV. These biomimetics can optionally include a fourth Fc-domain, where the fourth Fc domain comprises a constant region 4 (CH4) fourth �of immunoglobulin IgM. In this molecule, the Fc-hinge region may contain at least one cysteine.

[00248] In another embodiment of the present invention includes selected immunologically active biomimetic, which comprises: (a) the first Fc-domain or the corresponding partial Fc domain, wherein the first Fc-domain includes the Fc-hinge region (H) the domain of the first immunoglobulin, where the Fc-hinge region includes at least one cysteine, wherein the first Fc domain contributes to binding specificity with FcγRx, where x denotes I, II, III, or IV; and at least one of: (i) a second Fc-domain or the corresponding incomplete domain, wherein the second Fc domain comprises a constant region 2 (CH2) from a second immunoglobulin, which may be, or may not be the same as the first immunoglobulin, wherein the second Fc domain contributes to binding specificity with FcγRx, where x denotes I, II, or III, IV; and optionally ii) of the third Fc-domain or the corresponding partial domain, the Fc domain comprises a constant region 3 (CH3) of the third immunoglobulin, where is the third Fc domain contributes to binding specificity with FcγRx, where x denotes I, II, III, or IV; and (b) optionally, a fourth Fc-domain or the incomplete domain specificity is the fourth Fc-domain due to the constant region 4 (CH4) from immunoglobulin IgM.

[00249] In another VA�iante implementation of selected immunological active biomimetic is strathmere, where immunoglobulin, which served as the source Fc-domains, is the same or different, and includes the isotypes IgA isotypes IgG, IgD, IgE and IgM. Another variant of implementation of strathmere is selected immunologically active biomimetic comprising a secretory signal sequence.

[00250] In one of preferred embodiments a therapeutically effective amount of videlennih of immunologically active biomimetics of the present invention is the amount sufficient for the binding of biomimetics with two or more FcγRx, where x denotes I, II, III or IV, on the surface of immunocyte, wyzwala, thus, the aggregation FcγRx. Immunocyt be immune effector cell, e.g., a monocyte, a dendritic cell, a macrophage, an osteoclast, or NK cell. Maturation effector of immunocyte can be modulated immunological active biomimetic. On immunocyte can also be changed in relation to FcγRIIa FcγRIIb. Immunocyt can be located in the plasma, bone marrow, intestine, bone, lymphoid tissue, thymus, brain, site of infection or tumor. The functional activity of macrophage, dendritic cells, osteoclast or NK cells can be modulated.

[00251] a Therapeutically effective amount of immunologically active biomimetics, OPIE�specified above in the present application, you can typeex vivoin immunocyt for the purpose of obtaining a processed immunocyte, with the subsequent stage of the introduction of processed immunocyte the individual. Processed immunocyt may be a dendritic cell, a macrophage, an osteoclast or a monocyte.

[00252] Additional immunotherapy can be performed in combination with any of the selected immunologically active biomimetics described in the present application, administered in a therapeutically effective amount of the individual. Additional immunotherapy may include, for example, one or more costimulatory molecules, monoclonal antibody, polyclonal antibody, a protein, bispecific antibody, cytokine, immunologically recognizable antigen, a small molecule antitumor agent or an antiproliferative agent. Additional immunotherapy can be carried out simultaneously with or separately from the administration of immunologically active biomimetics.

[00253] the Levels of cytokines (including the aforementioned cytokines) may be modified, for example, by introducing one or more target cytokines, one or more other cytokines that modulate the level of one or more target cytokines and/or antibodies (any of the types and classes described in this application) that are specific to one or more of any cytokines from dvuhmesyachnik categories.

[00254] the Immunologically active biomimetics described in the present application, can be used to modulate the expression of costimulatory molecules in immunocyte comprising a dendritic cell, a macrophage, an osteoclast, a monocyte or NK-cell, or to inhibit the same immune cell differentiation, maturation, or secretion of cytokines, including interleukin-12 (IL-12), or to increase the secretion of cytokines, including interleukin-10 (IL-10) and interleukin-6 (IL-6). A qualified professional can also assess the effectiveness of immunologically active biomimetics, exposing immunocyt contact with immunologically active biomimetics with subsequent measurement of the modulation function immunocyte where immunocyt is a dendritic cell, a macrophage, an osteoclast or a monocyte. In one embodiment, the implementation of immunocyt subjected to contact with immunologically active biomimeticsin vitrofollowed by the stage of determining the number of receptors on the cell surface or level of production of cytokines, where the change in the number of receptors on the cell surface or level of cytokine production determines the modulation function immunocyte. In another embodiment, the implementation of immunocyt subjected to contact with immunologically active biomimeticsin vivoin the animal serving as a model of autoimmune disease, with an additional stage �the TsENKI the degree of improvement in autoimmune disease.

[00255] "Capable of specific contact FcγRx" as used in this application, refers to the binding of FcγR, such as FcγRIII. Specific binding is generally defined as the amount of labeled ligand, superseded later by an excess of unlabeled ligand in the analysis of binding. However, this does not preclude other methods of evaluation-specific binding, which are well known in the prior art (for example, Mendel CM, Mendel DB, 'Non-specific' binding. The problem, and a solution. Biochem J. 1985 May 15;228(1):269-72). Specific binding may be measured in various ways known in the prior art, for example, using the technology of surface plasmon resonance (SPR) (available through BIACORE®), enabling the characterization of binding constants and dissociation of immunologically active biomimetics (Asian K, Lakowicz JR, Geddes C. Plasmon light scattering in biology and medicine: new sensing approaches, visions and perspectives. Current Opinion in Chemical Biology 2005, 9:538-544).

Methods using immobilized Fc

[00256] to understand the role of Fc:Fc-gamma receptor (FcγR, Fc-receptor for IgG Fc) interaction and the importance of IVIG with respect to its Fc, biologically immobilized in the immunoglobulin molecule, the inventors compared the effect of IVIG and immobilized forms of recombinant Fc fragment of IgG1 (rFCF) and a soluble form of recombinant Fc fragment of IgG1 (sFc) containing carnero� region-CH2-CH3 domains on the function of monocytes in the process of differentiation from monocytes to immature dendritic cells (iDC).

[00257] the Impact on monocytes cultured in the presence of granulocyte-macrophage colony stimulating factor (GMCSF) and interleukin-4 (IL-4), immobilized rFCF and immobilized IVIG, not soluble IVIG in small doses, increased the expression of CD86, slowed the expression of CD11c and suppressed the expression of CD1a on the cell. In addition, these changes probably are not a result of non-specific protein immobilization rFCF on the polymer, since soluble thermogravimetry (sHA) IVIG, sHA rFCF or IVIG in large doses (as it is known, containing multimeric Fc) caused changes similar to the observed for immobilized rFCF.

[00258] In aggregate, these data indicate that the impact on iDC IVIG immobilized on the surface of a solid, semi-solid or gel-like substrate that results in a unique population of dendritic cells (with a high content of CD86 and low CD1a), able to induce the development of immune tolerance, and that the immobilized molecules which include the functional part of the Fc fragments of immunoglobulin G (IgG), can be used as IVIG mimetics for the treatment of local and systemic inflammation, as well as a wide range of other pathological conditions, which, directly and�and indirectly, mediated by cells derived from monocytes (MDC) such as iDC. In addition, the immobilization of the functional Fc part of IgG on the devices described in the present application as "device with a coating, which is implanted in the body or attached to the bodies of animals (e.g., humans), with molecules containing functional part of the Fc-fragment of IgG, can reduce, if not prevent, the inflammatory response to such device.

[00259] the Invention provides a method of inhibiting activity of a cell derived from a monocyte (MDC). The method comprises contact of the cell with a composition comprising a framework with which is associated the Fc reagent. Contact can occurin vitro,in vivoorex vivo. In the alternative, the cell may be in the body of the animal. The animal may be an animal that has, or is at risk of developing the disease mediated by cells derived from monocytes (MDCMC). MDC may be, for example, dendritic cell, a macrophage, a monocyte, or osteoclast.

[00260] the Invention also provides a method of treatment or prevention. The method comprises administering to the animal a composition containing a base, which is associated with the Fc-reactant, where the animal is an animal that has, or is at risk for MDCMC.

[00261] Used in the present application, the term "disease operados�TES cells derived from monocytes (MDCMC)" refers to a pathological condition which, directly or indirectly, partially or fully caused by the activity of cells derived from monocytes, or to the factors that caused them. Cells derived from monocytes, include, among others, monocytes, macrophages, interdigitating dendritic cells (usually referred to in the present application as "dendritic cells, including dendritic cells and follicular destinygodley cells (Mature and immature), osteoclasts, microglial-like cells, the insulin-producing monocyte-derived insulatio-like cells, monocyte-derived immature mast cells and monocyte-derived microparticles.

[00262] On the application of methods using immobilized Fc, the term "Fc-reagent" refers to any molecule or molecular complex, which includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20 or more of the functional parts of the Fc-fragment of immunoglobulin G (IgG). Fc-fragment of IgG consists of C-terminal parts of the two heavy chains of IgG, United together, which in turn consist of hinge regions, CH2 domain and CH3 domains of the two heavy chains are joined together. "Functional portion of the Fc-fragment of IgG consists of hinge regions, CH2 domain and optionally all or some of (e.g., 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49) of the first 50 (from N-end) amino acids CH3 domains of both United heavy chains. (A) the hinge region of IgG1 contains 15 amino acids, CH2 domain contains 110 amino acids and a CH3 domain contains 106 amino acids; (b) the hinge region of IgG2 contains 12 amino acids, CH2 domain contains 109 amino acids and CH3 domain contains 107 amino acids; (c) a hinge region of IgG3 contains 62 amino acids of the CH2 domain contains 104 amino acids and a CH3 domain contains 106 amino acids; and (d) the hinge region of IgG4 contains 12 amino acids, CH2 domain contains 109 amino acids and CH3 domain contains 107 amino acids.

[00263] As in the IgG molecules of the wild type in the above Fc-reagents two polypeptide chains derived from heavy chains of IgG, usually, but not necessarily, identical. Thus, the Fc-reactant may be, without limitation, full-size IgG molecule, a full-size IgG molecule linked to a polypeptide, which occurs not from the immunoglobulin Fc fragment of IgG, Fc-fragment of IgG, linked to a polypeptide, which occurs not from the antibody, functional part of the Fc-fragment of IgG, the Fc fragment of IgG associated with a polypeptide, which occurs not from the immunoglobulin, or multiparae (e.g., dimers, trimers, tetramer, pentamer, �examine, heptameron, ontamarama, enumerati or decamerone) of any of the above. Fc-reagents may also be described by strathmere and stradalli, provided they are within the definitions listed above Fc-reagent.

[00264] In immobilized Fcγ components of the heavy chains of immunoglobulin Fc-reagents may have the amino acid sequence of wild-type, or they can be amino acid sequences of wild-type, containing not more than 20 (for example, not more than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acid substitutions. Such substitutions preferably, but not necessarily, are conservative substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

[00265] "Fc-reagent" of the invention has at least 25% (e.g., at least: 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99,5% or 100%, or even more) of the ability of IgG molecules from which the IgG were derived components heavy chain Fc-reagent (basic molecule IgG) to contact the target Fc receptor. In the case where "Fc-reagent" contains the components of the heavy chains derived from molecules of more than one type of IgG, ishonamanki IgG is a IgG molecule, which is associated with a corresponding target Fc receptor with the greatest avidity.

[00266] Used in the present application "immobilized Fc" refers to the Fc-reagent that is associated with the "substrate", as defined below. The terms "immobilized Fc," related "Fc" and "stabilized Fc" are synonyms. Immobilized Fc consists of a functional Fc part (including, but not limited to, any polypeptide that comprises a functional Fc portion) that is attached to the substrate. Immobilized Fc includes, for example, as a direct binding and indirect binding through the Fc polymers with the substrate; the introduction of full-size IgG Fc in allocated; the introduction of a functional Fc domain of IgG or the introduction of full-size IgG Fc or functional domains of IgG Fc as part of a larger polypeptide, e.g., antibody, strathmere or Stradale.

[00267] in Relation to immobilized Fc, the term "substrate" refers to a solid, semi-solid or gel-like object. The substrate may be implanted in or attached (or attached) to the body surface of the animal. The substrate may include, for example, liquid or gaseous components, however, at least part of the substrate is solid, semi-solid, or gel. Thus, the substrate may be a material that is essentially insoluble� in an aqueous solvent, but soluble in non-aqueous solvent. Such substances include lipids (e.g., phospholipids), fatty acids and other fat-soluble, insoluble in aqueous solvents compounds. From real clear that the substrate include liposomes. The substrate may be porous or nonporous. In specific embodiments, the substrate is inert to the surface and/or the body into which it is implanted, attached, or affixed.

