Antibodies binding il-4 and/or bl-13 and their application

FIELD: biotechnologies.

SUBSTANCE: invention discloses a bispecific antibody or its functional fragment, specifically binding with IL-4 and IL-13, which contain variable domains of light and heavy chains with established amino acid sequence. The invention also includes use of antibodies or its functional fragments within a pharmaceutical composition for treatment of diseases or disturbances mediated by IL-4 and/or IL-13, including allergic diseases, asthma, cancer. The invention discloses a molecule of nucleic acid, which codes a bispecific antibody or its fragment, an expression vector and a master cell for production of a bispecific antibody and its functional fragment.

EFFECT: invention makes it possible to produce and use new inhibitors of cytokines, preserving stability in process of production and use in vivo.

17 cl, 2 dwg, 8 tbl, 8 ex

 

The SCOPE of the INVENTION

The present invention relates to novel antibodies, anti-IL-4 antibodies, anti-IL-13 and bespecifically antibody anti-IL-4/anti-IL-13 and their application to improve the condition, treatment, or prevention of diseases or disorders in mammals, including humans, caused by abnormal activity or abnormal metabolism of IL-4 and / or IL-13. Considering the antibody can block the binding and / or signaling ligand, such as IL-4 or IL-13 receptor or receptor complex, such as IL-4Rα, IL-13Rα1 and IL-13Rα2. Disclosed prophylactic immunotherapy and diagnostic preparations containing question antibodies, and their use within the methods of prevention or treatment of various diseases in mammals, including humans, are caused by abnormal metabolism and (or) activity of lymphoid and non-lymphoid cells, including monocytes, fibroblasts and endothelial cells. Such diseases include autoimmune diseases, and diseases caused or characterized by inflammation, such as allergic asthma and dermatitis.

The LEVEL of TECHNOLOGY

Interleukin-4 (IL-4) is a pleiotropic cytokine that has a wide range of biological effects in lymphoid b - and T-cells, as well as many non-lymphoid cells, misrepresented the number of monocytes, endothelial cells and fibroblasts. For example, IL-4 stimulates proliferation of several cell lines that are dependent on IL-2 and IL-3 induces the expression of molecules of the class II major histocompatibility complex in dormant In cells and enhances the secretion of IgG4 and IgE In human cells. IL-4 is associated with the immune response Th2-type and is produced by Th2 cells and promotes their differentiation. It is assumed that IL-4 is evident in the number of diseases such as allergies and asthma.

Was recently identified cytokine IL-13 (Minty, A. et al., Nature, 1993, 362, 248-250, and McKenzie, A. N. et al., Proc. Natl. Acad. Sci. U.S.A, 1993, 90, 3735-3739)containing 112 amino acids that is secreted activated T-lymphocytes, B-lymphocytes, as well as mastocytoma, after their activation.

On the basis of their diverse biological properties similar to IL-4, IL-13 has been attributed to IL-4-like cytokine. Its functions are, indeed, similar to the function of IL-4 in respect of b-cells (Defrance, T. et al., J. Exp. Med., 1994, 179, 135-143, Punnonen, J. et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 3730-3734, Fior, R. et al., Eur. Cytokine Network, 1994, 5, 593-600), monocytes (Giovanni, M. R. F. et al., Blood, 1994, 83, 1738-1743, De Waal Malefyt, R. et al., J. Immunol, 1993, 151, 6370-6381, Doyle, A. et al., Eur. J. Immunol. 1994, 24, 1441-1445, Montaner, L. J. et al., J. Exp. Med., 1993, 178, 743-747, Sozzani, P. et al., J. Biol. Chem., 1995, 270, 5084-5088) and other nonhematopoietic cells (Herbert, J. M. et al., Febs Lett., 1993, 328, 268-270, Derocq, J. M. et al., Febs Lett. 1994, 343, 32-36). On the other hand, unlike IL-4, he does not have the special is practical impact on dormant or activated T-cells (Zurawuki, G. et al., Immunol. Today, 1994, 15, 19-26).

The various manifestations of the biological activity of IL-13 in respect of monocytes/macrophages, b cells and certain hematopoietic precursors described in detail in A. J. Minty, and reviews on IL-13. In addition, some evidence suggests that this cytokine has pleiotropic effects on cells of other types. These nonhematopoietic cells that IL-13 has a direct impact include endothelial and microglial cells, keratinocytes and carcinoma cells of the kidney and colon.

One of the stages of analysis of the signal transmitted by the biological molecule inside the cell, provides for the identification of its membrane receptor. Research conducted for this purpose for IL-13 receptor, showed that IL-13 and IL-4 have a common receptor or at least some of the components of the common receptor complex, as well as common elements of a transmission circuit of the signal (Zurawski, S. M. et al., Embo Journal, 1993, 12, 2663-2670, Aversa, G. et al., J. Exp. Med., 1993, 178, 2213-2218, Vita, N. et al., Biol. Chem., 1995, 270, 3512-3517, Lefort, S. et al., Febs Lett., 1995, 366, 122-126). This receptor is present on the surface of different cell types in different quantities depending on the cell type. Comparative distribution of receptors IL-13 and IL-4 was seen in the works of A. J. Minty (Interleukin-13 for Cytokines in Health and Disease. Eds D. G. Remick and J. S. Fre, Marcel Decker, N.Y. 1996).

The cell surface receptors and receptor complexes bind IL-4 and/or IL - 13 with different affinity. The main components of receptors and receptor complexes that bind IL-4 and/or IL-13, include IL-4Rα, IL-13Rα1 and IL-13Rα2. These chains are expressed on the cell surface as monomers or heterodimeric IL-4Rα/IL-13Rα1 (type II IL-4R or IL-4Rα/γc (type I IL-4R). The monomer IL-4Rα and heterodimer IL-4Rα/γc bind IL-4, but does not bind IL-13. The monomers of IL-13Rα1 and IL-13Rα2 bind IL-13, but does not bind IL-4. Heterodimer IL-4Rα/IL-13Rα1 binds IL-4 and IL-13 (Murata et al., Int. J. Hematol., 1999, 69, 13-20).

Immune responses of Th2-type stimulate the production of antibodies and humoral immunity and developed to counteract extracellular pathogens. The Th2 cells are mediators of production Ig (humoral immunity) and produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 (Tanaka, et al., Cytokine Regulation of Humoral Immunity, 251 - 272, Snapper, ed., John Wiley and Sons, New York (1996)). Immune responses of Th2-type are characterized by the generation of certain cytokines (e.g. IL-4, IL-13) and the type-specific antibodies (IgE, IgG4) and typical allergic reactions, which may become watery eyes and asthmatic symptoms, for example inflammation of the Airways and reduce muscle cells of the Airways in the lungs.

And IL-4 and IL-13 are therapeutically important cytokine that is determined by their biological functions is, and play an important role in many diseases, including asthma (Curr Opin Allergy Clin Immunol 2005, Vo. 5, 161-166). It was shown that IL-4 can inhibit autoimmune disease, and demonstrated that IL-4 and IL-13 have the potential to enhance the antitumor immune response. Since both cytokine involved in the pathogenesis of allergic diseases, inhibitors of these cytokines may have a positive therapeutic effect.

There is therefore a need for improved agents that inhibit IL-4, inhibit IL-13, as well as agents that inhibit IL-4 and IL-13.

BRIEF description of the INVENTION

The present invention offers a new humanized monoclonal and bespecifically antibodies and their fragments and derivatives that specifically bind to IL-4 or IL-13. Some of the mono - or bespecifically antibodies anti-IL-4 or IL-13 and fragments thereof can be modified in order to prevent the formation of noticeplease disulfide bond, which leads to the formation of molecules, which is stable in the process of obtaining and using in vivo.Antibodies of the present invention neutralize the activity of IL-4 and / or IL-13 in a biological tests described in this document.

The invention includes amino acid after which outermost variable heavy and light chain antibodies and their corresponding nucleic acid sequences.

Another exemplary embodiment of the present invention includes cell lines and vectors containing sequences of the antibodies of the present invention.

Another exemplary embodiment of the present invention involves the use of antibodies for the preparation of a pharmaceutical preparation for the treatment of diseases and disorders related to the function and metabolism of IL-4 and / or IL-13. In particular, the present invention relates to the treatment of cancer, autoimmune diseases and diseases caused or characterized by inflammation, such as allergic asthma and dermatitis.

Additional features and advantages described herein will be apparent from the following complete description of the invention and figures.

BRIEF DESCRIPTION of FIGURES

In Fig. 1 presents a schematic representation of the molecule especifismo antibodies anti-IL-4/IL-13, containing the four polypeptide chains. Two more light chain contains N-VLhB-B13-linker-VLh8D4-8-CL-C (CL) constant region light chain), two more heavy chain contains N-VHhB-B13-linker-VHh8D4-8-CH1-CH2-CH3-C Linker sequence (G4S)2presents GGGGSGGGGS (SEQ ID NO:6).

In Fig. 2 provides the amino acid sequences of humanized variable domains of antibodies B-B13 anti-IL-13 (SEQ ID NO:1 and 2) and humanized in riebeling domain antibodies 8D4-8 anti-IL-4 (SEQ ID NO:3, 4 and 5). The underlined changes in amino acid sequence. Bold letters CDR.

DETAILED description of the INVENTION

The present invention is not limited to the particular methodology, protocols, cell lines, vectors or reagents described herein because they can be changed without affecting the main idea and scope of the invention. Further, as used herein, the terminology is intended to describe the specific accomplishments and do not purport to limit the scope of the present invention. If not stated otherwise, all technical and scientific terms and abbreviations used herein have the same meaning, which is well known those of ordinary skill in this field that applies the present invention. Any methods and materials similar or identical to those described herein can be used in the practical application of the present invention, and is described below only examples of the methods, devices, and materials.

All publications and patents mentioned herein are incorporated herein in by reference for the purpose of describing and disclosing driven him, proteins, enzymes, vectors, host cells and methodologies that could is t be used in conjunction with the present invention and in him. However, nothing in this document should not be interpreted as recognition that the present invention is not entitled to an earlier priority on the basis of previous inventions.

Before describing formulations and application methods associated with IL-4 or IL-13, and consider products for information professionals listed below are definitions of some terms and phrases.

The term "interleukin-4" (IL-4) refers to existing in nature or endogenous proteins of mammalian IL-4, and to proteins having amino acid sequence, which coincides with the existing in nature or the corresponding endogenous protein mammalian IL-4 (for example, recombinant proteins, synthetic proteins (i.e obtained using the methods of synthetic organic chemistry)). Accordingly, as indicated in this document, the term includes Mature protein IL-4, polymorphic or allelic variants, and other isoforms of IL-4 and modified or nemodulirovannyj forms mentioned above (for example lipids, glycosylated). Existing in nature or endogenous IL-4 include the Mature proteins, such as native IL-4, polymorphic or allelic variants, and other isoforms and mutant forms that are present in vivo in mammals (EmOC is emer, man, nonhuman primates). Such proteins can be removed or released from a source, which in natural conditions produces, for example, IL-4. Such proteins and proteins having the amino acid sequence, which coincides with the existing in nature or the corresponding endogenous IL-4, determine the name of the corresponding mammal. For example, if the corresponding mammal is a human, the protein is designated as IL-4 people. Specialists in this field there are several mutant proteins, IL-4, for example, as described in the application WO 03/038041.

The term "interleukin-13" (IL-13) applies to existing in nature or endogenous proteins of mammalian IL-13, as well as to proteins having amino acid sequence, which coincides with the existing in nature or the corresponding endogenous protein mammals IL13 (e.g., recombinant proteins, synthetic proteins (i.e obtained using the methods of synthetic organic chemistry)). Accordingly, as defined herein, the term includes Mature protein IL-13, polymorphic or allelic variants, and other isoforms of IL-13 (e.g., obtained by the method of alternative splicing or other cellular processes), and modified or nemodulirovannyj forms mentioned above (e.g., lipids,glycosylated). Existing in nature or endogenous IL-13 include the Mature proteins such as Mature IL-13, polymorphic or allelic variants, and other isoforms and mutant forms that are present in vivo in mammals (e.g. human, nonhuman primates). For example used here of IL-13 include the variant IL-13 human, in which Arg at position 110 of Mature IL-13 human is replaced by Gin (position 110 of Mature IL-13 corresponds to the position 130 protein precursor), which is associated with asthma (atopic and diatopically asthma) and other variants of IL-13. (Heinzmann et al., Hum MoI Genet. 9:549-559 (2000).) Such proteins can be removed or released from a source, which in natural conditions produces, for example, IL-13. Such proteins and proteins having the amino acid sequence, which coincides with the existing in nature or the corresponding endogenous IL-13, determine the name of the corresponding mammal. For example, if the corresponding mammal is a human, the protein is designated as IL-13 human. Specialists in this field there are several mutant proteins, IL-13, for example, as described in the application WO 03/035847.

The term "essentially identical" to the sequence of polypeptide chain antibodies can be interpreted as the identity on at least 70%, 80%, 90%, 95% or b is the larger of the sequence chain antibodies, and mapping the polypeptide sequence. The same term in relation to the nucleic acid sequence can be interpreted as a sequence of nucleotides, showing the identity of at least 85%, 90%, 95%, 97% or more sequence with respect to the matched nucleic acid sequence.

The terms "identity" or "homology" can mean percentage of nucleotide bases or amino acid residues in the sequence of the candidate, which is identical to the remainder of the respective sequence with which it is compared, after comparing the sequences and introducing gaps, if necessary, to ensure the maximum percent identity for the entire sequence and without considering any conservative substitutions as part of sequence identity. Neither the N-terminal or C-terminal extension segments or inserts should not be seen as reducing identity or homology. Methods and computer programs are available for mapping and well-known experts in this field. The sequence identity can be measured using software for analyzing sequences.

Phrases and terms "functional fragment, variant, derivative or analogue" and the like, as well as their form, applied to antibodies or antigen described is to see the connection or molecule, having a qualitative biological activity characteristic of a given antibody or full-antigen. For example, a functional fragment or analog of an antibody anti-IL-4 can be contacted with a molecule of IL-4 or with a molecule, which can prevent or substantially reduce the ability of the ligand or agonist or antagonist antibodies to contact with IL-4.

"Substitutionary" options are those where at least one amino acid residue in the native sequence removed and replaced with another amino acid, is introduced in its place in the same position. Replacement can be a single in which is replaced by only one amino acid in the molecule, or multiple, if in the same molecule are substituted with two or more amino acids. Multiple substitutions can be made in a sequential sites. In addition, one amino acid may be replaced by multiple residues, this version contains both the substitution and insertion. "Insertion" options are those where one or several amino acids were introduced in such a way that was directly adjacent to one or another amino acid in a particular position of the native sequence. Under the immediate neighbors of a particular amino acid is the amino acid that is associated with α-carb is Xylenol or α-amino functional group of amino acids. "Deletion" options are those where the removed one or more amino acids of the native amino acid sequence. Usually in deletion variants remove one or two amino acids in a specific region of the molecule.

The term "antibody" is used in its broadest sense, and, in particular, includes monoclonal antibodies (including full-size monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example, bespecifically antibodies), antibody fragments or synthetic polypeptides containing one or more CDR sequences or derived from CDR, provided that the polypeptides exhibit the desired biological activity. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins having the same structural features. Typically, antibodies are considered as Ig with a specific or recognizable specificity. Thus, the antibodies exhibit binding specificity to a specific target, while the immunoglobulins are antibodies and other antibody molecules that lack the specificity to the target. Antibodies of the present invention may belong to any class (for example,IgG, IgE, IgM, IgD, IgA, etc) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA etc.) (the terms "type" and "class"and "subtype" and "subclass" equivalent are used in this document). Native or natantia, that is obtained from a representative population without artificial manipulation of antibodies and immunoglobulins are usually heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has at one end a variable domain (VH), followed by more constant domains. Each light chain has at one end a variable domain (VL), and the other end is a constant domain. The phrase "without artificial manipulation" understand the absence of treatment, after which the elements contain or Express a foreign antigen-binding molecule. The definition of "native" can refer to the most dominant alleles or species registered in the population, or to an antibody obtained from an animal without manipulation, in comparison with the allele or polymorphism or variant or derivative, obtained through some form of manipulation, such as mutagenesis, recombinant techniques, etc. to change the amino acids of the antigen-binding molecule.

Used herein, the term "antibody anti-IL-4 oznacza the t antibody or polypeptide, received from him (derived)that specifically bind to IL-4 in accordance with the definition in this document, including, among others, molecules that inhibit or significantly reduce the level of binding of IL-4 to its receptor or inhibit the activity of IL-4.

Used herein, the term "antibody anti-IL-13" means an antibody or a polypeptide derived from it (derived)that specifically bind to IL-13 in accordance with the definition in this document, including, among others, molecules that inhibit or significantly reduce the level of binding of IL-13 to its receptor or inhibit the activity of IL-13.

The term "variable" in the context of the variable domain of the antibody refers to specific parts of the respective molecules, which largely differ in their sequence from an antibody and used for specific recognition and binding of specific antibody with its specific target. This variability is not evenly distributed across the variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e. CDR1, CDR2 and CDR3), also known as hypervariable sites, which are located in the variable domains, both light and heavy chains. is blasti variable domains with higher levels of conservation called spanning regions (FR) or sequences. The variable domains of native heavy and light chains contain four areas FR, largely have the conformation of β-sheet and connected by three CDRs, which form loops connecting, and in some cases forming part of the structure of β-sheet. CDRs in each chain are often held close to each other provinces FR, and together with the CDR of the other chain, contribute to the formation of the desired (epitope or determinant) of the binding site of antibodies (see, Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). Unless otherwise specified, as used herein, the numbering of amino acid residues of the immunoglobulin produced in accordance with the system of numbering amino acid residues of the immunoglobulin from the work of Kabat et al. One CDR may possess the ability to specifically bind cognacy epitope.

The term "hinge"or "hinge region"used in the present invention refers to a flexible polypeptide containing amino acids between the first and second constant domains of the antibody.

The definition of "antibody fragment" refers to a piece of intact or full chain or antibody, as a rule, the field of binding targets or variable regions. Examples of fragments of antibodies, among others, include fragments of the FabFab'F(ab')2and Fv. "Functional fragment" or "analogue and is Titel anti-IL-4 or IL-13" refers to a fragment, that may hamper the ability of the receptor, or substantially reduce this ability to contact the ligand or to initiate a signal. Used herein, the term "functional fragment" is synonymous with the term "antibody fragment" when used to refer to antibodies can relate to such parts as FvFabF(ab')2etc. that are able to inhibit the ability of the receptor, or substantially reduce this ability to contact the ligand or to initiate a signal. Fragment "Fvconsists of a variable domain of the dimer of one heavy and one light chain, formed by non-covalent Association (dimer VH-VL). In this configuration, the three CDRs of each variable domain interact, forming the desired binding site on the surface of the dimer VH-VLas well as the intact antibody. Collectively, these six CDRs provide the binding specificity of the target on an intact antibody. However, even a single variable domain (or half of the Fvcontaining only three CDRs specific to the target) may have the ability to recognize and bind the target.

