Affinity-matured crig versions
SUBSTANCE: invention refers to biochemistry, particularly to affinity-matured CRIg versions. Declared is the CRIg versions, which is an alternative complement pathway inhibitor at least twice stronger than human CRIg with a native sequence, and optionally possesses C3b-bindingaffinity at least twice as much. There are also declared a chimeric molecule and a pharmaceutical composition, both containing the above CRIg version. The CRIg version can be used for preparing a therapeutic agent for treating a complement-associated disease or condition.
EFFECT: invention enables improving the therapeutic effectiveness of CRIg polypeptides.
17 cl, 17 dwg, 4 tbl, 1 ex
OPTIONS CRIg WITH RIPE AFFINITY
The technical field to which the invention relates
The present invention relates to CRIg variants with ripe affinity. In particular, the present invention relates to CRIg variants having higher binding affinity to C3b and retaining the ability of the selective binding to C3b compared with C3.
The level of technology
The complement system
The complement system is a complex cascade of enzymes consisting of a series of serum glycoproteins, which are usually in the form of inactive proenzymes. There are three main ways of complement activation: the classical pathway, alternative pathway and the pathway mediated mannose-binding lectins, which are connected at the level of C3, where two similar convertase splits C3 education S3a and C3b.
Macrophages are specialized cells that possess a natural ability to recognize subtle differences in the structure expressed on the surface of cells identifying marks, so-called molecular patterns (Taylor, et al., Eur. J. Immunol. 33, 2090-1097 (2003); Taylor, et al., Annu. Rev. Immunol. 23, 901-944 (2005)). Although direct detection of such surface structures is a Central feature of innate immunity, opsonization call�ing receptors generic macrophages to mediate the uptake by the cell particles in the process of phagocytosis, increasing the efficiency and expanding the range of phagocyte recognition (Stuart and Ezekowitz, Immunity 22, 539-550 (2005)). The process of phagocytosis involves numerous interactions of receptors with ligands, and it is now known that different opsonins, including immunoglobulins, collective and components of complement, control the activity of cells required for the internalization of pathogenic microorganisms as a result of interaction with receptors of macrophages on the surface of cells (review in publication Aderem and Underhill, Annu. Rev. Immunol. 17, 593-623 (1999); Underhill and Ozinsky, Annu. Rev. Immunol. 20, 825-852 (2002)). Despite the fact that natural immunoglobulins encoded by the genes of germ cells, can detect a range of pathogens, most of opsonizing IgG is formed as a result of acquired immunity, so the effective clearance mediated by Fc-receptors, is not immediate (Carroll, Nat. Immunol. 5, 981-986 (2004)). The complement, on the other hand, quickly recognize molecules on the surface of the pathogen and stimulates the absorption of such particles of complement receptors (Brown, Infect. Agents Dis. 1, 63-70 (1991)).
The complement consists of more than 30 serum proteins that opsonizing a number of pathogenic microorganisms, so they can recognize the receptors of complement. Depending on original releaser Cass�hell can distinguish three ways (review publication Walport, N. Engl. J. Med. 344, 1058-1066 (2001)). All three paths share a common stage of activation of the Central component C3, but differ in the nature of recognition and the initial stages of the biochemical processes that cause the activation of C3. The classical pathway is activated by antibodies bound to the surface of the pathogen, which in turn are associated with the component of complement C1q, activating a cascade of serine proteases that eventually decompose with the formation of C3 to its active form C3b. Way, mediated by lectin-activated after recognition of the carbohydrate fragments of the proteins of the lectin. Currently identified three members of the specified path is: mannose-binding lectin (MBL), a family of SIGN-R1 lectins and picoline (Pyz et al., Ann. Med. 38, 242-251 (2006)). As MBL and picoline associated with serine proteases, which act as C1 in the classical way, activating components C2 and C4, causing the main stage C3. An alternative path different from the classic way and the path is mediated by the lectin, the fact that its activation occurs as a result of direct interaction of internal ester of C3 with recognizable fragments on the surface of the pathogen. Initial binding of C3 with an activating surface causes rapid amplification of C3b deposition under the influence of proteases alternatives�wow way factor b and factor D. it is Important to note that the classical path, or a path, mediated by lectins can also cause amplification of C3b deposition under the influence of factors b and D. In all three activation pathways of complement the main stage in opsonization is the transformation component C3 into C3b. Cleavage of C3 by enzymes of the complement cascades causes a nucleophilic attack complex thioether providing covalent binding of C3b to the surface antigen using domain thioether complex. The specified binding is the initial stage in opsonization complement. Subsequent proteolysis of the bound C3b causes the formation of fragments iC3b, S3s and C3dg, which are recognized by different receptors (Ross and Medof, Adv. Immunol. 37, 217-267 (1985)). This splitting eliminates the ability of C3b to continue to amplificate the deposition of C3b and activates downstream components of the complement cascade, including membranoatacking complex, is able to directly destroy the membrane. However, phagocytic macrophage receptors preferentially recognize C3b and its fragments; with a variety of education ester linkages C3-mediated opsonization is Central to recognition of pathogens (Holers et al., Immunol. Today 13, 231-236 (1992)), therefore, the receptors for different C3 cleavage products play an important role in the immune response of the host�.
C3 is a complex and flexible protein consisting of 13 different domains. The core of the molecule consists of 8 so-called domains macroglobulin (MG), which form a tightly Packed α - and β-chains of C3. In this structure embedded domains CUB (C1r/C1s, Uegf and Bone morphogenetic protein-1) and TED, with the latter domain contains complex thioether linkage that allows the covalent binding of C3b to the surface of pathogens. Other domains contain S3a or act as linkers and spacers internal domains. Comparison of the structures of C3b and S3a with C3 shows that the molecule undergoes significant conformational restructuring with each proteolysis, resulting in exposing not only the domain of TED, but also additional new surface molecules that can interact with cellular receptors (Janssen and Gros, Mol. Immunol. 44, 3-10 (2007)).
The C3 complement receptors on phagocytic cells
Known three receptor gene superfamily of complement: modules short consensus repeat (SCR) encoding CR1 and CR2, the members of CR3 and CR4 of the family of beta-2 integrins and member of CRIg of the superfamily of immunoglobulins Ig.
CR1 is a glycoprotein with a molecular weight of 180-210 kDa, consisting of 30 short consensus repeats (SCR), and plays an important role in the clearance of immune complex. Short to�sensously repeats (SCR) are modular structures consisting of about 60 amino acids, each of which has two pairs of disulfide bonds, providing the rigidity of the structure. High affinity binding to C3b and C4b by two separate sites, consisting of 3 SCR (review publication Krych-Goldberg and Atkinson, Immunol. Rev. 180, 112-122 (2001)). The structure of the C3b binding site, located within short consensus repeats (SCR) 15-17 glycoprotein CR1 (site 2), was determined from MRI (Smith et al., Cell 108, 769-780 (2002)) showed that the three modules are arranged in the direction from the globular phase to the end phase, if this flexibility ensures that the articulation on the websites 16-17. Determined by the structure of mutagenesis allowed us to identify positively charged surface area on the module 15, which is crucial for C4b binding. This plot together with the basic side chains of the module 16, which is located on the same surface CR1 required to bind C3b. The main function of CR1, originally described as a receptor of the immune adhesion (Rothman et al., J. Immunol. 115, 1312-1315 (1975)), is to capture the IC on the red blood cells to migrate to the liver for clearance (Taylor et al., Clin. Immunol. Immunopathol. 82, 49-59 (1997)). In the process of phagocytosis affects CR1 on neutrophils, but not on tissue macrophages (Sengelov et al., J. Immunol. 153, 804-810 (1994)). In addition to participating in the clearance of immune complexes, glycoprotein CR1 is strong in�ibition activation of the classical and alternative pathways as a result of its interaction with the relevant convertase (Krych-Goldberg and Atkinson, 2001, see above; Krych-Goldberg et al., J. Biol. Chem. 274, 31160-31168 (1999)). In a mouse CR1 and CR2 are the two products of the same gene generated by alternative splicing that are associated mainly with b-lymphocytes and follicular dendritic cells and generally regulate the response of b-cells (Molina et al., 1996). Murine functional equivalent CR1, Crry, inactivates the enzymes of the classical and alternative pathways and acts more as a natural regulator of complement activation, but not phagocytic receptor (Molina et al., Proc. Natl. Acad. Sci. USA 93, 3357-3361 (1992)).
CR2 (CD21) binds iC3b and C3dg and is the main receptor of complement, enhancing b-cell immunity (Carroll, Nat. Immunol. 5, 981-986 (2004); Weis et al., Proc. Natl. Acad. Sci. USA 81, 881-885 (1984)). The uptake of C3b-coated antigen cognate b-cells causes an increase in the signal transmitted by the receptor b-cell antigen. Thus, binding of co-receptor CD21-CD19-CD81 with the receptor b-cell antigen reduces the activation threshold of b cells and forms an important survival signal (Matsumoto et al., J. Exp. Med. 173, 55-64 (1991)). The binding site on CR2 iC3b was partially mapped at the boundary between domains TED and MG1 (Clemenza and Isenman, J. Immunol. 165, 3839-3848 (2000)).
CR3 and CR4 are the transmembrane heterodimers consisting of alpha-subunit (CD11b, respectively, or αMand CD11c or αX) and a common beta chain (CD18 or β2), to�which are involved in adhesion to extracellular matrix and other cells, and in recognition of iC3b. These receptors belong to the family of integrins and perform certain functions not only in phagocytosis, but also in the process of directed migration and movement of white blood cells, the formation and the co-stimulation of the synapse (review publishing Ross, Adv. Immunol. 37, 217-267 (2000)). Adhesiveness of integrin is regulated by a process called inverted transmission signal, transforming integrins from discouraging in high-affinity state of the binding (Liddington and Ginsberg, J. Cell Biol. 158, 833-839 (2002)). In addition, the binding of ligand causes transmission of signals from the extracellular domain to the cytoplasm. The iC3b binding sites have been mapped in several domains of the alpha-chain of CR3 and CR4 (Diamond et al., J. Cell Biol. 120, 1031-1043 (1993); Li and Zhang, J. Biol. Chem. 278, 34395-34402 (2003); Xiong and Zhang, J. Biol. Chem. 278, 34395-34402 (2001)). Several ligands for CR3: iC3b and beta-glucan and ICAM-1 appears to bind to partially overlapping sites in the I domain of CD11b (Balsam et al., 1998; Diamond et al., 1990; Zhang and Plow, 1996). Specific recognition proteoliticeski inactivated form of C3b, iC3b, was predicted on the basis of structural studies, which allowed to localize the binding site of CR3 in the residues that are opened when you deploy the CUB domain in C3b (Nishida et al., Proc. Natl. Acad. Sci. USA 103, 19737-19742 (2006)) that occurs with the cleavage of the α'-chain regulatory protease complement factor I.
CRIg �is associated with macrophage receptor, homologous antigen A33 and JAM1, which are necessary for the clearance of pathogens from the bloodstream. Protein human CRIg was originally cloned from a library of fetal human cDNA using degenerate primers recognizing conservative Ig domains JAM1 person. Sequencing of several clones revealed an open reading frame consisting of 400 amino acids. Studies using Blast confirmed the similarity of Z39Ig, a transmembrane protein type 1 (Langnaese et al., Biochim. Biophys. Acta 1492 (2000) 522-525). It was found that the extracellular region of the molecule consists of two Ig-like domains, including the N-terminal domain of the V-population and the C-terminal C2 domain-populations. New human protein was originally defined as ”a single transmembrane member of the Ig superfamily that is associated with a macrophage (huSTIgMA)”. Then, when the 3'- and 5'-end of primers was cloned playeronly option huSTIgMA, in which there is no close to the membrane domain IgC and which is shorter than 50 amino acids. Shorter playeronly variant of this human protein has been called huSTIgMA. Amino acid sequence huSTIgMA (defined as PRO362) and encoding a polynucleotide sequence described in U.S. patent No. 6410708, issued June 25, 2002 in addition, huSTIgMA and huSTIgMA and murine STIgMA protein (muSTIgMA) and�sequence of nucleic acids are described in PCT publication WO 2004031105, published April 15, 2004
Crystal structure of CRIg and the complex C3b:CRIg described in the publication of the patent application U.S. No. 2008/0045697 published February 21, 2008
Kupffer cells (KC) in the sinusoidal cavity liver, form the largest population of macrophages in the body. Despite the fact that Kupffer cells have markers similar to other tissue macrophages, they perform special functions that ensure the efficient clearance of intestinal bacteria, microbial residues, bacterial endotoxins, immune complexes and dead cells present in the blood of the portal vein coming from the microvascular system of the digestive tract (Bilzer et al., Liver Int. 26, 1175-1186 (2006)). Efficient binding of pathogenic microorganisms to the surface of Kupffer cells is an important stage in the primary immune defense against pathogenic microorganisms (Benacerraf et al., J. Exp. Med. 110, 27-48 (1959)). The Central role of Kupffer cells in the rapid clearance of pathogens from the bloodstream is illustrated a significant increase in mortality in mice with absence of Kupffer cells (Hirakata et al., Infect. Immun. 59, 289 to 294 (1991)). Identification of CRIg even more confirmed the important role of complement and Kupffer cells in primary immune protection against pathogenic microorganisms circulating in the bloodstream.
The only receptors of complement C3, IDA�tapicerowane in the Kupffer cells of mice are CRIg and CR3 (Helmy et al., Cell 124, 915-927 (2006)), while in the Kupffer cells of the person revealed additional expression of CR1 and CR4 (Hinglais et al., 1989). The receptor CRIg and CR3 in Kupffer cells contribute to the binding opsonization particles iC3b in vitro (Helmy et al., Lab. Invest. 61, 509-514 (2006)). In vivo role of the COP-expressed receptor CR3-binding iC3b-coated pathogens is less clear. It has been suggested that CR3 indirectly contributes to the clearance of pathogens by neutrophils recruitment and interaction with expressing neutrophils ICAM1 (Conlan and North, Exp. Med. 179, 259-268 (1994); Ebe et al., Pathol. Int. 49, 519-532 (1999); Gregory et al., J. Immunol. 157, 2514-2520 (1996); Gregory and Wing, J. Leukoc. Biol. 72, 239-248 (2002); Rogers and Unanue, Infect. Immun. 61, 5090-5096 (1993)). In contrast, CRIg is directly involved in the capture of pathogens passing through the sinusoidal cavity liver (Helmy et al., 2006, see above). Distinction in biology and CRIg CR3 partially reflected in the variation in the characteristics of binding to these two receptors. CRIg, expressed on Kupffer cells, is associated with constitutive Monomeric fragments of C3, while CR3 is associated only with iC3b-opsonization particles (Helmy et al., 2006, see above). The ability of CRIg to effectively trap the monomer components C3b and iC3b and C3b/iC3b-coated particles reflects the improved avidity, of a spec�Yoo multiple interactions between molecules CRIg, focused on the end extensions of the membrane of macrophages (Helmy et al., 2006, see above), and mnogomerna C3b and iC3b, present on the surface of the pathogen. While CR3 is associated only with iC3b-coated particles, CRIg additionally binds to C3b, the first C3 cleavage product derived from pathogenic microorganisms, opsonizing serum (Croize et al., Infect. Immun. 61, 5134-5139 (1993)). Since a large number of C3b molecules associated with the surface of the pathogen, protected from splitting by factors H and I (Gordon et al., J. Infect. Dis. 157, 697-704 (1988)), recognition of ligands C3b receptor CRIg provides rapid binding and clearance. Thus, despite the fact that the Kupffer cells expressed both CRIg and CR3, these receptors are characterized by different specificity for ligands with different binding properties and different kinetics of clearance of pathogens.