[00268] the Substrate may contain or be manufactured from a synthetic polymer, e.g., nylon, Teflon, Dacron, PVC, PES (polyetherurethane), PTFE (polytetrafluoroethylene), PMMA (polymethylmethacrylate), PEEK, thermoplastic elastomers, radioveronica polymers, polyethersulfone, silicones, polycarbonates, polyurethanes, polyisobutylene and its copolymers, polyesters, polyolefins, polyisobutylene, copolymers of ethylene - alpha-olefins, acrylic polymers and copolymers, vinylchloride polymers and copolymers, such as polyvinyl chloride, polyvinyl ethers, polivinilovogo ether, polyvinylidenechloride, polyvinylidene fluoride, polyvinylidene chloride, polyacrylonitrile, polivinilklorid, polivinilatsetatnyh compounds, polystyrene, complex, polyvinyl esters, polyvinyl acetate, copolymers of vinyl monomers with�polymers of vinyl monomers and olefins, ethylene-methyl methacrylate copolymers, copolymers of Acrylonitrile-styrene, ABS resins, ethylene-vinilacetata copolymers, polyamides, Nylon 66, polycaprolactone, alkyd resins, polyoxyethylenes, polyimides, polyethers, epoxy resins, triacetate rayon, cellulose, cellulose acetate, cellulose butyrate, acetate butyrate cellulose, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, carboxymethyl cellulose, collagens, chetinov, polylactic acid, polyglycol acid, copolymers of polylactic acid and polyethylene oxide, polysiloxanes, substituted polysiloxanes, copolymers of ethylene and vinyl acetate, polyolefin elastomers, and ethylene-propylene-montenevoso rubber, and combinations thereof.

[00269] the Substrate may also contain or be made of metal or metal alloy, e.g., stainless steel, platinum, iridium, titanium, tantalum, nickeltitanium alloy or cobalt alloy. In addition, the substrate may include or be cloth animal or product of animal tissue, e.g., tissue or organ transplant. The animal tissue may be, for example, bone (e.g., osteogenic tissue) or HaShem. In addition, the substrate may contain a protein, e.g., collagen or keratin. The substrate may also be or contain�AMB tissue matrix, for example, acellular tissue matrix. Dispersive and non-dispersive acellular matrices is described, for example, in U.S. patents 5,336,616 and 6,933,326, the descriptions of which are fully incorporated into the present application by reference. The substrate may also be, or include animal cell (e.g., cell, regenerative tissue, such as fibroblasts or mesenchymal stem cell), and can represent, for example, the complex forming pores in the membrane. The substrate may contain or be a polysaccharide, such as agarose. It may also contain or be a salt, preferably, a relatively insoluble salt, such as calcium sulfate. The substrate may be a gel or cream. In addition, it may contain silicone or Silastic. The substrate may also contain natural fiber such as silk, cotton or wool.

[00270] in addition, the substrate may be an implantable medical device. It may be, for example, stents (e.g., vascular stents, such as coronary stent; a stent placed in the airway such as an endotracheal or nasal stent, gastrointestinal stent, such as biliary or pancreatic stent; or ureteral stent, such as urethral stent) or surgical thread (for example, silk thread, chromium�with catgut suture, nylon, polymer or metal thread), or surgical clip (e.g., clip aneurysm). The substrate may be, for example, an artificial hip, an artificial hip, an artificial knee, an artificial knee joint, an artificial shoulder, an artificial shoulder joint, an artificial finger joint (hands or feet), bone plate, bone pin, the implant at bone fractures, intervertebral disc implant, bone cement or a layer of bone cement. It may be an arterial-venous shunt, implantable wire, a pacemaker, an artificial heart, a device that supports the heart, cochlear implant, implantable defibrillator, a spinal cord stimulator, a Central nervous system stimulant, implant perifericheskogo nerve. Other substrates are dentures or dental crowns.

[00271] In other embodiments, the substrate may be a device or mesh to protect larger vessels from embolism, subcutaneous device, a skin patch or patches in the submucosa, or implantable device for drug delivery. The substrate may also be a graft of a large blood vessel where the blood vessel is, for example, carotid �steria, femoral artery or the aorta. It can also be a subcutaneous implant, corneal implant, intraocular lens or contact lens.

[00272] the Substrate may be in the form of a sheet, pellets, meshes, particles, powder, filaments, granules or fibers. The substrate may contain or be a solid, semi-solid or gel-like substance.

[00273] the Polymers used in the invention are preferably such polymers that are biostability, biocompatible, particularly during the introduction or implantation of the device into the body and do not cause irritation of body tissues.

[00274] Fc-reagents can be deposited (i.e., immobilized or stabilized) to the substrate in any way. For example, they can be applied directly to the surface of the substrates to which they are attached, for example, through hydrophobic interactions. Here are a few other methods ((a)-(e)), including polymers application:

(a) Fc-reactant is mixed with a compatible polymer mixture, which was then layers applied to the surface of the implantable synthetic material, stabilizing, thus, the Fc-reactant. Monomers typically used in the prior art to obtain polymer mixtures include PLMA [poly(laurenmarie)]; PEG [polyethylene glycol], PEO [polietileno�d]; alkylated methacrylate functionalized polymers PMMA, PEMA, PBMA and PBMA; itaconate; fumarate and styrene polymers.

(b) a Polymeric primer layer or film of nanometer thickness fixed on the surface of the substrate, and then a polymer primer layer or film of nanometer thickness is fixed Fc-reagent, stabilizing, thus, the Fc-reactant.

(c) a Thin layer of monomer of the polymer coated on the surface of an implantable substrate, after which the monomer polymerizes. Such monomers include, for example, methane, tetrafluoroethylene, benzene, methanol, ethylene oxide, tetralin, acrylic acid, allylamine, hydroxyethylmethacrylate, N-vinylpyrrolidone and mercaptoethanol. Then the Fc-reactant is attached to the monomer.

(d) the Substrate is coated with a protein such as A protein or albumin, which is attached to the Fc-reagent, stabilizing, thus, Fc on the surface of the substrate.

(e) Fc-reagent can be labeled with a chain of hydrophobic amino acids that are associated with implantable synthetic materials and provide uni-directional Fc.

[00275] the Methods of the invention can be applied to any kinds of animals, and IgG molecules, which received IgG-derived Fc portion of the reactants can be from any animal species. Usually suitable species include those species in the cat�of which are IgG or IgG-like molecules. Mainly species to which the methods used, and species of which occur IgG-derived Fc portion of the reagents used in the methods are the same. However, they are not necessarily the same. Suitable animals are preferably mammals, and they include, without limitation, humans, all primates except humans (e.g., monkeys, baboons and chimpanzees), horses, cattle (such as bulls, cows or oxen) pigs, goats, sheep, dogs, cats, rabbits, gerbils, hamsters, rats and mice. Non mammal species include, for example, birds (e.g., chickens, turkeys and ducks) and fish.

[00276] the Terms "treatment" and "prevention" have the same meaning with respect to immobilized Fc, as described above for strathmere and stratatel.

[00277] In the case where the immobilized Fc are implantable devices coated Fc-reagents, they can be implanted into, attached to or mounted on respective internal organs or tissues or surfaces of a body of relevant individuals, using methods known in the prior art. When they are prepared in such form, as, for example, suspensions, powders, they can be prepared and injected as described above for strathmere and stratatel.

[00278] Immobilized Fc-reagents present�present invention can be used for the treatment or prevention of pathological conditions, including, among others, cancer, coronary heart disease (CHD), vasculitis, rosacea, acne, eczema, myocarditis and other lesions of the myocardium, systemic lupus erythematosus, diabetes, spondylopathies, synovial fibroblasts and bone marrow stroma; bone loss; Paget's disease of, hypertrophic osteopathy; dysfunctional osteopenia; malnutrition, periodontal syndrome, Gaucher's disease, histiocytosis of Langerhans cells, the lesion of the spinal cord, acute septic arthritis, osteomalacia, the syndrome Itsenko-Kushinga, manasalu fibrous dysplasia, pelissolo fibrous dysplasia, reconstruction of the periodontium and bone fractures, the regulation of pain in the bones, and humoral malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathies; graft rejection and viral infection.

[00279] All autoimmune diseases, in part or in whole, may constitute MDCMD. The term "autoimmune disease" as used in this application, refers to a large group of more than 80 different chronic diseases. The main problem with these diseases is that the immune system attacks your own body. Autoimmune diseases affect all major systems of the body, including connective tissue, nerves, muscles, endocrine�th system, skin, blood, respiratory and gastrointestinal system.

[00280] an Autoimmune disease or pathological condition can be hematolymphopoietic process, including, but not limited to, idiopathic thrombocytopenic purple, alloimmune/autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, Parvovirus B19-associated red cell aplasia, acquired autoimmunity against factor VIII acquired the disease von Willebrand, multiple myeloma, and a monoclonal gammopathy of unknown etiology, sepsis, aplastic anemia, pure red cell aplasia, anemia diamond-I am, hemolytic disease of the newborn, immunopositive neutropenia, refractoriness to platelet transfusion, neonatal posttransfusion purple, hemolytic uremic syndrome, systemic vasculitis, Trombitsky thrombocytopenic purple, or Evan's syndrome.

[00281] the Autoimmune disease or pathological condition can be a neuro-process, including but not limited to, Guillain-Barre syndrome, chronic inflammatory demyelinizing the polyradiculoneuropathy, paraproteinemic IgM demyelinizing polyneuropathy, myasthenic si�Drome Lambert-Eaton, myasthenia gravis, multifocal motor neuropathy, amyotrophic lateral sclerosis, associated with antibodies against GM1, demyelination, multiple sclerosis and optic neuritis, a syndrome of muscle stiffness, paraneoplastic degeneration of the cerebellum, called anti-Yo antibodies, paraneoplastic encephalomyelitis, sensory neuropathy caused by anti-Hu antibodies, epilepsy, encephalitis, myelitis, myelopathy especially associated with T-lymphotropic virus human first type, autoimmune diabetic neuropathy, or acute idiopathic autonomic neuropathy.

[00282] the Autoimmune disease or pathological condition can be a rheumatic process, including, but not limited, Kawasaki disease, rheumatoid arthritis, felty's syndrome, anti-neutrophil cytoplasmic antibodies-positive vasculitis, spontaneous polymyositis, dermatomyositis, antiphospholipid syndromes, recurrent spontaneous abortions, systemic lupus erythematosus, juvenile idiopathic arthritis, Raynaud's syndrome, CREST syndrome, or uveitis.

[00283] an Autoimmune disease or pathological condition can be dermatovenerologicheskiy process, including, among other things, toxic epidermal necrolysis, gangrene, granuloma, autoimmune skin disease with the development of abscesses, including vulgar pemphigus, bullous pemphigoid exfoliative pemphigus, vitiligo syndrome, streptococcal toxic shock, scleroderma, systemic sclerosis including diffuse and local cutaneous systemic sclerosis and atopic dermatitis (especially dependent atopic dermatitis).

[00284] an Autoimmune disease or pathological condition can be musculoskeletal immunological process involving, among other things, myositis with included cells, necrotizing fasciitis, inflammatory myopathies, myositis, anti-decorin (BJ antigen) myopathy, paraneoplastic necrotizing myopathy, aqualicious myopathy coupled to the X-chromosome, penicillamine-induced polymyositis, atherosclerosis, coronary heart disease or cardiomyopathy.

[00285] an Autoimmune disease or pathological condition can be gastrointestinal immunological process involving, among other things, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, celiac enteropathy, dermatitis herpetiformis, cryptogenic cirrhosis, reactive arthritis, Crohn's disease, Whipple's., ulcerative colitis or sclerosing cholangitis.

[00286] the Autoimmune disease or pathological condition may be homologous disease (GVHD), antibody-mediated graft rejection, and rejection of transpl�ntata bone marrow postinfectious inflammation, lymphoma, leukemia, neoplasia, asthma, diabetes mellitus type I with antibodies against beta cells, Sjogren's syndrome, mixed lesions of the connective tissue, Addison disease, syndrome Vogt-Koyanagi-Harada, membranosa-proliferative glomerulonephritis, goodpasture's syndrome, graves ' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropolarization, syndrome Cerca-Strauss, nodular nodosa or multiple organ failure.