"Single-chain Fv"fragments of antibodies sFv" or "scAb" include domains of the antibody VHand VLmoreover , these domains are present in the Dina polypeptide chain. Typically, the polypeptide Fvfurther comprises a polypeptide linker, often a flexible molecule, between domains VHand VLthat enables the sFv to form the desired structure for binding a target.

The term "diately" refers to fragments of antibodies with two antigen-binding sites, which fragments may include variable domain of the heavy chain (VH)associated with the variable domain of the light chain (VL) in the same polypeptide chain. Through the use of a linker that is too short to ensure the unification of the two variable domains of the same chain, the domains diatel forced to unite with the binding domains of another chain for forming two antigen-binding sites.

Fragment Fabcontains the variable and constant domains of the light chain and the variable and first constant domain (CH1) the heavy chain. Fragments of Fab'different from fragments Fabthe addition of a few residues at the carboxyl terminal end of domain CH1so that it contains one or more cysteines from the hinge region of the antibody. Fragments of Fab'can be obtained by cleavage of disulfide bonds in cysteine the hinge area of the product destruction pepsin F(ab')2. Additional enzymatic and chemical treatment of the antibody can bring the ü education other interest functional fragments.

The term "linear Fab" refers to a tetravalent antibody, as described in Miller et al. (2003), J Immunol. 170: 4854-4861. "Linear Fab consists of tandem identical domains CH1-VH, United identical light chain at each position CH1-VH. Such molecules were created in order to increase the valency of the antibody to improve its functional affinity due to the avidity effect, but they are monospecific.

The term "bespecifically antibodies (BsAb)" refers to molecules that combine the antigen-binding sites of the two antibodies in a single molecule. So, bespecifically antibody able to bind two different antigens simultaneously. In addition to diagnostic applications, BsAb open the door to new therapeutic applications due to reorientation of the powerful effector system on the affected area or by increasing neutralizing or stimulating functions of antibodies.

During the first attempts of combining the specificity of binding of two antibodies against different antigens are targets for medicinal purposes was used chemically fused heteroconjugate molecules (Staerz et al.(1985)Nature 314: 628-631).

Bespecifically antibodies were producirovanie from the original hybrid with heterohybridomas techniques, and in vitro have been demonstrated properties similar to those observed for heteroconjugate is s (Milstein & Cuello (1983) Nature 305:537-540).

Despite the promising results obtained using heteroconjugate or bespecifically antibodies produced as a result, as mentioned above, merge cells, a number of factors makes them inapplicable for large-scale therapeutic applications. These factors include: rapid elimination heteroconjugate in vivo, time-consuming techniques necessary to obtain any of the forms of the molecule, the need for thorough cleaning heteroconjugate from homoconjugated or monospecific antibodies and low outputs.

Genetic engineering is increasingly used for design, modification and production of antibodies or derivatives of antibodies with the desired set of properties of binding and effector functions.

We developed a variety of recombinant techniques for the efficient production BsAb, in both cases as fragments of antibodies (Carter et al. (1995), J. Hematotherapy 4:463-470; Pluckthun et al. (1997) Immunotechology 3:83-105; Todorovska et al. (2001) J. Immunol. Methods 248:47-66) and a full-sized formats IgG (Carter (2001) J. Immunol. Methods 248: 7-15).

Combining two different scFv leads to the formats BsAb with a minimum molecular weight, which is called sc-BsAb or Ta-scFv (Mack et al. (1995), Proc. Acad. Sci.USA. 92:7021-7025; Mallender et al. (1994) J. Biol. Chem. 269:199-206). BsAb was constructed by a genetic fusion of two scFv at the expense of providing functional dimerization is STI, for example latinboy fasteners (Kostelny et al. (1992) J. Immunol. 148:1547-53; de Kruif et al. (1996) J. Biol. Chem. 271:7630-4).

As mentioned above, diately called small fragments of bivalent and bespecifically antibodies. The fragments comprise the VH associated with the VL of the same polypeptide chain at the expense of the linker that is too short in length (less than 12 amino acids)to provide the link between the two domains on the same chain. The domains are forced intermolecular be combined with complementary domains of another chain for forming two antigen-binding sites. Such dimeric antibody fragments, or "diately"are bivalent and bespecifically. (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA. 90: 6444-6448). Diately have a size close to the Fab fragment. Polypeptide chain domain VH and VL linked by linker between 3 and 12 amino acids, consists of dimers (diatel), whereas in the case of the linker between 0 and 2 preferred amino acid residues are trimers (trifala) and tetramer (tetrathele). In addition to the length of the linker, the exact model of oligomerization obviously depends on the composition, but also on the orientation of the V-domains (Hudson et al. (1999), J Immunol Methods 231: 177-189). The predictability of the final structure of molecules diatel exceptionally low.

Although sc-BsAb and designs based on diatel have interesting clinical potential, which was shown, such ecovalence associated molecules are not sufficiently stable under physiological conditions. The overall stability of the scFv fragment depends on the internal stability domains VL and VH, as well as the stability of the domain interface. Lack of stability of the interface VH-VL scFv fragments often mentioned among the main causes irreversible inactivation of scFv as interim disclosure interface, which allows the peptide linker, provides access to hydrophobic areas that promote aggregation, and therefore determining the instability and low yield (Wörn and Plückthun (2001), J. Mol. Biol. 305: 989-1010).

An alternative method of preparing bespecifically bivalent antigen-binding protein of the domains VH and VL is described in the patent US 5989830. Such fragments of antibodies with two heads, get expression dvuhsostavnogo vector encoding two polypeptide chains, each polypeptide chain contains twice the VH sequence of the peptide linker (VH1-linker-VH2),

and the other polypeptide chain contains complementary VL domains connected in the sequence of the peptide linker (VL1-linker-VL2). In the patent US 5989830 it was shown that the linker should comprise at least 10 amino acid residues.

Polyvalent protein complexes (PPC) with increasing valency described in U.S. Pat is the US 2005/0003403 A1. PPC contain two polypeptide chains, which are usually located laterally with respect to each other. Each polypeptide chain usually consists of 3 or 4 "v-region containing amino acid sequences that are able to form a binding site of the antigen when compared with the corresponding v-region on the opposite polypeptide chain. Each polypeptide chain may be employed up to about 6 "v-regions". V-region of each polypeptide chain are related linearly to each other and can connect intersected by linker regions. When the organization in the form of PPC v-region of each polypeptide chains form the individual binding sites of the antigen. The complex may contain one or more binding specificdate.

However, when using such molecules was demonstrated propensity for aggregation, instability and low output expression (Wu et al. (2001) Prot. Eng. 14: 1025-1033). All this is typical stability problems, which can be observed when the expression of single-chain antibodies. (Wörn and Plückthun (2001), J. Mol. Biol. 305: 989-1010).

Therefore, the aim of the present invention to provide especifismo polyvalent antibodies, which you can use to avoid the formation of aggregates. In addition, it must have stability, which determines its pripadnost is for use in therapeutic purposes.

Used herein the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical, if not take into account potential natural mutations that may be present in minor amounts.

In this case, a monoclonal antibody that specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass (type or subtype) antibodies, while the rest of the chain (chain) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, provided that they exhibit the desired biological activity upon binding with IL-4 and (or) IL-13 or the impact on the activity or metabolism of IL-4 and / or IL-13 (U.S. patent No. 4816567; and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984)). Therefore, the CDR of the same class of antibodies can be introduced into FR antibodies of another class or subclass.

Monoclonal antibodies have a high specificity, they are focused on a single site is a target epitope or determ is Nantes. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes) antigen, each monoclonal antibody is directed against a single determinant in the target. In addition to specificity, the advantage of monoclonal antibodies is that they are produced by the host-cell without admixtures of other immunoglobulins, which provides the cloning of the corresponding gene and the mRNA that encodes the antibody of his chains. The modifier "monoclonal" indicates the characteristic of the antibody, which was obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in the present invention can be isolated from phage libraries of antibodies using well-known techniques or can stand out from the polyclonal preparation. The original monoclonal antibodies for use in accordance with the present invention can be prepared using the hybridoma method described in Kohler et al., Nature256:495 (1975), or may be obtained by recombinant methods, which are well known to specialists in this field.

The term "polyvalent the antibodies is about", used in the present invention refers to an antibody that contains two or more binding sites of the antigen, and therefore is able to simultaneously bind two or more antigens with the same or different structure. The term "bivalent" means that the antibody has two antigen-binding site. The term "bonds" means that the antibody has four antigen-binding site.

The term "antigen-binding site"as used in the present invention, refers to the part of the antibody containing a region specifically binding portion of an antigen or a complete antigen and complementary part of the antigen, or the antigen. In the case of a large amount of antigen antibody can bind only with a certain part, and this part is called the epitope. The antigen-binding domain may be provided by one or more variable domains of antibodies. Preferably, the antigen-binding domain was formed by binding variable domain of the light chain of the antibody (VL) and variable domain heavy chain antibodies (VH).

Used in the present invention, the term "antigen" refers to a molecule or part of a molecule that is able to communicate with antibodies of the present isoberlinia. The antigen may contain one or more epitopes. To primeramente, recognized by the antibodies of the present invention, among others, include serum proteins, such as cytokines, such as IL-4, IL5, IL9 and IL-13, biologically active peptides, cell surface molecules, such as receptors, carriers, ion channels, viral and bacterial proteins.

Used in the present invention, the term "monospecific" means that the multivalent antibody of the present invention recognizes only one antigen, the antigen-binding sites are identical.

Used in the present invention, the term "bespecifically" means that the multivalent antibody of the present invention recognize two different epitopes on the same antigen, or two different antigens.

Used in the present invention, the term "multispecificity" means that the multivalent antibody of the present invention recognizes many different epitopes on the same antigen or multiple antigens.

Used in the present invention, the term "linker" means a peptide which is adapted for connection variable domains of the structures of the antibodies of the present invention. The peptide linker may contain any amino acids, preference is given to the amino acids glycine (G) and serine (S). The linkers connecting the polypeptide heavy chain and p is lipacid light chain and within these chains, can be the same or different from each other. In addition, the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. Preferred peptide linker domain of the heavy chain and light chain domains is GGGGS. The number of linker units in the heavy chain and light chain can be the same (symmetric) or different from each other (asymmetric).

The peptide linker is preferably long enough to provide an appropriate degree of flexibility, excluding the impact of the functional areas of the antibodies on the activity of each other, for example because of steric hindrance, to allow for proper protein folding and, if necessary, to allow the antibody molecules to interact with two or more, possibly spatially remote from each other receptors on the surface of the same cells; however, it should preferably be short enough to guarantee the stability of the functional areas antibody inside the cell.

Therefore, the length, composition and/or conformation of peptide linkers can easily vary by specialists in this field in order to optimize the desired properties of polyvalent antibodies.

Compared with the antibody man "humanitarian the e form of inhuman (for example, murine) antibodies are chimeric immunoglobulins, chains of immunoglobulins or fragments thereof (e.g., FvFabFab'F(ab')2or other binding target subsequences of antibodies)that contain sequences derived from nonhuman immunoglobulin. As a rule, humanitariannet antibody will comprise essentially the entire sequence of one, and typically two, variable domains, in which all or substantially all of the CDR field correspond to nonhuman immunoglobulin and all or substantially all areas FR represent the matrix sequence of human immunoglobulin. Humanitariannet antibody may also include at least a portion of constant region of immunoglobulin (Fc), as a rule, the selected matrix sequence of human immunoglobulin. Usually, the main purpose is to obtain molecules are antibodies, which minimally immunogen for a person. Therefore, there is an opportunity to replace one or more amino acids in one or more CDR on those that are less immunogenic to the host body of a man, not significantly reducing specific binding of one or more CDR with IL-4 or IL-13. Alternatively, you can use inhuman FR, but in nenablue immunogenic amino acids are replaced by less immunogenic. However, as discussed above embedding in CDR is not the only method of obtaining gumanitarnogo antibodies. For example, modification of only the CDR regions may not be sufficient, because quite often spanning residues can play an important role in determining the three-dimensional structure of the CDR loops and the overall affinity of the antibody to its ligand. So you can apply any methods of modification of the molecule inhuman original antibody, so as to be less immunogenic to humans, and complete identity with the human antibody is not always required. So, humanization can also be achieved, for example, a simple replacement of only a few residues, in particular, those that are on the surface of the molecule antibodies and not hidden inside its structure, and therefore are not easily accessible to the immune system of the host. A similar method is proposed in this document in relation to the substitution of "mobile" or "flexible" residues of the molecule antibodies, with the aim to reduce or suppress the immunogenicity of the resulting molecules, without affecting the specificity of the antibodies against the epitope or determinant. See, for example, Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol Imm 44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia et al., J Mol Biol 196:901 (1987); Carter et al., Proc Natl Acad Sci USA 89:4285 (1992); Presta et al., J Immunol 151:2623 (1993), WO 2006/04333 and U.S. patent No. 5869619.

The proposed method of humanization based on the effects of flexibility of the antibody molecules in the process and at the time of immune recognition. The flexibility of the protein is associated with the molecular motion of its molecules. Under the protein flexibility implies the ability of a protein portion of the protein or individual amino acid residues to form the ensemble of conformations that are significantly different from each other. Data on the flexibility of the protein can be obtained by experimental results x-ray crystallography (see, e.g., Kundu et al. 2002, Biophys J 83:723-732.), nuclear magnetic resonance (see, for example, Freedberg et al., J Am Chem Soc 1998, 120(31):7916-7923) or the results of molecular dynamics simulation (MD). The MD simulation of the protein is carried out on the computer and allows you to detect motion of all atoms of the protein for a certain period of time by calculating the physical interactions of the atoms with each other. The result of MD simulation is the pathway for protein over a period of time modeling. The trajectory is an ensemble of conformations of a protein, also called static configurations, which are periodically recorded during the simulation, for example each PS (psec). It is through the analysis of the ensemble of static configurations, you can define the flexibility of the amino acid residues of the protein. P is this flexible balance can be which may have an ensemble of different conformations in the structure of the polypeptide, where the balance is. Methods MD-known to experts in this field, see, for example, Brooks et al. “Proteins: A Theoretical Perspective of Dynamics, Structure and Thermodynamics (Wiley, New York, 1988). Some products allow the MD simulation, including Amber (see Case et al. (2005) J Comp Chem 26:1668-1688), Charmm (see Brooks et al. (1983) J Comp Chem 4:187-217; and MacKerell et al. (1998) in “The Encyclopedia of Computational Chemistry” vol. 1:271-177, Hamburg airport yesterday et al., eds. Chichester: John Wiley & Sons) or Impact (see Rizzo et al. J Am Chem Soc; 2000; 122(51):12898-12900.)

The majority of protein complexes has a relatively long and planar hidden surfaces, and it has been shown that the flexibility of the binding partners provides the basis for their plasticity, which allows them to conformationally adapt to each other (Structure (2000) 8, R137-R142). Accordingly, it was shown that the examples of "induced compliance" play a dominant role in the interaction of protein-protein. In addition, there is an ever-growing set of data that show that proteins actually bind ligands of different shapes, sizes and structures (Protein Science (2002) 11:184-187) and that conformational diversity, obviously, is an essential component of the ability to recognize the different partners (Science (2003) 299, 1362-1367). Flexible residues involved in the binding of PA is the partners in a pair of protein-protein (Structure (2006) 14, 683-693).

Flexible residues can adopt a variety of conformations that form the ensemble of the areas of interaction, which, most likely, will be recognized In the memory cells and to cause immunogenic response. Thus, antibodies can be humanitarian by modifying the number of residues spanning sequence, so that the ensemble of conformations and accessible areas of recognition in a modified antibody as possible resembled existing antibody man.

This can be achieved by modification of a limited number of residues through: (1) build a homology model of the original monoclonal antibodies (mAb) and a molecular dynamics simulation (MD); (2) analysis of flexible residues and identification of the most flexible residues in the molecule nonhuman antibodies, and detection of residues or motifs that likely source of heterogeneity or the decay reaction; (3) identification of human antibodies, which demonstrates the presence of similar ensemble zones recognition as a source of antibodies; (4) identification of flexible residues for mutation of residues or motifs, which, most likely, will be a source of heterogeneity and decay and will also undergo mutation; and (5) test for the presence of known epitopes to T - or b-cells. Flexible residues could the t to be identified on the basis of calculations MD, offered in this document, using the model estimated solvent, which takes into account the interaction of the aqueous solvent with the protein atoms over a period of time modeling. After identifying the set of flexible residues in the variable light and heavy chains determine the set of spanning sequences of the heavy and light chain variable regions of a human, which have close similarities with existing in the antibody. For example, you can use the BLAST search for a set of flexible residues in the database for sequences of antibody germline person. This can also be achieved by mapping the dynamics of the original mAb with dynamics library canonical structures of the germ line. Residues and neighboring CDR residues are excluded from the search, in order to maintain high affinity for antigen.

Then replacing the flexible residues. When several residues man showed similar homology, the choice was also determined by the nature of the residues that most likely had an influence on the behavior gumanitarnogo antibodies in solution. For example, the preference was given to polar residues available in flexible loops, in contrast to hydrophobic residues. Residues, which are potential IP is a source of instability and heterogeneity was also subjected to mutation, even if they were found in the CDR. This includes the available methionine, since the formation of sulfoxide may be due to interaction with oxygen radicals, proteolytic cleavage of the acid-unstable relationships, such as the dipeptide Asp-Pro (Drug Dev Res (2004) 61:137-154), sites deliciouse present in the available residue asparagine, followed by a small amino acid such as Gly, Ser, Ala, His, Asn or Cys (J Chromatog (2006) 837:35-43), as well as sites of N-glycosylation, such as the site Asn-X-Ser/Thr. Usually available methionine will be replaced by latinae, available asparagine will be replaced by glutamine or aspartate, or be replaced followed by the rest. In the case of a glycosylation site (Asn-X-Ser/Thr) will be replaced or Asn, or the remainder of Ser/Thr.

The resulting composite sequence is checked for the presence of known epitopes In cells or linear epitopes of T cells. The search is conducted, for example, publicly accessible database of immune epitopes (IEDB). If inside the composite sequence recognized epitope is searched for a different set of sequences person, inside of which is substitution.