Examples of pathogens that use receptors on the cell surface for penetration into the cell, are viruses such as human immunodeficiency virus (HIV), intracellular bacteria such asMycobacterium tuberculosum, Mycobacterium leprae, Yersinia pseudotuberculosis, Salmonella typhimurium and Listeria Monocytogenesand parasites, such as prostigmatid Leishmania major (Cossart and Sansonetti, Science 304:242-248 (2004); Galan, Cell 103:363 to 366 (2000); Hornef et al., Nat. Immunol. 3:1033-1040 (2002); Stoiber et al., Mol. Immunol. 42:153-60 (2005)).
As mentioned above, CRIg is a recently identified receptor of complement C3, expressed in subpopulations of tissue macrophages. In addition to functioning as a receptor for proteins of the complement C3, extracellular IgV domain of the receptor CRIg selectively inhibits the alternative pathway complement activation by binding to C3b and inhibiting the proteolytic activation of C3 and C5. However, the affinity of binding of CRIg with subunit convertase C3b is low (IC50>1 μm), requiring a relatively high concentration of protein to achieve almost complete inhibition of complement. Therefore there is a need in the CRIg polypeptides having improved therapeutic efficacy. The present invention relates to such polypeptides.
Summary of the invention
The present invention is at least partially relates to the creation of a CRIg variant with high affinity binding. Protein CRIg-ECD that contains a combination of substitutions of amino acids Q64R and M86Y, characterized by a 30-fold increase in the affinity of binding and 7-fold improvement of activity of inhibition of complement compared to the CRIg variant wild type. In addition, treatment of hybrid CRIg protein with enhanced affinity svyazyvaniya in a mouse model of arthritis caused significant reduction in clinical evaluations comparing treatment with a protein CRg wild type.
Thus, the present invention relates to CRIg variants.
One object of the present invention is a CRIg variant comprising the amino acid changes in the field, selected from the group consisting of E8-K15, R41-T47, S54-Q64, E85-Q99 and Q105-K111 amino acid sequence SEQ ID NO:2.
In one embodiment the variant selectively binds to C3b compared to C3 or a fragment.
In another embodiment the CRIg variant has a higher affinity binding to C3b compared with CRIg person with a native sequence SEQ ID NO:2, wherein the affinity of binding can be increased, for example, at least 2 times in at least 3 times in at least 4 times in at least 5 times in at least 6 times in at least 7 times in at least 9 times in at least 10 times at least 15 times in at least 20 times, at least 30 times in at least 40-fold, at least 50 times in at least 70 times in at least 80 times in at least 90 times or at least 100 times.
In another embodiment the variant CRIg is a stronger inhibitor of the alternative pathway complement activation compared with CRIg person with a native sequence SEQ ID NO:2.
In another embodiment the variant CRIg VC�uchet replacement of amino acids at one or more positions, selected from the group consisting of provisions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105 and 110 in the amino acid sequence SEQ ID NO:2.
In yet another embodiment the CRIg variant includes a substitution of amino acids at one or more positions 60, 64, 86, 99, 105 and 110 in the amino acid sequence SEQ ID NO:2.
In an additional embodiment the CRIg variant comprises one or more substitutions selected from the group consisting of E8W, W14F, E84Y/W14F; P45F; G42D/D44H/P45F; Q60I; Q64R; Q60I/Q64R; M86Y; M86W, M86F, M86W/Q9R; M86F/Q99R; K110D, K11N; Q105R/K110N; Q105R/K110Q and Q105K/K110D.
In another embodiment the CRIg variant comprises one or more substitutions selected from the group consisting of Q64R/M86Y; Q60I/Q64R/E8Y; Q60I/Q64R/G42D; Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F; Q60I/Q64R/M86Y; Q60I/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N; Q60I/Q105R/K110N; M86Y/E8Y; M86Y/G42D/D44H/P45F; M86Y/P45F; M86Y/G42D/D44H/P45F and M86Y/Q99K/M86Y/Q99R/M86Y/Q105R/M86Y/Q105K/M86Y/Q105R/K110N.
In yet another embodiment the CRIg variant comprises one or more substitutions selected from the group consisting of Q60I, Q64R, Q60I/Q64R, M86Y, Q99L, Q105K/K110D, E8W/Q105R/K110N, Q64R/M86Y, Q60I/Q64R/E8Y, Q60I/Q64R/G42D, Q60I/Q64R/P45F, Q60I/Q64R/G42D/D44H/P45F, Q60I/Q64R/M86Y, Q60I/Q64R/Q105R, Q60I/Q64R/Q105K, Q60I/Q64R/K110N, M86Y/P45F and M86Y/Q105K.
In a more specific embodiment the variant CRIg includes replacement Q60I/Q64R/M86Y or Q60I/Q64R/G42D/D44H/P45F.
Another object of the present invention is a chimeric molecule comprising a variant CRIg and really�independence.
In one embodiment, the chimeric molecule is immunoadhesins.
In another embodiment of the invention immunoadhesin includes variant CRIg, which is shorter than reprocessing CRIg of SEQ ID NO:2.
In yet another embodiment, the chimeric molecule includes the extracellular domain of CRIg.
Another object of the present invention is a pharmaceutical composition comprising a CRIg variant or chimeric molecule, for example, immunoadhesin of the present invention, in admixture with a pharmaceutically acceptable excipient.
Another object of the present invention is a method for the prevention or treatment of complement-associated disease or condition, which comprises administering to the subject, those in need of such treatment, a prophylactically or therapeutically effective amount of CRIg variant or chimeric molecules, such as immunoadhesin that includes this option.
In one embodiment, the complement-associated disease is an inflammatory disease or autoimmune disease.
In another embodiment, the complement-associated disease is selected from the group including rheumatoid arthritis (RA), respiratory distress syndrome in adults (ARDS), loss of tissue removed after ischemia � reperfusion, activation of complement during surgery with extracorporeal circulation, dermatomyositis, pemphigus, lupus nephritis and its resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, coronary dysfunction of the endothelium induced by cardioplegia, membranosa-proliferative glomerulonephritis type II, IgA-associated nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, age-related macular degeneration, uveitis, diabetic retinopathy, allograft, Verhoture rejection, hemodialysis, chronic obstructive pulmonary disease (COPD), asthma, aspiration pneumonia, utricaria, chronic idiopathic utricaria, hemolytic uremic syndrome, endometriosis, cardiogenic shock, ischemic reperfusion injury and multiple sclerosis (MS).
In another embodiment, the complement-associated disease is selected from the group including inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), xeroderma (sjögren's syndrome), systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, night �paroxysmally the hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombotic purple, immune thrombocytopenia), thyroiditis (graves ' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the Central and peripheral nervous systems such as multiple sclerosis, idiopathic polyneuropathy, gall kidney diseases such as infectious hepatitis (hepatitis A, b, C, D, E and other nagapattanam viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung diseases (e.g., cystic fibrosis), patentability enteropathy, Whipple's disease, autoimmune or immune skin diseases including bullous skin diseases, polymorphic erythema, and contact dermatitis, psoriasis, allergicasthma diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and allergic pneumonia (pneumonia with hypersensitivity), diseases caused by transplantation, including graft rejection, graft-versus-host disease, Alzheimer's disease, the paroxysmal nocturnal hemoglobinuria, legacies�nny angioedema, atherosclerosis and membranosa-proliferative glomerulonephritis type II.
In a preferred embodiment, the complement-associated disease is rheumatoid arthritis (RA).
In another preferred embodiment, the complement-associated disorder is a complement-associated eye disease.
In another embodiment, the complement-associated eye disease is selected from the group including all stages of age-related macular degeneration (AMD), uveitis, diabetic and other caused by ischemia, retinopathy, endophthalmitis, and other intraocular diseases caused by formation of new vessels.
In another embodiment, the intraocular disease caused by the formation of new vessels, which are selected from the group including diabetic edema yellow spots, pathological myopia, disease von Hippel-Lindau, histoplasmosis of the eye, occlusion of the Central retinal vein (CRVO), the formation of new vessels in the cornea and the formation of new vessels in the retina.
In yet another embodiment, the complement-associated eye disease is selected from the group comprising age-related macular degeneration (AMD), the formation of new vessels in the choroid of the eye (NV), diabetic retinopathy (DR) and endophthalmitis, age-related macular degeneration (AMD) includes wet, dry or atrophic AMD.
In one embodiment, the subject is a mammal, preferably a human.
Another object of the present invention is a method of inhibiting the production of a fragment of the complement C3b in a mammal, which includes introduction to the specified mammal an effective amount of CRIg variant of the present invention or immunoadhesin that includes this option.
Another object of the present invention is a method for selective inhibition of alternative pathway complement activation in a mammal, which includes introduction to the specified mammal an effective amount of CRIg variant of the present invention or immunoadhesin that includes this option.
Brief description of the drawings
In the figures 1A-1B shows the nucleotide and amino acid sequence reprezentirovannoe long form of native human CRIg of 399 amino acids (huCRIg, SEQ ID NO:1 and 2, respectively).
In the figures 2A-2B shows the nucleotide and amino acid sequence of the short form of native human CRIg (huCRIg-short, SEQ ID NO:3 and 4, respectively).
In the figures 3A-3C shows the nucleotide and aminodimethylaniline native CRIg mouse from 280 amino acids (muCRIg, SEQ ID NO:5 and 6, respectively)
Figure 4. Activity CRIg mutants as determined by performing analysis of binding and analysis of inhibition. The affinity of binding of CRIg was measured in the form of competitive displacement C3b (A), and the biological activity was measured using the analysis of inhibition of hemolysis. PUR10680 was a control substance wild-type (red), RIL 41 (blue) and RL41 (green) were the two mutants (B). (C) Stepwise optimization of the boundaries of the CRIg binding.
Figure 5. The correlation between competitive-binding ELISA analysis and the analysis of inhibition of hemolysis.
Figure 6. Mutant CRIg Q64R/M86Y characterized by improved binding affinity by Biacore analysis results. (A) Sensogram SPR resulting from the introduction of C3b in increasing concentrations compared to wild-type CRIg with membrane proteins and CRIg Q64R M86Y. (B) Stationary data analysis of binding indicates that the value of Kd equal to 0.2 micromol for mutant Q64R/M86Y and 1.1 micromol for CRIg wild-type.
Figure 7. CRIg with improved affinity of binding selectively binds to C3b. Using a competitive-binding analysis of the Alpha Screen analyzed purified C3 and C3b.
Figure 8. Improved activity of inhibition of complement mutant CRIg Q64R/M86Y compared with wild-type CRIg. (A) Inhibition of complement CRIg wild-type and mutant CRIg Q46R/M86Y were compared using the analysis ingabire�ing hemolysis, specific to alternative ways, using rabbit erythrocytes and human serum, containing C1q. (B) Inhibition of complement CRIg wild-type and mutant CRIg Q46R/M86Y were compared using ELISA alternative ways microplate sensitized to LPS and serum of man, not containing C1q.
Figure 9. Mutant CRIg Q64R/M86Y best characterized activity in vivo compared with wild-type CRIg.
(A) Clinical scores of mice that were administered the KRN serum and were treated with different recombinant proteins CRIg human and mouse wild-type and affinity matured at different concentrations. Data represent the mean value for 4-7 mice per group. (B) Graphs of the dispersions clinical evaluations for individual mice on day 6 after injection of serum. (C) hematoxylin and eosin sections mice, which were injected with CRIg wild-type or mutant CRIg Q64R/M86Y 6 days after injection of serum. (D) Graphs of the dispersions histological evaluations for mice, which were injected with CRIg wild-type or mutant CRIg Q64R/M86Y 6 days after injection of the serum.
Table 1. Library of phages. For coverage of the contact area between CRIg and C3b was created five randomized libraries.
Table 2. Staged development of the CRIg with a higher affinity binding method display on phage. CRIg mutants against C3b, �Sobranie of five randomized libraries. In each block shows the clones that were selected from each library based on the affinity of binding to C3b. The sequence denoted by single letter codes of amino acids. In each block a separate mutants compared with a consensus sequence and the original sequence of wild-type (WT). Remains of painted accordingly: blue - randomized position; grey - not randomized; yellow - selected residues that differ from residues of wild-type (WT). Table 2 presents respectively SEQ ID NO:21-63 and 63-67 in the order listed.
Table 3. Comparison of binding affinity, determined using a competitive-binding ELISA and inhibition of hemolysis in vivo selected mutants. Mutants with more than 5-fold increase in the binding affinity and higher activity in vivo are colored yellow.
Table 4. Comparison of binding affinity and inhibition of hemolysis in vivo mutants of the second generation (the source sequence is colored in gray). Mutants with more than 5-fold increase in the binding affinity compared to the original mutant are highlighted in blue, mutants with more than 90-fold increase in the affinity of binding are highlighted in yellow. Similarly, mutants with higher activity in vivo compared with baseline after�euteleostomi highlighted in orange.
Detailed description of the invention
1. Definition of terms
The terms ”CRIg”, “PRO362”, “JAM4” and “STIgMA” have interchangeable meanings and are used to determine the CRIg polypeptides with a native sequence and variants.
“With native CRIg sequence” means a polypeptide having the same amino acid sequence as natural CRIg polypeptide regardless of the method of receipt. Thus, with native CRIg sequence can be isolated from a natural source or obtained through recombinant DNA and/or by synthesis. In the definition of “native CRIg sequence” includes natural truncated or secreted forms of CRIg (for example, the sequence of the extracellular domain), natural options (e.g., alternative spliced forms) and natural allelic variants CRIg. CRIg polypeptides with a native sequence includes a full-sized (reprezentirovanii) CRIg polypeptide of a man with a length of 399 amino acids represented by SEQ ID NO:2 (huCRIg shown in figures 1A and 1B), with and without N-terminal signal sequence, with and without the initiating methionine at position 1 and has or has not any or all of the transmembrane domains in the provisions of amino acids 277-307 SEQ ID NO:2. In another embodiment, the floor�peptide with native CRIg sequence consists of 305 amino acids and is a short form human CRIg (huCRIg-short, SEQ ID NO:4, shown in figures 2A and 2B), with and without N-terminal signal sequence, with and without the initiating methionine at position 1 and has or has not any or all of the transmembrane domains in the provisions 183-213 SEQ ID NO:4. In another embodiment, the CRIg polypeptide with native sequence has a length of 280 amino acids and is a full-sized polypeptide mouse CRIg of SEQ ID NO:6 (muCRIg shown in figures 3A-3C), with and without N-terminal signal sequence, with and without the initiating methionine at position 1 and has or has not any or all of the transmembrane domains in the provisions of amino acids 181-211 SEQ ID NO:6. In the definition of this term includes the CRIg polypeptides of other animals than man, including higher primates and mammals.