[00287] In the present application is "a malignant tumor" refers to or describes the physiological condition in mammals that is typically characterized by unregulated growth of cells. Examples of malignant tumors include carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteoblastic bone sarcoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, myxosarcoma, chondrosarcoma), osteoclastoma, neuroendocrine tumor, mesothelioma, chordoma, synovioma, sandamu, meningioma, adenocarcinoma, melanoma, and leukemia or lymphocytic leukemia. More specific examples of such forms of malignant tumors include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer, including epithelial plascak�mocny cancer), lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, small cell carcinoma of the lung, cancer of the abdominal cavity, hepatocellular cancer, gastric cancer, including cancer of the gastro-intestinal tract, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial carcinoma or uterine carcinoma of the salivary gland, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, the liver carcinoma, anal carcinoma, carcinoma of the penis, testicular cancer, esophageal cancer, tumors of the biliary tract, Ewing's tumor, basal cell carcinoma, adenocarcinoma, carcinoma of the sweat glands, carcinoma of the sebaceous glands, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, počečnokletočnyj carcinoma, hepatoma, carcinoma of the bile duct, choriocarcinoma, seminoma, teratocarcinoma, Wilms tumor, a tumor of the testis, carcinoma of the lung, carcinoma of the bladder, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, cranyopharyngioma, ependymoma, pinealoma, hemangioblastoma, the auditory nerve neuroma, oligodendroglioma, meningioma, melanoma,�Robusta, the retinoblastoma, leukemia, lymphoma, multiple myeloma, waldenstrom's macroglobulinemia, myelodysplastic disease, heavy chain disease, neuroendocrine tumors, Sandamu and other carcinomas, head and neck cancer, myeloid neoplasias such as acute myelogenous leukemia, including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute mielomonocitarnyi leukemia and acute monocytic leukemia, myelodysplastic syndromes and chronic myeloproliferative diseases, including chronic myelogenous leukemia, tumors of the Central nervous system, e.g., brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancer, ovarian cancer, primary liver cancer or endometrial cancer, tumors of the vascular system (angiosarcoma and hemangiopericytoma)), hematological neoplasia and diseases like neoplastic, for example, Hodgkin's lymphoma; non-hodgskins lymphoma (Burkitt's lymphoma, small cell lymphocytic lymphoma/chronic lymphocytic leukemia, mushroom granuloma, lymphoma cells, mantle zone, follicular lymphoma, diffuse�th large b-cell lymphoma, lymphoma boundary zone, histiocytic and reticuloendotheliosis lymphoplasmacytic leukemia), tumors of precursor cells of lymphocytes, including B-cell acute lymphoblastic leukemia/lymphoma and T-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of the Mature T - and NK-cells, including nodal T-cell leukemia, T-cell leukemia/T-cell lymphoma adult T-cell leukemia of large granular lymphocytes, osteolytic cancers and bone metastasis.

[00288] Used in the present application, the individual is "at risk of developing the disease mediated by cells derived from monocytes (MDCMD)" is an individual who has a predisposition to the development MDCMD, that is a genetic predisposition to the development MDCMD, or who has been subjected to conditions that can lead to MDCMD. An individual suspect the presence MDCMD" is an individual having one or more symptoms MDCMD. From the foregoing it is obvious that neither individuals with risk of developing MDCMD" or individuals, "suspects the presence MDCMD" are not all individuals in the target framework.

[00289] In any of the ways described above MDCMC can be a disease caused by the substrate, and the Fc-reactant is used for preventing MDCMC or improving the condition when MDCMC.

Example 1 - Design of immunologically active biomim�ticks

[00290] the Sequence encoding the monomer Fc fragment of human IgG1 (SEQ ID NO:1), was cloned in the expression vector (pCDNA 3.1 D/V5-His TOPO Invitrogen), including selected restriction sites, the signal sequence IgK (further defined below), and epitope tags, receiving the sequence of IgG1 monomer {restriction sites-IgK signal-restriction sites-(hinge region-CH2-CH3) IgG1-restriction sites-epitope tags (V5 and His)-STOP} shown in Fig.17 (SEQ ID NO:19). The design was transfusional in CHO cells (CHO-002) for the purpose of production of the protein. Additionally, I created several strathmere structures General structures:

a) {restriction sites-IgK signal-restriction sites-(hinge region-CH2-CH3)IgG1-XbaI site (hinge region-CH2-CH3)IgG1-STOP} (SEQ ID NO:21) (see also Fig.4A and Fig.18);

b) {restriction sites-IgK signal-restriction sites-(hinge region-CH2-CH3)IgG1-XbaI site (hinge region-CH2-CH3)IgG1-restriction sites-epitope tags (V5 and His)-STOP} (SEQ ID NO:23) (see also Fig.19);

c) {restriction sites-IgK signal-EcoRV site-(hinge region-CH2-CH3)IgG3-(hinge region-CH2-CH3)IgG1-restriction sites-epitope tags (V5 and His)-STOP} (SEQ ID NO:25) (see also Fig.21); and

d) {restriction sites-IgK signal-EcoRV site-(CH2) IgE (hinge region-CH2-CH3)IgG1-(hinge region-CH2)IgG1-(CH4) IgE-STOP} (SEQ ID NO:27) (see also Fig.22).

[00291] Strathmere design based on IgG1a) (SEQ ID NO:21; Fig.18) created using P�R. The primers complementary to the sequence of the hinge region (5'-end) IgGi (SEQ ID NO:29) and C-Terminus IgG1(3'end) (SEQ ID NO:30) was used for amplification of the hinge-Fc region of IgG. The restriction sites introduced in the primers to ensure the cloning inside the frame of the second Fc-domain series with the first, which was cloned in pcDNA cloning vector (pCDNA 3.1 D/N5-His TOPO, Invitrogen). A stop codon was built before the restriction site of the C-terminal primer, to prevent the reading of the flanking sequences of this design.

[00292] Stregoneria design (b) (SEQ ID NO:23; Fig.19), obtained in a similar manner, contained IgG Fc1- IgGi Fc, as described above, as well as two epitope tag added to the C-Terminus of the structure. These epitope tags used for identification or purification of the protein. In this second design, two epitope tag, V5 and His tag present in the reading frame before the stop codon.

[00293] the Proteins that are normally secreted, contain, as a rule, a hydrophobic signal sequence at N-end of the protein. For strathmere designs used signal sequence IgK METDTLLLWVLLLWVPGSTG (SEQ ID NO:35), which is removed from the protein during secretion in mammalian cells, such as ovarian cells of Chinese hamsters. The expected cleavage site was determined on the basis of algorithms to forecast�of the cleavage site of the signal sequence (SignalP 3.0).

[00294] has Constructed additional strathmere constructions like (a) and (b) above, which contained the structure of the Fc IgG1- Fc IgG1as described above (with and without the epitope tag), but with the use of the hinge domain of IgG3in design: Fc IgG1- the hinge region of IgG3-(CH2-CH3)IgG1.

Example 2 - Design and testing of immunologically active biomimetics

IVIG and Fc applied in the form of coatings, cause similar phenotypic changes

[00295] IVIG and Fc, when applied on the walls and the bottom of wells of a sterile tablet, cause almost identical changes in levels of CD1a and CD86 on immature DC and slow down the activation of CD11c. Because of the recognized critical role of DC in ITP these data provide a rational model for assessing the functions of IVIG-mimetics, such as strathmere. Also concluded that the fact that the phenotypic changes caused by IVIG, fully reproduced recombinant Fc, suggests that the effect of IVIG on DC, it is likely, is Fc-mediated.

Getting strathmere

[00296] Constructed strathmere four different classes, which reproduces the effects of IVIG on immature DC. Got all the serial strathmere, cluster strathmere the units making up the cluster strathmere, cor-strathmere units, vkljuchajuwih� cor-strathmere, and Fc-fragment-strathmere below in Table 3, except in designated cases. To obtain the appropriate sequences for each of the constructs of man, listed below, were synthesized cDNA from total RNA isolated from the IPC (peripheral blood mononuclear cells). To retrieve these sequences from other species RNA was isolated from tissues of the species concerned. To obtain cDNA used random primers. cDNA was used for amplification of target fragments using PCR for synthesis, cloning, and sequence analysis of DNA fragments. The final design received either compound overlapping PCR fragments (Horton RM, Hunt HD, Ho SN, Pullen JK and Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61-68, 1989), or by using the appropriate present restriction sites, to obtain relevant merged fragments.

[00297] for Example, clone G-007 CH4 domain of IgE leaked directly from the CH2 domain of IgG1 at the 3'-end of the protein. It followed, having primers that contained overlapping sequences IgG1CH2 (C-end) with N-terminal amino acids IgECH4. In one case hybrid primer used for amplification of the 5' sequences of IgG1 and a complementary primer for amplification of 3' p�aimerai with C-end IgECH4. The products of the two reactions were mixed and used flanking primers for the amplification fused protein. Sequence analysis confirmed the correctness of the design.

[00298] In many cases used the restriction sites, which are conveniently located on the ends of the molecules. When the restriction sites are embedded, associate at the ends of the molecules are determined by the residual restriction sequence. This approach was used for most of the designs are shown below in Table 3. The amino acid sequence of strathmere shown in Table 3, are shown in Fig.24. Some sequences shown with His-tags, known in the prior art, which are used in the purification of proteins.

Table 3
Serial strathmere
N-endChurn.CH2CH3Churn. CH2CH3
G-003IgG1IgG1IgG1IgG1IgG1IgG1
G-004IgG1IgG1IgG1IgG1IgG1IgG
G-007IgECh2IgG1IgG1IgG1IgG1IgG1IgECh4
G-011IgECh2IgG1IgG1IgG1IgG1IgG1
G-012IgECh2IgG1IgG1IgG1IgG1IgG1IgG1IgECh4
G-012IgECh2IgG1IgG1IgG1IgG1IgG1IgG1IgECh4
G-014IgG1IgG1IgG1IgG1IgG1IgG1IgG1IgG1IgG1
G-016IgG3IgG3IgG3IgG1 IgG1IgG1
(x-b)Re-AETR
G-017Lin-kerIgG1IgG1IgGlx-bIgG1IgG1IgG1
G-023IgG1IgG1IgG1IgG1IgG1IgG1Ig3H 32/62

CH3
G-024IgG3IG3 IgG3IgG1IgG1IgG1IgG1IgG1IgG1
G-025AAIgG1IgG1IgG1
G-026AAIgG1IgG1IgG1IgG1IgG1IgG1
The components of the Fc-fragment-strathmere and the cor-strathmere
N-endChurn.CH2CH3Churn.CH2CH3
G-002IgG1IgG1IgG1
G-022IgG1IgG1IgG1IgG3
Cluster strathmere units
N-endChurn.CH2Churn.CH2CH3
G-008IgG1IgG1IgG1IgG1IgG1IgG1IgM
CH3-CH4-TP
G-009IgG1IgG1IgG1IgMCH3-CH4-TP
G-010IgECh2IgG1IgG1IgG1
G-018 IgG2IgG2IgG2IgG1IgG1IgG1
G-019IgG2IgG1IgG1IgG
G-020IgG2IgG1IgG1IgG1IgG1IgG1IgG1
G-021IgG2IgG1IgG1IgG1
G-027IgECh2-IgECh2IgG1IgG1IgG1
G-028ILZIgG1IgG1IgG1
G-029ILZ-IgECh2IgG1IgG1IgG1
G-030ILZIgG2 IgG2IgG2IgG1IgG1IgG1
G-031ILZ-IgG2IgG1IgG1IgG1
G-032ILZ-ILZIgG1IgG1IgG1
G-033IgG2-IgECh2IgG1IgG1IgG
G-034IgG2-ILZIgG2IgG2IgG2IgG1IgG1IgG1
G-035IgG2-IgG2IgG1IgG1IgG1
G-036IgG2-ILZIgG1IgG1IgG1

Have to get the
N-endHCH2CH3HCH2CH3HCH2CH3
401IgG1IgG1IgG1IgG3IgG3IgG3
402IgG3IgG1IgG1IgG3IgG1IgG1
403IgG1IgG3IgG1IgG1IgG3IgG1
404IgG3IgG3IgG1IgG3IgG3IgG1
406IgG1IgG1noIgG3IgG3no
407IgG3IgG3IgG3IgG3IgG3IgG3IgG3IgG3IgG3
408IgG1IgG1IgG1IgG3IgG3IgG3IgG1IgG1409IgG3IgG3IgG3IgG1IgG1IgG1IgG3IgG3IgG3
410IgG3IgG1IgG1IgG3IgG1IgG1IgG3IgG1IgG1
411IgG3IgG3IgG1IgG3IgG3IgG1IgG3IgG3IgG1
412IgG1IgG1IgG4CH4IgG3IgG3IgG4CH4IgG1IgG1 IgGCH4
413IgG1IgG1IgG3IgG3IgG1IgG1
414IgG1IgG1IgG1IgG1IgG1IgG1IgG1IgG1IgG1
415IgG3IgG3IgG3IgG3IgG3IgG3IgG3IgG3IgG3

The protein expression of strathmere

[00299] For protein expression of strathmere plasmid DNA encoding strathmere described above, transtitional in suspension culture of CHO cells (expression system FreestyleTMMAX CHO, Invitrogen SA). After protein expression of expressionand�e strathmere was isolated from a cultivation environment using affinity column chromatography using resins with A-protein or G-protein. Peeled strathmere was analyzed by electrophoresis in LTOs-page (polyacrylamide gel with sodium dodecyl sulphate) with reconstruction, with subsequent staining of Kumasi blue, confirming the presence of bands of Monomeric proteins of the expected size, as shown: G-002: band of approximately 35KD, G-004 band of approximately 70KD, G-010: a band of approximately 45KD, G-011: a band of approximately 80KD, G-012: the band approximately 85KD, G-018 band of approximately 70KD, G-019: approximately 35KD, G-028: stripe approx 37KD. Plasmid DNA encoding the described strathmere, can also be transfected by in other mammalian cells such as HEK 293, BHK cells, mouse cells and mouse NSO cells SP2/0.