Unlike proposed in U.S. patent No. 5639641 method of changing the surface (resurfacing), in the proposed method teaches who are also immunogenic responses, mediated by b - and T-cells. The method also allows to circumvent the problem of loss of activity, which is sometimes observed in the case of inserts in CDR (U.S. patent No. 5530101). In addition, in process engineering and selection also takes into account the aspects of stability and solubility, which allows you to create an antibody that is optimized in terms of low immunogenicity, high affinity to the antigen and more acceptable biophysical characteristics.

Strategies and ways to modify the surface of the antibody, as well as other methods of reducing the immunogenicity of antibody in different host organisms described, for example, in U.S. patent No. 5639641. In General, the preferred method: (1) a comparison of the provisions of the set of variable regions of heavy and light chains of antibodies in order to determine the available surface position of the heavy and light chain variable regions spanning sequence, with associated provisions for all variable fields should be at least about 98% identical; (2) the definition of a set inhuman available surface amino acid residues of the heavy and light chain variable regions spanning sequence, for example, antibodies rodents (or its fragment); (3) is determined by the available surface amino acid residues of the heavy and light chains areabelow region spanning sequence, which is closest to the set of available surface amino acid residues of rodents; and (4) the available surface amino acid residues of the heavy and light chain variable regions spanning the sequence identified in step (2)is replaced by the set of available surface amino acid residues of the heavy and light chain variable regions spanning the sequence identified in step (3), except for those amino acid residues that are within 5Å from any atom of any residue of CDR antibody rodents, in order to obtain humanitariannet antibody, for example a rodent antibody that retains the binding specificity.

Antibodies can humanservices based on a variety of other techniques, including insert into CDR (EPO 0239400; WO 91/09967; and U.S. patent No. 5530101 and 5585089), changes in surface antibodies or restore surface access (EPO 0592106; EPO 0519596; Padlan, 1991, Molec Imm 28(4/5):489-498; Studnicka et al., 1994, Prot Eng 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973) and permutation circuit (U.S. patent No. 5565332). Human antibodies can be prepared using various methods known to experts in this field, including, among others, methods, phage display, see U.S. patent No. 4444887, 4716111, 5,545,806 and 5814318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, using a transgene, the s animals for example rodents, using chimeric cells, etc.

The terms "homolog antibody" or "homolog" refers to any molecule that specifically binds IL-4 and / or IL-13 in accordance with the contents of this document. Thus, homologues of antibodies are native or recombinant antibody, a modified or unmodified; parts of an antibody that retain the interest of the biological properties; for example, the binding of IL-4 or IL-13; for example, molecules of Fabor Fv; single-chain antibody; a polypeptide containing one or more CDR regions, etc. Amino acid sequence of the homologue does not have to be identical sequence existing in nature antibodies, but may be changed or modified by the inclusion of the replacement amino acids introduced amino acids deleted amino acids, amino acids that are not included in the number of twenty, which are found in proteins, etc. in order to obtain a polypeptide with improved or other useful properties.

Antibodies with homologous sequences called antibodies with amino acid sequences that have sequence homology with the amino acid sequences of the antibodies of IL-4, IL-13 or especifismo antibodies IL-4/IL-13 of the present invention. P is edocfile, homology exists with the amino acid sequence of the variable regions of the antibodies of the present invention. "Sequence homology" refers to the amino acid sequence of the present document, which is defined as a sequence with a homology of about 90%, 91%, 92%, 93%, 94% or higher and, more preferably, the homology is at least about 95%, 96%, 97%, 98% or 99% relative to another amino acid sequence, which is determined, for example, by the method of FASTA search in accordance with Pearson &Lipman, Proc Natl Acad Sci USA 85, 2444-2448 (1988).

Chimeric antibody called antibody containing different parts of an antibody derived from a variety of sources, such as different antibodies, different classes of antibodies, various species of animals, such as antibodies having a variable region derived from a murine monoclonal antibodies, combined with the constant region of human immunoglobulin, etc. So humanitariannet antibody is a chimeric antibody. Methods for the preparation of chimeric antibodies known to specialists in this field, see, e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J Immunol Methods 125:191-202; and U.S. patent No. 5807715, 4816567 and 4816397.

Artificial antibodies include fragments of scFv, a chimeric antibody, diately, Triatel, tetrathele and mru (see Winter & Milstein, 1991, Nature 349:293-299; and Hudson, 1999, Curr Opin Imm 11:548-557), each of them has the ability to bind an antigen or epitope. In the single-stranded fragment Fv(scFv), the domain of VHand VLantibodies are linked by a flexible peptide. Typically, the linker is a peptide with a length of about 15 amino acids. If the linker is substantially less, for example a length of 5 amino acids, are formed of diately. The lowest binding unit antibody is a CDR, usually CDR2 of the heavy chain, which has sufficient capacity for recognition and binding. This fragment is called molecular recognition area, or mru. Several such mru can be linked together with a short linker peptides, thereby forming an artificial binding protein with higher avidity than individual mru.

Within the scope of the present invention also includes functional equivalents of the considered antibodies. The term "functional equivalents" includes, for example, antibodies with homologous sequences, homologues of antibodies, chimeric antibodies, synthetic antibodies and modified antibodies, such as those in which every functional equivalent is defined by the ability to bind to IL-4 or IL-13, inhibiting the ability or function of IL-4 and / or IL-13 to transmit a signal, or inhibiting the binding of IL-4 and / or IL-13 to its receptor. Experienced specialist in this field will obviously overlap of terms denoting groups of molecules, "antibody fragments" and "functional equivalents". Methods for the preparation of functional equivalents, which retain the ability to bind IL-4 and / or IL-13, known qualified specialists in this field and described, for example, in WO 93/21319, EPO Ser. No. 239400, WO 89/09622, EPO Ser. No. 338745 and EPO Ser. No. 332424.

Functional equivalents of the present application also include modified antibodies, such as antibodies, modified through the covalent bond of a molecule of any type of antibody. For example, the modified antibodies include antibodies that have been modified, for example by glycosylation, acetylation, PEG, deliberatorium, phosphorylation, amidation, introduction of the famous defensive/blocking groups, proteolytic cleavage, linkage to a cellular ligand Association with toxins or cytotoxic group or other protein, etc. Covalent binding does not necessarily lead to the antibody that is protected from the formation of the anti-idiotypical response. The modification may be carried out using known techniques, including, among others, specific chemical cleavage, azetilirovanie, formirovanie, metabolic synthesis and tpol, the modified antibodies may contain one or more non-classical amino acids.

Those of ordinary skill in this field there are many methods that allow to optimize the affinity of binding. Typically, such methods include substitution of different amino acid residues in the site with subsequent screening analysis the affinity of binding of the mutant polypeptide with cognatum antigen or epitope.

After identification and isolation of antibodies is often useful to have a variant or mutant antibodies, or mutein, where the modified one or more amino acid residues, for example, in one or more hypervariable regions of the antibodies. Alternatively or additionally, the structure of antibodies can be made one or more changes (e.g., substitutions) in the residues spanning the sequence that leads to an improvement in the affinity of binding of the mutant antibodies with IL-4 or IL-13. Examples of residues spanning the areas that can be modified are those that directly ecovalence associated antigen (Amit et al., Science 233:747-753 (1986)); interact with the conformation of CDR or affect it (Chothia et al., J Mol Biol 196:901-917 (1987)); and (or) part of the interface VL-VH(EP 239 400). In some embodiments, the modification of the real the or more of these residues spanning region leads to an improvement in the affinity of binding of the antibody with cognatum antigen. For example, in the present embodiment, of the invention can vary from one to about five residues spanning region. Sometimes this can be enough to get the mutant antibodies suitable for use in preclinical trials, even if no changes are made in any of the residues of the hypervariable region. However, as a rule, the mutant antibody can include one or more changes in the hypervariable region. The constant region may also vary in order to achieve the desired or more than desired effector properties.

The remains of the hypervariable region, which are subject to substitution, may change randomly, especially if the original binding affinity of the original antibody is that randomly the resulting mutant antibodies can easily be analyzed in the screening process to change the binding according to the method of analysis given in this document.

One method of obtaining mutants antibodies, such as mutants CDR is "mutagenesis scanning alanine" (Cunningham & Wells, Science 244:1081-1085 (1989); and Cunningham & Wells, Proc Nat Acad Sci USA 84:6434-6437 (1991)). One or more residues of the hypervariable region are replaced by alanine or polyalanine remains. Such a balance (the remains of) a hypervariable region that exhibits functional senses is required to override, then modify by introducing further or other mutations in the sites of substitution. Thus, despite the fact that the site for the introduction of variations in the amino acid sequence is predetermined, the nature of the mutation optional pre-specified. You can try to introduce a similar substitution with other amino acids, depending on the desired properties of the scanned residues.

A more systematic method of identifying amino acid residues for modification involves selecting residues hypervariable region involved in the binding of IL-4 and / or IL-13, as well as those residues of the hypervariable region, which is poorly involved or not involved in the binding of IL-4 and / or IL-13. Performed alanine scanning residues neisvaziuosiu hypervariable region, each Ala mutant is tested to improve the binding of IL-4 and / or IL-13. In another example implementation for modification of the selected residue (residues), which are significantly involved in the binding of IL-4 and / or IL-13. The modification may include a deletion of the residue or the introduction of one or more residues in the immediate vicinity of the considered residue. However, as a rule, the modification involves the replacement of the residue by another amino acid. The first substitution may be conservative. If such a replacement is riodic to changes in biological activity (e.g., the affinity of binding), then you can spend the next conservative substitution to determine whether to make more substantial changes.

Even more significant change in the line of antibodies and the manifestation of the biological properties can be achieved when selecting amino acids, which is more markedly different properties from that normally found on this website. Therefore, such a substitution can be made while maintaining: (a) structure of the polypeptide backbone in the area of the substitution, for example in the form of a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule in misheneva the website, or (b) the volume of the side chain.

For example, naturally occurring amino acids can be subdivided into groups based on common properties of the side chain:

(1) hydrophobic: methionine (M or Met), alanine (A or Ala), valine (V or Val), leucine (L or Leu) and isoleucine (I or Ile);

(2) neutral hydrophilic: cysteine (C or Cys), serine (S or Ser), threonine (T or Thr), asparagine (N or Asn) and glutamine (Q or Gln);

(3) acidic: aspartic acid (D or Asp) and glutamic acid (E or Glu);

(4) basic: histidine (H or His), lysine (K or Lys) and arginine (R or Arg);

(5) residues that influence chain orientation: glycine (G or Gly) and Proline (P or Pro);

(6) aromatic: tryptophan (W or Trp), tyrosine (Y or Tyr) and phenylalanine (F or Phe).

Under conserve the active substitutions may involve replacement of one amino acid to an amino acid from a different group. Under conservative substitutions can be the replacement of one amino acid to another within the same group.

For your preferred substitutions of amino acids include those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity of and (4) provide or modify other physicochemical or functional properties of such analogs. Among the analogs can be different mutiny sequence that is different from the existing in the nature of the peptide sequence. For example, single or multiple substitutions of amino acids (preferably conservative substitutions of amino acids) can be produced in existing in nature sequence (preferably in the portion of the polypeptide outside the domain (domain)responsible for intermolecular contacts). Conservative replacement amino acid should not substantially change the structural characteristics of the original sequence (for example the replacement amino acid should usually not violate helical conformation, which was present in the original sequence, or disrupt other secondary structure elements, which characterize the original sequence), with the exception of changes in the overall volume or change the conformation of R-groups or side C is PI, Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (Branden &Tooze, eds., Garland Publishing, New York, N. Y. (1991)); and Thornton et al. Nature 354:105 (1991).

Typically, the mutant antibodies with improved biological properties will have an amino acid sequence that is at least 75% identical or similar amino acid sequence of the heavy or light chain variable domain of the original anti-human antibody to IL-4 or IL-13, or has at least 80%, at least 85%, at least 90%and often at least 95%identity. Identity or similarity in respect to the sequence of the original antibody is defined herein as the percentage of amino acid residues in the sequence of the candidate that are identical (i.e. the same residue) or similar (i.e. amino acid residue from the same group on the basis of the closest properties of the side chain, see above) remains a source of antibodies after comparing the sequences and introducing gaps, if necessary, to ensure the maximum percent sequence identity.

In an alternative embodiment, the mutant antibodies can be obtained by systematic mutation regions FR and CDR of the heavy and light chains or region Fcantibodies anti-IL-4, anti-IL-13 or especifismo antibodies IL-4/IL-13. The other is Oh method of obtaining mutants antibodies involves the use of affinity maturation using phage display (Hawkins et al., J Mol Biol 254:889-896 (1992) and Lowman et al., Biochemistry 30(45):10832-10838(1991)). The slit membrane proteins of bacteriophage (Smith, Science 228:1315 (1985); Scott &Smith, Science 249:386 (1990); Cwirla et al. Proc Natl Acad Sci USA 8:309 (1990); Devlin et al. Science 249:404 (1990); Wells & Lowman, Curr Opin Struct Biol 2:597 (1992); and U.S. patent No. 5223409) is known as a convenient objects for linking phenotype proteins or peptides display with genotype particles of the bacteriophage, which encode them. When using the phage-display also shows the domains of Fabantibodies (McCafferty et al., Nature 348: 552 (1990); Barbas et al. Proc Natl Acad Sci USA 88:7978 (1991); Garrard et al. Biotechnol 9:1373 (1991)).

Monovalent phage display provides a display of a set of variants of the protein in the form of a fused protein with the protein shell of bacteriophage on the phage particles (Bass et al., Proteins 8:309 (1990). The affinity maturation, or improvement of the equilibrium affinity of binding of different proteins, previously achieved through consistent application of mutagenesis, monovalent phage display and functional analysis (Lowman & Wells, J Mol Biol 234:564 578 (1993); and U.S. patent No. 5534617), for example, special attention was paid to the CDR regions of the antibodies (Barbas et al., Proc Natl Acad Sci USA 91:3809 (1994); and Yang et al., J Mol Biol 254:392 (1995)).

Library set (for example, 106or more) variants of proteins, which differ in certain provisions of the sequence, you can build on the bacteriophage particles, each of which contains DNA encoding specific options, the ant protein. After cycles of affinity purification using immobilized antigen, isolate individual clones of bacteriophages and on the basis of DNA determine the amino acid sequence of the displayed protein.

After receipt of the mutant antibodies can determine the biological activity of this molecule compared to the original antibody, in accordance with the information set out in this document. As noted above, this may require to determine the affinity of binding and / or other biological activity or physical properties of the antibodies. In a preferred implementation of the present invention prepares a set of mutant antibodies with subsequent screening of affinity of binding to antigen. One or more mutant antibodies are selected based on the screening results, can be validated using one or more of the other methods of analysis of biological activity, in order to confirm that the mutant (mutant) antibodies have new or improved properties. In preferred embodiments, the mutant antibody retains the binding ability of IL-4 and / or IL-13 with a binding affinity of close or better/higher in comparison with the characteristics of the original antibody.

Selected mutant (mutant) antibodies can podvergat the Xia further modifications, which often depend on the intended use of the antibody. Such modifications may include further changes in amino acid sequence, fusion with a heterologous polypeptide (or polypeptides) and / or covalent modification. For example, the cysteine residue which is not involved in maintaining the proper conformation of the mutant antibodies can be replaced, usually to serine, to improve stability of the molecule oxidation and prevent aberrant crosslinking. In contrast, the cysteine may be introduced into the structure of the antibody to improve its stability (particularly, if the antibody is selected fragment of the antibody, for example, a fragment of the Fv).

Another type of mutant antibodies has an altered structure of glycosylation. This purpose can be removed one or more carbohydrate groups present in the antibody, and (or) to add one or more glycosylation sites not found in the molecule of the antibody. Glycosylation of antibodies is typically either N-linked to Asn or O-linked to Ser or Thr. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are common recognition sequences for enzymatic binding of the carbohydrate group with the side chain of asparagine. N-acetyll cozamin, galactose, fucose or xylose, for example, contact hydroxyquinolinato, generally with serine or threonine, although it can be used and 5-hydroxyproline or 5-hydroxylysine. The addition or substitution of one or more residues of serine or threonine to the sequence of the original antibody can increase the probability of O-linked glycosylation.

It may be desirable to modify the antibody of the present invention in terms of effector function, so as to increase the efficiency of antibody. For example, in the region Fcyou can enter the balance (remainder) of cysteine, which provides education miaocheng disulfide bond in this area. Thus obtained homodimeric antibody can be characterized by a high capacity for internalization and / or complement-dependent lysis and antibody-dependent cellular cytotoxicity (ADCC), see Caron et al., J Exp Med 176:1191-1195 (1992) and Shopes, Immunol 148:2918-2922 (1993). In an alternative embodiment, can be constructed antibody, which has double the area of Fcand so can provide higher complement-dependent lysis and the ability to ADCC, see Stevenson et al., Anti-Cancer Drug Design 3: 219 230 (1989).

Covalent modifications of the antibody are included in the scope of the present invention. They can be implemented either by chemical synthesis or, if this is possible, enzymatic or chemical cleavage of antibodies. Other forms covalent modifications of the antibody are introduced into the molecule by interaction of selected amino acid residues of the antibody with an organic agent used to obtain the derivative, which is capable of reacting with selected side chains or the N-terminal or C-terminal residue.

Caseinline residues can react with α-galvanically (and corresponding amines), such as Chloroacetic acid or chloroacetamide, education carboxymethyl or carboxylatomethyl derivatives. Derivatives containerindex residues can also be obtained, for example, by reaction with BROMOTRIFLUOROMETHANE, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetylation, N-alkylamide, 3-nitro-2-pyridyldithio, methyl-2-pyridyldithio, p-chloromercuribenzoate, 2-chloromercuri-4-NITROPHENOL, or chloro-7-nitrobenzo-2-oxa-1,3-diazoles.

Derivatives histidinemia residues can be obtained by reaction with diethylpyrocarbonate at pH 5.5-7.0. p-Brompheniramine can also be used, the reaction is preferably carried out in a 0.1 M solution of cacodylate sodium at pH of 6.0.

Libinline residues and α-terminal residues can react with succinic anhydride or with other anhydrides of carboxylic acids, in order to change the shape of the charge remains. Other convenient reagents for obtaining derivatives of α-aminecontaining residues are imediately, such as methylphenidate, pyridoxal phosphate, pyridoxal, chlorproguanil, trinitrobenzenesulfonic acid, O-methylisoleucine and 2,4-pentandiol, and the amino acid may kataliziruetsa transaminase in the presence of glyoxylate.

Residues of arginine can be modified by reaction with one or more standard reagents, for example phenylglyoxal, 2,3-butanedione, 1,2-cyclohexandione and ninhydrin. To obtain the derivatives in the case of arginine residues is often required to carry out the reaction in an alkaline environment. In addition, the reagents can interact with lysine, as well as with the ε-amino group of arginine.