“Extracellular domain” CRIg or “ECD” CRIg means in the form of a CRIg polypeptide, which largely lacking transmembrane and cytoplasmic domains of the respective full-sized molecules. CRIg ECD typically contains less than 1% of such transmembrane and/or cytoplasmic domains and preferably less than 0.5% of such domains. CRIg ECD may include amino acid residues in position 1 or about 21 to X of SEQ ID NO:2, 4 or 6, where X means any amino acid in position from about 271 to 281 in SEQ ID NO:2, any amino acid�in the position from about 178 to 186 in SEQ ID NO:4 and any amino acid in position from about 176 to 184 in SEQ ID NO:6.
The term ”variant CRIg” used here value means the active CRIg polypeptide, described below, in which the amino acid sequence at least about 80% identical to the CRIg polypeptide with native sequence and which includes, but is not limited to, reprezentirovanii huCRIg (SEQ ID NO:2), huCRIg-short (SEQ ID NO:4) and muCRIg (SEQ ID NO:6), each of which is N-terminal initiating methionine, with and without N-terminal signal sequence, is all or part of the transmembrane domains and has or does not have an intracellular domain. In a specific embodiment the CRIg variant has an amino acid sequence that is at least about 80% homologous to the amino acid sequence of Mature, reprocessing peptide, represented by SEQ ID NO:2. In another embodiment the CRIg variant has an amino acid sequence that is at least about 80% homologous to the amino acid sequence of Mature, reprocessing peptide, represented by SEQ ID NO:4. In yet another embodiment the CRIg variant has an amino acid sequence that is at least about 80% homologous to the amino acid sequence of Mature, reprocessing peptide, n�establing SEQ ID NO:6. Variant CRIg usually has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% identical to the Mature amino acid sequence represented by SEQ ID NO:2, 4 or 6. In the description of the invention, including the examples, the term ”wild-type” or “WT” means Mature, reprezentirovannoe short form human CRIg (CRIg(S)) (SEQ ID NO:4), wherein the numbering of amino acid residues in CRIg variants corresponds to the sequence SEQ ID NO:4.
Options CRIg of the present invention are CRIg agonists, as described below. In particular, CRIg variants retain the ability to selectively bind to C3b compared to C3, where the term ”selective binding” is used to denote binding to C3b and lack of binding to C3. In addition, in a preferred embodiment the CRIg variants of the present invention have a higher affinity binding to C3b compared with CRIg polypeptide with native sequence, such as the long form of human CRIg (SEQ ID NO:2). In various embodiments, the binding affinity is increased by at least about 2 times, at least about 3 fold, at least about � 4 times at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25 fold, at least about 30-fold, at least about 35-fold, at least about 40 times, at least about 45-fold, at least about 50-fold, at least about 55 times in at least about 60-fold, at least about 65 times in at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85, at least about 90 times at least about 95-fold, or at least about 100 times compared to the CRIg polypeptide of a person with a native sequence SEQ ID NO:2. In other embodiments, the affinity of binding to C3b compared with CRIg polypeptide of a person with a native sequence SEQ ID NO:2 increased approximately 5-10 times, about 5-15 times, about 5-20 times, about 5-25 times, about 5-30 times, about 5-35 times, about 5-40 times, about 5-45 time, about 5-50 times, about 5-55 time of about 5-60 times, about 5-65 times, about 5-70 times, about 5-75 times, about 5-80 times about 5-RS, about 5-90 times, about 5-95 time or about 5-100 times.
“Percent (%) amino acid sequence identity” with CRIg variants in the present description of the invention is defined as the percentage of amino acid residues in the sequence CRIg variant, compare identical amino acid residues in the native sequence CRIg after aligning the sequences and introducing, if necessary, gaps to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. For sequences of different lengths, the percentage sequence identity is determined relatively long sequences throughout the length of the longer sequence. Comparative analysis to determine the percentage identity of amino acid sequences can be accomplished in various ways known to specialists in this field, for example, using publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in this oblasti can determine the relevant parameters needed to perform the comparative analysis, including any algorithms needed to achieve maximal alignment with�animemix sequences over the entire length. Identity of sequences is then computed relative to a longer sequence, i.e., even if the shorter sequence is 100% identical to part of a longer sequence, the common identity of the sequences will be less than 100%.
“Percent (%) sequence identity of the nucleic acid sequences encoding CRIg variant of the present invention, is defined as the percentage of nucleotides in the sequence of the candidate that are identical with the nucleotides in the sequence encoding the variant CRIg, after aligning the sequences and introducing, if necessary, gaps to achieve the maximum percent sequence identity. Comparative analysis in order to determine the percent identity of the nucleic acid sequence may be performed by various methods known in this field, for example, using publicly available software, such as BKAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in this field can determine the relevant parameters needed to perform the comparative analysis, including any algorithms needed to achieve maximal alignment of the compared sequences over the entire length. Identity sequences for�compute eat relatively long sequences, that is, even if the shorter sequence is 100% identical to part of a longer sequence, the common identity of the sequences will be less than 100%.
In the definition of variant CRIg include all amino acid sequence variants discussed above in the present description of the invention, regardless of how you identify or obtain. In this definition particularly includes the options, modified by introducing a part of the non natural amino acids, chemical, enzymatic, or other acceptable methods, provided that these variants retain the biological properties with native CRIg sequence. Examples of substitutions of natural amino acids include the following replacement.
Amino acid residues are divided into four main groups:
Acidic: the residue has a negative charge due to the loss of ion N at physiological pH and is attracted by aqueous solution, occupying a surface state in conformation containing peptide when the peptide is in aqueous solution.
Basic: the residue has a positive charge due to Association with ion N at physiological pH and is attracted by aqueous solution, occupying a surface state in conformation containing peptide in the presence of this peptide in water�th medium at physiological pH.
Neutral/nonpolar: the residue has no charge at physiological pH and is repelled by aqueous solution, occupying the internal situation in conformation containing peptide when the peptide is in aqueous medium. These residues are also referred to as ”hydrophobic residues”.
Neutral/polar: the residue has no charge at physiological pH, but is attracted by aqueous solution, occupying an external position in conformation containing peptide when the peptide is in aqueous medium.
Amino acid residues can be further classified as cyclic or acyclic, aromatic or non-aromatic based groups in the side chain, and such designations are well known to qualified professionals.
Usually occurring amino acids that are not encoded by the genetic code, include 2-aminoadipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminoadamantane acid (Aib) for Gly; cyclohexylamine (Cha) for Val, Leu and Ile; homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-utilisation (EtAsn) for Asn and Gln; hydroxylysine (Hyl) for Lys; �logicboxes (AHyl) for Lys; 3-(and 4 -) hydroxyproline (3Hyp, 4Hyp) for Pro, Ser and Thr; allosaurid (AIle) for Ile, Leu and Val; amidinohydrolase for Ala; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala; N-methylisoleucine (MrIle) for Ile; Norvaline (Nva) for Met and other aliphatic amino acids; norleucine (Nle) for Met and other aliphatic amino acids; ornithine (Orn) for Lys, Arg and His; citrulline (Cit) and methanesulfonate (MSO) for Thr, Asn and Gln; N-methylphenylamine (MePhe), trimethylphenylammonium, halogen (F, Cl, Br, and I)phenylalanine, trifluorophenylboronic for Phe.
Used here meaning the term ”immunoadhesin” means antitelomerase molecules that combine the binding specificity of a heterologous protein (”adhesin”) with the effector functions of the constant domain of immunoglobulin. Structurally immunoadhesin represent a hybrid amino acid sequence with the desired binding specificity which is not indigenousness and the center of the antigen-binding antibody (i.e. is ”heterologous”) with the sequence of the constant domain of immunoglobulin. Adhesin, which is part of the molecule immunoadhesin, typically is a contiguous amino acid posledovatelnosti comprising at least the binding site of the receptor or ligand. The sequence of constant region of immunoglobulin in immunoadhesin can be �allowed to receive from any immunoglobulin, such as subtypes of IgG-1, IgG-2, IgG-3 or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
“Treatment” means intervention with the aim of preventing the development or altering the pathology of a violation. Thus, the term ”treatment” means both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment, are subjects that already have a violation and subjects, which is necessary to prevent the occurrence of violations.
The term “reducing the intensity of symptoms” in used here value means improvement.
The term ”mammal” is used here in the meaning means any animal classified as a mammal, which includes, but is not limited to, humans, primates except humans, domestic and farm animals, animals kept in the zoo, sporting animals or Pets such as horses, pigs, cattle, dogs, cats, ferrets, etc. In predpochtitelno embodiment, a mammal is the highest Primate, most preferably a human.
The term ”complement-associated disease” used in the present description of the invention in its broadest sense and includes all diseases and pathological conditions, the pathogenesis of which �suggests the violation of activation of the complement system, such as, for example, the absence of complement. This term is used for the identification of diseases and pathological conditions for which a beneficial effect of inhibition of C3 convertase. In the definition of the term additionally includes diseases and pathological condition for which a beneficial effect of inhibition, including selective inhibition of the alternative pathway activation of complement. Complement-associated diseases include, but are not limited to, inflammatory diseases and autoimmune diseases, such as rheumatoid arthritis (RA), respiratory distress syndrome in adults (ARDS), loss of tissue removed after ischemia and reperfusion, complement activation during surgery with extracorporeal circulation, dermatomyositis, pemphigus, lupus nephritis and its resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, coronary dysfunction of the endothelium induced by cardioplegia, membranosa-proliferative glomerulonephritis type II, nephropathy is caused by IgA, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, age-related macular degeneration, uveitis, diabetic retinopathy, allotransplantation, Verhoture rejection, hemodialysis, chronic obstructive �zabolevanie lung (COPD), asthma, aspiration pneumonia. In a preferred embodiment, the complement-associated disease” is a disease in which an important role is played by the alternative pathway complement activation, and includes rheumatoid arthritis (RA), a complement-associated eye diseases such as age-related macular degeneration, antiphospholipid syndrome, intestinal damage and kidney failure caused by ischemia and reperfusion, and membranosa-proliferative glomerulonephritis type II.
The term ”complement-associated eye disease” used in the present description of the invention in its broadest sense and includes all eye diseases, pathology which is caused by complement, including the classical and alternative pathways, and in particular, the alternative path of complement activation. This group includes all eye diseases associated with alternative manner, occurrence, development or progression of which can be controlled by inhibiting alternative pathway complement activation. Complement-associated eye diseases include, but are not limited to, degenerative diseases of the macula, such as all stages of age-related macular degeneration (AMD), including dry and wet (nonexudative and exudative) forms, education but�'s vessels in the choroid of the eye (CNV), uveitis, diabetic and other caused by ischemia, retinopathy, endophthalmitis, and other intraocular diseases caused by formation of new vessels such as diabetic edema yellow spots, pathological myopia, disease von Hippel-Lindau, histoplasmosis of the eye, occlusion of the Central retinal vein (CRVO), the formation of new vessels in the cornea and the formation of new vessels in the retina. In a preferred group of complement-associated eye diseases include age-related macular degeneration (AMD) including nonexudative (wet) and exudative (dry or atrophic) AMD, the formation of new vessels in the choroid of the eye (CNV), diabetic retinopathy (DR) and endophthalmitis.
The terms ”inflammatory disease” and “inflammatory violation” are interchangeable and mean values for a disease or disorder in which the component of the immune system of a mammal causes, mediates or otherwise contributes to the occurrence of the inflammatory response that causes the disease of a mammal. In this group are also diseases in which the attenuation of the inflammatory response has beneficial effects on the development of the disease. In the definition of this term are immune inflammatory diseases including autoimmune diseases.
�Ermin ”mediated T-cell” disease means a disease, in which T cells directly or indirectly mediate or in any other way contribute to disease of a mammal. Mediated T-cell disease may be associated with cretaceouspaleogene the impact, lymphokine-mediated effects, etc., and even effects associated with b cells if the b cells are stimulated, for example, lymphokines, sekretiruemyi T-cells.
Examples of immune and inflammatory diseases, some of which are mediated by T-cells include, but are not limited to, inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), xeroderma, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, the paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombotic purple, immune thrombocytopenia), thyroiditis (graves ' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes, immune disease of the kidneys (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the Central and peripheral nervous system, such�to multiple sclerosis, idiopathic polyneuropathy, gall kidney diseases such as infectious hepatitis (hepatitis A, b, C, D, E and other nagapattanam viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung diseases (e.g., cystic fibrosis), patentability enteropathy, Whipple's disease, autoimmune or immune skin diseases including bullous skin diseases, polymorphic erythema, and contact dermatitis, psoriasis, allergicasthma diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and allergic pneumonia, diseases of, due to transplantation, including graft rejection, graft-versus-host disease, Alzheimer's disease and atherosclerosis.
Introduction ”in combination with” one or more other drugs means simultaneous and sequential introduction in any order.
The terms ”active” or “activity” in relation to embodiments of the CRIg polypeptides of the present invention means the forms of such polypeptides, which retain a biological and/or immunological activity of native or natural polypeptide according to the present invention. A preferred biological activity of�Yu is the ability to bind to C3b and/or affect the complement fixation or activation of complement, in particular, to inhibit alternative pathway complement activation and/or C3 convertase. Inhibition of C3 convertase can, for example, be determined by measuring the inhibition of the turnover of C3 in normal serum in the case of arthritis induced by collagen or the antibody, or the inhibition of C3 deposition in the joints affected by arthritis.
The term ”biological activity” as applied to polypeptide that mimics the biological activity of CRIg, partially refers to the ability of these molecules to contact with C3b and/or affect the complement fixation or activation of complement, in particular, to inhibit alternative pathway complement activation and/or C3 convertase.
The term ”agonist” CRIg used in its broadest sense and includes any molecule that mimics a biological activity (defined above) CRIg polypeptide with native sequence.
The term ”functionally linked” means adjacent the position at which the components to function properly. Thus, the coding sequence functionally linked with regulatory sequences, is a configuration in which the coding sequence can be expressed under the control of these sequences that are linked DNA sequences are contiguous and in the case of a secretory leader sequence, are adjacent and are in the phase of reading. For example, DNA for predpolagavshegosja or secretory leader sequence functionally linked to DNA for a polypeptide if it is expressed in the form of predella involved in secretion of the polypeptide; a promoter or enhancer functionally linked to the coding sequence if it affects the transcription of the sequence; or the binding site of the ribosome is functionally linked to the coding sequence, if its location facilitates streaming. Linking is accomplished by ligation at convenient restriction sites. In the absence of such sites using a synthetic oligonucleotide adapters or linkers in accordance with known practice.