Education multimer

[00300] it Was observed that these structures during transfection, culturing and purification, can give proteins of the expected size in sedentarism and denaturing protein analysis. Furthermore, it was observed that some compounds also showed bands of larger size, which in accordance with the criteria for determining the size are multimarine protein expected in dimeric form.

[00301] the Formation of compounds of higher order from the selected strathmere were analyzed by electrophoresis in LTOs-page with subsequent Western-blotting without recovery (A) and recovery (B). Anal�W by electrophoresis in LTOs-page revealed the formation of high-molecular compounds of strathmere G-002, G-010 G-019 without recovery compared to recovery:

• G-002: band of approximately 35KD recovery - strips approximately 70KD (dimer) and 135KD (tetramer) without recovery.

• G-010: a band of approximately 45KD a recovery in strips approximately 90KD (dimer) and 180KD (tetramer) without recovery.

• G-019: a band of approximately 35KD recovery - strips approximately 70KD (dimer), 140 KD (tetramer) without recovery.

[00302] it is Assumed that the tetramers and other multimer higher order, formed from dimeric protein, will significantly increase the biological activity of the compound when measured in the analysis on immature dendritic cells (see below).

The monomers of strathmere, strathmere and multimer of strathmere higher order preserve the sites of recognition

[00303] Each of the proteins in Table 3 were recognized rabbit antibody against human IgG (Fc) [Thermo Scientific 31789]. From this he concluded that each of these proteins keeps the recognition sites for the antibody.

Visualization method plasmon resonance

[00304] the Ability of strathmere shown in Table 3, to bind FcγRIIIa was assessed using the method of surface plasmon resonance (SPR) (commercially available as Biacore®). People FcγRIIIa was immobilizovana. the�but by linking amine on the chip CM5 Biacore, diluted ligand in acetate pH 5.0 to a concentration of 5 μg/ml. Ligands was passed through a certain flow cell at a flow rate of 5 µl/ml, has not yet been achieved 250 RU (resonance units Biacore, ACC. ~1 PG of protein per mm2biosensor). Then the flow cell was blocked with ethanolamine. Strathmere and IVIG was diluted to 1000 nm in HBS-EP (0.01 M HEPES, pH of 7.4; 0.15 M NaCl; 3 mm EDTA; 0.005% of surface-active additives P20) and got a serial dilution of 500 nm, 250 nm, 125 nm and, finally, to 62.5 nm. Also included is a background sample containing only buffer (HBS-EP). A flow rate of 20 µl/min was used for all samples. In total was injected 60 ml of the sample for 3 minutes. The restoration was provided by passing a current of buffer through the flow cell over extended period of time of approximately 10 minutes.

[00305] At 500 nm measured value Req (equilibrium) relative to the baseline for strathmere design G-010 was 24.9 EN when passing over human FcγRIIIa, and KD was 1.95 e-7 using the model binding 1:1. IVIG at 500 nm over FcγRIIIa person showed Req 63,6 RU and KD of 1.89 e-7 using the model binding 1:1. Thus, it was determined that G-010 was associated with FcγRIIIa. Similar binding capacity identified from other biomimetic compounds. Below are some other examples:

1:1 w Mass transferBivalent compliance
RmaxChi2KD(M)KA(I/M)RmaxChi2
Control:2,050,4514,7 e-92,1e85,210,39
Neg.(Mouse IgG2a)87,76,81,9 e-75,3e6
Paul.(IVIG)
Biomimetics:
002of 6.461,12of 2.2 e-84,9e716,70,82
00430,21,744,8 e-82,5e788,22,47
01125,90,361of 5.5 e-61,8e757,40,15

[00306] concluded that these proteins differ in ability to bind to the recombinant FcγRIIIa in the analysis with the method of plasmon resonance, and that some compounds, such as G-010, consistent with bivalent curve similar to the curve for bivalent antibodies, which indicates that streamer can show multi-valent binding to FcγRIIIa.

�teradomari reproduce the biological effect of IVIG

[00307] Evaluated the biological function of these strathmere. For the purpose of determining the ability of each strathmere from Table 3 to reproduce the functional properties of IVIG for patients with ITP, werein vitroanalysis using immature dendritic cells (iDC). The basis for iDC as the target cells was published data that demonstrated that adoptive transfer of DC from mice treated with IVIG, induced protection against the development of ITP in intact animals (Siragam, V. et al. Intravenous immunoglobulin ameliorates ITP via activating Fc [gamma] receptors on dendritic cells. Nat Med 12, 688 (2006)). In their initial studies, the inventors evaluated the effects applied, i.e. immobilized on the plate, recombinant Fc (rFc) and IVIG on the expression of various markers of activation, maturation and costimulation on human CD14+ cells cultured in the presence of IL-4 and GMCSF. When compared with cells grown in the presence of cytokines, the cells exposed to the coating IVIG or rFc, demonstratively a marked increase in the expression of CD86 and the suppression of the expression of CD1a and the delay of activation of CD11c.

[00308] it is Then determined whether the reproduced strathmere shown in Table 3 described the effect of immobilized IVIG or Fc against iDC. These compounds when applied on the walls and bottom of the plate well indeed in�proizvodite this effect: G-002, G-004, G-005, G-014, G-and G 018-019. The following compounds when applied on the walls and bottom of the wells did not reproduce this effect: G-010 G-011 G-012.

[00309] These compounds in solution reproduced the effect of immobilized IVIG or Fc against iDC: G-002, G-010, G-014, G-and G 018-019. The following compounds in solution did not reproduce this effect: G-004, G-005, G-011 G-012.

[00310] it is Possible to check whether the processing of iDC immobilized IVIG affects the subsequent response to proinflammatory stimuli.

[00311] the data obtained made the following observations:

i. selected strathmere applied to cultural tablet, reproduce the functional ability of immobilized IVIG to activate the expression of CD86 and to suppress the expression of CD1a on immature DC,

ii. selected strathmere, administered at low dose in a soluble form, reproduce the functional ability of immobilized IVIG to activate the expression of CD86 and to suppress the expression of CD1a on iDC,

iii. some strathmere can induce phenotypic changes both in soluble and immobilized form, and other strathmere, for example, G-010, can induce phenotypic change in an instant, but not in immobilized form,

iv. strathmere different patterns can be biologically active, as evidenced by the example of Fc-fragment-Stra�Omer, formed from G-002, and cluster strathmere formed from G-010,

v. protein analysis revealed larger structures than would be expected from the dimerization of monomers of strathmere, and these multimeric structures can be consistent with biological activity comparable to the activity of IVIG, and

vi. strathmere formed from demonizovana monomers of strathmere, may exhibit bivalent compliance with the conditions of plasmon resonance, which is consistent with linking multiple Fcγ-receptors and suggests the presence of multimeric tertiary structures of strathmere.

Example 3 - Termoaktivirovannye biomimetics more effective than IVIG

[00312] Streamer is a biologically active mimetic aggregated immunoglobulin, and in particular of aggregated Fc fragments of immunoglobulin. In some cases, turboagregata of biomimetics described in the present application, can enhance the biological activity. It can be concluded that termoaktivirovannye biomimetics, as described in the present application, can be just as effective as IVIG.

Example 4 - Fc-fragment shows a few activities

[00313] the Fc-fragment was used as a positive control in the experiments described above in which a protein that was applied and t�Kim, was immobilizovana on the polymer, showed such biological properties, which reproduced the properties of immobilized IVIG. Fc-fragment can also be used as a cor-strathmere unit when it is attached to cor-groups, such as liposomes, particles or albumin. In addition, the inventors have demonstrated that the Fc-fragment by culturing in some expression systems and certain types of cells, such as system transient transfection Invitrogen FreestyleMax based cells CHO-S, can form multimeric higher in protein analysis, show bivalency the binding profile when rendering in terms of plasmon resonance and show potent biological activity in a soluble form, comparable with the activity of immobilized IVIG, in the analysis on immature DC. Thus, we can conclude that under certain carefully controlled conditions Fc-fragment forms the Fc-fragment-streamer. This effect may be the result of posttranslational modifications, such as changes in glycosylation.

Example 5 - Cor-streamer, which is a Fc-coated particle may be changed by phagocytic capacity compared to particles without coating

[00314] the IPC was isolated from dakotabilities layer of healthy donors using centrifugation � density gradient ficoll-vipaka. After separation of the IPC were washed twice with PBS. Then CD14+ cells were purified using a separation column (MACS (Miltenyi). Purified cells were counted and were resuspended to a density of 2x105/ml in complete RPMI medium containing 800 μg/ml GMCSF and 5 ng/ml IL-4. Then cells were seeded into the wells of conventional (not for tissue culture), but sterile 6-well plates. After sowing CD14+ cells in the plates were added in the ratio 1:1, polystyrene FITZ-microbeads (0.52 mm) coated with saturating or nancymai number of Fc or IVIG, and incubated for 6 days at 37°C, 5.0% of CO2and then analyzed for phagocytosis of microspheres by measuring the fluorescence of sorted cells.

[00315] the Particles are covered as IVIG and Fc, functioned as a cor-strathmere and, therefore, could modify the phagocytic capacity in comparison with the uncoated particles.

Example 6 - Design of immunologically active biomimetics of the modified affiniscape FcγRIII binding

[00316] it Was shown that a common set of IgG1 residues is involved in binding with all Fcγ receptors. Also showed that the additional residues in IgG1 molecules are involved in binding and FcγRII, and FcγRIII. Some remains when you change inhibited the binding of one or more receptors. It is noteworthy that the specific double mutation S28A/K334A increased the binding FcγIIIa and reduced binding to FcγIIb. These residues were marked on the design of strathmere shown in Fig.16 (with an asterisk on both amino acids). Thus, it is possible to use site-directed mutagenesis to obtain strathmere molecule with the structure encoded in accordance with SEQ ID NO:17, but with the corresponding mutations S298A/K334A.

Example 7 - Expression of recombinant proteins

[00317] There are numerous expression systems that are suitable for use in preparation of the compositions described above. In particular, expression systems based on eukaryotic cells can be used to obtain nucleotide sequences or the corresponding polypeptides, proteins and peptides. Many such systems are widely available for sale.

[00318] In a preferred embodiment of the implementation of strathmere described in the present application is obtained using the ovary cells of Chinese hamsters (CHO), widely used for the production of recombinant immunoglobulins in accordance with standard methods. In an alternative embodiment, to obtain human strathmere described in the present application may be applied, for example, transgenic animals, typically through expression in the milk of the animal, using well-known methods of expression in transgenic animals (Lonberg N. Human antibodies from transgenic animals. Nat Bioechnol. 2005 Sep;23(9):1117-25; Kipriyanov SM, Le Gall F. Generation and production of engineered antibodies. Mol. Biotechnol. 2004 Jan;26(l):39-60; see also Ko K, Koprowski H. Plant biopharming of monoclonal antibodies. Virus Res. 2005 Jul;111(1):93-100.

[00319] the System based on the baculovirus in insect cells can provide a high level of expression of proteins with fragment of heterologous nucleic acid, as described in U.S. patents 5,871,986 and 4,879,236 included in full in the present application by reference, and can be purchased, for example, under the name MAXBAC®2.0 from Invitrogen®and BACPACK™ Baculovirus Expression System from Clontech®.

[00320] Other examples of expression systems include a system for inducible expression in mammalian cells from Stratagene®that uses synthetic Edison-induced receptor. Another example of the inducible expression system is a system of T-REX™ from Invitrogen®tetracycline-regulated inducible expression system in mammalian cells that uses a full-sized CMV promoter. Invitrogen®also provides a yeast expression system,Pichia methanolicawhich is designed to obtain high levels of recombinant proteins in the methylotrophic yeastPichia methanolica. Specialist in the art knows how to make expression vectors, such as expression constructs described in the present application, get them coding�Yu nucleotide sequence or the polypeptide protein or peptide. Cm. reviews Recombinant Gene Expression Protocols, Rocky S. Tuan, Humana Press (1997), ISBN 0896033333; Advanced Technologies for Biopharmaceutical Processing, Roshni L. Dutton, Jeno M. Scharer, Blackwell Publishing (2007), ISBN 0813805171; Recombinant Protein Production With Prokaryotic and Eukaryotic Cells By Otto-Wilhelm Merten, Contributor, European Federation of Biotechnology, Section on Microbial Physiology Staff, Springer (2001), ISBN 0792371372.