Specific modification of tyrosine residues can be made using aromatic diazonium compounds or tetranitromethane. For example, N-acetylimidazole, tetranitromethane used for the formation of O-acetyltyrosine compounds and 3-nitro-derivatives, respectively. Residues of tyrosine can idiomatic using125I or131I to get labeled proteins for use in radioimmunoassay analysis.

Carboxyl side groups (aspartyl or glutamyl) can be modified by reaction with carbodiimides (R-N=C=C-R', where R and R' may be different and the alkyl groups, for example, 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-asoni-4,4-dimethylpentyl)carbodiimide. In addition, the remains of aspartyl and glutamyl can be transformed into asparaginase and glutaminovuyu residues by reaction with ammonium ions.

Glutaminovuyu and asparaginase remains often diamidino to the appropriate glutamine and aspartyl residues, respectively, in neutral or basic environment. Deliciously the form of these residues included in the scope of the present invention.

Other modifications include hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups of the residue serenil or threonyl, methylation of the α-amino groups of the side chains of lysine, arginine and histidine (Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Another form of covalent modification is a chemical or enzymatic binding of glycosides with the antibody. Such procedures do not require the production of antibodies in the cell host, which manifests glycosylase properties in N-linked or O-linked glycosylation. Depending on the method used combinations of sugar (sugar) may contact: (a) arginine and histidine; (b) free carboxyl GRU is the groups; (C) free sulfhydryl groups such as cysteine; (d) free hydroxyl groups, such as serine, threonine, or hydroxyproline; (e) aromatic residues such as phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. Such methods are described in WO 87/05330 and in Aplin & Wriston, CRC Crit Rev Biochem, pp. 259-306 (1981).

Removal of any carbohydrate groups present in the structure of antibodies, can be chemical or enzymatic method. For example, for chemical deglycosylation may need to act on the antibody such a reagent as triftormetilfullerenov acid or equivalent connection, which leads to the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-atsetilgalaktozamin), while the antibody remains intact. Chemical deglycosylation described, for example, in Hakimuddin et al. Arch Biochem Biophys 259:52 (1987) and Edge et al., Anal Biochem 118:131 (1981). Enzymatic cleavage of carbohydrate groups on the antibodies can be performed by any of many endoglycosidase and ectoparasites, as described, for example, Thotakura et al., Meth Enzymol 138:350 (1987).

Another form of covalent modification of the antibody comprises linking the antibody to one of the many non-protein polymers, such as polyethylene glycol, polypropyleneglycol or polio what silkolene, based on the method described in U.S. patents№№ 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337.

Another preferred method for producing mutants or Malinov represents the affinity maturation using phage display (Hawkins et al., J Mol Biol 254:889-896 (1992) and Lowman et al., Biochemistry 30(45):10832-10838(1991)). In a nutshell, is the mutation of several sites hypervariable region (e.g., 6-7 sites)to get all possible amino acid substitutions at each site. Thus obtained mutant antibodies monovalent appear on the particles of phage as a fusion protein on the particles. Phage expressing the various mutants can cycle out procedures for the selection of binding with subsequent isolation and sequencing of those mutants that exhibit high affinity.

The method of selection of new binding polypeptides can rely on a library of structurally related polypeptides. Library of structurally related polypeptides, such as fused with the protein shell of the phage obtained through mutagenesis and manifests itself on the surface of the particles. Then the particles come into contact with the molecule-target, and the particles with the highest affinity to the target are separated from possessing lower affinity. Bind with high affinity particles then it provided amplification by infection with the right bacterial host, and the stage of competitive binding repeat. The process is repeated, until will not be received polypeptides with the desired affinity.

Alternatively, multivalent phage (McCafferty et al. (1990) Nature 348:552-554; Clackson et al. (1991) Nature 352:624-628) can also be used for the expression of random point mutations (for example resulting from the use of flawed DNA polymerase) to obtain ragovoy libraries of fragments of antibodies, which can then be screened to verify the affinity to IL-4 or IL-13, Hawkins et al., (1992) J Mol Biol 254:889-896.

Preferably, the maturation process of the affinity of a replicable expression vector remained under the strict control of the control of transcription, and the culturing conditions are adjusted so that the quantity or number of particles that carry more than one copy of the fused protein was less than about 1%. In addition, it is preferable that the number of particles that carry more than one copy of the fused protein was less than 10% of the number of particles carrying a single copy of the fused protein. Preferably, this number was less than 20%.

Functional equivalents can be obtained by changing the various CDR in different antibody chains in spanning or composite FR received from many antibodies. For example, it is possible to obtain various Klas is s antibodies for a given set of CDR by substitution of various heavy chains, for example, IgG1-4, IgM, IgA1-2or IgD in order to obtain different types and isotypes of antibodies to IL-4 or IL-13. Similarly, artificial antibodies included within the scope of the present invention can be obtained by including a given set of CDR in fully synthetic spanning region.

The antibodies and fragments of the functional equivalents of the present invention include molecules that have registered with the level of binding to IL-4 or IL-13. The recorded level of binding includes all values in the range of at least 10-100%, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99% of binding ability of interest antibodies. Within the scope of the present invention also includes equivalents affinity of more than 100% compared to the antibody.

CDR usually are of great importance for the recognition of epitopes and antibody binding sites. However, this may change in the residues, which are part of the CDR, while no effect on the ability of antibodies to recognize cognacy epitope and to contact him. For example, alterations may be made which do not affect the recognition of epitopes, but nevertheless increase the binding affinity of ant the body epitope. A number of studies provides an overview of the effects of replacing one or more amino acids in different positions of the sequence antibodies, based on knowledge of the primary sequence of the antibody on their properties, such as binding and expression level (Yang et al., 1995, J Mol Biol 254:392-403; Rader et al., 1998, Proc Natl Acad Sci USA 95:8910-8915; and Vaughan et al., 1998, Nature Biotechnology 16, 535-539).

Thus, the considered equivalents of the antibodies can be obtained by changing the gene sequences of the heavy and light chains in the CDR1, CDR2 or CDR3, or spanning regions using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, PCR low accuracy, rearrangement in the DNA or mutator strains ofE. coli(Vaughan et al., 1998, Nat Biotech 16:535-539; and Adey et al., 1996, Chap. 16, pp. 277-291, in Phage Display of Peptides and Proteins, eds. Kay et al., Academic Press). Methods for modifying nucleic acid sequence of the primary antibodies can lead to the formation of antibodies with high affinity (Gram et al., 1992, Proc Natl Acad Sci USA 89:3576-3580; Boder et al., 2000, Proc Natl Acad Sci USA 97:10701-10705; Davies &Riechmann, 1996, Immunotech 2:169-179; Thompson et al., 1996, J Mol Biol 256:77-88; Short et al., 2002, J Biol Chem 277:16365-16370; and Furukawa et al., 2001, J Biol Chem 276:27622-27628).

Repeated cycles polypeptide of selection can be applied for the selection of the systems with the higher binding affinity of, for example, by selection changes several amino is the slot, sampled during multiple cycles of selection. After the first cycle of selection, including the first breeding of amino acids in the ligand or polypeptide antibodies, are additional cycles of selection in other areas or the amino acid ligand. The breeding cycles are repeated to achieve the desired characteristics of affinity.

To improved antibodies also include antibodies with more advanced characteristics, prepared using standard methods of immunization of animals, education hybrid and selection of antibodies with defined characteristics.

The term "antagonist" refers to a molecule capable of inhibiting one or more manifestations of the biological activity of a target molecule, such as signal transmission, IL-4 or IL-13. The antagonist can inhibit binding of the receptor with the ligand and Vice versa, incapacitating or destroying cells activated by the ligand, and (or) blocks the activation of the receptor or ligand (for example the activation of tyrosine kinase), or the transmission signal after binding of the ligand to the receptor. Antagonist capable of blocking the interaction of the receptor-ligand or may significantly inhibit such interactions.

The term "agonist" refers to a compound, including a protein, polypeptide, peptide, antibody, antibody fragment, Kon is what, a large molecule, a small molecule that activates one or more manifestations of the biological activity of IL-4 and / or IL-13. Agonists may interfere with the binding of the receptor with the ligand and Vice versa, acting as a mitogen cells activated by the ligand, and (or) preventing inactivation of cells or inhibition of the signal after binding of the ligand to the receptor. For the purposes of the present invention all such aspects of the impact of agonist are equivalent.

Used herein, the terms "cell", "cell line" and "cell culture" include their offspring. This also implies that all progeny may not be precisely identical, such as DNA content, due to deliberate or accidental mutations. This includes the option of offspring, which has the same features or interest biological characteristic, which is screening in the source cell.

The term "vector" means a construct nucleic acid, the medium containing the nucleic acid, the transgene, the foreign gene or gene of interest, which may be functionally linked to appropriate control sequences for expression of the transgene in a suitable host organism. Such control sequences include, for example, the promoter for p is Ogadenia transcription optional operator sequence to control such transcription, a sequence encoding suitable mRNA binding sites on the ribosome, as well as sequences that control the stop of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. After transformation into a suitable host, the vector may replicate and function independently of the genome of the host, or may in some cases be integrated into the genome of the host cell. In the following description, the terms "plasmid" and "vector" are used interchangeably as the plasmid is a widely used form of vector. It is expected however, that the invention includes such other forms of vectors which serve equivalent functions of the media, which are known or become known to the experts in this field, such as viruses, synthetic molecules that contain nucleic acids, liposomes, etc.

Under "mammal" for purposes of treatment refers to any animal classified as a mammal, including man, domestic and farm animals, nonhuman primates, and animals from the zoo, sports or pet animals, such as dogs, horses, cats, cows, etc.

Representing interests the EU antibodies can be subjected to screening or may be used in methods of analysis, described herein or known to the skilled in this field. Often such analyses requires a reagent, which can be detected with conventional methods, for example containing the label. Used herein, the term "label" refers to detectivemisa the compound or composition, which directly or indirectly may conjugates with a molecule or protein such as an antibody. You can label itself is detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate or composition, which are amenable to detection.

Used herein, the term "solid phase" means a non-aqueous matrix to which may be attached structure or molecule, for example, the antibody of the present invention. Examples of solid phases included in the scope of the present invention are entirely or partially formed of glass (such as glass with controlled porosity), polysaccharides (such as agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In some embodiments, depending on the context, the solid phase may be a hole in the analytical tablet; in other cases it can be used in a column for purification (on the example in the column for affinity chromatography). Thus, the solid phase may be paper, granules, plastic surface, chip and the like, it may be manufactured from various materials, such as nitrocellulose, agarose, polystyrene, polypropylene, silicone and the like, and may also have various configurations.

As is well known to specialists in this field, a gene or cDNA encoding IL-4 and IL-13, can be cloned into a plasmid or other expression vector and expressed in any of several systems of expression in accordance with methods well-known qualified specialists in this field, see, for example, below.

The nucleic acid molecule encoding the amino acid sequence mutants, can be obtained using various methods well-known experts in this field. To such a method, among others, include oligonucleotide (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis previously prepared mutated or neutropenia option given molecule (see, for example, Kunkel, Proc Natl Acad Sci USA 82:488 (1985)).

Recombinant expression of the antibodies of the present invention or its fragment, derivative or analogue (e.g. heavy or light chain of the antibody of the present invention, single-stranded antibodies of the present invention or antibodies mutein this is the future of invention) involves the construction of expression vector, containing polynucleotide that encodes the antibody or antibody fragment as described herein. After receiving polynucleotide encoding the antibody molecule, it is possible to prepare the vector for the production of antibodies using recombinant DNA technology known to specialists in this field. Constructed an expression vector containing the coding antibody sequences, as well as markers of control transcription and translation. Such methods include, for example, methods of recombinant DNA in vitro, synthetic methods and genetic recombination in vivo.

The expression vector is transferred into the cell of a host using standard techniques, and transfetsirovannyh cells are then cultured using standard techniques to obtain the antibody of the present invention or its fragment. In one aspect of the invention in the cell-the owner may be held co-expression vectors encoding the heavy and light chains, in order to ensure the expression of the entire immunoglobulin molecules, as described in this document.

When the expression of antibody molecules presented in this invention can be used in a variety of systems of host/expression vector. Such expression systems represent vehicles for the receipt and follow what she purification of interest coding sequences, but also represent cells which upon transformation or transfection of the corresponding coding nucleotide sequences able to Express the antibody molecule of the present invention in situ. Bacterial cells, such asE. coliand eukaryotic cells are widely used for the expression of molecules of recombinant antibodies, especially for the expression of all molecules of recombinant antibodies. For example, mammalian cells such as CHO cells, in combination with the vector, for example, carrying the main element intermediate early gene promoter of human cytomegalovirus is an effective expression system antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology8:2 (1990)). As is well known to specialists in this field, for the preparation of proteins of interest can also be used by plants and culture of plant cells, insect cells, etc.

In addition, you may get a host cell, which modulates the expression of inserted sequences, or modifies and ensures the processing of the gene product of a particular set. Such modifications (e.g. glycosylation) and processing (for example, cleavage) of protein products may be important for the functioning of the protein. Different cell owners have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or system owners can be selected to provide the required modification and processing expressed interest antibodies. Therefore, it can be used eukaryotic cell host, which has a cellular device for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. These cells masters mammals, among others, include CHO, COS, 293, 3T3, or myeloma cells.

Stable expression is preferred for long-term high production of recombinant proteins. For example, can be designed cell line, which stably Express the antibody molecule. Rather than using expression vectors which contain viral replication sources, a host cell can be transformed with DNA in the regulatory environment, which is provided corresponding elements control expression (for example a promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc. and breeding marker. After the introduction of foreign DNA, engineered cells within one or two days to give grow up in an enriched environment, they are then transferred to selective medium. Breeding marker in recombinant the Noah plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into the chromosome with the subsequent development of the cell line. Such engineered cell lines are suitable for the production of antibodies, but also suitable for screening and evaluation of compounds that directly or indirectly interact with the antibody molecule.

Can be used a number of breeding systems, including, among others, thymidine kinase of virusHerpes simplex(Wigler et al., Cell 11:223 (1977)), gipoksantin-guaninephosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA 48:202 (1992)), selection glutamines in the presence of methanesulfonamide (Adv Drug Del Rev 58, 671, 2006, see also the web site or literature Lonza Group Ltd.), as well as genes adrinfo.standortstr (Lowy et al., Cell 22:817 (1980)) in cells tk -, hgprt-or aprt, respectively. Also resistance to antimetabolites can be used as the basis for selection for the following genes: dhfr, which provides resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77:357 (1980); O'hare et al., Proc Natl Acad Sci USA 78:1527 (1981)); gpt, which provides resistance to mycophenolate acid (Mulligan et al., Proc Natl Acad Sci USA 78:2072 (1981)); neo, which provides resistance to the aminoglycoside G-418 (Wu et al., Biotherapy 3:87 (1991)); and hygro, which provides resistance to hygromycin (Santerreet al., Gene30:147 (1984)). Methods known to experts in the field of recombinant DNA technology, can a standard way to apply for selection of the desired recombinant clone, and such methods are described, nab is emer, in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press (1990); Dracopoli et al., eds., Current Protocols in Human Genetics, John Wiley & Sons (1994); Colberre-Garapin et al., J Mol Biol 150:1 (1981).

The levels of expression of antibody molecules can be increased by vector amplification (for example, see Bebbington et al., in DNA Cloning, Vol. 3. Academic Press (1987)). If amplification of the marker in the vector system expressing antibody, an increase in the level of inhibitor present in culture, will lead to an increase in the number of copies of the marker gene. Because amplificatory region associated with the gene of the antibody, production of the antibody will also increase (Crouse et al., Mol Cell Biol 3:257 (1983)).

A host cell may jointly transliterates two or more expression vectors of the present invention, for example, the first vector encoding derived from the heavy chain polypeptide and the second vector encoding derived from the light chain polypeptide. Two vectors can contain identical breeding markers that provide equal expression of the polypeptides of the heavy and light chains. Alternatively can be used a unit vector that encodes and able to Express the polypeptides in both heavy and light chains. In such cases, the light chain should be placed before the heavy to avoid and what Bytca toxic free heavy chain (Proudfoot, Nature 322:52 (1986); and Kohler, Proc Natl Acad Sci USA 77:2197 (1980)). Coding sequences for the heavy and light chains may include cDNA or genomic DNA.

Once an antibody molecule of the present invention obtained in the animal body, synthesized chemically or by recombinant expression, it can be cleaned using any of the methods known to experts in this field, used for purification of immunoglobulin molecules, for example, chromatography (e.g. ion exchange, affinity, particularly based on the affinity of IL-4 and / or IL-13 protein A, as well as gel filtration and the like), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, to facilitate purification of the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or well-known experts in this field.

Antibodies of the present invention can be obtained by any convenient method known to specialists in this field. The antibodies of the present invention can be polyclonal antibodies, although due to the modification of antibodies to optimize the use of human subjects as well as to optimize the use of the antibodies themselves, monoclone the performance communications antibodies are preferred because of the ease of obtaining and processing of specific proteins. Methods of preparing polyclonal antibodies are known qualified specialists in this field (Harlow et al., Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988)).

Antibodies of the present invention preferably include monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, such as described in Kohler et al., Nature 256:495 (1975); U.S. patent No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) and Hammerling etal., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981), methods of recombinant DNA, for example the preparation and use of transfection, or other methods known to experts in this field. Other examples of methods that can be used for the production of monoclonal antibodies, among others, include, hybridoma techniques In human cells (Kosbor et al., Immunology Today4:72 (1983); Cole et al., Proc Natl Acad SciUSA80:2026 (1983)), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss (1985)). Such antibodies can refer to any class of immunoglobulins, including IgG, IgM, IgE, IgA and IgD, and any of their division. Hybridoma producing mAb of the present invention, can be cultured in vitro or in vivo.

Within the hybridoma model of the host organism, such as a mouse, humanized mice, transgenic mice with the genes of the human immune system, hamster, rabbit is, rat, camel or any other suitable animal host, immunities in order to activate the lymphocytes that produce or are capable of producing antibodies, specific binding with IL-4 or IL-13. Alternatively, the lymphocytes may be immunized in vitro. Then lymphocytes merge with myeloma cells using a suitable reagent for the merge, such as polyethylene glycol, to form a cell hybridoma (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp.59-103 (1986)).

Typically, in the preparation of hybridomas producing antibodies used peripheral blood lymphocytes (PBL), if it is desirable to obtain cells of human origin, or spleen cells or lymph nodes, if there are cells derived from mammals, except man. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells derived from rodents, cattle or humans. Usually use a line of myeloma cells in rats or mice. Cell hybridoma may be cultured in a suitable culture medium that preferably contains one or more compounds that inhibit the growth or survival nesmith, immortalized cells. For example, if the parental cells lack the enzyme gipoksantin-guanine is storybuildinternet (HGPRT or HPRT), the culture medium for the hybridomas typically will contain gipoksantin, aminopterin and thymidine ("environment NAT) - substances that prevent the growth of HGPRT-deficient cells.