The term ”regulatory sequence” means a DNA sequence required for expression of the functionally linked coding sequence in a particular organism, the host. Regulatory sequences suitable for prokaryotic organisms include, for example, a promoter, optionally an operator sequence and the binding site of the ribosome. It is known that in eukaryotic cells are used as promoters, polyadenylation signals, and enhancers.
The term ”expression system” means a DNA sequence containing t�buemoy encoding sequence and a regulatory sequence, functionally related to each other, allowing the hosts transformed with these sequences, can produce kodirovanie proteins. For transformation of the host expressing the system can be introduced into the vector; however, the relevant DNA may also be integrated into the chromosome of the host.
In the value used here, the terms “cell”, “cell line” and “cell culture” are used interchangeably values and are used to denote the offspring. Thus, ”transformants” or “transformed cells” refers to primary cells of the subject, and obtained from them the culture regardless of the number of passages. In addition, it should be noted that all progeny may not be completely identical on the DNA because of the possibility of intentional or accidental mutations. In the definition of this term includes mutant progeny that have the same functional ability as that of the originally transformed cell. The use of other notation will be clear from the context.
“Plasmids” are designated by a lower case letter ”p” preceded and/or followed by capital letters and/or numbers. The original plasmids available through commercial channels on an unlimited basis or create such publicly known methods plasmids. In addition, in this area things�there are other equivalent plasmids, to be a well-known expert.
The term ”library displayed on phage” means a protein expression library, in which is expressed a collection of cloned protein sequences in the form hybrids with the protein shell of the phage. This obrazom, the phrase ”library displayed on phage” means a collection of phage (e.g., filamentous phage), expressing external (typically heterologous) protein. Outer protein can interact (communicate) with other parts, which contacts the phage. Each phage displaying outer protein, is a ”member” of the library displayed on phage.
The term ”filamentous phage” refers to a viral particle capable of displaying heterogenous polypeptide on its surface, and includes, but is not limited to, f1, fd Pf1 and M13. Filamentous phage may contain a selectable marker such as tetracycline (e.g., ”fd-tet”). Specialists in this area is well known for different systems display on the filamentous phage (see, for example, publications Zacher et al., Gene 9:127-140 (1980), Smith et al., Science, 228:1315-1317 (1985); and Parmley and Smith, Gene, 73: 305-318 (1988)).
The term ”panning” means several cycles of screening process for identification and selection of phages carrying compounds, such as antibodies, with high affinity and specificity to the target.
The phrase ”conservative amino acid residues” oz�ACHAT amino acid residues which are identical in two or more amino acid sequences, subjected to comparative analysis.
II. Detailed description of the invention
Complement is an important component of innate and acquired immune response, wherein the cleavage products of complement, formed as a result of the activation of each of the three ways of complement activation (classical, alternative and mediated by lectin), can cause inflammation and tissue destruction. Thus, uncontrolled complement activation due to the lack of regulation causes a variety of chronic inflammatory diseases. In the specified inflammatory cascade is dominated by cleavage products of complement S3a and S5a, which act as chemoattractants and activators of neutrophils and inflammatory macrophages through receptors S3a and S5a (Mollnes, T. E., W. C. Song, and J. D. Lambris. 2002. Complement in inflammatory tissue damage and disease. Trends Immunol. 23:61-64).
CRIg is recently detected by the complement receptor that is expressed on sedentary subpopulations of tissue macrophages. As a functional receptor, the extracellular IgV domain of the receptor CRIg is a selective inhibitor of the alternative pathway complement activation (Wiesmann et al., Nature, 444(7116): 217-20, 2006). It is established that the soluble form of CRIg can eliminate in�the Palen and bone loss in experimental models of arthritis by inhibiting alternative pathway complement activation in the joint. In addition, it was found that the alternative path of complement activation is required not only for the occurrence of disease, but also for the progression of the disease. Thus, inhibition of the alternative pathway receptor CRIg creates a promising therapeutic approach for the prevention and treatment of diseases and disorders, the pathogenesis of which determines the alternative path of complement activation. For more information on this topic see, for example, the publication Helmy et al., Cell, 125(1):29-32, 2006) and Katschke et al., J. Exp. Med. 204(6):1319-1325 (2007).
However, the affinity to the CRIg subunit convertase C3b is low (in the micromolar range). To create a stronger inhibitor for the purpose of obtaining drugs was used for crystal structure of CRIg in complex with C3b, and method of display on the phage were created CRIg variants with improved affinity binding to C3b.
Thus, the present invention relates to CRIg variants with improved properties, such as improved binding affinity to C3b and higher efficiency of inhibition.
Identification of CRIg variants with ripe affinity
As described in more detail in the example, the display of protein or peptide libraries on phage is a useful method for the selection of CRIg variants with improved binding affinity to C3b �/or other improved properties such as higher biological activity (Smith, G. P., (1991) Curr. Opin. Biotechnol. 2:668-673). High-affinity monovalent proteins that are displayed in the form of a hybrid protein with the membranes of the gene III of M13 (Clackson, T., (1994) et al., Trends Biotechnol. 12:173-183), can be identified by cloning and sequencing the corresponding DNA packaged in phage particles after several cycles of selection characteristics of the binding.
The affinity maturation using display on the phage is described, for example, Lowman et al., Biochemistry 30(45):10832-10838 (1991), see also Hawkins et al., J. Mol. Biol. 254: 889-896 (1992) and in the example below. Not strictly adhering to the following description, this process can be summarized as follows: multiple sites in a predefined area are mutated with the implementation of all possible substitutions of amino acids at each site. The thus obtained mutants antibodies display in the form of monovalent antibodies on the particles of filamentous phage, forming a hybrid with the product of the gene III of M13 packaged within each particle, the Phage expressing the various mutants can be subjected to several cycles of selection characteristics of binding followed by isolation and sequencing of mutants with high affinity. The method also described in U.S. patent No. 5750373, issued may 12, 1998
Modified method, enabling�th display total affinity, described in the publication Cunningham, B. C. et al., EMBO J. 13(11), 2508-2515 (1994). The method to select new binding polypeptides, includes (a) establishing replicate expressing vector comprising a first gene encoding the polypeptide, a second gene encoding at least a portion of the natural or wild coat protein of the phage, wherein the first and second genes are heterologous, and the item that regulate transcription, functionally linked to the first and second genes with the formation of a hybrid gene encoding a fusion protein; (b) mutating the vector at one or more selected positions within the first gene with the formation of a family of related plasmids; (C) acceptable transformation of host cells indicated plasmids; (d) infecting the transformed host cells by phage-helper containing the gene encoding the protein shell of the phage; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phage particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so as to small number of phage particles displaying more than one copy of the hybrid protein on the surface of the particle; f) contacting the phage particles with the molecule-target, so that at least a portion of phage frequent�C associated with the molecule-target, and (g) separation of phage particles that bind from those that do not bind. The method further preferably includes the acceptable transformation of host cells recombinant fagbemi particles that communicates with the molecule-target, and repeating stages (d) - (g) one or more times.
It should be noted that, although the CRIg variants of the present invention were identified by display on phage, to identify the CRIg variants with improved properties, including CRIg variants with ripe affinity, can also be used other methods of display.
Options CRIg with ripe affinity of the present invention have been created to fill the area of contact between CRIg and C3b, which was identified using the crystal structure of CRIg and complex C3b:CRIg described in the publication of the patent application U.S. No. 20080045697. In particular, as shown in table 1, the library of 1-5 were established, respectively, for residues E8-K15, R41-T47, S54-Q64, E85-Q99 and Q105-K111 reprezentirovannoe molecules with native CRIg sequence of SEQ ID NO:2.
In one embodiment the CRIg variants include amino acid changes at one or more positions of the amino acids selected from the group consisting of provisions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105 and 110 in the amino acid sequence SEQ ID NO:2.
Ti�ary CRIg variants of the present invention are shown in table 3.
The replacement is preferably performed in one or more positions of the amino acids 60, 64, 86, 99, 105 and 110 the amino acid sequence reprocessing native CRIg of SEQ ID NO:2.
Options CRIg with ripe affinity include, but are not limited to, one or more of the following substitutions in SEQ ID NO:2: E8W, W14F, E84Y/W14F, P45F, G42D/D44H/P45F, Q60I, Q64R, Q60I/Q64R, M86Y, M86W, M86F, M86W/Q9R, M86F/Q99R, K110D, K11N, Q105R/K110N, Q105R/K110Q and Q105K/K110D.
Other options with native CRIg sequence of SEQ ID NO:2 with two or more substitutions of amino acids shown in table 3. In this group are Q64R/M86Y, Q60I/Q64R/E8Y, Q60I/Q64R/G42D, Q60I/Q64R/P45F, Q60I/Q64R/G42D/D44H/P45F, Q60I/Q64R/M86Y, Q60I/Q64R/Q105R, Q60I/Q64R/Q105K, Q60I/Q64R/K110N, Q60I/Q105R/K110N, M86Y/E8Y, M86Y/G42D/D44H/P45F, M86Y/P45F, M86Y/G42D/D44H/P45F; M86Y/Q99K/M86Y/Q99R/M86Y/Q105R/M86Y/Q105K/M86Y/Q105R/K110N.
Preferred CRIg variants of the present invention include mutations selected from the group consisting of: Q60I, Q64R, Q60I/Q64R, M86Y, Q99L, Q105K/K110D, E8W/Q105R/K110N, Q64R/M86Y, Q60I/Q64R/E8Y, Q60I/Q64R/G42D, Q60I/Q64R/P45F, Q60I/Q64R/G42D/D44H/P45F, Q60I/Q64R/M86Y, Q60I/Q64R/Q105R, Q60I/Q64R/Q105K, Q60I/Q64R/K110N, M86Y/P45F; M86Y/Q105K.
Particularly preferred variants include mutations Q60I/Q64R/M86Y or Q60I/Q64R/G42D/D44H/P45F.
In the scope of the present invention preferably includes the options that contains one or more mutations described above or shown in tables 3 and 4, but otherwise preserving the native sequence CRIg of SEQ ID NO:2. Such options are presented in the present description of the invention with the decree�of specific mutations previous ”CRIg”. For example, an option that differs from native CRIg sequence of SEQ ID NO:2 only by mutation E8W, designated as ”E8W CRIg” version, which differs from the native CRIg sequence of SEQ ID NO:2 only by mutations Q60I/Q64R/M86Y designated as Q60I/Q64R/M86Y CRIg” etc.
Getting CRIg variants
DNA, which may encode proteins that are used for recombinant synthesis of CRIg variants of the present invention, can be obtained by different methods. For example, DNA can be obtained on the basis of natural DNA sequences that encode changes in amino acid sequence of the obtained protein. Such mutant DNA can be used to obtain CRIg variants of the present invention. When presenting in a simplified form, these methods include obtaining a gene encoding CRIg polypeptide with native sequence, the modification of the genes obtained by the methods of recombinant DNA, as described below, the insertion of genes into appropriate expression vector, introducing the vector into an appropriate host cell, culturing the host cell for expression of the desired variant CRIg and purification of the resulting molecules.
More specifically, the DNA sequence encoding CRIg variant of the present invention, is produced by the artificial creation of a sequence� DNA in accordance with the description, given in standard textbooks such as, for example, Sambrook, J. et al., Molecular Cloning (2nded.), Cold Spring Harbor Laboratory, N.Y., (1989).
and. Mutagenesis using oligonucleotides
Mutagenesis using the oligonucleotides is predpochtitelno method of obtaining options of CRIg polypeptide with native sequence or its fragment by substitutions, deletions and insertions. This method is well known in this field and are described in publications Zoller et al., Nucl Acids Res. 10:6487-6504 (1987). In accordance with the summary of the specified method of nucleic acid encoding the original polypeptide sequence, change, hybridizer oligonucleotide encoding the desired mutation, with matrix DNA, in which the matrix is a single-stranded form of the plasmid containing the unaltered or native DNA sequence of the encoding nucleic acid. After hybridization using DNA polymerase to synthesize the entire second complementary strand of the matrix, which, therefore, will incorporate the oligonucleotide primer, and to encode the selected change of the original nucleic acid.
Usually used the oligonucleotides comprising at least 25 nucleotides. Optimal oligonucleotide contains from 12 to 15 nucleotides that is fully complementary to the matrix on either side of the nucleotide(s) coding�th mutation. This ensures the correct hybridization of the oligonucleotide with single-stranded molecule of the matrix DNA. The oligonucleotides can be easily synthesized by methods known in this field, which are described in the publication Crea et al., Proc. Natl. Acad. Sci. USA 75:5765 (1978).
When you use the display on the phage matrix DNA can only be generated by those vectors that are obtained from vectors based on bacteriophage M13 (suitable public vectors M13mp18 and M13mp19), or vectors containing the origin of replicatio single-stranded phage, which are described in the publication Viera et al., Meth. Enzymol. 153:3 (1987). Thus, the desired DNA mutations required to enter into one of these vectors to generate single-stranded matrix. Obtaining single-stranded matrix is described in sections 4.21-4.41 publications Sambrook et al., see above.
To change the native DNA sequence of the oligonucleotide hybridizing with single-stranded matrix in a suitable hybridization conditions. Then add DNA polimerizarii enzyme, usually a fragment maple DNA polymerase I to synthesize the complementary strand of a matrix, using the oligonucleotide as a seed for the synthesis. Thus formed heteroduplex molecule in which one strand of DNA encodes the mutated form of CRIg and the other chain (the original matrix) encodes the native, unchanged�th sequence CRIg. Specified heteroduplex molecule is then transferred into appropriate host cell, typically in a prokaryotic cell, such as E. coli JM-101. The grown cells are put on cups with agarose and examined using an oligonucleotide primer labeled with a radioactive isotope32phosphate to identify the bacterial colonies that contain the mutated DNA.
The above method can be modified to create homoduplexes molecules in which both strands of the plasmid contain the mutation. The modification is performed as follows: single-stranded oligonucleotide hybridizing with single-stranded matrix in accordance with the above description. A mixture of three deoxyribonucleotides, namely desoxyephedrine (dATP), deoxyribofuranosyl (dGTP) and desoxyepothilone (dTTP), is combined with the modified timezonebias called dCTP-(aS) (Amersham). The resulting mixture was added to the complex matrix of the oligonucleotide. After adding DNA polymerase to the mixture formed a chain of DNA that is identical to the matrix except for the mutated bases. In addition, the new strand of DNA will contain dCTP-(aS) instead of dCTP, which protects it from cleavage with restriction endonuclease. Matrix double-stranded chain of heteroduplex, after she cut the appropriate restriction enzyme, can be split well�leasau ExoIII or another appropriate nuclease behind the field, contains the site subjected to mutagenesis. Then the reaction is stopped while gaining a molecule that is only partially single-stranded. Then form a complete homoduplex double-stranded DNA using DNA polymerase in the presence of all four deoxyribonucleotide-triphosphates, ATP, and DNA ligase. The specified molecule homoduplex can then be moved into appropriate host cell, such as E. coli JM101, as described above.