Example 8 - Expression and purification of immunologically active biomimetics

[00321] Construction of nucleic acids described in Examples 1 and 2, transtitional in cell lines, which in the natural state do not Express Ig. The encoded polypeptides expressibility in the form of secreted proteins due to the presence of secretory leader sequences, which are usually removed by endogenous proteases in the process of transport from cells or can be subsequently cleaved and removed by methods known in the prior art. The data is synthesized, and immunologically active biomimetics was purified by chromatographic methods based on A-protein or His-tags, widely known in the prior art, and the size was confirmed by electrophoresis in LTOs-page (polyacrylamide gel with sodium dodecyl sulphate) in reducing and/or non-reducing conditions.

Example 9 - Expression and purification of immunologically active biomimetics for large scale production

[00322 as] whereas for larger Koli�EU ETS specific protein can be used with different systems including bacteria, insect cells or yeast, expression in mammalian cells can minimize problems due to altered glycosylation of proteins. Mammalian cells such as CHO cells, used to overproduce different proteins, merged with Ig basis. Fc-domain to design becomes a tag, which provides subsequent purification from cell supernatant using purification on affinity column (Harris, CL, Lublin DM and BP Morgan Efficient generation of monoclonal antibodies for specific protein domains using recombinant immunoglobulin fusion proteins: pitfalls and solutions., J. Immunol. Methods 268:245-258, 2002). Many fusion proteins were directly cloned in frame with the constant region of Ig, namely monomers incomplete CH2 and CH3 Fc domains. A specific example of the expression of the extracellular domain of the receptor gamma-interferon, with a murine Ig, was used to obtain large quantities of functionally active proteins (Fountoulakis, M, C. Mesa, G. Schmid, R. Gentz, M. Manneberg, M. Zulauf, Z. Dembic and G. Garotta, Interferon gamma receptor increasing interest among domain expressed as IgG fusion protein in Chinese hamster ovary cells: Purification, biochemical, characterization and stoichiometry of binding, J. Biol. Chem. 270:3958-3964, 1995).

Example 10 - Construction of immunologically active biomimetics with a modified Fc glycosylation

[00323] using a method essentially the same as described by Shields et al. for Homo-antibodies, dealseriously Fc domains can be obtained in a mutant CHO cell�x with a deficiency of the enzymatic activity, provides tools for adding fucose protein to carbohydrates. They are used for the expression of strathmere with higher affinity to FcγRIII compared to fokusirovannyi form of the same molecule. (Robert L. Shields, et al. Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human Fc RIR and Antibody-dependent Cellular Toxicity. J. Biol. Chem., Jul 2002; 277:26733-26740 (doi:10.1074/jbc.M202069200)).

[00324] it Was shown that changes in Valerevna Fc N-glycan may increase the biological activity (Kaneko Y, Nimmerjahn F, Ravetch JV. Science. 2006 Aug 4;313(5787):627-8). Thus, using such methods can be derived molecule strathmere with changed Valerevna.

[00325] Alternative methods for modifying the glycosylation of the Fc-domain of strathmere include chemoenzymatically methods for the production of polypeptides with a specific structure of glycosylation. See, Li, B., Song, H., Hauser, S., and Wang, L. X. 2006. A Highly Efficient Chemoenzymatic Approach Toward Glycoprotein Synthesis. Org. Lett. 8:3081-3084; Cm. also, the application for international patent PCT/US07/70818.

Example 11 - Merged design with (176 V/F) polymorphism FcγIIIa

[00326] As described previously, anti-inflammatory activity of IVIG is dependent on the primary interactions between the Fc domain and FcγIIIa. These interactions can be effectively assessed by quantitative analysis using FBP method for determining the constants of binding and dissociation of immunological active biomimetic�in two uznavaemymi polymorphic variants FcγIIIa (176 V/F). To determine the affinity of binding and dissociation of the monomer control Fc-domain and strathmere structures of the present izobreteniya in CHO cells need to get merged FcγIIIa-His-tag proteins both V (SEQ ID NO:33) and F (SEQ ID NO:31), is polymorphic at position 176, variants (Fig.20). These sequences can be embedded into pCDNA 3.1 and transfected by in CHO cells. These fusion proteins FcγIIIa was purified from the supernatants of the transfected cells, using an affine Nit'-column for protein purification. All fusion proteins FcγIIIa characterized by cDNA sequencing and electrophoresis in LTOs-page.

[00327] For expression FcγIIIa and describing the interaction with the immunological active biomimetics can use other methods known in the prior art. See, for example, the section "materials and methods" in Robert L. Shields, et al. High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR. J. Biol. Chem., Feb 2001; 276: 6591-6604 (doi:10.1074/jbc.M009483200).

Example 12 Screening functions of immunologically active biomimeticsin vitro

[00328] To check the function of immunologically active biomimetics, such as that shown in Example 1, developedin vitrothe analysis to determine a possible mechanism whereby native Fc domains reduce inflammationin vivo. Recently it was demonstrated that hIVIG inhibits with�revenue dendritic cells and alters the secretion profile of IL-10, IL-12 and TNF-alpha (Bayry J, et al., Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin, Blood 101(2):758-765(2003)). Strathmere of the present invention mediate the effects regarding DC similarly hIVIG. The inhibition of DC maturation and changes in the secretion of cytokinein vitrocan be an effective way to determine some of the biological activities of many designs of strathmere. Design strathmere described above, can also be tested using the following experimental parameters:

Table 4
GroupExperimental conditionMeasurement of outcome 1 (FAGS)The measurement result 2 ELISA/Elispot
1NoCD1a,14,40,80,83,86,HLADRIL-10,IL-12,TNFa,IL-23
2Soluble IVIGCD1a,14,40,80,83,86,HLADRIL-10,IL-12,TNFa,IL-23
3Immobilized IVIGCD1a,14,40,80,83,86,HLADRIL-10,IL-12,TNFa,IL-23
4CD1a,14,40,80,83,86,HLADRIL-10,IL-12,TNFa,IL-23
5Immobilized FcCD1a,14,40,80,83,86,HLADRIL-10,IL-12,TNFa,IL-23
6Soluble streamerCD1a,14,40,80,83,86,HLADRIL-10,IL-12,TNFa,IL-23
7Immobilized streamerCD1a,14,40,80,83,86,HLADRIL-10,IL-12,TNFa,IL-23

[00329] In one preferredin vitrothe analysis, shown in Table 4, measured the impact on human DC phenotype soluble immunologically active biomimetics with the necessary affiniscape binding. Soluble not cross-linked structures based on the natural sequence Fc-domain can serve as a control. Evaluated specific DC markers on the surface of DC, including activation markers (CD80, CD83 and CD86), and Fcγ receptors. Cm. Prechtel AT, Turza NM, Theodoridis AA, Steinkasserer A. CD83 knockdown in monocyte-derived DCs by small interfering RNA leads to a diminished T cell stimulation. J Immunol. 2007 Sep 1;178(9):5454-64. In addition, multiplex analysis can be used to assess the impact of immunologically active biomimetic� of the present invention on the production of cytokines in DC (Jongbloed, Sarah L., et al. Enumeration and phenotypic analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis. Arthritis Res Ther. 2006;8(1):R15 (Published online December 16, 2005 doi: 10.1186/ar1864)). Finally, to confirm the interaction of dendritic cells with monocytes, as expected, the control dendritic cells and dendritic cells, treated immunologically active biomimetics, were cultured with purified monocytes and using flow cytometry was evaluated by changes in levels of activation of FcγRIIa receptors and other cell surface determinants that are associated with the activation status of monocytes.

[00330] In specific embodiments, strathmere can reduce the number of FcγRIIa receptors on the immune cell, increasing, thus, the ratio of receptor FcγRIIb, the inhibitory receptor FcγRIIa, which leads to inhibition of the functions of immunocyte.

Example 13 Screening functions of immunologically active biomimeticsin vivo

[00331] Numerous autoimmune diseases such as idiopathic thrombocytopenic purpura, multiple sclerosis, asthma and inflammatory bowel disease are recognized, well-known models for animal testingin vivo(Wu GF, Laufer TM. The role of dendritic cells in multiple sclerosis. Curr Neural Neurosci Rep. 2007 May;7(3):245-52; Targan SR, Karp LC. Defects in mucosal immunity leading to ulcerative colitis. Immunol Rev. 2005 Aug;206:296-305). For example, in a real�the time available numerous models, ETC. See, for example, Crow AR, et al. IVIG inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity. Br J Haematol. 2001; 115:679-686. Immunologically active biomimetics intended for modulation of the immune system, as appropriate for each specific autoimmune diseases, can be evaluated in suchin vivomodels. Importantly, in many of the models, the introduction of hIVIG is likely to result in immune response to foreign (e.g., in mice) human antibodies that can drown out or give a false positive result associated with anti-inflammatory effects.

[00332] a Murine model of idiopathic thrombocytopenic purpura was developed according to the following method: the number of thrombocytes in C57BL6 mice was measured periodically producing an incision of the tail vein, 10 µl of blood was diluted in 15 μl of citrate buffer. Then the samples were analyzed by the absolute number of platelets using flow cytometer HemaVet 950. In mice in ITP the control group, starting from the 2nd day, daily reduced the number of platelets, making intraperitoneal injection of 2 µg anti-rat platelet antibodies against CD41 mice (MWReg30) (BD Biosciences pharmingen). Mouse in prophylactic IVIG control group each morning I/p injection of 2 g/kg (40 mg/mouse) of human IVIG and MWReg30 at the same dose as in ITP the control group. Some�or, that IVIG provides effective protection in the number of platelets in this model induced ITP, and concluded that this model can be used to test strathmere in comparison with IVIG on the subject of the relative degree of protection from the reduction in the number of platelets. Streamer can be evaluated in this model at different concentrations to evaluate the protection compared to IVIG, as follows:

Group in an experiment

1) Control - no ITP, no IVIG

2) ITP control group - 2 µg MWReg30 every evening, starting from the 2nd day

3) Group IVIG prophylaxis 40 mg IVIG every morning and 2 µg MWReg30 every evening, starting from the 2nd day

4) Streamer equivalent to 10112 Fc-domains IV every morning

5) Streamer equivalent to 10111 Fc-domains IV every morning

6) Streamer equivalent to 10110 Fc-domains IV every morning

7) Streamer equivalent to 1019 Fc-domains IV every morning

8) Streamer equivalent to 1018 Fc-domains IV every morning

9) Streamer equivalent to 1017 Fc-domains IV every morning

10) Streamer equivalent to 1016 Fc-domains IV every morning

Example 14 - assessment of the effectiveness of immunologically active biomimeticsin vivoin the treatment of ITP

[00333] In another murine model of ITP can be used the mouse with deficiency of normal function of B-cells. The deficit in the normal function of B-cells is used to eliminating idiotype- �antiidiotypic effects of murine antibodies against human Fc-fragment, which are produced with the introduction of mouse Fc-fragment or incomplete Fc-fragment of human rights and lead to false-positive results. A deficiency in the function of B-cells can be caused, for example, by the introduction of antibodies against B-cells or obtained in genetically engineered mouse strains, such as knock-out mice (Jackson Labs, line B6.129S2-Igh6tmlCgn/J) with a deficit of Mature B-cells.

[00334] the Immune system of immunodeficient mice regenerate by either unmodified or purified B-cells from IPC immunocompetent animals. Then these animals are treated with antibodies to platelets, reproducing ETC using well-known methods in the art. After that, animals are treated immunologically active biomimetics according to the following pattern:

Table 5
In vivothe effectiveness of immunologically active biomimetics [Fc-domain of hIgG1- Fc-domain hIgGi] (SEQ ID NO: 22) in the treatment of ITP
GroupNumber of animalsThe IPC used to restore the mouseTreatmentMeasured parameter
15 NoFc IgG1The number of platelets
25NoStreamer IgG1 Fc-IgG1 FcThe number of platelets
35UnmodifiedFc IgG1The number of platelets
45UnmodifiedStreamer IgG1 Fc-IgG1 FcThe number of platelets
55Without b-cellsFc IgG1The number of platelets
65Without b-cellsStreamer IgG1 Fc-IgG1 FcThe number of platelets
75UnmodifiedhIVIGThe number of platelets

[00335] Expected in groups 1 and 2 ITP nebudet to evolve with the introduction of antibodies, as these mice have no B-cells to produce antibodies against platelets, necessary for the destruction of platelets. Expected in groups 3 and 4 and streaminy polypeptide {hIgG Fc1-HIgG Fc1} and Monomeric polypeptide hIgG Fc1will effectively improve the state with ITP because of the endogenous mouse antibodies, epitopes interacting with the Fc-domain hIgGi, cross-linked Monomeric Fc polypeptides hIgG1. On the contrary, in the absence of endogenous mouse antibodies streaminy polypeptide {hIgG Fc1-HIgG Fc1} (group 6) was more effective than non-cross-linked Monomeric Fc polypeptide IgG1(group 5) to improve ETC. Group 7 served as the positive control treatment effect.