My favorite lines immortalized cells are those that are effectively involved in a merger, support stable high-level production of antibodies selected antibody-producing cells, and are sensitive to the environment, such as environment, NAT. Such cell lines myeloma lines are murine myeloma, for example derived from murine tumors MOPC-21 and MPC-11, supplied Salk Institute Cell Distribution Center, San Diego, Calif. and cells SP2/0, FO or X63-Ag8-653, supplied by American Type Culture Collection, Manassas, VA.

Documented production of monoclonal human antibodies from cell lines of human myeloma and mouse-human cell heteromyinae (Kozbor, J Immunol 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc, pp. 51-63 (1987)). Can also be used cell line murine myeloma NSO (European Collection of Cell Cultures, Salisbury, Wilshire, UK).

Alternatively, to obtain a hybrid encouraged to use electric merge instead of chemical. Together merge-cells can be immortality using, such as Epstein-Barr or other transforming gene, see, for example, Zurawaki et al., in Monoclonal Antibodies, ed., Kennett et al., Plenum Press, pp. 1933. (1980). Can also be used transgenic mice and mice with severe combined immunodeficiency (SCID), which is transplanted Into a human lymphocytes.

Cultural environment in which to grow cells hybridoma is examined for production of monoclonal antibodies directed against IL-4 or IL-13. The binding specificity of monoclonal antibodies produced by cells of hybridoma can decide on thus or analysis of binding in vitro, for example radioimmunological assay (RIA), fluorocytosine analysis (FACS) or enzyme-linked immunosorbent assay (ELISA). The mentioned methods known in this field, and owns any specialist in this field. The binding affinity of monoclonal antibodies to IL-4 or IL-13 can, for example, be determined by analysis Scatchard (Munson et al., Anal Biochem 107:220 (1980)).

After identification of cells hybridoma producing antibodies with the desired specificity, affinity and / or activity, the clones may be subcloned using procedures of serial dilutions and cultured using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). Suitable culture media, for example, is modified, Dulbecco Wednesday Needle (D-MEM) or medium RPMI-1640. In addition, cells hybridoma can virusheat is camping in vivo in the form of ascitic tumors in the animal body.

Monoclonal antibodies produced by the subclones, properly separated or distinguished from the culture medium, ascitic fluid or serum using standard procedures purification of immunoglobulin, such as protein a-sepharose, protein G-sepharose, chromatography on hydroxyapatite, gel filtration chromatography, gel electrophoresis, dialysis, or affinity chromatography.

In this area there is a wide variety of techniques for production of monoclonal antibodies, and therefore the invention is not limited only to their production in hybridomas. For example, monoclonal antibodies can be prepared using methods of recombinant DNA, for example, such as described in U.S. patent No. 4816567. In this context, the term "monoclonal antibody" refers to an antibody obtained from a single eukaryotic, phage or prokaryotic clone.

DNA encoding the monoclonal antibodies of the present invention, is easily detected, and sequeiros using standard procedures (for example with the use of oligonucleotide probes that are specific to contact with the genes encoding the heavy and light chains of murine antibodies or the same chain of a human, humanized or from other sources) (Innis et al. in PCR Protocols. A Guide to Methods and Applications, Academic (1990), and Sanger et al., Proc Natl Acad Sci 74:563 (1977)). The hybrid cells serve as a source of such DNA. After DNA extraction may be injected into expression vectors, which are then transfairusa in cell host, such as cellsE. colicells , NS0 cells, COS cells, Chinese hamster ovary (CHO) or myeloma cells that in other situations not produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant cell host. DNA can also be modified, for example by substituting the constant domains of the coding sequence of the heavy and light chains of the person instead of the homologous sequences of the mouse (U.S. patent No. 4816567; and Morrison et al., Proc Natl Acad Sci USA81:6851 (1984)) or covalent binding of the coding sequence of the immunoglobulin with the whole or part of the coding sequence nimmanahaeminda polypeptide. This nimmanahaeminda polypeptide can be used instead of the constant domains of the antibodies of the present invention or the variable domains of one of the sites of the combination of IL-4 or IL-13 antibodies of the present invention, so as to form a chimeric bivalent antibody.

Antibodies may be monovalent. Methods of preparing monovalent antibodies are well known to specialists in this field. For example, one method involves recombinant the expression of light chain immunoglobulin and a modified heavy chain. Heavy chain usually clipped anywhere in the region Fcin order to prevent the formation of cross-links in the heavy chain. Alternatively, the relevant cysteine residues are replaced by other amino acid residue or are deleted so as to prevent cross-linking.

Fragments of antibodies that recognize specific epitopes may be obtained using known techniques. Such fragments are traditionally produced using proteolytic destruction of intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys Methods24:107 (1992); and Brennan et al., Science 229:81 (1985)). For example, fragments of Faband F(ab')2the present invention can be obtained by proteolytic cleavage of immunoglobulin molecules under the action of enzymes such as papain (to produce fragments Fab) or pepsin (to produce fragments F(ab')2). Fragments of F(ab')2contain the variable region, the constant domain of the light chain domain and CH1the heavy chain. However, these fragments can be obtained directly from recombinant host cells. For example, fragments of antibodies can be isolated from ragovoy library of antibodies. In an alternative embodiment, fragmani F(ab')2-SH can be directly isolated fromE. coliand chemical contact with the formation of fragments F(ab') (Carter et al., Bio/Technology 10:163 (1992). In accordance with another approach, the fragments of F(ab')2can be directly isolated from the culture of the recombinant host cells. Experienced experimenter will be apparent, other methods of obtaining fragments of antibodies. In other preferred embodiments the antibody is a single-stranded fragment Fv(Fv) (WO 93/16185).

For some applications, including the use of antibodies in humans in vivo and analytical methods for the detection in vitro, it may be preferable to use chimeric, humanized or human antibodies. Methods of producing chimeric antibodies are known to specialists in this area, see, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J Immunol Methods 125:191 (1989); and U.S. patent No. 5807715; 4816567 and 4816397.

Humanized antibodies derived from antibody molecules, which are produced by organisms than humans, and are associated with IL-4 or IL-13, and one or more of their CDR entered in the field FR molecules of the immunoglobulin. Antibodies can humanservices on the basis of a variety of techniques known to experts in this field, including, for example, insert into CDR (EPO 239400; WO 91/09967; and U.S. patent No. 5225539; 5530101 and 5585089), changes in surface antibodies (resurfacing) (EPO 592106; EPO 519596; Padlan, Molecular Immunology 28:489 (1991); Studnicka et al., Protein Engineering 7:805 (1994); the Roguska et al., Proc Natl Acad Sci USA91:969 (1994)), and changes in the circuit (U.S. patent No. 5565332).

Humanitariannet antibody contains one or more amino acid residues that originate from any organism, except human. Not present in human amino acid residues are often referred to as "imported" residues, which are typically taken from an "import" variable domain. Humanization can mainly be carried out using methods of winter with employees (Jones et al., Nature321:522 (1986); Riechmann et al., Nature332:323 (1988); and Verhoeyen et al., Science 239:1534 (1988)), by the substitution of nonhuman CDR or sections of the CDR sequences corresponding to the sequences of human antibodies. Accordingly, such "humanized" antibodies are chimeric (U.S. patent No. 4816567), in which a much smaller area as compared with intact variable domain of human substituted by the corresponding sequence of an organism that are not human. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues substituted similar sites rodent antibodies. The constant region of the heavy chain and the hinge region can be taken from any class or subclass in order to achieve the desired effect, for example konkretnej effector functions.

Often spanning residues spanning regions may be replaced with an appropriate balance of the CDR donor antibody to alter, and, if possible, to improve binding to the antigen. Spanning substitution determined by methods known to experts in this field, for example, by modeling of the interactions of the CDR and spanning residues in order to determine the spanning residues important for antigen binding, as well as by comparison of the sequences for the identification of unusual spanning residues in the specific provisions, see, for example, U.S. patent No. 5585089; and Riechmann et al., Nature 332:323 (1988).

In addition, preferably, humanized antibodies retained high affinity to IL-4 or IL-13, as well as retained or acquired other favorable biological properties. Thus, humanized antibodies are prepared as part of the process of analysis of the source sequences and various conceptual humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulins are widely available and well-known experts in this field. There are computer programs that visually represent and indicate the possible three-dimensional conformational structures of selected candidate immune the globulin sequences. The study of images allows for an analysis of the likely role of different residues in the functioning of the immunoglobulin sequences of the candidate, i.e. the analysis of residues that influence the ability of the immunoglobulin candidate to bind IL-4 and / or IL-13. Thus, it can be selected and combined residues of FR of the recipient and import sequences so as to maximize the desirable characteristics of antibodies, such as increased affinity to the target antigen, although it is the CDR residues are directly and most substantially affect the binding of IL-4 or IL-13. Region CDR can also be modified so as to contain one or more amino acids that differ from those obtained from the original antibody, which was taken CDR, in order to ensure that the gain or the appearance of new and interesting properties, such as binding with greater affinity or greater avidity.

It is possible to manipulate and make changes to certain parts of the constant regions of antibodies, in order to give the homologues, derivatives, fragments and other elements of the antibody properties that differ from the observed in the original antibody or better compared with him. For example, many antibodies IgG4 form noticeplease disulfide bond in the vicinity of the hinge region. Noticeplease St. the z able to destabilize the original bivalent molecule with the formation of monovalent molecules, containing a heavy chain with an associated light chain. Such molecules can again be associated, but randomly.

It was noted that modification of amino acids in the hinge region of IgG4 molecules can reduce the probability of formation of noticeplease communication, thereby stabilizing the IgG4 molecule that will minimize the probability of formation bespecifically molecules. Such modification may be useful, if used for the treatment of the antibody is of the IgG4 molecule, as increased stability to minimize the probability of dissociation of molecules in the process of reception and production, as well as in vivo. Monovalent antibody may not have the same efficiency that bivalent original molecule. For example, if the patient is introduced bivalent IgG4, the percentage of bivalent IgG4 drops to about 30% over a two week period. The substitution of amino acid at position 228 increases the stability of IgG4. Serine, which is in position 228, may be substituted to another amino acid, for example one of the other 19 amino acids. Such a change can be made, in particular, in the case of recombinant antibodies, where the coding sequence of the nucleic acid can be metirovan in order to get a replacement amino acid at position 228. For example, the component S can be substitutes the n on Proline.

A different set of amino acids suitable for the modification includes the amino acids in the hinge region, which affect the binding of a molecule containing the heavy chain of the receptor, to bind to the Fcand the internalization of the bound antibodies. In IgG1 molecules such amino acids are residues from about 233 for approximately 237 (Glu-Leu-Leu-Gly-Gly); (SEQ ID NO:49) with about 252 for approximately 256 (Met-Ile-Ser-Arg-Thr) (SEQ ID NO:50) and with approximately 318 (Glu) at about 331 (Pro), including, for example, Lys320, Lys322and Pro329.

The use of fully human antibodies are particularly desirable for therapeutic effect on people-patients. Human antibodies can be prepared using various methods known to experts in this field, including methods phage display described above, using a library of human antibodies derived from immunoglobulin sequences of human, see U.S. patent No. 4444887 and 4716111; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741. Methods of work of Cole et al. and Boerder et al. can also be used to prepare monoclonal antibodies man (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); and Boerner et al., J Immunol 147:86 (1991)).

Human antibodies can also be produced using transgenic mice which are incapable of expression of functional endogenous immunoglobulins, but which is s also Express certain genes of human immunoglobulin. For example, gene complexes of the heavy and light chains of human immunoglobulin can be introduced into embryonic stem cells mouse randomly or by homologous recombination. In an alternative embodiment, the variable region of the human constant region and the D-region can be introduced into mouse embryonic stem cells in addition to the genes of the heavy and light chain of a human. Mouse genes of the heavy and light chain immunoglobulin can be translated in non-functional condition separately or simultaneously with the introduction of the loci of the immunoglobulin via homologous recombination. In particular, homozygous deletion in the JH region prevents endogenous production of antibodies. Modified embryonic stem cells multiply and spend their microinjection into blastocysts to obtain chimeric mice. From chimeric mice then derive homozygous offspring which expresses human antibodies, see, for example, Jakobovitis et al., Proc Natl Acad Sci USA 90:2551 (1993); Jakobovitis et al., Nature 362:255 (1993); Bruggermann et al., Year in Immunol 7:33 (1993); and Duchosal et al., Nature 355:258 (1992)).

Transgenic mouse as usual undergo immunization cytokine IL-4 or IL-13, such as the full sequence of IL-4 or IL-13 or a part of it. Monoclonal antibodies directed against IL-4 and IL-13, can be obtained from immunized Tr is shinnyh mice using standard hybridoma technology. The transgenes of the human immunoglobulin accumulated in the transgenic mice, reorganized during the differentiation of b-cells and then undergo class switching and somatic mutation. Thus, using this technique is the ability to produce therapeutically applicable antibodies IgG, IgA, IgM and IgE. Review, see Lonberg et al., Int Rev Immunol 13:65-93 (1995). Discussion the production of human antibodies and monoclonal human antibodies and protocols for producing such antibodies, see, for example,WO 98/24893; WO 92/01047; WO 96/34096 and WO 96/33735; EPO No. 0 598 877; and U.S. patent№№5413923; 5625126; 5633425; 5569825; 5661016; 5545806; 5814318; 5885793; 5916771 and 5939598. In addition, companies such as Amgen (Fremont, CA), Genpharm (San Jose, CA) and Medarex, Inc. (Princeton, NJ) can be utilized for delivery of human antibodies, directed against IL-4 or IL-13, using technology that is similar to the above.

In addition, mAb person can be prepared by immunization of mice that transplanted leukocytes in human peripheral blood, splenocytes or bone marrow (for example, the method trim XTL Biopharmaceuticals, Israel). Completely human antibodies which recognize a selected epitope can be obtained using a technique called directional selection. In this approach, selected nonhuman monoclonal antibody, such as a mouse antibody, is used to order the military selection of fully human antibodies recognizing the same epitope (Jespers et al., Bio/technology 12:899 (1988)).

When using recombinant techniques variant antibodies can be produced intracellularly, in periplasmic space, or directly to secretariats on Wednesday. If the antibody is produced intracellularly, in the first stage it is possible to separate the remains of particles, cells masters or fragments after lysis, for example, by centrifugation or ultrafiltration. In the work of Carter et al., Bio/Technology 10:163 (1992) described a procedure for isolating antibodies which are secreted into periplasmatic spaceE. coli. Vkratze, the cell paste is treated with sodium acetate (pH 3.5) and EDTA. The remains of the cells can be removed by centrifugation. If the option to secrete antibody into the medium supernatant from such expression systems generally first concentrated using a commercially available filters for the concentration of proteins, such as ultrafiltration system Amicon or Millipore Pellicon. For inhibition of proteolysis can be added protease inhibitor such as PMSF, and you can also include antibiotics to prevent the growth of random pollutants.

The preparation of antibodies prepared from the cells can be purified using, for example, chromatography on hydroxyapatite, gel electrophoresis, dialysis, and affinity chromatography. The suitability of protein a or B. the LCA G as an affinity ligand depends on the species and isotype of any domain F cimmunoglobulin, which is contained in the variant antibodies. Protein a can be used for purification of antibodies, which are based on the heavy chain IgG1, IgG2 or IgG4 human (Lindmark et al., J Immunol Meth 62:1 (1983)). Protein G can be used to isotypes of mouse and human IgG3 (Guss et al., EMBO J 5:1567 (1986)). As a matrix, which is bound affinity ligand, most commonly used electric agarose, but there are other matrices. A mechanically stable matrices such as glass with adjustable porosity or poly(styrene-butadiene)benzene, allow the use of higher flow rates and can achieve shorter processing times compared with agarose. If the variant antibodies included CH3 domain, cleaning is convenient to use the resin Bakerbond ABXTM (JT Baker; Phillipsburg, NJ). Depending on the secreted antibody and its variants can also be applied to other methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin-agarose, chromatography on an anion-exchange or cation-exchange resin (for example on a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.

After any stage (stages) pre-treatment can be chromatography with hydrophobic interaction when n is skih pH for the mixture, contains interest of the antibody or variant and impurities, in terms of elution buffer at a pH in the range of about 2.5 to 4.5, preferably carried out at low salt concentrations (for example in the range of about 0-0,25 M salt).

Antibodies of the present invention can be bespecifically. Bespecifically antibodies may be monoclonal, preferably human antibodies or humanitarianism antibodies that have the binding specificity of at least two different antigens. In the preferred implementation bespecifically antibody, fragment, etc. demonstrates the binding specificity directed to IL-4 and IL-13.

Methods for the preparation bespecifically antibodies is well known to specialists in this field. Typically, recombinant products bespecifically antibodies based on a joint expression of pairs of heavy chain/light chain of two immunoglobulins, and two heavy chains have different specificnosti (Milstein et al., Nature 305:537 (1983)). Due to the random distribution of heavy and light chains of immunoglobulin hybridoma (quadroma) produce a potential mixture of ten different antibody molecules, of which only one has the correct bespecifically structure. Purification of the desired molecule is usually done through several stages affine x is omatography. Similar procedure as described in the application WO 93/08829 and work Traunecker et al., EMBO J 10:3655 (1991). Other methods for the preparation bespecifically antibodies are, for example, in Kufer et al., Trends Biotech 22:238-244, 2004.

The variable domains of the antibodies with the desired specificnosti binding may be incorporated into the sequences of the constant domains of immunoglobulins. The fusion preferably is with the constant domain of the heavy chain of immunoglobulin containing at least a portion of the hinge regions CH2and CH3. In at least one of the products of merger may contain a constant region of the first heavy chain (CH1including the site required for binding to the light chain. DNA encoding the products of the merging of the heavy chain of the immunoglobulin, and, if desirable, the light chain immunoglobulin entered into various expression vectors together and transformed into a suitable host organism. Learn more about getting bespecifically antibodies can learn, for example, Suresh et al., Meth Enzym 121:210 (1986).

Heteroconjugate antibodies are also part of the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies, for example, have been proposed to Orient the immune system cells to unwanted cells (U.S. patent No. 4676980). It is assumed that antibodies can the be prepared in vitro using known methods in synthetic protein chemistry, including the use of agents for crosslinking. For example, immunotoxins may be constructed using the reaction of disulfide exchange or due to the formation of thioester linkages. Examples of suitable reagents are aminothiols and methyl-4-mercaptopyrimidine, as well as proposed, for example, in U.S. patent No. 4676980.