Mutants with replacement of more than one amino acid can be created in one of several ways. Amino acids located near each other in the polypeptide chain, can be motroway simultaneously using one oligonucleotide that encodes all of the desired amino acid substitutions. However, if amino acids are located some distance from each other (separated by more than about ten amino acids), are more difficult to generate a single oligonucleotide that encodes all of the desired changes. In this case you can use one or two alternative methods.
In accordance with the first method creates a separate oligonucleotide for each replaced amino acid. The oligonucleotides simultaneously hybridizing with single-stranded matrix DNA, the second DNA strand synthesized from the matrix will encode all of the desired amino acid substitutions. Alternative�second method involves two or more cycles of mutagenesis to produce the desired mutant. The first cycle is similar to that described for the single mutants: wild-type DNA is used as template and oligonucleotide encoding the first amino acid substitution(s), hybridizing with the matrix, resulting in a DNA molecule of heteroduplex. In the second cycle of mutagenesis mutated DNA obtained in the first cycle of mutagenesis, are used as matrix. Thus, this matrix already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution, then hybridizing with the matrix, whereby the resulting strand of DNA now encodes mutations obtained in both the first and second cycle of mutagenesis. The obtained DNA can be used as a matrix in the third round of mutagenesis, and so on.
b. Clustered mutagenesis
This method is also the preferred method of obtaining CRIg variants with the substitution, deletion and insertion. The basis of this method is the method described in the publication of Wells et al., Gene 34:315 (1985). The starting material is the plasmid (or other vector) comprising the gene 1, the gene is subject to mutation. Identify the codons susceptible to mutations in the nucleic acid molecule encoding the source CRIg. On each side of the identified mutation sites must be clearly defined website restricts�and endonuclease. If these restriction sites are not available, they can be created by the method described above mutagenesis using oligonucleotides to introduce relevant provisions in the gene 1. The plasmid after the introduction of restriction sites is cut at these sites for linearization. Standard methods synthesize double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation. The two strands are synthesized separately and then hybridizing together by standard methods. This double-stranded oligonucleotide is referred to as a cluster. The specified cluster has 3'- and 5'-ends compatible with the ends of the linearized plasmid with which the cluster can be directly Legerova with the plasmid. Now the plasmid contains the mutated DNA sequence of the receptor CRIg.
C. Obtain CRIg variants by methods of recombinant DNA
DNA encoding the variants, is introduced into an appropriate plasmid or vector. The received vector to transform a host cell. In these hosts typically use plasmid vectors containing replicating and regulatory sequences, isolated from species compatible with the host cell. The vector typically contains a replication site, as well as sequences that encode proteins capable of producing a selection on phenotype in �transformirovannykh cells.
For example,E. colican be transformed using the plasmid pBR322 isolated from the species E. coli (Mandel, M. et al., (1970) J. Mol. Biol. 53:154). Plasmid pBR322 contains genes for resistance to ampicillin and tetracycline, and, thus, are a simple means of screening. Other vectors may contain different promoters, which often are important for expression. For example, plasmids pKK223-3, pDR720 and pPL-λ represent expression vectors with the tac promoter, trp or PLwhich currently can be purchased commercially (Pharmacia Biotechnology).
Other preferred vectors can be generated using standard methods by combining related signs vectors considered in the present description of the invention. Related signs vectors include a promoter, the binding site of the ribosome, variant or hybrid gene, a signal sequence, markers of antibiotic resistance, the number of copies and the appropriate oridzhiny replicable.
Acceptable the host cell suitable for cloning or expression of the DNA in the vectors include prokaryotic cells, yeast cells, or higher eukaryotic cells. Prokaryotic cells suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, still� as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serrafia, e.g., Serratia marcescans, and Shigeila, as well as Bacilli such as B. subtilis and B. Bacillus licheniformis (e.g., B. Bacillus licheniformis 41P described in the patent DD 266710, published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred host for cloning of E. coli is E. coli 294 (ATCC 31446), although, other strains such as E. coli B, E. coli X 1776 (ATCC 31537) and E. coli W3110 (ATCC 27325). The examples above are illustrative and do not limit the scope of the invention.
In addition to prokaryotic cells, acceptable cloning or expression hosts for antitelomerase vectors are eukaryotic microbes such as filamentous fungi or yeast. Saccharomyces cerevisiae, or common Baker's yeast are the most commonly used lower eukaryotic microorganisms-hosts. However, in the present invention can be used a number of other genera, species and strains, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC price has been 56500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K. marxianus; yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
The host cell, p�ielemia for the expression of glycosylated antibodies, isolated from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Identified numerous baculovirus strains and variants and corresponding valid cells-the hosts of insects released from such owners as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. Different viral strains is also available for transfection, for example, variants of L-1 Autographa californica NPV and the strain Bm-5 Bombyx mori NPV, and such viruses may be used in the present invention, particularly for transfection of cells Spodoptera frugiperda. As hosts of the can also be used plant cell culture of cotton, corn, potato, soybean, Petunia, tomato and tobacco.
However, of most interest are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) is a common practice. Examples of acceptable cell lines of mammalian hosts are cell line CV1 monkey kidney transformed by the virus SV40 (COS-7, ATCC CRL 1651); the line of human embryonic kidney cells (293 cells or 293 cells, sublimirovanny for growing in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977); kidney cells baby hamster (BHK, ATCC CCL 10); the cells of the Chinese hamster ovary/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); cell� mouse Sertoli (TM, Mather, Biol. Reprod. 23:243-251 (1980)); kidney cells of monkeys (CV1 ATCC CCL 70); kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); cancer cells human cervical (HELA, ATCC CCL 2); cells dog kidney (MDCK, ATCC CCL 34); liver cells buff rats (BRL 3A, ATCC CRL 1442); lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); breast tumor mouse (MMT 060562, ATSS CCL51); cell TRI (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); the cells, MRC 5; FS4 cells and cell line human hepatoma (Hep G2).
The host cell is transformed above expressing or cloning vectors to obtain the CRIg variants of the present invention, cultured in standard nutrient media modified as appropriate for inducing promoters, selected transformants or amplificateur genes encoding the desired sequences.
The host cell used to obtain CRIg variants of the present invention, can be cultured in different media. For the cultivation of host cells suitable commercially available medium, such as medium ham's F10 (Sigma), minimal maintenance medium ((MEM), (Sigma), RPMI-1640 (Sigma) and modified according to the method of Dulbecco Wednesday Needle(DMEM), Sigma). In addition, as culture media for the host cells can be used in any environment that is described in the publication Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biohem. 102:255 (1980), U.S. patent No. 4767704, 4657866, 4927762, 4560655 or 5122469; WO 90/03430; WO 87/00195 or a U.S. patent with registration number 30985. Any of these media, if necessary, may contain hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium phosphate and magnesium), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. You can also enter other necessary additives in appropriate concentrations, known to specialists in this field. Culturing conditions, such as temperature, pH and the like, correspond to the previously used for the host cells selected for expression, and should be obvious to a person skilled in this field.
When using methods of recombinant DNA variant CRIg can be obtained inside the cell, the periplasmic space or secreted directly into the environment. When you receive a CRIg variant within the cells as the first stage removes debris of host cells or lysed cells, for example, by centrifugation or ultrafiltration. If CRIg variant secretyou� Wednesday, the supernatants from such expression systems are generally first concentrated using a commercially available filter for concentrating the protein, for example, ultrafiltrate device Amicon or Millipore Pellicon. A protease inhibitor such as PMSF may be used on any of the above mentioned stages to inhibit proteolysis and antibiotics may be used to prevent the growth of accidental contaminants.
Variant CRIg obtained from cells can be purified by known methods, for example, using chromatography on a column of hydroxyapatite, gel electrophoresis, dialysis, and/or affinity chromatography.
Other modifications of the CRIg variants
Options CRIg of the present invention may also be modified with the formation of chimeric molecules comprising a variant CRIg, including its fragments, hybridized with another a heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a hybrid variant CRIg or its fragment labeled with a polypeptide constituting the epitope, which can selectively bind an antibody against a given label. Tag epitope is typically located at the amino - or carboxylic polypeptide variant CRIg. The presence of such forms CRIg variant tagged with the epitope can be detected, �using a labeled antibody against the polypeptide. In addition, the presence of the epitope tag enables you to easily clean the CRIg polypeptide by affinity purification using antibodies against the tag or another type of affinity matrix that binds to epitope tag. In this area known variously labeled polypeptides and corresponding antibodies. Examples include polyhistidine (poly-his) or polyesterlining (poly-his-gly) tags; the labeled polypeptide flu HA and antibody to it SA [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; label c-myc and antibody 8F9, 3C7, E, G4, B7 and E thereto (Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)); glycoprotein D of herpes simplex virus (gD) and the antibody to the label (Paborsky et al., Protein Engineering, 3(6):547-553 (1990)). Other labeled polypeptides include the Flag-peptide (Hopp et al., Bio Technology, 6:1204-1210 (1988)); a peptide epitope CT (Martin et al., Science, 255:192-194 (1992)); a peptide epitope tubulin with Quaternary structure [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; protein and peptide 10 T7 gene (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)).
In another embodiment, the chimeric molecule may include a hybrid variant CRIg or its fragment with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a hybrid can be obtained with the Fc-region of IgG molecules. Such hybrid polypeptide are antitelomerase molecules that combine the binding specificity heterological� protein (”adhesin”) with the effector functions of the constant domain of the immunoglobulin, and often defined as immunoadhesin. Structurally immunoadhesin represent a hybrid amino acid sequence with the desired binding specificity which differs from indigenousness and binding center of an antibody (i.e. is ”heterologous”), and the sequence of the constant domain of immunoglobulin. Adhesin, which is part of the molecule immunoadhesin, typically is a contiguous amino acid sequence comprising at least the binding site of the receptor or ligand. The sequence of the constant domain of immunoglobulin in immunoadhesin can be obtained from any immunoglobulin, such as subtypes of IgG-1, IgG-2, IgG-3 or IgG-4, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.
In the simplest structure immunoadhesin combined binding protein ”adhesin” hinge region and the Fc-region of the heavy chain of immunoglobulin. Upon receipt of CRIg-immunoglobulin chimeras of the present invention With the end of the nucleic acid encoding the extracellular domain of CRIg, usually hybridizing with nucleic acid that encodes the N-end sequence of the constant domain of immunoglobulin, however, also possible N-terminal hybrids.
In such hybrids, the encoded chimeric polypeptide generally retains at least functionally �active hinge region, CH2 - and CH3-domey constant region of the heavy chain of immunoglobulin. Also get hybrids with C-end of the Fc portion of a constant domain or N-end with CH1 of the heavy chain or the corresponding region of the light chain.
The exact site on which can be obtained hybrid, is not critical; particular sites are well known and can be chosen to optimize the biological activity, secretion or binding characteristics CRIg-immunoglobulin chimeras.
In some embodiments, CRIg-immunoglobulin chimeras are obtained in the form of monomers, hetero - or romanogobio and, in particular, in the form of dimers or tetramers, similar to that shown in the publication WO 91/08298.
In a preferred embodiment, the sequence of the extracellular domain of CRIg hybridizing with N-end C-terminal portion of an antibody (in particular the Fc domain), which perform effector functions of an immunoglobulin, e.g. immunoglobulin G. sub.1 (IgG 1). Can be hybridized entire constant region of the heavy chain with the sequence of the extracellular domain of CRIg. However, more preferably, in the hybrid use a sequence beginning in the hinge region at the top from the site of cleavage by papain (which is chemically defines the Fc-region of IgG; residue 216, wherein the first residue of konstantinoupoli the heavy chain is the residue 114, or analogous sites of other immunoglobulins). In a particularly preferred embodiment the amino acid sequence CRIg hybridizing with the hinge region, CH2 and CH3 or CH1, hinge region, CH2 and CH3 domains of the heavy chain of IgG1, IgG2 or IgG3. The exact site on which can be obtained hybrid, is not critical, and the optimal site can be determined by ordinary experimentation.
In some embodiments, CRIg-immunoglobulin chimeras are obtained in the form of Magomedov and, in particular, in the form of homodimers or homotetramers. Such collected immunoglobulins usually are known basic structure. Main chetyrehetapnogo structural unit is the form in which IgG, IgD and IgE. Chetyrehetapnogo link is repeated in the immunoglobulins with higher molecular weight; IgM generally exists in the form of pentamera main chetyrehtomnik links connected by disulfide bonds. The IgA globulin, and occasionally globulin IgG may also exist in a multidimensional form in serum. In the case of megaera all chetyrehtrubnye the links can be the same or different.
Alternative sequence of the extracellular domain of CRIg can be inserted between the sequences of the heavy chain and light chain immunoglobulin, to thereby produce an antibody, comprising himem�Yu heavy chain. In this embodiment, the sequence CRIg hybridizing with 3'-end of the heavy chain of immunoglobulin in each spacer between immunoglobulin hinge region and CH2 domain, or between the CH2 and CH3 domains. Similar designs have been presented in the publication Hoogenboom et al., Mol. Immunol., 28:1027-1037 (1991).
Although the presence of light chain immunoglobulin is not required in immunoadhesin of the present invention, the light chain of an immunoglobulin may be present, being covalently bonded hybrid CRIg polypeptide and heavy chain immunoglobulin or directly hybridized with the extracellular domain of CRIg. In the former case, DNA encoding a light chain of an immunoglobulin, usually coexpression with DNA encoding the hybrid protein of CRIg and the heavy chain of immunoglobulin. In the process of secretion of heavy chain and light chain hybrid covalently swjasiwatsa, forming immunoglobulinovogo structure including two pairs of heavy chain-light chain immunoglobulin, linked by disulfide bonds. Methods for such structures, for example, described in U.S. patent No. 4816567, issued March 28, 1989
The pharmaceutical composition
Options CRIg of the present invention can be introduced for the treatment of diseases, the pathology of which is caused by alternative complement activation.
Therapeutic of prep�get with ATA storage capability, mixing the active molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. ), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers must not be toxic to recipients at the used doses and concentrations, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as chloride of octadecyltrimethoxysilane; hexamethonium chloride; benzalkonium chloride, chloride benzene; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechin; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; helatoobrazovateli such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; metal complexes (�of primer, Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLYRONIC™ or polyethylene glycol (PEG).