Example 15 Treatment of patients with ITP with intravenous forms strathmere protein (SEQ ID NO: 18 and 22)

[00336] In the treatment of ITP with application examples strathmere proteins encoded by SEQ ID NO.: 17 and 21, used the methodology based on regulatory guidelines for hIVIG therapy for ITP, such as practical guidance of the Executive Committee of the American society of Hematology for diagnosis and therapy of primary immune thrombocytopenic purpura. Cm. George JN, et al. Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology. Blood. 1996 Jul 1;88(l):3-40; Cm. also guide pediatricians heme�of olegov Italy (2000), leadership hematologists UK (2003) and management of pediatric hematologists Japan (2006). In the alternative, the scheme of application of strathmere IV with ITP may include the initial phase of the introduction of dosages comprising from about 0.1 to about 0,001 from the specified dosing regimen. The initial phase with a low dose is designed to minimize any short-term Pro-inflammatory effects when administered strathmere, still being sufficient to cause long-term anti-inflammatory effect, which is subsequently amplified and supported in the second phase of the standard dosage described above. The rationale for this alternative approach is that some embodiments of strathmere can provide short-term inflammatory effect, and prolonged anti-inflammatory effect by reducing the expression of FcγRIIa. Initial low dose (or initial low dose) can be used to stimulate prolonged anti-inflammatory effect, minimizing short-term inflammatory effect.

[00337] the Effective dose of strathmere usually ranges from approximately 0.01% to approximately 15% of the effective dose hIVIG, more preferably, from about 0.1% to about 3% of the effective dose hIVIG. Effective dose hIVIG at ITP �typically is in the range of approximately 100 mg/kg to about 2 grams/kg, enter every 10-21 days.

[00338] a Dosage form of strathmere for intravenous administration is essentially the same as dosage forms hIVIG, approved by the FDA, however, may not contain stabilizers, prisutstvie in some dosage forms hIVIG. See, for example, the leaflet of the drug Gammagard S/D, distributed by Baxter Healthcare Corporation and FDA approved for the treatment of ITP.

Example 16 Treatment of patients with ITP using intraperitoneal administration of cor-strathmere

[00339] the scheme of treatment for ITP with application examples strathmere proteins, which are Fc-fragments attached to KOR group, such as a liposome, include intraperitoneal administration with doses constituting from about 1% to about 0.001% of standard dosages in schemes for intravenous IVIG. The rationale for this alternative approach is that cor-strathmere consisting of related fragments Fc, delivered in a stable dosage form inside the abdominal cavity, make available the impact of multiple Fc-domains on monocyte-derived effector cells, like IVIG, but at significantly lower doses.

Example 17 - a Design of immunologically active biomimetics (stradal)

[00340] was Designed and transfusional two Stradale. For each Stra�calving on the basis of total RNA, derived from hybrid cell lines, expressindia target antibody, synthesized cDNA encoding. The establishment of hybrid cell lines known in the prior art. Amplification of the target cDNA encoding the heavy and light variable regions of the antibody was performed using a kit for amplification of BD SMART™ RACE (Clontech, CA). To obtain the cDNA encoding the heavy and light chain variable regions of antibodies, numerous other available methods (Sassano, M. et. al., 1994. Nucl Acids Res. May 11;22(9):1768-9; Jones, S. T., Bendig, M. M., 1991. Biotechnology (NY) Feb:9(1):88-9). To obtain stratatel heavy variable region chain merge with strdomainname designs either by PCR with elongation of overlapping fragments (Hutton and Pease), or present use compatible restriction sites for fusion of the respective fragments. Stratatel expressible in cells CHO-S and was isolated from cell supernatants by affinity purification on a column And protein. Binding of purified Stradale with the target antigen was confirmed by testing for binding using flow cytometry, using cell lines expressing the antigen.

[00341] the Standard analysis of ADCC using NK cells as effectors and antigen-expressing tumor cells as targets at various ratios of effector/target, was used for CP�of Vania efficiency Stradale and monoclonal antibodies (Mab), having the same Fab-domain in the induction of ADCC against tumor cell lines expressing the antigen with high and low level. To further improve chose Stradale, which showed results similar to the results of the coupled Mab to NK-analysis against cell lines with high level of expression of epitopes, but excellent results compared with paired Mab to NK-analysis against cell lines with low expression of epitopes.

Example 18 Treatment of patients with breast cancer using intravenous dosage forms stratatel containing the antigen-binding domain of Trastuzumab

[00342] In the treatment regimens for breast cancer with the use of example stratatel containing Fab similar to Fab from commercial drug trastuzumab, which has activity against the epitope of her2/neu, used the methodology based on regulatory guidelines for the treatment of breast cancer. Cm. Romond EH et. al. Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer. NEJM. 2005 Oct. 20; 353:1673-1684; Seidman, et AD. al. Weekly Trastuzumab and Paclitaxel Therapy for Metastatic Breast Cancer With Analysis of Efficacy by HER2 Immunophenotype and Gene Amplification. Journal of Clinical Oncology. Vol 19, Issue 10 (May), 2001: 2587-2595; Vogel, CL et. al. Journal of Clinical Oncology. Vol 20, Issue 3 (February), 2002:719-726

[00343] it is Expected that the effective dose stratatel varies in the range from about 1% to about 500% e�effective effective dose of a monoclonal antibody Fab in which similar Fab Stradale, more preferably, from about 50% to about 100% of the effective dose of a monoclonal antibody. In clinical cancer therapy effective dose of a monoclonal antibody varies. For monoclonal antibodies to Her-2 neu dose usually ranges from approximately 2 mg/kg to about 4 mg/kg, administered every 7-21 days.

Example 19 Treatment of patients with head and neck cancer or colon cancer using intravenous dosage forms stratatel containing the antigen-binding domain of Cetuximab

[00344] it is Expected that the scheme of treatment of breast cancer with the use of example stratatel containing Fab, or similar which is Fab commercial cetuximab with activity against EGFR epitope, include methods based on regulatory guidelines for the treatment of head and neck cancer and colon cancer. Cm. Robert, F et. al. Phase I Study of Anti-Epidermal Growth Factor Receptor Antibody Cetuximab in Combination With Radiation Therapy in Patients With Advanced Head and Neck Cancer. Journal of Clinical Oncology, Vol 19, Issue 13 (July), 2001:3234-3243; Bonner, JA et. al. Cetuximab prolongs survival in patients with locoregionally advanced squamous cell carcinoma of head and neck: A phase III study of high dose radiation therapy with or without cetuximab. Journal of Clinical Oncology, 2004 ASCO Annual Meeting Proceedings (Post-Meeting Edition).Vol 22, No 14S (July 15 Supplement), 2004:5507; Shin, DM et. al. Epidermal Growth Factor Receptor-targeted Therapy with C225 and Cisplatin in Patients with Head and Neck Cancer. Clinical Cancer es Vol. 7, 1204-1213, May 2001; Cunningham, D et al. Cetuximab Monotherapy and Cetuximab plus Irinotecan in Irinotecan-Refractory Metastatic Colorectal Cancer. NEJM. Volume 351:337-345, 2004.

[00345] it is Expected that the effective dose stratatel to EGFR/HER1 varies in the range from about 1% to about 500% of the effective dose of a monoclonal antibody Fab which is similar to Fab Stradale, more preferably, from about 50% to about 100% of the effective dose of a monoclonal antibody. In clinical cancer therapy effective dose of a monoclonal antibody varies. For monoclonal antibodies, EGFR dose is usually in the range of approximately 250-400 mg/square meter, which is approximately 5 mg/kg 25 mg/kg, administered every 7-21 days.

Example 20 is an Enlarged multimerization as a result of altered glycosylation can increase the activity of immunologically active biomimetics

[00346] the glycosylation Profiles of a murine protein-dependent cell line in which the protein is expressed. The cell is Chinese hamster ovary (CHO cell), typically used for protein expression and purification, gives the glycosylation profile that is different from, for example, glycosylation in cells HEK 293, which are of human origin and are also widely used for expression of endogenous proteins. Because the profile of glycosylation can affect with�STS bind Fc fragments and the cor-strathmere units increased multimerization and, as a consequence, the increased biological activity of the expressed peptides can be achieved by expression in other cell lines than CHO, or cell lines, including genetically modified CHO with a modified glycosylation profile on N-glycans, which contributes to increased aggregation between Fc-fragments or peptides containing a Fc-domain. Increased multimerization Fc-fragment or selected cluster strathmere units by modifying the glycosylation profiles can enhance the ability of immunologically active biomimetics to reproduce the effects of hIVIG.

Example 21 - Changes to IVIG or rFcF (recombinant Fc-fragments) phenotype of Mature DC (mDC) as they are processed?

[00347] the Fragments rFCF of human IgG1 used in this experiment were obtained using standard techniques of genetic engineering of proteins. Each of the two chains of the human rFCF consisted of a hinge region (15 amino acids), CH2 domain (110 amino acids) and CH3 domain (106 amino acids) to the heavy chain of human IgG1.

[00348] CD14+ cells can be isolated from peripheral blood mononuclear cells (IPC), obtained from the blood of healthy human donors using separation columns Miltenyi MACS. Cells were cultured at a final concentration of 2×105/ml in the presence of GMCSF (80 IU/ml) and IL-4 (5 ng/ml) for 5 days at 37°C. Environment in all cultures was updated on the 3rd day of cultivation. On the 5th day in a suitable culture was added lipopolysaccharide (LPS; 10 µg/ml) to induce maturation into Mature dendritic cells. Mature dendritic cells are known in the prior art, does not Express significant levels of CD16, CD32 or CD64. Then cells were cultured for another two days and aliquots were analyzed on the expression of CD11c, CD80, CD83, CD86, CD1a and CD14 by two-dimensional fluorescence flow cytometry (FFC). Then the remaining cells cultivated with LPS, were placed in the wells with soluble or immobilized IVIG or human rFcF (all at a concentration of 10 μg/ml) for 24 hours at 37°C, then collected and analyzed the expression of the above markers using two-dimensional FFC.

[00349] the Experimental groups were as follows:

(1) CD14+ cells; GMCSF; IL-4; without LPS ("7d-FSC")

(2) CD14+ cells: GMCSF; IL-4; LPS ("7d+LPS")

(3) CD14+ cells; GMCSF; IL-4; LPS; immobilizovannyi IVIG ("cIVIG")

(4) CD14+ cells; GMCSF; IL-4; LPS; soluble IVIG ("sIVIG")

(5) CD14+ cells; GMCSF; IL-4; LPS; immobilizovannyi rFcF ("cFc")

(6) CD14+ cells; GMCSF; IL-4; LPS; soluble rFcF ("sFc")

(7) CD14+ cells; GMCSF; IL-4; LPS ("Control")

Example 22 - does the processing iDC immobilized IVIG to inhibition of phagocytosis opsonization of red blood cells?

[00350] CD14+ cells were purified from human� IPC healthy human donor, as described in Example 21, and were cultured at 37°C for 6 days in the presence of GMCSF and IL-4 at concentrations indicated in the previous examples and in the presence or absence of immobilized or soluble IVIG. Then the cells were collected and incubated at 37°C or at 4°C for two hours with human rhesus-positive (Rho+) erythrocytes, coated or not coated with antibodies against D-factor (Rho), by fluorescein isothiocyanate conjugated (FITZ). After incubation with red blood cells CD14+ cells were stained APC-conjugated CD1a. Then phagocytosis was assessed using two-dimensional FFC, measuring the side scattering (SSC-A), forward scattering (FSC-A), fluorescence FITZ (FITC-A) and fluorescence AFC (CDIa).

Example 23 - does the treatment of immobilized IVIG to iDC reduced capacity to stimulate allogeneic reaction of the mixed culture of lymphocytes

[00351] CD14+ cells were isolated from blood of a healthy human donor, as described in the previous examples. Then they were cultured at 37°C for 6 days with GMCSF and IL-4 in the presence or absence of soluble and immobilized IVIG. The concentrations of all of these reagents are the same as described above. Then the cells were collected and seeded into the wells of 96-well microplates for tissue culture in different density (the maximum dose was 2.5×104cells � hole). CD3+ T-cells were purified from the IPC of the second person-the donor, who was HLA-incompatible with the donor, from which were derived CD14+ cells. T-cells were introduced into each well of 96-hole tablet for tissue culture (105T-cells per well). After five days of co-cultivation in each well was added 1 μci3H-thymidine. Then cultures were incubated for 6 hours, and then measured the uptake of3H-thymidine (counts/min) as an indicator of the degree of cell proliferation in cultures. Tested three different iDC stimulant populations: one obtained by culturing only with GMCSF and IL-4, one obtained when cultured with GMCSF, IL-4 and immobilized IVIG, and one obtained when cultured with GMCSF, IL-4 and soluble IVIG.