In addition, it is possible to obtain single-domain antibodies to IL-4 or IL-13. Examples of such technologies are described in the application WO 9425591 for the case of antibodies derived from the heavy chain Ig camel, as well as in the US 20030130496 discussing the allocation of a single domain of fully human antibodies from phage libraries.

Alternatively, techniques described for obtaining single-chain antibodies (U.S. patent No. 4946778; Bird, Science 242:423 (1988); Huston et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al., Nature334:544 (1989))can be adapted to obtain single-chain antibodies. Single-chain antibodies are formed by linking the fragments of the heavy and light chains of the region Fvthrough amino acid bridge with the formation of single-chain polypeptide. You can also use methods of Assembly of functional fragments of the FvinE. coli(Skerra et al., Science242:1038 (1988)).

The present invention includes antibodies, recombinante fused or chemically conjugated (including covalently and ecovalence the conjugated) with the polypeptide. Fused or conjugated to the antibodies of the present invention can be used for easy cleanup, see, for example, WO 93/21232; EP 439095; Naramura et al., Immunol Lett 39:91 (1994); U.S. patent No. 5474981; Gillies et al., Proc Natl Acad Sci USA 89:1428 (1992); and Fell et al., J Immunol 146:2446 (1991). Amino acid sequence of a marker can be exegetically peptide, for example similar to a marker in the vector pQE (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available, Gentz et al., Proc Natl Acad Sci USA 86:821 (1989). Other peptide markers, easy to clean, among others, include the token "ON", which corresponds to the epitope derived from hemagglutinins protein of influenza (Wilson et al., Cell 37:767 (1984)) and the marker flag.

You can also create single-stranded peptide binding molecules in which the heavy and light chain Fvconnected to each other. Single-chain antibody (scFv") and the method of their construction is described, for example, in U.S. patent No. 4946778. Alternatively, in a similar way it is possible to construct and Express Fab. All fully or partially human antibodies may be less immunogenic than fully murine monoclonal antibodies, fragments and single-chain antibodies can also be less immunogenic.

Antibodies or antibody fragments can be released from phage libraries of antibodies, polucen the x using the techniques, described in McCafferty et al., Nature348:552 (1990). Clarkson et al., Nature 352:624 (1991) and Marks et al., J Mol Biol 222:581 (1991) consider the selection of antibodies mouse and human antibodies, respectively, using phage libraries. In subsequent publications described the production of high affinity (nm interval) antibodies through changes in chain (Marks et al., Bio/Technology10:779 (1992)), as well as a method of combinatorial infection and recombination in vivo as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl Acids Res 21:2265 (1993)). Thus, such methods are practical alternative to traditional hybridoma techniques in obtaining monoclonal antibodies.

Potential antibodies anti-IL-4 or IL-13 was analyzed by the methods of enzyme-linked immunosorbent assay (ELISA), FACS, Western blotting, or other immunochemical methods known to experts in this field.

To determine associated with whether the specific homolog antibody to human IL-4 or IL-13, you can use any standard method of analysis of binding. To convenient methods of analysis of binding IL-4 and IL-13 are the FACS analysis, ELISA, surface plasmon resonance (Biacore), radioimmunoassay methods of analysis and the like, which detects the binding of an antibody to IL-4 or IL-13, and the resulting function. For such is the analyses convenient discussed here have full-length and soluble forms of IL-4 and IL-13 human. The binding of an antibody or homolog to IL-4 or IL-13, or soluble fragments, you can easily register through the use of second antibody specific to antibodies of the species from which the antibody or homolog.

To install, blocked in an appreciable degree of a particular antibody or homolog binding to IL-4 or IL-13, you can use any convenient method of competitive analysis. Suitable methods of analysis, for example, include methods ELISA, FACS methods, radioimmunoassay methods of analysis and the like, which allow to quantify the ability of the antibody or homolog to compete with IL-4 or IL-13. Preferably, evaluates the ability of a ligand to block the binding of labeled IL-4 or IL-13 human with immobilized antibody or homolog.

Antibodies of the present invention may be described or characterized from the point of view of the epitope (epitope) or part (s) of IL-4 and / or IL-13, which antibody recognizes or with which it specifically binds. The epitope (epitope) or part (s) of the polypeptides, as described herein, may be characterized, for example, by N-terminal and C-terminal positions, the length of a continuous chain of amino acid residues, conformational epitopes, etc.

Antibodies of the present invention can also is to be described or characterized by cross-reactivity. Within the scope of the present invention also includes antibodies that bind polypeptides of IL-4 and / or IL-13, characterized by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% identity (which is calculated using methods known to experts in this field and described in this document) compared with IL-4 or IL-13.

Antibodies of the present invention may also be described or characterized by the affinity of binding with interest IL-4 or IL-13. Antibodies anti-IL-4 and (or) anti-IL-13 can contact KDless than about 10-7M, less than about 10-6M or less than about 10-5M. higher binding affinity of the considered antibodies may be more desirable, for example, the equilibrium dissociation constant or KDfrom about 10-8up to about 10-15M, from about 10-8up to about 10-12M, from about 10-9up to about 10-11M, or from about 10-8up to about 10-10M. In the present invention also provides antibodies that inhibit competitive binding of an antibody to an epitope of the present invention as determined by any method known to the specialists in the given field for registration of competitive binding, for example described in this document, methods of immunological analysis. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50%.

Within the scope of the present invention also includes conjugates containing an antibody. Conjugates consist of two main components: this antibody and the second component, which may be binding cell agent, cytotoxic agent, etc.

Used herein, the term "linking cell agent" refers to a substance that specifically recognizes and binds to a molecule on the cell surface. So, as connecting cells of the agent can be an antigen CD, pathogenic antigen such as a viral antigen, a differentiating antigen, a cancer antigen, cleocinonline antigen, tissue-specific antigen, Ig or similar Ig molecule, etc.

Linking cells agents can be of any type currently known or becomes known in the future, these include peptides, non-protein compounds, saccharides, nucleic sour is s, the ligands, receptors and the like, or combinations thereof. Linking cells agent can be any compound which binds to the cell both specific and non-specific way. Generally, as the agent can be an antibody (especially monoclonal antibody), lymphokines, hormones, growth factors, vitamins, molecules are carriers of nutrients (such as transferrin), or any other linking cell molecule or substance.

Other examples of linking cell agents that can be used in the present invention include polyclonal antibodies, monoclonal antibodies and antibody fragments, for example, FabFab'F(ab')2and Fv(Parham, J. Immunol. 131:2895-2902 (1983); Spring et al., J. Immunol. 113:470-478 (1974); and Nisonoff et al., Arch. Biochem. Biophys. 89: 230-244 (1960)).

The second component may also be a cytotoxic agent. Used herein, the term "cytotoxic agent" refers to a substance that inhibits or blocks the functioning or growth of cells and (or) causes the destruction of cells. So, as a cytotoxic agent can be a Taxol, maytansinoid, such as DM1 or DM4, CC-1065, or a CC-1065 analog, ricin, mitomycin C, etc. In some embodiments cytotoxic agent, like any binding agent conjugate of the present invention, covalently linked to predstavlyayuschim interest antibody directly or via split or necessasary the linker.

Examples of suitable maytansinoids are maytansines and analogues maytansine. Maytansinoid inhibit the formation of microcannulas and highly toxic to mammalian cells.

Examples of suitable analogues maytansine are having a modified aromatic ring, as well as analogues with modifications at other positions. These are suitable for use maytansinoid proposed in U.S. patent№№4424219; 4256746; 4294757; 4307016; 4313946; 4315929; 4331598; 4361650; 4362663; 4364866; 4450254; 4322348; 4371533; 6333410; 5475092; 5585499 and 5846545.

Examples of suitable analogues maytansine with modified aromatic ring include: (1) C-19-Dehler (U.S. patent No. 4256746) (obtained, for example, LAH-recovery ansamitocins P2); (2) C-20-hydroxy (or C-20-demethyl) +/- C-19-Dehler (U.S. patent No. 4361650 and 4307016) (obtained, for example, by demethylation using streptomycete or actinomycetes or dechlorination using sociallyengaged (LAH)); and (3) C-20-dimethoxy, C-20-acyloxy (-OCOR), +/-Dehler (U.S. patent No. 4294757) (obtained by acylation under the action of acylchlorides).

Examples of suitable analogues maytansine with modifications in other provisions include: (1) C-9-SH (U.S. patent No. 4424219) (obtained by the reaction maytansine with H2S or P2S5); (2) C-14-alkoxymethyl (dimetoxy/CH2OR) (U.S. patent No. 4331598); (3) C-14-hydroxymet the l or acyloxymethyl (CH 2OH or CH2OAc) (U.S. patent No. 4450254) (derived from Nocardia); (4) C-15-hydroxy/acyloxy (U.S. patent No. 4364866) (obtained by the conversion maytansine the streptomycete); (5) C-15-methoxy (U.S. patent No. 4313946 and 4315929) (allocated fromTrewia nudiflora); (6) C-18-N-demethyl (U.S. patent No. 4362663 and 4322348) (obtained by demethylation maytansine the streptomycete); and (7) 4,5-deoxy (U.S. patent No. 4371533) (produced by restoration maytansine under the action of the titanium trichloride/LAH).

Cytotoxic conjugates can be obtained by the methods in vitro. To bind a cytotoxic agent, drug, or prodrugs by antibody, typically used linker group. Suitable linker groups are known to specialists in this area, these include disulfide groups, thioester groups, kislotoneustoichiv group, photolabile group, group unstable to the effects of peptidases, as well as group unstable to the effects of esterase. For example, the conjugates can be constructed using the reaction of disulfide exchange or by forming a thioester link between interest antibody and the drug or prodrug.

As discussed above, the present invention offers a sequence of isolated nucleic acids encoding described in this document antibody or its functional fragm the t or option construction of vectors comprising a nucleotide sequence encoding a binding part of the antibody or functional fragment of IL-4 and / or IL-13 of the present invention, the cells of the host, carrying a vector, and recombinant methods of production of such polypeptide.

The vector typically consists of components that are known to specialists in this area and usually, among others, includes one or more of the following elements: a signal sequence, the starting point of replication, one or more markers or genes selection, sequence, and contributing (or) stimulating broadcast, enhancer, etc. Thus, the expression vectors contain a nucleotide sequence that is functionally connected with such suitable transcriptional or translational regulatory nucleotide sequences, for example derived mammalian genes, microbes, viruses, or insects. For additional examples of regulatory sequences include operators, ribosomal binding sites, mRNA and / or other appropriate sequences which control transcription and translation, for example, initiation and termination. Nucleotide sequences are "functionally linked"when the regulatory sequence which is functional with respect to the nucleotide sequence of the corresponding polypeptide. Thus, the nucleotide sequence of the promoter functionally linked, for example, with a sequence of heavy chain antibodies, if the nucleotide sequence of the promoter controls the transcription of such a nucleotide sequence.

In addition, the expression vectors can be integrated sequences encoding appropriate signal peptides that are not usually natural way associated with sequences of the heavy and / or light chains of the antibody. For example, the nucleotide sequence of the signal peptide (secretory leader) may be fused inside the frame with a polypeptide sequence, so that the antibody secretarials in periplasmatic space or environment. Signal peptide, which acts alleged in the cells of the host, enhances extracellular secretion of the corresponding antibody or part thereof. The signal peptide can be chipped off from the polypeptide upon secretion of antibodies from the cell. Examples of such secretory signals are well known and include, for example, described in U.S. patent No. 5698435; 5698417 and 6204023.

As the vector may be a plasmid, single-stranded or double-stranded viral vector, single-stranded or double-stranded RNA or DNA phage vector, fahmid, cosmid or any other medium of interest is ranchera. Such vectors may be introduced into cells as polynucleotide, using well known methods for introducing DNA and RNA in cells. The vectors, in the case of phage or viral vectors may also be introduced into cells in the form of a virus in the shell or capsule using well-known techniques of infection and transduction. Viral vectors can be competent or defective with respect to replication. In the latter case, the virus will usually take place only in the complementary cells masters and using multiple vectors carrying different viral components required for the production of particles. Cell-free system broadcast can also be used for production of the protein using RNA derived from existing designs of DNA (see, for example, WO 86/05807 and WO 89/01036; and U.S. patent No. 5122464).

The expression of the antibodies of the present invention may be implemented in any cell, the owner. Examples of host cells suitable for use in the present invention include prokaryotic, yeast or higher eukaryotic cells, and here, among others, includes microorganisms such as bacteria (includingE. coli,B. subtilis,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia and Shigella, as well as rod-shaped bacteria found and strap oomycete), transformed using expression vectors recombinant DNA bacteriophage, plasmid DNA or kosmidou DNA containing the coding sequences of interest antibodies; yeast (for example Saccharomyces cerevisiae, Pichia,actinobacteria, kluveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora and hyphomycetes, such as Neurospora, pencil, tolypocladium and aspergilli)transformed with recombinant expression vectors of yeast containing encoding the antibody sequence; cell system of insects infected with the recombinant expression vectors of the virus (e.g. baculovirus)containing the sequence encoding the antibody; plant cell systems infected with recombinant expression vectors of the virus (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with expression vectors recombinant plasmids (for example,the Ti plasmid)containing the coding sequences of the antibody; or a cellular system mammalian (e.g., COS cells, CHO, BHK, 293, or 3T3)carrying the recombinant constructs of the expression containing promoters derived from the genome of mammalian cells (for example, the promoter metallothionein) or from mammalian viruses (e.g., the late promoter of adenovirus is whether 7.5K promoter of vaccinia virus).

The expression vectors for use in prokaryotic cells-owners usually include one or more phenotypic breeding marker genes. Phenotypic breeding marker gene, for example, is a gene that encodes a protein that provides resistance against antibiotics or satisfies autotrophy requirements. Examples of suitable expression vectors for prokaryotic host cells are obtained on the basis of commercially available plasmids such as a series of vector pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, WI), pET (Novagen, Madison, WI) and pRSET (Invitrogen, Carlsbad, CA) (Studier, J Mol Biol 219:37 (1991); and Schoepfer, Gene 124:83 (1993)). To promoter sequences commonly used for recombinant expression vectors prokaryotic host cells include T7, (Rosenberg et al., Gene 56:125 (1987)), β-lactamase (penicillinase), promotor system of lactose (Chang et al., Nature275:615 (1978); and Goeddel et al., Nature281:544 (1979)), the promoter system of tryptophan (Trp) (Goeddel et al., Nucl Acids Res 8:4057 (1980)), and tac-promoter (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990)).

In yeast vectors often contain a sequence of the starting point of replication, for example, from plasmids yeast 2μ, Autonomous can replicate the sequence (ARS), a promoter region, sequences polied is melirovanie, the sequence of the termination of transcription and gene breeding marker. Suitable promoter sequences for yeast vectors, among others, include the promoters of metallothionein, 3-phosphoglycerate (Hitzeman et al., J Biol Chem 255:2073 (1980)) or other glycolytic enzymes (Holland et al., Biochem 17:4900 (1978)), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, glucokinase, piruvatcarboksilazy, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, piruwatkinaza, triazolopyrimidine, phosphoglucomutase and glucokinase. Other suitable vectors and promoters for use in yeast expression are described in detail in the work Fleer et al., Gene 107:285 (1991). Other suitable promoters and vectors for yeast and protocols for transformation of yeast is well known to specialists in this field. Protocols for the transformation of yeast is also well known. One of these protocols is described in Hinnen et al., Proc Natl Acad Sci 75:1929 (1978), which is the selection of transformants Trp+in the selective medium.

Applicable to any cell culture eukaryotes, as from a culture of vertebrates and invertebrates. Examples of invertebrate cells include the cells of plants and insects (Luckow et al., Bio/Technology 6:47 (1988); Miller et al., Genetic Engineering, Setlow et al., eds., vol. 8, pp. 277-9, Plenum Publishing (1986); and Maeda et al., Nature 315:592 (1985)). For example, for the production of heterologous white is s can be used baculovirus systems. In the system of the insect nuclear polyhedrosis virusAutographa californica(AcNPV) can be used as a vector for expression of foreign genes. The virus grows in the cells ofSpodoptera frugiperda. The coding sequence of the antibody may be cloned under control of an AcNPV promoter (for example the polyhedrin promoter). Other established masters like mosquitos,Drosophila melanogasterandBombyx mori. Widely available variety of viral strains for transfection, such as option L-1 AcNPV and strain Bm-5Bombyx moriNPV. In addition, as is well known to specialists in this field, cell culture of plants, such as cotton, maize, potato, soybean, Petunia, tomato, and tobacco, can also be used as hosts.

The use of vertebrate cells, and propagation of vertebrate cells in cell culture (tissue culture) can be a standard procedure, although in reality there are whimsical line cells, which require, for example, a specialized environment with unique factors that feed the cells and the like, see Tissue Culture, Kruse et al., eds., Academic Press (1973). Examples of suitable cell lines of mammalian hosts include cells of monkey kidney; cells mesonephros person; cells of the kidneys of the newborn hamster; cells Chinese hamster ovary/-DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 77:4216 (1980)); mouse is by Sertoli cells; carcinoma cells human cervical (e.g. HeLa)cells of the kidney of the dog; human lung cells; liver cells human; tumor cells of the mammary gland of mice and NS0 cells.

Cell host transformed with vectors for the production of antibodies and cultured in standard culture medium containing growth factors, vitamins, minerals, etc. and inductors that are suitable for your cells and vectors. Widely used sequence of the promoter and enhancer sequence derived from virus polyoma, adenovirus 2, monkey virus 40 (SV40), and human cytomegalovirus (CMV). DNA sequences derived from the SV40 viral genome, can be used for preparing other genetic elements for expression of structural genetic sequence in the cells of the host mammal, for example derived from the SV40 early and late promoters, enhancer, splicing sites and polyadenylation. Viral early and late promoters are particularly useful because both are easy enough to obtain from a viral genome as a fragment, which can also contain the viral replication origin. In commercially available commercially available standard expression vectors for use in the cells of the host mammal.

For cell culture-x is Zaev suitable commercially available environment for example, the environment of ham F10, minimum maintenance medium (MEM), RPMI-1640 and modified Dulbecco Wednesday Needle (DMEM). In addition, any media described in Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al., Anal Biochem 102:255 (1980), and in U.S. patents№№4767704; 4657866; 4560655; 5122469; 5712163 or 6048728, can be used as culture media for the host cells. Any of these media may be supplemented with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as chlorides, in particular the chlorides of sodium, calcium or magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations lying in the micromolar range), and glucose or an equivalent energy source. Depending on the objectives can be included and any other necessary additives in appropriate concentrations. Culturing conditions, such as temperature, pH, etc. as known to experts in the field, suitable for cell and provide the desired transgene expression.