Lipofectin or liposomes can also be used for delivery into cells of polypeptide, antibody or antibody fragment. When using fragments of antibodies preferred is the smallest fragment that specifically binds to the binding domain of the target protein. For example, on the basis of sequences of the variable regions of the antibody can be obtained peptide molecules that retain the ability to bind to the sequence of the target protein. Such peptides can be synthesized by chemical methods and/or obtained by recombinant DNA methods (see, for example, the publication Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 ).
The drug of the present invention may also contain several active compounds that are beneficial for treating specific diseases, preferably having a complementary activity and no known influence on each other. Such molecules are present in combination in amounts that ensure the efficient achievement of the target.
Active molecules can also be enclosed in microcapsules obtained, for example, methods of coacervation or interfacial polymerization, such as, for example, hydroxymethyl�lulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal systems drug delivery (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such methods are described in the publication Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Drugs intended for administration in vivo, must be sterile. Sterility is easily achieved by filtration through sterile filtration membranes.
Can be obtained preparations with prolonged action. Acceptable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which have the form of shaped articles, e.g. films, or microcapsules. Examples of matrices with a slow release include polyesters, hydrogels (e.g., poly(2-hydroxyethylmethacrylate) or poly(vinyl alcohol)), polylactic acid called PLA (U.S. patent No. 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, unsplittable ethylene-vinyl acetate, degradable copolymers of lactic acid and glycolic acid, such as LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid and glycolic acid and leuprolide), and poly-D-(-)-3-hydroxybutyric acid. Although such polymers as ethylene vinyl acetate and a copolymer of lactic acid and Pikalevo� acid, enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate under the influence of moisture at 37°C, losing, thus, the biological activity and causing possible changes in immunogenicity. The drug can be efficiently stabilized depending on the mechanism. For example, if it is established that the mechanism of aggregation involves the formation of intermolecular S-S bonds in the result ridiculing of interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using appropriate additives, and the creation of special compositions based on the polymer matrix.
Methods of treatment
Due to its ability to inhibit activation of complement, in particular, alternative pathway complement activation, CRIg variants of the present invention find application in the prevention and/or treatment of complement-associated diseases and pathological conditions. Such diseases and conditions include, but are not limited to, complement-associated inflammatory and autoim�unnie disease.
Specific examples of complement-associated inflammatory and immune diseases and disorders that can be directed to influence CRIg variants of the present invention described above.
The present invention will be further illustrated in more detail below by examples, not limiting the scope of the invention.
Getting CRIg variants with ripe affinity
Materials and methods:
Materials - Enzymes and phage-M13 helper-KO (New England Biolabs); tablets for immunoassay Maxisorp (Nunc. Roskilde, Denmark); 96-well polypropylene tablet with U-shaped wells (COSTAR; catalog number 3365); 96-well plate with flat bottom wells, non binding (NUNC; catalog number 269620); the conjugate of horseradish peroxidase/antibody against M13 (Pharmacia); the substrate of 3,3',5,5'-tetramethylbenzidine, substrate peroxidase H2O2(TEV) (Kirkegaard and Perry Laboratories, Inc);Escherichia coliXL1-blue andE. coliBL21 (DE3) (Stratagene); bovine serum albumin (BSA) and tween 20 (Sigma); agarose Ni-NTA (Qiagen); red blood cells (RBC) rabbit (Colorado Serum Company; catalog number CS1081); vernally buffer with gelatin (GVB) [100 ml veronlough buffer (Bio Whittaker; No. 12-E); gelatin (from bovine skin type V; SIGMA; catalogue number G9391-100G); serum with the absence of C1q (CompTech; catalogue number A300); fH-protein (Complement Technology; catalogue number A); anti�lo against FLAG-HRP in 50% glycerol, Sigma, catalogue number A-8592, 1.1 mg/ml.
Create libraries display CRIg on the phage
The DNA fragment encoding CRIg, was ligated with rahovym vector, split XhoI and SpeI (p3DvlzDZ-gD) (Kunkel et al., Methods Enzymol. 154:367-382 (1987)), and was used as a control sample wild-type and matrix to create a CRIg variants. Then from E. coli cells CJ239 received matrix with termination codon TAA in each residue for the directed randomization (Kunkel et al., 1987, supra). Options CRIg were selected by the method of soft randomization, whereby in the selected position were asked the frequency of mutations equal to about 50%, using a method based on oligonucleotide poison and mixtures grounds 70-10-10-10 favourable for wild-type nucleotides. In libraries: 5 = 50% A, 10% G, 10% and 10%; 6 = 50% G, 10% A, 10% C and 10% T; 7 = 50% C, 10% A, 10% G and 10% T; 8 = 50% T, 10% A, 10% G and 10% S.
It has established five libraries.
BCR1, ATC CTG GAA GTG CAA 656 (SEQ ID NO:7) AGT GTA ACA GGA CCT 866 (SEQ ID NO:8) 555 GGG GTG GAT AAT CTT (SEQ ID NO:9) in the library 1;
BCR2, AAG TGG CTG GTA CAA 768 (SEQ ID NO:10) 668 TCA 657 775 688 577 ATC TTT (SEQ ID NO:11) 786 CGT 657 TCT TCT GGA GAC CAT (SEQ ID NO:12) in the library 2;
BCR3, TTT CGT CTA GAC TCT (SEQ ID NO.:13) 877 668 657 757 588 756 756 678 555 TAS 756 GGC CGC CTG CAT GTVG (SEQ ID NO:14) in the library 3;
BCR4, CAA AGC TTG ACC CTG (SEQ ID NO:15) 586 656 657 GAC 768 AGC CAC TAC ACG TGT 656 (SEQ ID NO:16) GTC ACC TGG 756 (SEQ ID NO:17) ACT CCT GAT GGC AAC (SEQ ID NO:18) in the library 4;
BCR5, ACT CCT GAT GGC AAC 756 (SEQ ID NO:19) GTC 688 768 657 555 ATT ACT GAG CTC CGT (SEQ ID NO:20) in the library 5
Site-directed mutagenesis for of point mutations was performed by the method described above using the appropriate codons for the mutations, the correct clones were identified by sequencing.
Sorting and screening of libraries for the selection of CRIg variants
Tablets for immunoassay Maxisorp were sensitized overnight at 4°With complement C3b (5 μg/ml) and blocked for 1 hour at room temperature saline solution with phosphate buffer (PBS) and 0.05% (wt./about.) bovine serum albumin (BSA). Library phages were applied to C3b-sensitized plates and incubated at room temperature for 3 hours. The plates were washed ten times, the bound phage were suirable 50 mm HCl and neutralized with an equal volume of 1.0 M Tris-base (pH 7.5). Selected phages were amplified, Passera onE. coliXL1-blue, and used to perform additional cycles of selection binding. After performing 5 cycles were selected 12 individual clones from each library, which were grown on 96-well plates in 500 μl of 2YT broth containing carbenicillin and phage-M13 helper-CO. Culture supernatants were subjected to twofold serial dilution, were applied to 384-well plates, sensitized C3b, an antibody against gD, BSA and an unrelated protein demand�x positions. To determine the concentration of phage was used to measure binding affinity, which C3b was administered in much larger quantities than antilo against gD that did not belong to BSA and an unrelated protein. The concentration of phage was fixed, exploring approximately 200 clones from each library is the same method, resulting in the selected 24-48 clones, which were largely associated with C3b compared with the antibody against gD in each library were then sequenced for analysis.
Competitive binding analysis of the phage ELISA
To determine the affinity of binding was performed a modified analysis of the phage ELISA. 96-well titration microplates were sensitized with 2 mg/ml 3Cb in 50 mm carbonate buffer (pH 9,6) over night at 4°C. the plates were Then blocked with PBS, 0,5% BSA for 1 hour at room temperature. Phage displaying options CRIg, serially diluted in PBT buffer and measured the binding to determine the concentration of phage, feeding 50% of the signal at saturation. The concentration of phage at partial saturation was fixed, the phage were pre-incubated for 2 hours with serial dilutions of C3b and transferred the mixture on analytical plates, sensitized C3b. After incubation for 15 minutes the plates were washed with PBS, 0,05% tween 20 and incubated for 30 minutes with the conjugate perk�of easy horseradish/antibodies against M13 (dilution 1:5000 in PBT buffer). The plates were washed, showed substrate TMB was quenched with a 1.0 m solution of N3RO4and read with a spectrophotometer to read the tablets at a wavelength of 450 nm. The affinity (IC50) was calculated in the concentration of competing C3b, providing half of the maximum binding of the phage.
The protein purification
A single colony ofE. coliBL12 (DE3) containing the expression plasmid were inoculable in 30 ml of LB medium containing 50 μg/ml karbenicillina (Wednesday LB/carbs) and grown over night at 37°C. the Bacteria were collected, washed, were resuspended and inoculable in 500 ml LB medium/carbs. The culture was grown at 37°C to the average log-phase (A600= 0,8). Protein expression was induced with 0.4 mm isopropyl-1-thio-D-galactopyranoside and the culture was grown for 24 hours at 30°C. Bacteria were centrifuged at acceleration 4000 g for 15 minutes, washed twice with saline phosphate buffer (PBS) and frozen within 8 hours at -80°C. the Precipitate was resuspended in 50 ml PBS and bacteria were literally by passing them through microfluidizer or ultrasonic equipment. Proteins CRIg variants were cleaned in 2 ml of agarose NI-NTA and gel filtration.
Competitive binding analysis mutCRIg-huFc in the liquid phase method ELISA
huCRIg(L)-LFH was diluted to 2 μg/ml in PBS, pH 7.4, were applied to 384-well plates with flat bottom wells Maxisorp(Nunc, Neptune, NJ) and incubated overnight (16-18 hours) at 4°C (25 µl/well). The plates were washed three times in wash buffer (PBS, pH 7.4, with 0.05% tween-20) and to each well was added 50 μl/well blocking buffer (PBS, pH 7.4, with 0.5% BSA). The plates were blocked for 1-3 hours; this and all subsequent incubation was performed on a rotary shaker at room temperature. At the stage of blocking C3b (purified in the company Genetech) was diluted to 20 nm in analytical buffer (PBS, pH 7.4, with 0.5% BSA, 0,05% tween-20) and molecules mutCRIg-huFc also serially diluted in analytical buffer in the concentration range 20000 - 0.34 nm. Then C3b molecules mutCRIg-huFc were mixed in the ratio 1:1 and pre-incubated for 0.5-1 hour. Blocked plates were washed three times (as described above) and the reaction tubes were applied C3b complexes:mutCRIg-huFc (25 µl/well). After incubation for 1-2 hours tablets for ELISA were washed three times (as described above) and were found associated with the tablet C3b, adding the antibody against human C3b (clone 5F202, US Biological, Swampscott, MA; 600 ng/ml, 25 μl/well). The plates were incubated for 1-2 hours and washed with the above described method. Then add HRP-conjugated IgG anti-mouse Fc (Jackson ImmunoResearch, West Grove, PA) diluted to 1:2000, (25 µl/well) and the plates were incubated for 1-2 hours. After final washing tablets DL� ELISA analysis was applied 25 ál/well of TMB substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). After about 8 minutes stopped the manifestation of color by adding 25 μl/well of 1.0 M solution of phosphoric acid. The optical density at 450 nm and 650 nm were determined using a spectrophotometer to read the titration microplates SpectraMax 250 (Molecular Devices, Sunnyvale, CA).
Analysis of complement activation
The ability ofmutCRIg-Fc to inhibit activation of complement was determined, using a set of alternative pathway complement activation Wieslab™ (Alpco Diagnostics, Salem, NH). Serially diluted mutCRIg-Fc (400 to 0.2 nm) and human serum with the absence of C1q (5%) (Complement Technology, Tyler, TX) was obtained at a concentration that was twice the desired final concentration, in the form of a mixture with a ratio of 1:1, pre-incubated for 5 minutes on a rotating shaker at a speed of 300 Rev/min and were applied to LPS-sensitized tablets for ELISA analysis (100 µl/well). The rest of the analyses were performed according to the manufacturer's instructions. Briefly, the samples tablets for ELISA were incubated for 60 to 70 minutes at 37°C and washed three times in wash buffer (PBS, pH 7.4, with 0.05% tween-20). On the tablet for ELISA analysis was applied 100 μl/well of conjugate antibodies against C5b-9. After incubation for 30 minutes at room temperature, the tablet for ELISA analysis were washed with the above described method, in CA�blow hole was added 100 µl of the substrate and the plates were incubated at room temperature for another 30 minutes. The manifestation of color was stopped by adding 50 μl/well of 5 mm EDTA. Optical density at 405 nm was determined using a spectrophotometer to read the titration microplates MultiSkan Ascent (Thermo Fisher Scientific, Milford, MA).
Analysis of inhibition of hemolysis
The rabbit erythrocytes (Colorado Serum Company, Denver, CO) were washed three times veronalum buffer (Sigma, St. Louis, MO) containing 0.1% bovine skin gelatin (Sigma) (GVB), and after each washing, centrifugally at a speed of 1500 Rev/min at 4°C for 10 minutes. After the last stage of centrifugation the cells were resuspended in GVB at a final concentration of 2×109cells/ml Inhibitors of complement, serially diluted in GVB, were applied to 96-well polypropylene plates with U-shaped wells (Costar, Cambridge, MA) in an amount of 50 μl/well and then added 20 μl/well of rabbit erythrocytes diluted in the ratio 1:2 in 0.1 M MgCl2/0.1 M EGTA/GVB. The cascade of complement activation on the tablet was started by adding 30 μl/well of the serum with the absence of C1q (Complement Technology, Tyler, TX), which had previously bred GVB in the ratio of 1:3. The tablet(s) were incubated by gently shaking for 30 minutes at room temperature, after which the reaction was stopped by adding 100 μl/well of 10 mm EDTA/GVB. The tablet(s) were centrifuged at a speed of 1500 Rev/min for 5 min, then the supernatants were transferred to h�simple 96-well plates with flat bottom wells, not causing binding (Nunc, Neptune, NJ) and determined the optical density at 412 nm using a spectrophotometer for reading the microplate (Molecular Devices, Sunnyvale, CA).
Competitive binding analysis of the Alpha Screen
Potential cross-reactivity of the mutant molecules CRIg in respect of C3 was determined using a kit for the detection of histidine (Nickel chelate) AlphaScreen® (PerkinElmer, Waltham MA). Serially diluted complements human C3 and C3b (from 3000 to 0.7 nm), biotinylating iC3b in constant concentration (30 nm) and mutant molecules CRIg (mutCRIg) and wild-type CRIg (15-60 nm) was obtained at a concentration that was three times above the desired final concentration, in the form of a mixture with a ratio of 1:1:1 and pre-incubated at room temperature for 30 minutes on a rotary shaker with a speed of 3000 Rev/min the Mixture donor pellet streptavidin and acceptor pellets chelate of Nickel with a ratio of 1:1 (0.1 mg/ml each) was obtained in the concentration, which was four times the desired final concentration, and added to the reaction mixture. The reaction plate was incubated at room temperature for 60 minutes on a rotary shaker with a speed of 3000 Rev/min in the dark. The tablet was analyzed using the analyzer for microplates AlphaQuest®-HTS (PerkinElmer, Waltham, MA).