Example 24 - iDC treatment Effect of immobilized and soluble rFcF and IVIG on the expression of cytokines in iDC and mDC

[00352] Culture containing CD14+ cells, GMCSF and IL-4, and either rFcF (immobilized or soluble) or IVIG (immobilized or soluble), was maintained at the conditions described in the previous examples. Instead of testing cells for the expression of cell surface markers was assessed by phagocytic ability or ability to stimulate allogeneic SCR-reaction and the production of cytokines produced by cells. It was expected that immobilize�this rFcF modulates cytokine production in cells similar to IVIG, but not likewise soluble rFcF. Thus, it was expected that the level of cytokines, which inhibit inflammatory responses (e.g., interleukin-4, interleukin-6 and interleukin-12) will increase in the handling of immobilized cells rFcF. In addition, it was expected that treatment of cells immobilized rFcF will reduce the output level by the cells of cytokines that amplify the inflammatory response (e.g., interferon, interleukin-23 and tumor necrosis factor-a (I).

Example 25 - Recombinant murine Fc-fragments

[00353] the Recombinant Fc-fragments (rFcF) from mouse IgG2a was obtained, using standard methods of cloning and expression of recombinant proteins. Each of the two chains of the murine rFcF consisted of a hinge region (21 amino acid), CH2 domain (110 amino acids) and CH3 domain (107 amino acids) to the heavy chain of mouse IgG2a. Mouse IgG2a was active in the analysis at iDC man, when he was deposited on the sides and bottom of the wells.

[00354] Although the present invention and its advantages have been described in detail, it should be understood that the present invention may be made various changes, substitutions and modifications without deviation from the essence and scope of the invention defined in accordance with the attached formula. In addition, the volume of this statement should not be limited to specific variants of implementation�Oia ways devices, methods of manufacture, compositions, means, methods and stages set forth in the description. The average specialist in the art from the description of the present invention, it is obvious that the methods, devices, methods of manufacture, compositions, means, methods or stages that are present as of the moment, or may be later developed that perform essentially the same function or reaching essentially the same result as the corresponding embodiments of described in this application can be used according to the present invention. Thus, the attached formula of the invention includes methods, devices, methods of manufacture, compositions, means, methods or stages. All U.S. patents and foreign patents, patent applications and non-patent literature (including, but not limited to, abstracts, articles in scientific journals, books, shipping literature and guidelines) referred to or cited in this application are hereby fully incorporated by reference.

The LIST of references

The following references are fully incorporated by reference.

1. Smiley, D. & MG, T. Southwestern internal medicine conference: High dose intravenous gamma globulin therapy: How does it work?Am JMed Sci309, 295-303 (1995).

2. Nimmerjahn, F. &Ravetch, J. V. The antiinflammatory activity of IgG: the intravenous IgG paradox.J. Exp. Med.204, 11-15 (2007).

3. Samuesson, A., Towers, T. L. &Ravetch, J. V. Anti-inflammatory Activity of IVIG also been other ideas where Through the Inhibitory Fc Receptor.Science291, 484-486 (2001).

4. Follea, G. et al. Intravenous plasmin-treated gamma globulin therapy in idiopathic thrombocytopenic purpura.Nouv Rev Fr Hematol27, 5-10 (1985).

5. Solal-Celigny, P., Bernard, J., Herrera, A. & Biovin, P. Treatment of adult autoimmune thrombocytopenic purpura with high-dose intravenous plasmin-cleaved gammaglobulins.Scand J Haematol31, 39-44 (1983).

6. Made significant gains, M. & Bonnet, M.-C. Infusion of Gcgamma fragments for treatment of children with acute immune thrombocytopenic purpura.Lancet342, 945-49 (1993).

7. Burdach, S. E., Evers, K. & Geurson, R. Treatment of acute idiopathic thrombocytopenic purpura of childhood with intravenous immunoglobulin G: Comparative efficacy of 7S and 5S preparations.J Pediatr109, 770-775 (1986).

8. Siragam, V. et al. Intravenous immunoglobulin ameliorates ITP via activating Fe[gamma] receptors on dendritic cells.Nat Med12, 688 (2006).

9. Clarkson, S. et al. Treatment of refractory immune thrombocytopenic purpura with an anti-Fe gamma-receptor antibody.N Engl JMed314, 1236-1239 (1986).

10. Bleeker, W. K. et al. Vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase.Blood95, 1856-1861 (2000).

11. Teeling, J. L. et al. Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia.Blood98, 1095-1099 (2001).

12. Augener, W., Friedman, B. & Brittinger, G. Are aggregates of IgG from the effective part of high-dose immunoglobulin therapy in adult idiopathic thrombocytopenic purpura (ITP)?Blut50, 249-252 (1985).

13. Tankersley, D. L., Preston, M. S. & Finlayson, J. S. Immunoglobulin G dimer: An idiotypeanti-idiotype complex.Molecular Immunology25, 41 (1988).

14. Robert L. Shields, Angela K. Namenuk, Kyu Hong, Y. Gloria Meng, Julie Rae, John Briggs, Dong Xie, Jadine Lai, Andrew Stadlen, Betty Li, Judith A. Fox, and Leonard G. Presta. High Resolution Mapping of the Binding Site on Human IgG1 for FcγR1, FcγRI, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FOR J. Biol. Chem., Feb 2001; 276: 6591 - 6604; doi:10.1074/jbc.M009483200

15. Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000)Nature406, 267-273

16. Robert L. Shields, Jadine Lai, Rodney Keck, Lori Y. O'connell, Kyu Hong, Y. Gloria Meng, Stefanie H. A. Weikert, and Leonard G. Presta Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity. J. Biol. Chem., Jul 2002; 277: 26733 - 26740 ; oi:10.1074/jbc.M202069200

17. Ann Wright and Sherie L. Morrison. Effect of C2-Associated Carbohydrate Structure on Ig Effector Function: Studies with Chimeric Mouse-Human IgG1 Antibodies in Glycosylation Mutants of Chinese Hamster Ovary Cells. J. Immunol., Apr 1998; 160: 3393 - 3402.

18. Crow AR, et al. IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of antiidiotype reactivity. Br J Haematol. 2001;115:679-686.

19. Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin Jagadeesh Bayry, Sebastien Lacroix-Desmazes, Cedric Carbonneil, Namita Misra, Vladimira Donkova, Anastas Pashov, Alain Chevailler, Luc Mouthon, Bernard Weill, Patrick Bruneval, Michel D. Kazatchkine, and Srini V. Kaveri Blood 2003 101: 758-765. Prepublished online August 29, 2002; DOI 10.1182/blood-2002-05-1447

20. R. Deng and J. P. Balthasar. Comparison of the effects of antibody-coated liposomes, IVIG, and anti-RBC immunotherapy in a murine model of passive chronic immune thrombocytopenia. Blood, March 15, 2007; 109(6): 2470 - 2476. Prepublished online as a Blood First Edition Paper on November 28, 2006; DOI 10.1182/blood-2006-04-018093.

21. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991)Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda

22. U.S. Published Patent Application 20060074225.

1. Dimeric compound for the formation of multimer able to reproduce the effector function of aggregated IgG by multimerization che�ithout multimeasures region, which contains identical monomers, where each monomer contains:
(a) a monomer multimeasure region, which forms the dimerization multimeasures the area where the monomer multimeasure region represents a monomer hinge region of IgG2 monomer or isolationof lightning, and
b) at least one monomer Fc-domain that contains the hinge region, CH2 domain and CH3 domain of IgG1, which forms the dimerization of Fc-domain, which is able mainly to the binding of Fcγ-receptor.

2. Dimeric compound according to claim 1, wherein the monomer multimeasure region is monomer a hinge region of IgG2.

3. Dimeric compound according to claim 1, in which multimerization through the specified multimeasures region capable of binding to at least two FcR selected from the group comprising: FcγRI, FcγRII, FcγRIII, FcγRIV, or their non-human equivalents.

4. Multimeric compound is able to reproduce the effector function of aggregated IgG containing two or more dimeric compounds according to claim 1, which is able to bind at least two FcR selected from the group comprising: FcγRI, FcγRII, FcγRIII, FcγRIV, or their non-human equivalents.

5. A method of modifying an immune response, wherein the subject in need this, is administered a pharmaceutical composition, containing�th a therapeutically effective amount of the dimeric compound according to claim 1 or multimeric compound according to claim 4.

6. A method of treating an inflammatory disease, wherein the patient in need this, enter the multimeric compound according to claim 4.

7. A method according to claim 6, where the inflammatory disease is an autoimmune disease.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Described is a bispecific antigen-binding protein which is heterodimeric with respect to binding protein A. The protein contains a first polypeptide comprising, from N-terminal to C-terminal, a first epitope-binding region that selectively binds a first epitope, an immunoglobulin constant region that comprises a first CH3 region of a human IgG selected from IgG1, IgG2, and IgG4, where the first CH3 region binds with protein A, and a second polypeptide comprising, from N-terminal to C-terminal, a second epitope-binding region that selectively binds a second epitope, an immunoglobulin constant region that comprises a second CH3 region of a human IgG selected from IgG1, IgG2, and IgG4, where the second CH3 region comprises a modification that reduces or prevents binding of the second CH3 domain to protein A.

EFFECT: invention enables rapid isolation of protein A.

9 cl, 10 dwg, 3 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of purifying antibodies, including human CD20 and VEGF binding antibodies, from a composition which contains an antibody and at least one impurity compound, said method comprising the following steps: (a) loading the composition on a cation-exchange material, where said composition has first pH value from 4.0 to 6.0; (b) washing the cation-exchange material with a first washing buffer at pH higher than that of composition (a), where pH of the first washing buffer ranges from 6.8 to 9.0; (c) washing the cation-exchange material with a second washing buffer at pH lower than that of the first washing buffer; where the second washing buffer has conductivity from 0.5 to 3.0 mS/cm and pH from 5.0 to 6.0; and (d) elution of the antibodies from the cation-exchange material with an elution buffer at conductivity which is at least 2 mS/cm higher than that of the second washing buffer, where pH of the second washing buffer and pH of the elution buffer are approximately equal, and where pH of the elution buffer ranges from 5.0 to 6.0. Described is a conjugation method, which involves conjugating the purified product obtained using said method with a heterologous molecule. Also disclosed is a method of producing a pharmaceutical composition, which involves combining said purified product with a pharmaceutically acceptable carrier.

EFFECT: invention improves purification of antibodies used for treatment.

16 cl, 3 dwg, 4 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a method for producing high-affinity polyclonal antibodies. The method involves the stages as follows: a) application of an antibody solution on an affine sorbent with an immobilised antibody at an excess molar quantity of antigen antigen-specific antibodies; b) removal of proteins and low-affinity antibodies; c) removal of the bound medium-affinity antibodies by means of a moderately chaotropic agent, such as LiCl, MgCl2, glycine, d) elution of the high-affinity antibodies by means of the chaotropic agent, e) removal of the latter and f) if needed, removal of antibody aggregates. The method provides producing the antibodies affinity and specificity of which substantially exceeds those of the polyclonal antibodies included in the state of the art.

EFFECT: use of the antibodies prepared by the method according to the invention enables prominently increasing sensitivity and specificity of immunoassay techniques.

13 cl, 5 dwg, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to liquid chromatography. Disclosed is a method for chromatographic extraction of immunoglobulin, involving dissolution of a blood plasma protein fraction in a buffer solution, said blood plasma protein fraction being a residue A of blood plasma alcohol fractionation via a Cohn process. The obtained solution undergoes preliminary purification in two series-connected columns filled with a hydrophobic sorbent and anionite, respectively, and then passing a buffer solution through said two columns. After collecting the purified liquid fraction, which contains immunoglobulin, it is taken for solvent-detergent viral inactivation and then for chromatographic purification, which is carried out in a system of three series-connected columns filled with anionite, hydrophobic sorbent and cationite, respectively. The immunoglobulin from the column filled with cationite is eluted and columns with the anionite and hydrophobic sorbent are taken for regeneration.

EFFECT: invention enables to extract immunoglobulin from crude material - a residue A, obtained from blood plasma alcohol fractionation via a Cohn process, with high output and purity of the end product.

8 cl, 2 ex

FIELD: medicine.

SUBSTANCE: method of monomer antibody recovery from a compound containing large molecular weight antibody aggregates provides contacting of the compound with hydroxyapatite resin and elution of purified antibody from resin. There are also disclosed versions of such method providing protein A affinity chromatography, ion-exchange chromatography and hydroxyapatite chromatography.

EFFECT: methods allow producing a compound with the lower content of large molecular weight antibody aggregates.