Using any of the methods known to experts in this field, you can get interest polynucleotide and to determine the nucleotide sequence that is tidow sequence such polynucleotides. For example, if the known nucleotide sequence of the antibody, polynucleotide encoding the antibody may be composed of chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., Bio/Techniques 17:242 (1994)), and then related ligands polynucleotide amplified, e.g. using PCR.

Alternatively, you can create polynucleotide encoding an antibody, a nucleic acid that expresses one or the other cell. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the molecule of the antibody is known, a nucleic acid encoding the immunoglobulin, can be obtained from a suitable source, such as a library, which can be specific for producing the antibody of cells, for example cells hybridoma selected for expression of the antibodies of the present invention. For PCR amplification can be prepared by the appropriate primers. Amplificatoare nucleic acid obtained by PCR, can then be cloned into replicable cloning vectors using any method known to specialists in this field.

Once the nucleotide sequence and the corresponding sequence of the antibody is established, the possible manipulation of the nucleotide sequence of the antibody in order to obtain interest equivalents, described in the present invention, using the methods of manipulation of nucleotide sequences, which are known to experts in this field, for example the techniques of recombinant DNA, site-directed mutagenesis, PCR and the like (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1998), in order to obtain antibodies containing different amino acid sequences, such as substitutions, deletions and / or insertions of amino acids.

Amino acid sequence of the heavy and or light chain variable domain can be analyzed for identification of CDR sequences using well-known methods, for example by comparison with the known amino acid sequences of other heavy and light chain variable regions, in order to identify areas of hypervariability sequence. Using standard techniques of recombinant DNA, you can enter one or more CDR in spanning region, such as spanning region of the protein man, in order to humanize the inhuman antibody as described above. Interest polynucleotide obtained by the combination of spanning regions and one or more CDRs encodes an antibody that specifically binds IL - and (or) IL-13 or at least his ED domain. Such methods, for example, can be used to perform substitutions or deletions of amino acids for one or more cysteine residues of the variable regions, which are involved in the formation of interchain disulfide bonds, in order to obtain the antibody molecule without one or more interchain disulfide bonds.

Antibodies or fragments of antibodies according to the present invention can be used to detect IL-4 or IL-13, and hence of cells expressing IL-4 or IL-13 in a biological sample, both in vitro and in vivo. In one example implementation antibody anti-IL-4 or IL-13 of the present invention is used to establish the presence and the level of IL-4 and / or IL-13 in tissues or cells isolated from a tissue. The levels of IL-4 and / or IL-13 in the tissue or biopsy sample can be determined, for example, by immunoassay for antibodies or fragments of antibodies of the present invention. Fabric or sample of her biopsy can be frozen or fixed. The same or other methods can be used to set other properties of IL-4 and / or IL-13, such as its level, cellular localization, mRNA levels, mutations, etc.

The above-described method, for example, can be used to diagnose cancer in a patient who is at risk of origin of cancer or who prefer Agueda cancer, the level of IL-4 and / or IL-13, is recorded in the aforementioned patient-matched normal healthy patient or with the standard. Consider the analysis can also be used for the diagnosis of arthritis or other autoimmune diseases, which are characterized by the infiltration and the concentration of cells along with the development of differentiated lymphoid tissue.

The present invention also provides monoclonal antibodies, humanized antibodies and fragments thereof that bind epitopes, in which you can type a label for subsequent use in research or diagnostic applications. In some embodiments the label is radioactive, as well as the label uses a fluorophore, a chromophore, a contrast agent or a metal ion.

There is a diagnostic method in which the aforementioned labeled antibodies or fragments thereof that bind the epitope is introduced to the patient, which is expected to cancer, arthritis, autoimmune disease or other mediated IL-4 or IL-13 disease, and is measured or monitored the distribution of labels in the body of the patient.

Antibodies or fragments thereof proposed in the present invention, can be used as agents for affinity purification. In this process, the antibodies immobilized on the solid phase, for example resins is with dextran or agarose or on filter paper, using methods known to experts in this field. The immobilized antibody is processed by a sample containing IL-4 and / or IL-13 or bearing its cells, which are cleaned, and then the substrate is washed with a suitable solvent that will remove almost all of the material from the sample, in addition to IL-4 or IL-13 or cells that are cleaned as they are bound to the immobilized antibody, which is of interest for the present invention. Finally, the substrate is washed with another suitable solvent, such as glycine buffer, pH 5.0, which provides the release of IL-4 and / or IL-13 or cells from the antibody.

For diagnostic applications considering the antibody will generally be marked contrast agent. There are many different labels, which, in General, can be grouped into the following categories: (a) radioisotopes, such as36S14C,125I3H and131I (antibody may be labeled with a radioisotope-based methods, such as described in Current Protocols in Immunology, vol. 12, Coligen et al., ed., Wiley-Interscience, New York (1991), and radioactivity can be measured on the basis of scintillation account); (b) fluorescent labels such as rare earth chelates metals (europium chelates), fluorescein and its derivatives, rhodamine and its derivatives, d is ncil, lissaman, phycoerythrin and Texas red, fluorescent labels can conjugates with the antibody using techniques described, for example, in Current Protocols in Immunology, as mentioned above, and the fluorescence can be quantitatively determined using fluorimetry; and (C) there are also labels as substrates for various enzymes (U.S. patent No. 4275149 contains an overview), and the enzyme generally catalyzes a chemical transformation of a chromogenic substrate that can be determined using various techniques, for example, the enzyme may catalyze a color change of the substrate, which may be logged by spectrophotometric, or the enzyme may influence on fluorescence or chemiluminescence substrate. Known methods of quantitative determination of changes in fluorescence, for example using a luminometer, or registration of transfer of energy from the fluorescent label to the acceptor. Examples of enzymatic labels include luciferase (such as Firefly luciferase and bacterial luciferase; U.S. patent No. 4737456), luciferin, 2,3-dihydropteridine, malatdegidrogenaza, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, SharedAccess (e.g. glucoseoxidase, galactosidase and glucose-6-phosphatedehydrogenase), heterocyclic is xidase (such as uricase and xanthine oxidase), lactoperoxidase, microbiocides etc. Methods of conjugating enzymes to antibodies are described in O'sullivan et al., Meth Enz, ed. Langone & Van Vunakis, Academic Press, New York, 73 (1981).

In case of using such labels are suitable substrates, for example: (i) for peroxidase from horseradish peroxide as a substrate, and the hydrogen peroxide oxidizes a dye precursor (e.g., orthophenylene (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB)); (ii) alkaline phosphatase (AP) as the chromogenic substrate is p-nitrophenylphosphate; and (iii) β-D-galactosidase (β-D-Gal) is used chromogenic substrate (e.g. p-nitrophenyl-β-D-galactose) or fluorogenic substrate, for example 4-methylumbelliferyl-β-D-galactosidase.

Qualified specialists in this field are available, and other combinations of enzyme substrates. A General overview, see U.S. patent No. 4275149 and 4318980.

In some cases, the label is indirectly conjugated with the antibody. For example, the antibody can conjugates with Biotin and any of the above reporters can conjugates with Avidya or Vice versa. Biotin is selectively associated with Avidya, and under these conditions, the label may indirectly to conjugates with the antibody. Alternatively, to obtain indirect conjugation of the label to the antibody binds with a small hapten (e.g. the digoxin), and one of the different types of labels or reporters, mentioned above, conjugated with the antibody anti-digoxin. Thus, by using a second antibody, it is possible to conduct indirect conjugation of the label with the antibody or muteena.

In another example embodiment of the invention the antibody optional label is introduced, and its presence may be recorded using any other form of second antibody - labeled antibody, which binds to the antibody.

Antibodies of the present invention can be used in any known method of analysis, for example analysis of competitive binding, direct and indirect sandwich assays and analyses thus. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc. 1987).

Analysis of competitive binding relies on the ability of a labeled standard to compete with the test sample in the process of binding with a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the number of standard binds to antibodies. To facilitate determining the amount of standard that becomes bound, the antibodies, usually before or after a competitive interactions translate into an insoluble form. As a result, standard and test samples that are associated with antibodies, can easily be separated from the article is ngarta and the test sample, which remain unbound.

Sandwich the analysis involves the use of two antibodies, each of which is able to communicate with different immunogenic parts, determinants or epitopes of the target, which is detected. In the sandwich method, the test sample binds to the first antibody, directly or indirectly immobilized on a solid substrate, after which the second antibody is labeled directly or indirectly associated with the recorded test pattern, thus forming an insoluble three-part complex, see, for example, U.S. patent No. 4376110. The second antibody may be in itself marked the registered label (direct sandwich method) or can be detected using antiimmunoglobulin antibodies or other suitable component associated pairs (such as antibody/antigen, receptor/ligand, enzyme/substrate), which is labeled detectable label (indirect sandwich method). For example, one type of sandwich assay is a method ELISA, in this case, the detectable label is an enzyme.

Within the scope of the present invention also includes kits, for example, including antibodies, their fragments, homologues, derivatives thereof and the like, such as a labeled or cytotoxic conjugates, as well as instructions for using the antibody to yugata for the destruction of certain types of cells, etc. The instructions may include instructions for using the antibody, conjugate, etc. in vitro, in vivo or ex vivo. The antibody may be in liquid or in solid form, as a rule, liofilizovannyh. A set can contain other suitable reagents, such as buffer, the recovered solution and other necessary ingredients for its intended use. It is assumed that the set will be a packaged combination of reagents in predetermined amounts with instructions for their use, such as for medicinal purposes for conducting the diagnostic tests. If the antibody is injected label such as an enzyme, the kit may include substrates and cofactors required for the enzyme (e.g., the predecessor of the substrate, which provides education registered chromophore or fluorophore). In addition, the kit can include other additives, such as stabilizers, buffers (for example, a block buffer or litany buffer), etc. Relative amounts of the various reagents can be changed, so that the set was attended by the concentrated reagent solutions, which provides the user the flexibility, space savings, reagents and other Reagents can be supplied in the form of solid powders, usually liofilizovannyh, together with fillers, which after dissolution form a reagent solution in the good the soup concentration.

Antibodies of the present invention can be used to treat mammals. In one example implementation, the antibody or its equivalent, of interest, are introduced to a mammal, except man, for example to obtain preclinical data. Examples of mammals besides humans that can get the drug, include nonhuman primates, dogs, cats, rodents and other mammals, which are conducted preclinical studies. These mammals can be proven animal model for a particular disease to be treated with antibodies, or they can be used for studying the toxicity in question antibodies. In each of these embodiments for a mammal is able to study increasing the dose.

As a medicinal product can be used antibody in the presence or in the absence of the second component, for example a therapeutic agent conjugated to him, shall be put without additives or in combination with cytotoxic factor (factors). The present invention is devoted to the antibody therapy, which involves the antibodies of the present invention to an animal, mammal or human for the treatment mediated by IL-4 or IL-13 disease, disorder or condition is.

Used in the present invention the term "treatment" refers to therapeutic treatment and prophylactic and preventive measures. It refers to the prevention, treatment, termination, smoothing, softening, minimizing, suppressing or stopping the destructive effects of a pathological state, disease progression, the causative agent of the disease (e.g., bacteria or virus) or other abnormal condition.

Therefore, the present invention also includes polyvalent antibodies, including bespecifically antibodies anti-IL-4/IL-13, with related diagnostically or therapeutically functional effector molecules, atoms, or other fragments. For example, the antibody may contain radioactive diagnostic label or cytotoxic radioactive atom, or metal, or cytotoxic components, for example a chain of ricin, which are associated with him for the purposes of in vivo diagnosis or treatment of cancer.

In addition, antibodies of the present invention can be used in immunoassay analysis, treatment methods, and other methods that use antibodies or fragments thereof. Such applications are well known to specialists in this field.

Accordingly, the invention also provides preparations containing antibodies anti-IL-13 and/or anti-IL-4 or afragment in accordance with the invention in a convenient combination with a pharmaceutically acceptable carrier, diluent or excipient, which are usually used in this field.

Used in the present invention, the term "pharmaceutical product" refers to the compositions of various drugs. Compositions containing therapeutically effective amount of polyvalent antibodies are sterile liquid solutions, liquid suspensions or liofilizovannye options and may contain stabilizers or fillers.

Used in the present invention, the term "violation" refers to any condition that may have a beneficial effect of treatment with antibodies of the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which determine the susceptibility of a mammal and, in particular, of the person to the violation. Among the many examples of disorders the treatment of which is assumed in the present invention, various forms of cancer, inflammation, autoimmune diseases, infections, cardiovascular diseases, respiratory diseases, neurological diseases and metabolic diseases.

Antibodies of the present invention can be used for the treatment, inhibition or prevention of various diseases, such as allergic diseases, Th2 mediated disease is s, mediated IL-13 diseases mediated by IL-4 diseases and/or mediated by IL-4/IL-13 diseases. Examples of such diseases are Hodgkin's disease, asthma, allergic asthma, atopic dermatitis, atopic Allergy, ulcerative colitis, scleroderma, allergic rhinitis, idiopathic pulmonary fibrosis HOSL, chronic graft rejection, caused by bleomycin pulmonary fibrosis, radiation-induced pulmonary fibrosis, pulmonary granulomas, progressive systemic sclerosis, schistosomiasis, liver fibrosis, kidney cancer, Burkitt's lymphoma, Hodgkin's disease, non-Jackinsky lymphoma, syndrome Cesari, asthma, septic arthritis, dermatitis herpetiformis, chronic idiopathic urticaria, ulcerative colitis, scleroderma, hypertrophic scarring, syndrome Whipple, benign prostatic hyperplasia prostate, lung disorder in which the role of the IL-4 receptor, a condition in which the role of the mediated IL-4 receptor breach of the epithelial barrier, disruption of the digestive system in which the role of the IL-4 receptor, an allergic reaction to a particular drug, Kawasaki disease, sickle cell anemia syndrome Cerca-Strauss, diffuse toxic goiter, preeclampsia, Sjogren syndrome, autoimmune limpop referativnyi syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, cystic fibrosis, allergic bronchopulmonary mycosis, chronic obstructive pulmonary disease, induced by bleomycin pneumopathy and fibrosis, pulmonary alveolar proteins syndrome slow respiratory failure, sarcoidosis syndrome Jobe, idiopathic hypereosinophilic syndrome, autoimmune education cutaneous abscesses, common bladderwort, bullous pemphigoid, malignant gravis, chronic fatigue syndrome, nephrosis.

The term "allergic disease" refers to a pathological condition in which the patient hypersensibility to the substance, which is usually not immunogenic and forms an immunological reaction to the substance. Allergic disease is usually characterized by activation of mastocytes by IgE, which leads to an inflammatory response (e.g., local response, system response), and its symptoms can range from harmless as the common cold, to life-threatening anaphylactic shock and death. Examples of allergic diseases, among others, include allergic rhinitis (e.g. hay fever), asthma (e.g., allergic asthma), allergic dermatitis (e.g., eczema), contact dermatitis, food Allergy and ur the Ikaria (hives).

Used herein, the term "Th2 mediated disease" refers to a condition in which the abnormality occurs (fully or partially) due to the immune response (immune response Th2-type), adjustable by means of T-lymphocytes CD4+Th2, which typically produce IL-4, IL-5, IL-9 and IL-13. The immune response to Th2-type is associated with the production of certain cytokines (such as IL-4, IL-13) and certain classes of antibodies (e.g., IgE), and is associated with humoral immunity. Mediated Th2 diseases are characterized by the presence of elevated levels of Th2 cytokines (eg, IL-4, IL-13) and/or certain classes of antibodies (e.g., IgE), and include, for example, allergic diseases (e.g. allergic rhinitis, atopic dermatitis, asthma (for example, atopic asthma), allergic airway disease (ASDP), anaphylactic shock, conjunctivitis), autoimmune diseases associated with elevated levels of IL-4 and/or IL-13 (e.g., rheumatoid arthritis, disease, transplant rejection by the host organism, disease kidney (e.g., nephritic syndrome, lupus nephritis)), as well as infections associated with elevated levels of IL-4 and/ or IL-13 (e.g., viral, parasitic, fungal (e.g.C. albicans) infection). Certain forms of cancer associated with elevated levels of IL-4 and/or IL-13 or associated with IL-4-induced and/or IL-13-induced proliferation of cancer cells (for example, B-cell lymphoma, T-cell lymphoma, multiple myeloma, carcinoma of the head and neck, breast cancer and ovarian cancer). Such forms of cancer can be treated, suppressed or prevented by the ligand of the present invention.

Used herein the term "cancer" refers to a physiological condition in mammals, in particular humans, which is typically characterized by unregulated cell growth. Examples of cancer, among other things, include carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

Used herein, the term "autoimmune disease" refers to a non-malignant disease or disorder that occurs in native tissue of a patient and directed against its own tissues. Examples of autoimmune diseases or disorders, among others, include inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis; allergic conditions such as eczema and asthma; other States, including infiltration of T cells and chronic inflammatory responses; atherosclerosis; diabetes mellitus (e.g., diabetes mellitus type I or insulin dependent diabetes), multiple sclerosis and inflammatory disorder of the Central nervous system (CNS).

Antibodies of the present invention can be used in quality is solid fuel compositions for individual introduction or in combination with other therapeutic agents. Antibodies can be used in combination therapy with existing drugs IL-13 (e.g. existing agents IL-13, such as anti-IL-13Rαl, trap IL-4/13, anti-IL-13) with the antibody anti-IL-4 and existing agents IL-4 (for example an anti-IL-4R, mutein IL-4, IL trap-4/13) with the antibody anti-IL-13 antibodies, IL-4 (for example WO 05/0076990 (CAT), WO 03/092610 (Regeneron), WO 00/64944 (Genetic Inst.) and WO 2005/062967 (Tanox)).