Affinity to C3b mutant CRIg and CRIg wild type was determined by measurement of the resonance of surface plasmons in the device A100 Biacore® (GE healthcare). Used immobilized antibody against the Fc and calculated the value ofKDby performing the measurement of equilibrium binding. Sensor chip obtained using a set of immobilized antibodies against muFc (BR-1008-38) according to the manufacturer's instructions. Mutant CRIg and CRIg wild type bred in the separating buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 0.01% tween 20) to a concentration of 1 μg/ml and injected 60 µl in such a way that one spot of the chip surface was captured ~100 rat units (RU) of the hybrid protein. Sensograms were recorded for solutions with different concentrations of C3b, which were applied on the stain CRIg 10 minutes, minus the reference signal for the spots containing immobilized antibody without CRIg. Data were obtained for a series of 2-fold dilutions of C3b in a concentration of from 4 μm to 15.6 nm at a flow rate of 10 µl/min and a temperature of 25°C. the Surface was regenerated between cycles of binding by administration of 10 mm Gly-HCl, pH of 1.7, for 30 seconds. Smooth the values obtained at the end of each insertion C3b, used to calculate theToDusing the affinity algorithm software Biacore A100, version 1.1 (Safsten et al. (2006) Anal. Biochem. 353:181).
Creating library�to phages
To create libraries of phages was used crystalline structurea CRIg together with C3b. It has established five libraries to cover the entire area of contact between CRIg and C3b (figure 4). Library CRIg were created in the form of a hybrid with minor protein shell g3p in the vector of monovalent display phage (Zhang et al., J. Biol. Chem. 281(31):22299-311 (2006)). By mutagenesis were introduced termination codons in CRIg-encoding part ragovoy plasmids in each position randomizirebul balance. All structures containing a termination codon, are then used to create a library display on the phage (see ”Materials and methods”). The method of ”soft randomization” was used for the selection of binder required to maintain the offset of the wild-type sequence so that selected provisions were motroway only for 50% of the time. All five libraries were obtained with an average variability of >1010independent sequences in the same library (table 1).
Selection using the library display CRIg on the phage
After the completion of four cycles of selection binding was obtained 38 unique clones from five libraries (table 2). In the library 1 was retained by lysine in position 15. Glutamic acid in position 8 has been replaced by aromatic residues, tyrosine and tryptophan. In position noodels source tryptophan or homologous phenylalanine. In the library 2 were sequenced 24 clone, and they all agree with each other. The provisions in 42, 46 and 47 were retained residues present in the wild-type sequence. The wild-type sequence at positions 43, 44 and 45 was replaced by asparagine, histidine and phenylalanine. In the library 3 were arbitrarily replaced by residues 10 provisions while preserving the residues in positions 54, 55, 56, 57, 58, 61, 62 and 63. In position 60 were isoleucine or lysine. Glutamine at position 64 has been replaced by arginine or saved. In the library 4 at position 86 was dominated by aromatic residues and in position 99 prevailed homologous basic residues, arginine and lysine. Residues at positions 85, 87 and 95 were also arbitrarily replaced, but remained highly conservative. In the library 5 in position 105 is glutamine were preferably used two significant homologous basic residue, lysine and arginine. At position 110 was dominated by negatively charged residues, aspartic acid or acidic residues, asparagine.
The affinity of some of the mutants were determined using competitive binding analysis of phage ELISA (data not available), it was found that the library 3 were clones, which is about eight times better than the bound C3b compared with wild-type CRIg.
Determination of the affinity of Saint�of zivania in vitro and biological activity in vivo
To determine residues are particularly important for increasing the affinity of binding to C3b and activity, when performing analysis of inhibition of hemolysis, was created by the second generation of CRIg variants by introducing a single dominant mutation and conservation in the other provisions of residues of wild-type or selection of 2-3 high-affinity clones from libraries of phages of the first generation that were identified using the analysis of phages by ELISA method. For accurate measurement of the affinity and activity of mutants all variants were expressed in the form of selected proteins. The results (table 3), obtained by analysis of inhibition of hemolysis showed that L12 from the library 1, L33 from library 2 and L41 from the library 4 showed a significant increase of activity in 4-10 times compared with wild-type clones, used in the performance analysis of inhibition of hemolysis. L32 from the library 3 was characterized by a 10-fold increase in the IC50 value compared with wild-type CRIg. The data also showed that the values of binding affinity and activity, measured using cell tests were not correlated.
The combination of mutants
On the basis of the results obtained in the study of second generation libraries were established by the third generation of mutants to further enhance actively�ti, installed when running the analysis of inhibition of hemolysis, and affinity binding. As the matrix were selected three mutant with the greatest biological activity (L12-8W, L33-Q60I/L32-Q64R and L41-M86Y) and one mutant with the highest binding affinity (L32-Q64R). Eventually, these mutants were combined with other biologically active clones obtained in the second generation libraries, to determine the optimal set increases the activity of mutations to perform the analysis of inhibition of hemolysis and affinity of binding. Options CRIg was expressed and purified for detailed analysis. The data showed (table 4) that the combined mutants obtained from L12, did not improve the activity of inhibiting and even were characterized by lower activity compared to the original mutant, despite a 3-6-fold increase in binding affinity of mutants and WL41 WL59. Among the mutants obtained from L32, mutant RL41 was characterized by the affinity of the knitting, which was 1.8 times higher than in the wild type, and a 6-fold increase in activity when performing analysis of inhibition of hemolysis. All the mutants from the group L33 was characterized by znachitelnym increase in the binding affinity of about 27-226-fold compared with wild type, although no significant increase of activity when performing analysis of inhibition g�of Molise. In addition, it was noted that the remains 60I 64R and 86Y are present in the majority of combined clones with improved binding affinity.
Improved binding affinity and increased activity of inhibition of complement mutant CRIg Q64R M86Y
For further analysis, we selected a mutant Q64R M86Y that showed the highest affinity binding when you perform a competitive-binding ELISA (Fig. 4 and table 3). To determine the affinity of binding of wild-type CRIg and CRIg Q64R M86Y against C3b was performed Biacore analysis wild-type CRIg and CRIg Q64R M86Y. The affinity of binding of CRIg Q64R M86Y was enhanced 5-fold compared with wild-type CRIg (Fig. 6). Previous studies showed that wild-type CRIg selectively binds to C3b, but not associated with native C3 (Wiesman et al., Nature, 444(7116):217-20, 2006). Because the mutation can change the selectivity, compared the affinity of binding of CRIg Q64R M86Y with C3b and C3 when performing competitive binding analysis in the liquid phase Alpha Screen. CRIg Q64R M86Y competed with soluble C3b, but not competed with soluble C3, which implies that the mutation did not affect the selectivity of CRIg in respect of the active component C3b (Fig. 7). This selectivity was further confirmed by analysis of residues in the structure of CRIg Q64R M86Y in complex with C3b (data not available).
To ensure that the best e�of effectiveness is the result of an improved binding affinity and retained selectivity against C3b, CRIg Q64R M86Y and wild-type CRIg experienced when performing analysis of inhibition of hemolysis on the basis of red blood cells, which has a selectivity in relation to alternative pathway activation complement. CRIg Q64R M86Y was characterized by a 4-fold increase in the IC50 value compared with CRIg wild type (Fig. 8A). For further confirmation of the presence of higher activity towards inhibition of complement alternative pathway inhibitory activity CRIg Q64R M86Y compared with wild-type CRIg using the analysis based on the LPS, with the selectivity in respect of the alternative pathway activation of complement. In this case, CRIg was characterized by a 180-fold increase in the IC50 value compared with recombinant wild-type protein. CRIg wild-type and CRIg Q64R M86Y did not affect activation of complement in the classical way. Thus, the replacement of two amino acids in the binding of CRIg-causes the formation of C3b molecules with improved binding affinity and higher activity of inhibition of complement when you perform two different analyses, with the selectivity in respect of the alternative pathway activation of complement.
To further determine that the enhanced binding affinity and activity provide better therapeutic efficacy, compared the protective effect of CRIg wild-type and vari�NTA Q64R M86Y receptor CRIg whey models of arthritis. Previous studies have shown that CRIg intensively inhibits inflammation and bone loss in arthritis induced by collagen and antibody (Katschke et al., J. Exp. Med. 204(6):1319-1325 (2007)).
The effectiveness of CRIg was experienced in the third preclinical models of arthritis mediated by immune complex. Spontaneous murine model of rheumatoid arthritis, K/BxN, mimics many of the clinical and histological signs of arthritis in humans. At day 0 mice were injected with 50 μl of serum obtained from mice suffering from K/BxN. The condition of the animals was monitored daily and the extent of disease was assessed by visual inspection. All mice were euthanized on day 6.
The mice daily subcutaneously injected with a specified number isotype control or recombinant proteins hCRIg-mIgG1 or hCRIg-RL41-mIgG1 in 100 μl of sterile saline, starting from the 1st day.
Monitoring and evaluation:
Evaluation for each foot.
0 = No erythema and swelling
1 = Erythema and slight swelling, limited middle part of the foot (Tarsus) or ankle joint
2 = Erythema and slight swelling from the ankle to the middle of the foot
3 = Erythema and moderate swelling from the ankle to the metatarsal joints;
4 = Erythema and severe swelling encompassing the ankle, one hundred�and fingers
Average score = sum of scores for 4 paws.
Stage of the disease include mild (rating 1-3), moderate (average score 4-8) and heavy (average 9 and up) form of the disease. Rating corresponds to the number of affected joints.
On the 6th day before the killing of animals they took samples of blood by intracardiac puncture performed under anesthesia. In serum measured the amount of hybrid protein hCRIg-Fc. Joints were collected for histological examination.
Transfusion serum of mice KRN mice in recipient Balb/c caused a rapid and sustained immune response, which is characterized by symmetric inflammation of the joints. The occurrence of arthritis mediated by autoantibodies against glucose-6-phosphate isomerases, which form a proinflammatory immune complexes in the joints (Kouskoff, V., Korganow, A. S., Duchatelle, V., Degott, C., Benoist, C. and Mathis, D. (1996). Organ-specific disease provoked by systemic autoimmunity. Cell 87, 811-822). The progression of arthritis is completely dependent on an intact alternative pathway complement activation and function of Fc receptors, as evidenced by the absence of disease in mice lacking components of the complement alternative pathway, and in mice lacking the gamma chain of Fc-receptor (Ji, H., Ohmura, K., Mahmood, U., Lee, M. D., Hofhuis, F. M., Boackle, S. A., Takahashi, K., Holers, V. M., Walport, M., Gerard, C., et al. (2002). Arthritis critically dependent on innate immune system players. Immunity 16, 157-168). Due to the rapid in�invention and severity of disease, treatment hybrid protein wild-type CRIg - Fc reduced the evaluation of arthritis only 22% (Fig. 9A, B). Treatment CRIg Q64R M86Y has lowered ratings of arthritis by 66%. Histological examination showed a significant decrease in the infiltration of immune cells, consisting mainly of neutrophils and macrophages in mice, which were injected with CRIg Q64R M86Y, compared with mice that were administered CRIg wild-type or control fusion protein (Fig. 9C, D). The concentration of wild-type CRIg and CRIg Q64R M86Y in serum were identical, indicating that the difference in the estimates of arthritis was not associated with different half-life of wild-type CRIg compared with CRIg protein Q64R M86Y. Thus, it was found that the best affinity binding of CRIg to C3b-target indicator is significantly higher therapeutic efficacy.
All patents and literature references cited in the present description of the invention, and fully incorporated by reference.
Although the present invention has been described with reference to specific embodiments of, it should be clear that the invention is not limited to these options for implementation. On the contrary in the scope of the present invention includes various modifications and equivalents, as defined by essence and scope of the attached claims.
1. Variant CRIg, which includes the replacement of amino acids in one or neskolkochasovyh amino acid sequence SEQ ID NO:2,
or its biologically active fragment, where the specified substitution selected from the group consisting of E8W, W14F, E8Y/W14F, G42D/D44H/P45F, Q60I, Q64R, Q60I/Q64R, M86Y, M86W, M86F, M86W/Q99R, M86F/Q99R, K110D, K110N, Q105R/K110N, Q105R/K110Q, Q105K/K110D, Q64R/M86Y, Q60I/Q64R/E8Y, Q60I/Q64R/G42D, Q60I/Q64R/P45F, Q60I/Q64R/G42D/D44H/P45F, Q60I/Q64R/M86Y, Q60I/Q64R/Q105R, Q60I/Q64R/Q105K, Q60I/Q64R/K110N, Q60I/Q105R/K110N, M86Y/E8Y, M86Y/G42D/D44H/P45F, M86Y/P45F, M86Y/G42D/D44H/P45F, M86Y/Q99K, M86Y/Q99R, M86Y/Q105R, M86Y/Q105K, M86Y/Q105R/K110N, and
where specified CRIg variant or a fragment thereof is at least 2 times more potent inhibitor of the alternative pathway complement activation than human CRIg with a native sequence SEQ ID NO:2, and optionally has increased at least 2 times the affinity of binding to C3b compared with CRIg person with a native sequence SEQ ID NO:2.
2. Variant according to claim 1, which selectively binds to C3b or its biologically active fragment compared to the C3.
3. Variant according to claim 1, wherein the binding affinity is increased by at least 5 times.
4. Variant according to claim 1, wherein the binding affinity is increased by at least 10 times.
5. Variant according to claim 1, wherein the binding affinity is increased by at least 90 times.
6. Variant according to claim 1, comprising the amino acid changes in one or several positions 60, 64, 86, 99, 105 and 110 in the amino acid sequence SEQ ID NO:2.
7. A chimeric molecule that is at least 2 times more potent inhibitor al�ernatives pathway activation of complement, than CRIg person with a native sequence SEQ ID NO:2, and optionally has increased at least 2 times the affinity of binding to C3b compared with CRIg person with a native sequence SEQ ID NO:2 comprising a variant according to any one of claims.1-6, hybridized with another, the heterologous polypeptide or amino acid sequence.
8. Chimeric molecule according to claim 7, representing immunoadhesin.
9. Chimeric molecule according to claim 8, where the specified variant CRIg shorter than reprezentirovanii CRIg of SEQ ID NO:2.
10. Chimeric molecule according to claim 9, comprising the extracellular domain of CRIg.
11. Pharmaceutical composition for use in the treatment of complement-associated disease in a subject in need, comprising effective concentrations of CRIg variant according to any one of claims.1-6 or immunoadhesin according to any one of claims.8-10 in a mixture with a pharmaceutically acceptable excipient.
12. A composition according to claim 11, where the specified subject is a mammal, and where the specified mammal is not necessarily the person.
13. The use of CRIg variant according to any one of claims.1-6 or immunoadhesin according to any one of claims.8-10 when getting medicines for the treatment of complement-associated disease or condition.