23 cl, 4 dwg, 14 tbl, 11 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining a human immunoglobulin based preparation, and can be used in medicine. The preparation is obtained via purification of class G, A and M immunoglobulins isolated from the blood of HIV infected patients through affinity chromatography on a column with integrase-sepharose.

EFFECT: invention enables to obtain class G, A and M immunoglobulins isolated from the blood of HIV infected patients, capable of selectively splitting HIV integrase only.

7 dwg, 4 ex

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

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to manufacturing of medical products. There is disclosed method for producing immunoglobulin G that involves ethanol fractionation of donor plasma to make sediment B and purification of prepared immunoglobulin. The sediment B is dissolved in 0.9% saline pH 5.15 and mixed with acetate buffer solution 2 M and 53% ethanol, and centrifugated. Centrifugate is mixed with sodium hydrocarbonate at solution pH 5.5. After centrifugation, the centrifugate is purified, mixed with acetate buffer solution at pH 5.4, 96% (vol/vol) ethanol and sodium bicarbonate at pH 7.2, centrifugated at minus (10-12)°C. The recovered sediment is dissolved in acetate buffer solution 0.05 M at pH 5.5, added with solvent detergent mixture containing acetate buffer solution 0.05 M at pH 5.5, and 1 wt % tri-n-butylphosphate and 1 wt % polysorbate 80. The mixture is stirred, then diluted 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. Immunoglobulin processed in such a way is immobilised and two-stage washed on sulphoprolylcation sorbent by column chromatography followed with elution, ultrafiltration and sterilisation filtration.

EFFECT: invention provides high-degree purification of immunoglobulin G, absence of viruses and stability of ready preparation.

3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and biotechnology, and method of obtaining immunoglobulin medicines. Method of obtaining immunoglobulin medicines involves a sequential processing of alcohol sediment B (Cohn III fraction) by tenfold volume of 0.9% sodium chloride solution and by chloroform in 0.1%-3.0% end concentration, centrifugation, addition of copper sulfate to centrifugate at pH 6-8, sediment removal by centrifugation, and further extraction of target product by ultrafiltration method in the presence of complex-forming compounds, product stabilisation, clearing and stabilising filtration. Copper sulfate is taken in 0.003-0.03% concentration.

EFFECT: increased content of separated immunoglobulin A fraction, improved product quality.

3 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns medicine, in particular, to immunology, and can be used for immunotherapy and immunologic prophylaxis of bacteriemic and virus diseases. The base for immunobiological preparations is offered; it contains IgG, IgM and IgA, and also subclasses IgG, IgG1, IgG2, TgG3 and IgG4, thus the content of immunoglobulins makes: IgG 70-80%, IgM 10-15% and IgA 10-15%. The way of reception of immunoglobulin marker base for immunobiological preparations is offered. Suppositories and ointment for prevention and therapy of bacteriemic and virus diseases are offered.

EFFECT: development of an economic and effective way of processing of a deposit A (II+III according Cohn), allowing to receive highly effective paste for immunoglobulin marker preparations.

4 cl, 5 ex, 2 tbl, 2 dwg

FIELD: veterinary medicine.

SUBSTANCE: product comprises Lycopodium clavatum, Acidum arsenicosum, Phosphorus, Podophyllum peltatum, Thuja occidentalis, Echinacea purpurea, Silybum marianum, Selenocysteine, and the components are taken in the dilutions described below in the following ratio, in parts: Lycopodium clavatum ⌀=D1 0.004, Podophyllum peltatum ⌀ 0.003, Acidum arsenicosum ⌀=D2 0.0001, Phosphorus ⌀=D3 0.001, Thuja occidentalis ⌀ 30, Echinacea purpurea ⌀ 30, Silybum marianum ⌀ 60, Selenocysteine 0.2.

EFFECT: product has an effective stress-protective and growth-stimulating effect, it regulates the metabolism in young farm animals.

3 cl, 10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method for producing an agent for stimulating body cells involving preparing a mixture of aqueous solution of selenious acid and PEG 400; that is followed by preparing a mixture of hydrazine hydrochloride and PEG 400; the prepared mixtures are combined; the solution is put to dialyse against distilled water; surplus of water is driven off; the produced solution is added with hexamethylene tetramine; pH is reduced to 7.2-7.4; the method is implemented in certain circumstances.

EFFECT: producing high-effective, ecologically safe agent by the synergism of colloidal selenium and hexamethylene tetramine on body cell stimulation.

1 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine and can be used for the treatment of radiation-thermal injury of an organism. For this purpose a single subcutaneous introduction of bifidumbacterin, irradiated by gamma-rays in a dose of 14.0 Gy, is carried out. Bifidumbacterin is introduced in a dose of 1.43·106 CFU/kg. After that, 10% hypericum oil is applied on the burnt region. Then a 10% hypericum cream is applied after 3-4 days.

EFFECT: method makes it possible to carry out the treatment of combined radiation-thermal injuries in an effective way with the application of available and cheap pharmacotherapeutic means.

1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: erythrocyte cell medium is added with an aqueous solution of sodium and potassium salts of humic acids prepared on brown coal of leonardite in a dose of 10.0 mg/kg. That is incubated at a temperature of 37°C for 40 minutes before treatment with acidic haemolytic.

EFFECT: invention enables normalising the cell membrane permeability and reducing the damaged cell count under acidic haemolytic.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a new intensifier of the antitumour effect, which is a uracil derivative of the general formula (I) or its pharmaceutically acceptable salt. In the general formula (I) X represents a C1-5-alkylene group, and wherein one of methylene groups making an alkylene group is optionally substituted by an oxygen atom; R1 represents a hydrogen atom or a C1-6-alkyl group; R2 represents a hydrogen atom or a halogen atom; and R3 represents a C1-6-alkyl group, C2-6-alkenyl group, C3-6-cycloalkyl group, (C3-6-cycloalkyl)-C1-6-alkyl group, halogen-C1-6-alkyl group or a 5-6-merous saturated heterocyclic group with an oxygen atom as a heteroatom, a uracil derivative presented by the following formula (I). The invention also refers to a method for potentiating the antitumour action or a method of treating tumours, involving administering an effective amount of a combination of the above uracil derivative or its pharmaceutically acceptable salt and an antimetabolite in an effective amount. The antimetabolite represents an agent specified in 5-fluoruracil (5-FU), potassium tegafur/gimeracil/oteracil (TS-1), tegafur/uracil (UFT), capecitabin, 5-fluor-2'-deoxyuridine (FdUrd) and Pemetrexed.

EFFECT: preparing the intensifier of the antitumour effect.

18 cl, 10 dwg, 10 tbl, 67 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to methods of purification and health improvement of an organism. For this purpose therapeutic starvation for not fewer than 5 days in case of a 7-day programme and for not fewer than 7 days in case of a 9-day programme is carried out. The duration of a recovery period constitutes two days. Food intake is realised nine times per day each day both in the process of therapeutic starvation and in the recovery period. In the period of therapeutic starvation the first food intake includes bee products "APIGRANULES 2" in a dose of one teaspoon, "KHINAZI balm" in a dose of one teaspoon, "A-P-V" in a dose of one teaspoon, "APITOK" in a dose of one teaspoon, "Antihelm phyto" - one capsule and still mineral water - one glass. The second food intake includes a drink with ginger, lemon juice, garlic and mint, honey - one teaspoon and one glass of apple-carrot juice. The third food intake supposes an intake of bee products "APIGRANULES 2" in a dose of one teaspoon, "KHINAZI balm" in a dose of one teaspoon, "A-P-V" in a dose of one teaspoon, "Antihelm phyto" - one capsule and still mineral water - one glass. The fourth food intake includes a drink with ginger, lemon juice, garlic and mint, honey - one teaspoon and one glass of apple-carrot juice. The fifth food intake consists of tea black or green with ginger, one teaspoon of honey and one glass of apple-carrot juice. The sixth food intake consists of bee products "APIGRANULES 2" in a dose of one teaspoon, "A-P-V" in a dose of one teaspoon, "Antihelm phyto" - one capsule and one glass of still mineral water. The seventh food intake includes tea black or green with ginger, one teaspoon of honey and one glass of apple-carrot juice. The eighth food intake supposes an intake of tea black or green with ginger, one teaspoon of honey and one glass of apple-carrot juice. The ninth intake of food includes depressant tea, including mint grass, valerian root, fennel seeds, cumin seeds, epilobium or strawberry leaves and one glass of apple-carrot juice. In the recovery period in 1-st, 2-nd, 3-rd, 4-th and 6-th intakes of food the composition of products remains the same as in the process of starvation. For the fifth intake of food the patients take tea black or green with ginger, one teaspoon of honey and vegetable soup. For the seventh intake of food the patients take tea black or green with ginger, "MILK COCKTAIL WITH CHITOSAN". The eighth intake of food includes tea black or green with ginger, one teaspoon of honey, "APICAMPA" cereal. The ninth intake of food for the recovery period includes a baked apple, depressant tea. In addition, a number of procedures are carried out after each food intake during the period of therapeutic starvation and the recovery period. After the first food intake gymnastics "5 Tibetan pearls" is realised. After the second food intake exercises on training apparatuses, procedures of press-therapy, electrolypolysis and myostimulation are realised. After the third food intake exercises of therapeutic physical training or aerobics are performed. After the fourth food intake a rest in form of a walk, a halotherapy session, combined with a session of relaxation therapy are realised. After the fifth food intake a course of strip-plastic is carried out. After the sixth food intake massage by manual application with a peloid-based mixture or manual massage with honey is carried out. After the seventh food intake an infra-red sauna, shower, phytobath with medicinal herbs, honey, ginger, lemon juice is taken. After the eighth food intake a shower is taken.

EFFECT: method ensures effective health improvement of the organism with the preservation of the full value life style and a sense of a comfortable state in the starvation period, reduction of recovery period term after starvation, purification of the organism without loading on the gastrointestinal tract, weight loss, and improvement of the general state and workability of the patients.

3 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, specifically to a fused protein containing a variant of rodostomin, and can be used in medicine. An ανβ3 integrin selective polypeptide consisting of an amino acid sequence SEQ ID NO:1 conjugated on the N terminal by a linker amino acid sequence containing a combination of the amino acids glycine and serine with a variant of a human serum albumin (HSA) with SEQ ID NO:4.

EFFECT: invention enables the higher therapeutic effectiveness in the diseases related to ανβ3 integrin.

12 cl, 14 dwg, 2 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for the correction of the individual's functional state and performance. That is ensured by administering Semax neuroactive peptide in a dose of two drops in each nasal passage. That is followed by the electric current exposure covering a frontal-mastoid region at pulse length 0.2ms, current intensity 0.8mA and pulse train 800Hz for 40 min. The exposure is combined with at least 10 sessions of hyperbaric oxygenation at pressure 1.6 atm.

EFFECT: method provides the rapid and effective increase of performance in sportsmen, military men and individuals involved in the other professions related to significant physical and mental stress by improving the functions of various regions of brain cortex as a result of the selected complex exposure enabling the substantial vasodilatation and maximum tissue oxygenation.

1 tbl

FIELD: medicine.

SUBSTANCE: using a compound representing a bicyclic pyrimidine derivative as Hrf2 transcription factor activators.

EFFECT: compounds of general formula can be used preventively to increase body defences before operations with toxic chemicals and high radiation doses.

3 cl, 7 tbl, 11 ex, 9 dwg

FIELD: medicine.

SUBSTANCE: invention refers to methods for cell and tissue protection against a hypoxic injury and can be used for developing remedies for hypoxic and ischemic injuries. The developed method for protection is based on cell treatment by substances increasing a level of glutathionylation of Na,K-adenosine triphosphatase catalytic subunit that reduces to its activation. The obtained results can be actual for biological research institutions, medical facilities, particularly cardiologic clinics, as well as biotechnological companies for transplantology and tissue engineering.

EFFECT: presented method can be used for research activities aiming at creating the therapeutic agents for relieving tissue injuries, particularly myocardial tissue in hypoxia and hypoxia/reoxygenation.

6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. Presented are variants of anti-CD20 modified antibody or its antigen-binding fragment. Each of the variants is characterised by the fact that it contains a variable light and heavy chain domain, and induces a higher apoptosis level as compared to anti-B-Ly1 chimeric antibody. There are presented: a mixture of antibodies, wherein at least 20% of oligosaccharides in Fc domain have a branched chain and are not fucosylated, as well as a pharmaceutical composition for producing a therapeutic agent for a malignant haematological or autoimmune disease by using the antibodies or the mixture of antibodies. Described are: an expression vector, a based host cell, variants of coding polynucleotides, as well as a method for producing the antibody in the cell.

EFFECT: using these inventions provides the new antibodies with the improved therapeutic properties, including with increased binding of Fc receptor, and with the increased effector function that can find application for treating the malignant haematological or autoimmune disease.

32 cl, 3 ex, 9 tbl, 26 dwg

Up!