14. The use according to claim 13, wherein said complement-associated disease:
(i) is in�sporitelny disease or autoimmune disease; or
(ii) selected from the group comprising rheumatoid arthritis (RA), respiratory distress syndrome in adults (ARDS), loss of tissue removed after ischemia and reperfusion, activation of complement during surgery with extracorporeal circulation, dermatomyositis, pemphigus, lupus nephritis and its resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, coronary dysfunction of the endothelium induced by cardioplegia, membranosa-proliferative glomerulonephritis type II, IgA-associated nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, age-related macular degeneration, uveitis, diabetic retinopathy, allograft, Verhoture rejection, hemodialysis, chronic obstructive pulmonary disease (COPD), asthma, aspiration pneumonia, utricaria, chronic idiopathic urticaria, hemolytic uremic syndrome, endometriosis, cardiogenic shock, ischemic reperfusion injury and multiple sclerosis (MS); or
(iii) selected from the group comprising inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), xeroderma, systemic vasculitis, sarcoidosis, and�tommorow hemolytic anemia (immune pancytopenia, night the paroxysmal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombotic purple, immune thrombocytopenia), thyroiditis (graves ' disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the Central and peripheral nervous systems such as multiple sclerosis, idiopathic polyneuropathy, gall kidney diseases such as infectious hepatitis (hepatitis A, b, C, D, E and other nagapattanam viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung diseases (e.g., cystic fibrosis), patentability enteropathy, Whipple's disease, autoimmune or immune skin diseases including bullous skin diseases, polymorphic erythema, and contact dermatitis, psoriasis, allergic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and allergic pneumonia, diseases caused by transplantation, including graft rejection, graft-versus-host disease, Alzheimer's disease, the paroxysmal nocturnal hemoglobinuria, a�tively angioedema, atherosclerosis and membranosa-proliferative glomerulonephritis type II; or
(iv) is rheumatoid arthritis (RA); or
(v) is the complement-associated eye disease where the complement-associated eye disease is not necessarily selected from the group comprising all stages of age-related macular degeneration (AMD), uveitis, diabetic and other caused by ischemia, retinopathy, endophthalmitis, and other intraocular diseases caused by formation of new vessels, and where the intraocular disease caused by the formation of new vessels, optionally selected from the group comprising diabetic edema yellow spots, pathological myopia, disease von Hippel-Lindau, histoplasmosis of the eye, occlusion of the Central retinal vein (CRVO), the formation of new vessels in the cornea and the formation of new vessels in the retina.
15. The use according to claim 14, part (v), wherein said complement-associated eye disease is selected from the group comprising age-related macular degeneration (AMD), the formation of new vessels in the choroid of the eye (CNV), diabetic retinopathy (DR) and endophthalmitis.
16. The use according to claim 14, in which the specified macular degeneration (AMD) is wet macular degeneration.
17. The use according to claim 14, in which the specified macular degeneration (AMD) �is dry or atrophic macular degeneration.
SUBSTANCE: invention relates to field of biotechnology, immunology and genetic engineering. Genetic modification contains deletion of endogenic low-affinity FcγR locus, where contain functional FcRγ-chain. Modified mice express up to five genes of low-affinity human FcγR on antigen-presenting cells of host immune system. Also described is method of screening substance, containing Fc site of human antibody by means of said mice. Invention can be used in the field of medicine.
EFFECT: presented are genetically modified mice, not related to man, and methods and compositions for their obtaining and application.
15 cl, 11 dwg, 6 tbl, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biochemistry and is intended for treatment or prevention of undesirable immune response. Claimed is novel soluble polypeptide CD83 (sCD83), containing SEQ ID NO:7, in which amino acid residues 12, 20, 92 and 114 represent cysteine, amino acid in position 85 represents serine and one or several of amino acids 1, 2, 3, 4 and 130 can be absent. Also claimed is nucleic acid, coding said sCD83, and application of said polypeptide in methods of treatment or prevention of transplant rejection or autoimmune diseases, such as type I diabetes, multiple sclerosis, Crohn's disease and ulcerative colitis.
EFFECT: invention makes it possible to increase stability and biological activity of sCD83 preparations.
15 cl, 39 dwg, 9 tbl, 10 ex
SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.
EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.
18 cl, 124 dwg, 10 ex
SUBSTANCE: invention refers to biochemistry, particularly to artificial immunogenic proteins having the properties of melanoma antigens. What is declared is an artificial gene coding MEL-TCI-A0201 polyepitope immunogenic protein containing multiple cytotoxic restricted HLA-A*0201 and T-helper epitopes of NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1 melanoma antigens, having a sequence of 1,535 base pairs presented in Fig. 3. There are also declared a recombinant plasmid DNA containing the above artificial gene, and MEL-TCI-A0201 immunogenic protein with the properties of the melanoma antigens.
EFFECT: invention enables providing higher immunogenicity of the artificial polyepitope T-cell immunogen inducing a higher level of cytotoxic T-lymphocyte response.
3 cl, 11 dwg, 3 tbl, 2 ex
SUBSTANCE: present invention relates to biotechnology and provides humanised synthetic analogues (R6313/G2) of variable domains of the anti-angiotensin II type 1 receptor monoclonal scFv antibody 6313/G2, i.e. which specifically bind the molecule which specifically binds with a peptide, having the amino acid sequence EDGIKRIQDD. The invention also discloses a method of treating cancer, use of specifically binding molecules according to the invention when preparing a drug for treating cancer, a combined preparation and pharmaceutical compositions containing a specifically binding molecule according to the invention and angiotensin II.
EFFECT: present invention enables to block the harmful effect of angiotensin II and use said analogues to treat cancer in humans.
9 cl, 15 dwg, 4 ex
SUBSTANCE: invention relates to biochemistry, particularly to recombinant fused protein dimers intended to inhibit or suppress immune response in a mammal, which bind human CD80 or human CD86 or the extracellular domain of any thereof, and has higher capacity for suppressing immune response than a dimer of the fused protein LEA29Y-Ig. Also disclosed are nucleic acids which code said dimers, expression vectors containing said nucleic acids, as well as recombinant host cells containing said nucleic acids and/or said vectors. Disclosed are pharmaceutical compositions for inhibiting or suppressing immune response in a mammal, which contain said fused protein dimers, as well as use of said dimers to produce drugs for inhibiting or suppressing immune response in a mammal, treating diseases or disorders of the immune system or treating organ or tissue transplant rejection in a mammal. Methods of producing said fused protein dimers are also disclosed.
EFFECT: invention provides effective inhibition or suppression of immune response in a mammal.
9 cl, 15 dwg, 11 tbl, 12 ex
SUBSTANCE: invention describes creation of an artificial conformational antigen including peptide fragments consisting of two extracellular loops of muscarine M2-receptor, which are cross-linked with disulphide bond, and which is able to fix autoantibodies to this receptor with sensitivity exceeding the sensitivity for a linear prototype or mechanical mixture of data of linear peptide fragments. Synthetic antigen represents an individual chemical compound containing peptide corresponding to the sequence of the 1-st extracellular loop and peptide of the 2-nd extracellular loop, which are linked with disulphide connection between Cys96 and Cys176 residues (structure of antigen is disclosed in the formula and description). As per the invention, there described are methods for obtaining and using an antigen for creation of a diagnostics set for reveal of antibodies to muscarine M2-receptor in blood of patients with idiopathic arrhythmia using an enzyme-linked immunosorbent assay (ELISA) method.
EFFECT: improvement of the method.
5 cl, 1 dwg, 3 tbl, 4 ex
SUBSTANCE: obtained is protein complex, possessing GPCRα1L affinity to ligand, which includes GPCRα1A and polypeptide with sequence of amino acids SEQ ID NO:1. Binding of said G-protein-conjugated receptor with polypeptide alters ligand affinity of the receptor. Also claimed are methods of screening agonists or antagonists of G-protein-conjugated receptor with application of transformant, in which said altered G-protein-conjugated receptor is expressed.
EFFECT: carrying out analysis of many supposed G-protein-conjugated receptors with still unknown structure.
12 cl, 3 dwg, 2 tbl, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, particularly to such binding elements specific for IgE as antibody molecules or its antigen-binding fragments.
EFFECT: antibody or its versions specific for IgE are effective for treating IgE-associated disorders, such as allergy and asthma.
17 cl, 17 dwg, 8 tbl, 9 ex
SUBSTANCE: what is presented is a method for making a panel of serums for the purpose of quality control of diagnosing hepatitis B with the certified low concentration of HBsAg subtypes AD and AY which involves the analysis of donor serums for the presence of anti-HIV, anti-HCV and anti-HBs antibodies and the nonspecific HBsAg binding; the selection of donor serums with said parameters not found; the use of the selected serums to prepare diluting solutions with a stabilising additive introduced; the certification of the monopreparations of HBsAg subtypes AD and AY by titration dilutions of international standards and the preparation of a reference panel from the certified monopreparations of HBsAg subtypes AD and AY within the range of 0.01 to 0.5 IU/ml; the lyophilisation of the serums followed by thermal degradation panel testing to determine a shelf life. The use of a new formulation of the stabilising additive containing proline 200-250 mM and benzoic acid 0.05-0.08 wt %, and the selection of an optimum temperature storage conditions promote the prolonged activity preservation at annual loss (at +4°C) making less than 0.5%.
EFFECT: higher shelf life.
3 cl, 5 tbl, 1 dwg, 1 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.
EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.
20 cl, 42 ex, 8 tbl, 3 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to biotechnology, namely to novel IL-17-inhibiting polypeptides, corresponding to fused proteins, to compositions and their application for medicinal purposes. Polypeptide contains amino acid sequence, which is selected from group, consisting of GVTLFVALYD YKAFWPGDLS FHKGEKFQIL RTSDGDWWEA RSLTTGETGY IPSNYVAPVD SIQ (SEQ ID NO: 39), GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWVA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 57) or GVTLFVALYD YKAFWPGDIS FHKGEKFQIL RTSDGEWWIA RSLTTGEEGY IPSNYVAPVD SIQ (SEQ ID NO: 107); amino acid sequence, which has, at least, 80%, preferably, at least, 90%, more preferably, at least, 95% identity of amino acid sequence with SEQ ID NO: 39, SEQ ID NO:57 or SEQ ID NO: 107; fragment or functional derivative of SEQ ID NO: 39, SEQ ID NO: 57 or SEQ ID NO: 107, obtained due to substitution, addition and/or removal of not more than 5 amino acids.
EFFECT: invention makes it possible to bind IL-17 with high specificity and affinity.
33 cl, 17 dwg, 3 tbl, 12 ex
SUBSTANCE: invention refers to immunology. What is disclosed is using a peptide with an amino acid sequence GLAGGSAQSQRAPDRVL for selecting CεmX-specific antibodies and antigen-binding fragments of these antibodies. What is presented is the CεmX-specific antibody, as well as a method providing selecting the antibodies specifically binding the peptide according to the invention, and a method of treating IgE-mediated diseases involving administering the antibody into an individual according to the invention.
EFFECT: it has been disclosed that the monoclonal antibodies specifically binding the CεmX segment GLAGGSAQSQRAPDRVL can effectively bind to mIgE in human B-cells and are applicable for targeting to these B cells for treating IgE-mediated diseases.
14 cl, 5 dwg, 4 ex
SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.
EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.
33 cl, 21 dwg, 3 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: group of inventions refers to the strain Lactobacillus rhamnosus CNCM I-3690 and to a dairy food product containing the above strain. The presented strain possesses the mannose-specific adhesive properties. The strain possesses the antimicrobial properties in relation to, e.g. Escherichia coli, Salmonella enteritidis and Lysteria monocytogenes.
EFFECT: strain possesses the immunomodulatory properties, particularly possesses an ability to inhibit an inflammatory reaction of HT-29 epithelial cells.
2 cl, 2 dwg, 5 tbl, 3 ex
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
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
SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.
EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.
23 cl, 74 dwg, 17 tbl, 33 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. There are presented an antibody and its antigen-binding fragment specifically binding human colony-stimulating factor-1 receptor (CSF-1R) characterised by sequences of complementary-determining regions (CDR). There are also disclosed a nucleic acid coding the antibody according to the invention or its antigen-binding fragment, a vector providing the expression of the antibody and its antigen-binding fragment, and a pharmaceutical composition applicable in treating the diseases associated with an inflammation or an autoimmunity, or cancer.
EFFECT: invention can find further application in diagnosing and therapy of the CSF-1 associated diseases.
23 cl, 18 dwg, 4 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to a compound of formula
wherein A represents 1,2,4-oxadiazol or 1,3,4-oxadiazol (of formula , or Ia), wherein star signs show a bond, which binds to a pyridine group of formula (I); R1 represents 3-pentyl, 3-methylbut-1-yl, cyclopentyl or cyclohexyl; R2 represents a methoxy group; R3 represents 2,3-dihidroxypropoxy group, -OCH2-CH(OH)-CH2-NHCO-CH2OH, -OCH2-CH(OH)-CH2N(CH3)-CO-CH2OH, -NHSO2CH3 or -NHSO2CH2CH3; and R4 represents ethyl or chlorine; or its pharmaceutically acceptable salts. The invention also refers to a pharmaceutical composition possessing S1P1/EDG1 receptor agonist activity, containing an effective amount of the compound of formula (I) or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
EFFECT: pyridine-4-yl derivatives for preventing or treating diseases or disorders associated with the activated immune system.
26 cl, 4 tbl, 33 ex
SUBSTANCE: invention refers to biotechnology, specifically to VEGF-A specific binding proteins, and can be used in medicine for treating pathological angiogenesis in mammals. The antiangiogenic protein contains one ankyrin recurrent domain consisting of a N-terminal capping module of ankyrin recurrence, a recurrent module presented by an ankyrin recurrent motif of the sequence 1D23G4TPLHLAA56GH7EIVEVLLK8GADVNA (SEQ ID NO:5), wherein 1 represents an amino acid residue specified in A, N, R, V, Y, E, H, I, K, L, Q, S and T; 2 is specified in S, A, N, R, D, F, L, P, T and Y; 3 is specified in T, V, S, A, L and F; 4 is specified in W, F and H; 5 is specified in P, I, A, L, S, T, V and Y; 6 is specified in W, F, I, L, T and V; 7 is specified in L or P and 8 is specified in A, H, N and Y; a recurrent module presented by an ankyrin recurrent motif of the sequence 1D23G4TPLHLAA56GHLEIVEVLLK7GADVNA (SEQ ID NO:1), wherein 1, 2, 3, 4, 5, 6 and 7 independently represents an amino acid residue specified in the group of A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y, and a C-terminal capping module.
EFFECT: invention enables producing an antiangiogenic binding VEGF-A165 with Kd less than 10-7 M protein, which inhibits binding VEGF-A165 to VEGFR-2.
12 cl, 4 dwg, 4 ex