Antibodies of the present invention may be introduced and/or be included in a composition together with one or more drugs or active agents. If the ligand is introduced together with an additional pharmaceutical agent, it can be entered before, concurrently or after administration of an additional agent. Typically, the ligand and the additional agent are introduced so as to allow an overlap between therapeutic effect. Additional agents that can be entered or included in a composition together with the ligand of the present invention, include, for example, various immunotherapy drugs, such as cyclosporine, methotrexate, adriamycin or cisplatin, antibiotics, fungicide, antiviral agents and immunotoxins. For example, if the antagonist is introduced for the prevention, suppression or treatment of inflammation of the lungs or respiratory disease (e.g. asthma), it can be entered together with what nhibitory phosphodiesterase (for example, inhibitors of phosphodiesterase-4), bronchodilators (such as β2-agonists, anticholinergics, theophylline), beta-agonists short-term actions (for example, albuterol, salbutamol, bambuterol, footer[Sigma]l, isoetharine, isoproterenol, leva[jot]buterol, metaproterenol, pirbuterol, terbutaline and Tornet), beta-agonists long-acting (such as formoterol and salmeterol), anticholinergics short-acting (e.g. ipratropium bromide and oxitropium bromide), anticholinergics long-acting (e.g tiotropy), theophylline (such as short-acting formulations, the compositions of the prolonged action), inhaled steroids use (such as beclomethasone, beclomethasone, budesonide, flunisolide, fluticasone propionate and triamcinolone), steroids oral administration (such as methylprednisolone, prednisone, prednisolone, and prednisone), the combination of beta-agonists short-acting with anticholinergics (such as albuterol/salbutamol/ipratopium and fenoterol/ipratopium), the combination of beta-agonists long-acting with inhaled steroids use (such as salmeterol/fluticasone and formoterol/budesonide), and mucolytic agents (e.g erdosteine, acetylcysteine, Bromhexine, carbocisteine, guiafenesin and audirovannyj glycerol).

Other suitable agents for joint therapy, which can be entered together with the antibody of the present invention for the prevention, suppression or treatment of asthma (e.g., allergic asthma), include corticosteroids (such as beclomethasone, budesonide, fluticasone), cromoglycate, nedocromil, beta-antagonists (such as salbutamol, terbutaline, bambuterol, fenoterol, reproterol, tulobuterol, salmeterol, Vomero), zafirlukast, salmeterol, prednisone, prednisolone, theophylline, zillatron, montelukast and leukotriene modifiers. The ligands of the present invention can be co-administered with a variety of agents for co-therapies, suitable for the treatment of diseases (such as diseases, mediated by Th-2 mediated YL-A-mediated IL-13-mediated IL-4, and cancer), including cytokines, analgesics/antipyretics, antiemetics and chemotherapy.

Antibodies of the present invention can be supplied in the form of pharmaceutically acceptable compositions, which are known to experts in this field or described herein. The term "physiologically acceptable", "pharmaceutically acceptable", etc. means approved by the regulatory authority of the Federal government or the state government or listed in the U.S. Pharmacopoeia or other generally accepted Pharmacopoeia for use in animals and, on the features, on the man.

Antibodies anti-IL-4, anti-IL-13 and bespecifically antibodies anti-IL-4/anti-IL-13 can be administered to a mammal, particularly a human, in any acceptable way. Methods of introducing, among others, include parenteral, subcutaneous, intraperitoneal, intra-lungs, intranasal, epidural, inhalation and oral route, and also, if this is desirable in the case of immunosuppressive treatment, provides for the introduction inside the affected area. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial, or intraperitoneal administration. Antibodies or compositions can be administered in any suitable way, for example by infusion or bolus injection, by absorption through epithelial or skin and mucous membranes (such as the mucosa of the oral cavity, the mucosa of the rectum and intestine and the like) and may be administered together with other biologically active agents. The administration can be systemic or local. In addition, it may be desirable to introduce therapeutic antibodies or compositions of the present invention into the Central nervous system by any convenient method, including through intraventricular injection and injection into the upper spinal canal; and intraventricular injection can facilitate intraventricularly catheter, for example connected with what Tervueren, for example, the tank Ommaya. In addition, the antibody is convenient to introduce a pulse infusion, particularly in the case of declining doses of the antibody. Preferably, the dosage is effected by injection, preferably by intravenous or subcutaneous injection, partly depending on, is the introduction of short-term or long-term in nature.

There are various delivery systems, and they can be used for the introduction of the antibodies of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules (see Langer, Science 249:1527 (1990); Treat et al., in Liposomes in therapy of Infectious Disease and Cancer; Lopez-Berestein et al., eds., p. 353-365 (1989); Lopez-Berestein, ibid., p. 317-327), and recombinant cells that can Express the connection-mediated receptor endocytosis (see, e.g., Wu et al., J Biol Chem 262:4429 (1987)); the construction of nucleic acid within a retroviral or other vector, etc.

The active ingredients can also be captured in the prepared microcapsules, for example using the techniques koatservatsii or interfacial polymerization, for example, microcapsules of hydroxymethylcellulose or gelatin and methyl methacrylate) microcapsules, respectively, in colloidal systems drug delivery (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such metadisciplinary in Remington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).

Can also be used pulmonary delivery, such as using an inhaler or an aerosol apparatus, and composition containing the agent for transfer to the aerosol. The antibody can also be administered into the lungs of the patient in the form of a dry powder composition, see, for example,U.S. patent No. 6514496.

In one of specific embodiments, it may be desirable to introduce therapeutic antibodies or compositions of the present invention locally to the area in need of treatment; this can be achieved by various methods, such as, among others, local infusion, by topical application, by injection, catheter, by means of a suppository or an implant, and mentioned the implant may be made of a porous, non-porous, or gelatinous material, including a membrane, for example, slastikov membrane or fiber. Preferably with the introduction of the antibodies of the present invention is to pay special attention to the use of materials that do not adsorb and not absorb protein.

In yet another embodiment, the antibody can be delivered in a controlled release system. In one example implementation may use a pump (see Langer, Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng14:201 (1987); Buchwald et al., Surgery88:507 (1980); and Saudek et al., N Engl J Med 321:574 (1989)). In the other embodiment can be applied to polymeric materials (see Medical Applications of Controlled Release, Langeret al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley (1984); Ranger et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al., Science228:190 (1985); During et al., Ann Neurol 25:351 (1989); and Howard et al., J Neurosurg 71:105 (1989)). In yet another example implementation of a controlled release system can be placed in proximity of therapeutic target.

Therapeutic polypeptide or antibody can be prepared for storage in the form liofilizovannyh formulations or aqueous solutions by mixing the polypeptide desired degree of purity with possible "pharmaceutically acceptable carriers, solvents, fillers or stabilizers that are commonly used in this field, such as buffer agents, stabilizing agents, preservatives, historification, non-ionic detergents, antioxidants and other various additives, see Remington''s Pharmaceutical Sciences, 16th ed., Osol, ed. (1980). Such additives generally are non-toxic to the host organism used in the doses and concentrations, and therefore, excipients, diluents, carriers, etc. are pharmaceutically acceptable.

"Isolated"or "purified" antibody sufficiently free of cellular material or other contaminating proteins from the cell or tissue source or media is, from which the resulting protein or sufficiently free from chemical precursors or other chemicals when received by chemical synthesis. The expression "sufficiently free of cellular material" includes preparations of antibodies, in which the polypeptide/protein is separated from cellular components of the cells from which it is selected or obtained by recombination. Thus, the antibody is sufficiently free of cellular material includes preparations of antibodies containing less than about 30%, 20%, 10%, 5%, 2,5% or 1% (dry weight) of contaminating protein. In the case where the antibody obtained using recombinant methods, it is also preferable if it is sufficiently free of culture medium, i.e., if the culture medium is less than about 20%, 10%, 5%, 2,5% or 1% of the drug is protein. In the case where the antibody obtained when chemical synthesis, preferably, that it was sufficiently free from chemical precursors or other chemicals and reagents, that is, to consider the antibody was separated from chemical precursors or other chemicals used in the synthesis of protein. Accordingly, such preparations of antibodies contain less than about 30%, 20%, 10%, 5% or 1% (dry the ECA) of chemical precursors or chemical substances, other than those addressed by antibodies. In the preferred embodiment of the present invention is the selection or purification of antibodies.

Herein, the expression "low to undetectable levels of aggregation" refers to samples containing no more than 5%, no more than 4%, no more than 3%, no more than 2%not more than 1% and often less than 0.5% aggregates by weight of protein as determined, for example, high-performance gel permeation chromatography (WAGP).

Used herein the term "low to undetectable levels of fragmentation" refers to samples containing at least 80%, 85%, 90%, 95%, 98% or 99%, of the total protein, for example in a single peak as determined VAGP, or in two (2) peaks (heavy chain and light chain), defined, for example, using restorative capillary gel electrophoresis (rCGE), and containing no other single peaks with more than 5%more 4%more 3%more 2%more 1% or more than 0.5% of total protein each. Used in this document, the term rCGE refers to capillary gel electrophoresis under the conditions of recovery sufficient to restore the disulfide bonds in the antibody or the molecule type antibody, or a derived molecule.

Used in this document, the terms "stability" and "stable" in the context of liquid drug, steriade what about the antibody Il-4 or IL-13 or its binding fragment, refer to the stability of the antibody or antigen-binding fragment in the product to thermal and chemical unfolding, aggregation, degradation or fragmentation under specific conditions of production, reception, transport and storage. Stable preparations of the present invention retain the biological activity of not less than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% in the specific conditions of production, reception, transport and storage. The stability of the mentioned drug antibodies can be assessed by degrees of aggregation, degradation or fragmentation using methods known qualified specialists in this field, including, among others, rCGE, gel-electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) and VAGP, compared with the standard.

The term "carrier" refers to a diluent, adjuvant, excipient or carrier with which the injected drug. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and similar oils. Intravenous drug preferred carrier is water. Physiological shall estuary and aqueous dextrose and glycerol can also be used as liquid carriers, special for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerylmonostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and similar substances. If desired, the composition may also contain small amounts of wetting agents or emulsifying agents, or buffering agents to maintain pH. These preparations may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, dosage forms with prolonged release of substances, depots and the like. The composition may be in the form of a suppository with standard binders and carriers such as triglycerides. Preparations for oral administration can include standard carriers such as related to pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate and the like, Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences,Martin. Such compositions will contain an effective amount of the antibody, preferably in purified form, together with the appropriate amount of carrier so as to provide the form for proper administration patient. As is well known to specialists in this area, the part will be creating the sterile so to match the way of introduction.

Buffer reagents help to maintain pH in the range, which is close to physiological conditions. The buffers are preferably present in concentrations in the range of from about 2 mm to about 50 mm. As a suitable buffer reagents of the present invention can be used both organic and inorganic acids and their salts, such as citrate buffers (e.g., a mixture of moonacre citrate-disodium citrate mixture of citric acid-trisodium citrate mixture of citric acid-monolatry citrate and the like), succinate buffers (for example, a mixture of succinic acid-monolatry succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate and the like), tartrate buffers (for example, a mixture of tartaric acid-sodium tartrate, tartaric acid, potassium tartrate, a mixture of tartaric acid-sodium hydroxide and the like), fumaric buffers (for example, a mixture of fumaric acid-monolatry fumarate, a mixture of fumaric acid-disodium fumarate, a mixture of moonacre fumarate-disodium fumarate and the like), gluconate buffers (for example, a mixture of gluconic acid, sodium gluconate, a mixture of gluconic acid-sodium hydroxide, the mixture of gluconic acid, a gluconate, potassium and the like), oxalate buffer (for example a mixture of oxalic acid-sodium oxalate mixture of oxalic acid-hydroxide intothree is, a mixture of oxalic acid-potassium oxalate and the like), lactate buffers (for example a mixture of lactic acid-sodium lactate, a mixture of lactic acid-sodium hydroxide, a mixture of lactic acid-potassium lactate and the like) and acetate buffers (for example a mixture of acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide and the like). Can be used phosphate buffers, carbonate buffers, his-tag buffers, salts of trimethylamine, such as Tris, HEPES, and other similar known buffers.

To slow down the growth of microorganisms can be added preservatives that can be used in amounts in the range of 0.2%to 1% (weight/about). The preservatives suitable for use in the present invention include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyltrimethylammonium, benzalkonium halides (such as chloride, bromide and iodide), hexamethylene, alkylarene, for example methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonically are introduced, in order to ensure physiological isotonicity liquid preparations of the present invention, these include polyhydric sugar alcohols, preferably trivalent or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can PR is to attend in amounts from about 0.1% to about 25%, by weight, preferably from 1% to 5% with respect to the relative amounts of the other ingredients.

Stabilizers are called broad category of fillers, whose functions vary from forming substances to additives, solubilizers drug or contributing to the prevention of denaturation or adhesion to the vessel wall. As is typical stabilizers can be polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine and the like, organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerin and the like, including cyclically, such as Inositol; polyethylene glycol; polymers of amino acids; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, thioglycolate sodium, thioglycerol, α-monothioglycerol and sodium thiosulfate; the polypeptides of low molecular weight (i.e. < 10 residues); proteins such as serum albumin human albumin production of whey, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, sugars, monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides, neprimerenog, maltose and sucrose; trisaccharide, such as raffinose; polysaccharides, such as dextran and the like, Stabilizers are present in the range from 0.1 to 10000 weight/weight to one part of the active protein.

Additional various fillers include forming agents (for example starch), chelating agents (e.g. EDTA), antioxidants (such as ascorbic acid, methionine or vitamin E) and auxiliary solvents.

Herein, the compositions can also contain more than one active ingredient, if it is necessary for individual indications for treatment, preferably components having complementary activity that does not adversely influence each other. For example, it may be desirable to additionally introduce an immunosuppressant. Such molecules is properly present in the composition in amounts that are effective for the intended purpose.

Used herein, the term "surfactant" refers to organic compounds with amphipatic structure, namely, composed of groups of opposing solubility characteristics, as a rule of fat-soluble hydrocarbon chain and a water-soluble ionic group. Depending on the charge of surface-active groups of the surface-and the active substances can be divided into anionic, cationic and nonionic. Surfactants are often used as wetting, emulsifying, solubilizing and dispersing agents for various pharmaceutical compositions and preparations of biological materials.

Nonionic surfactants or detergents (also known as "wetting agents") can be added to help solubilize the drug, as well as to protect medicinal protein from induced shaking of aggregation, which also allows you to expose a part of the impact surface of the shift without causing denaturation of the protein. Suitable nonionic surface-active substances include Polysorbate (20, 80, and so on), poloxamer (184, 188, etc.), Pluronic polyols®and monetary of polyoxyethylenesorbitan (TWEEN-20®TWEEN-80®and so on). Nonionic surfactants may be present in amounts in the range from about 0.05 mg/ml to about 1.0 mg/ml, preferably from about 0.07 mg/ml to about 0.2 mg/ml

Used herein, the term "inorganic salt" refers to any compound not containing carbon, which is formed by the replacement of some or all of the acidic hydrogens or acid metal or group serving as metal, and often used the WMD as a substance for adjusting toychest in pharmaceutical compositions and preparations of biological materials. The most common inorganic salts are NaCl, KCl, NaH2PO4etc.

The present invention encompasses liquid formulations having stability at temperatures typical for commercial refrigerators and freezers that are used in physician offices or laboratories, for example from about -20 to about 5ºC, and the aforementioned stability is estimated, for example, by high performance gel permeation chromatography (VEGP), for storage purposes, for example, within about 60 days to about 120 days, about 180 days, about the year, about 2 years or more. The liquid compositions of the present invention are also showing stability, which is estimated, for example, VAGP at room temperature at least for a few hours, e.g. one hour, two hours or three hours until use.

In the scope of the term "small molecule" and analogous terms, among others, include peptides, peptidomimetics, amino acids, analogs of amino acids, polynucleotide, analogs of polynucleotides, nucleotides, nucleotide analogues, organic or inorganic compounds (e.g., including hetero-organic and (or) ORGANOMETALLIC compounds)having a molecular weight less than about 10,000 grams per mole, organic or inorganic connection is to be placed, having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters and other pharmaceutical acceptable forms of such compounds.

Therefore, in the case of the antibodies of the present invention can be administered alone or in combination with other drugs for the treatment of cancer, including standard chemotherapeutic drugs (paclitaxel, carboplatin, cisplatin, and doxorubicin), anti-EGFR agents (gefitinib, erlotinib and cetuximab), antiangiogenic agents (bevacizumab and sunitinib), as well as immunomodulators such as interferon-α and thalidomide.

Used in the present invention the definition of "therapeutic agent" or "therapeutic agents" refer to any agent (agents)that can be used in the treatment, control or mitigating syndromes of diseases, disorders, disorders, etc. associated with aberrant metabolism and activity of IL-4 and / or IL-13.

In addition, antibodies of the present invention can conjugates with various effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins, see, in the example, WO 92/08495; WO 91/14438; WO 89/12624; U.S. patent No. 5314995; and EPO 396387. The antibody or its fragment can conjugates with a therapeutic agent such as a cytotoxin (e.g cytostatic or cytopathic agent), a therapeutic agent or a radioactive ion of the metal (for example α-active isotope, such as213Bi). To cytotoxins or cytotoxic agents include any substance that is devastating effect on the cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues. To therapeutic agents, among others, include antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-tioguanin, cytarabine, 5-fluorouracil and dacarbazine), alkylating agents (e.g. mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustin (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and CIS-dichlorodiammineplatinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin, daunomycin and doxorubicin), antibiotics (such asdactinomycin, actinomycin, bionic is h, mithramycin and astromicin (AMC)), and antimitoticescoe agents (e.g. vincristine and vinblastine).

Methods of conjugation of such therapeutic options with antibodies are well known, see, for example,Arnon et al., in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), p. 243-56, Alan R. Liss (1985); Hellstrom et al., in Controlled Drug Delivery, 2nd ed., Robinson et al., eds., p. 623-53, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., eds., p. 475-506 (1985); Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al., eds., p. 303-16, Academic Press (1985); and Thorpe, et al., Immunol Rev 62:119 (1982). Alternatively, the antibody may conjugates with the second antibody with the formation of heteroconjugate antibodies, for example, bifunctional antibodies, see, for example, U.S. patent No. 4676980.

The conjugates of the present invention can be used for modifying a given biological response, therapeutic agent or drug should not be construed as limited to classical chemical therapeutic agents. For example, as a medicinal product may be a protein or polypeptide possessing a desired biological activity. Such proteins may, for example, be a toxin, such as abrin, ricin A, exotoxin of Pseudomonas or difference toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, an act of wator tissue plasminogen the agent of apoptosis, such asTNF-α, TNF-β, AIM I (WO 97/33899), AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int Immunol, 6:1567 (1994)), VEGF (WO 99/23105); thrombotic agent, an antiangiogenic agent, e.g. angiostatin or endostatin; or biological response modifiers, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (GCSF) or other growth factors.

Preparations for the introduction of in vivomust be sterile. This can be achieved, for example, by filtration through sterile filtration membranes. For example, liquid preparations of the present invention can be sterilized by filtration through a filter of 0.2 μm or 0.22 μm.

Can prepare drugs with a slow release. Typical examples of drugs with a slow release include semi-permeable matrices of solid hydrophobic polymers containing the antibody, these matrices are moulded products, such as film or sensor. Examples of matrices with a slow release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate), poly(vinyl alcohol)), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable copolymers of lactic is islote-glycolic acid (such as injectable microspheres, consisting of a copolymer of lactic acid-glycolic acid) and poly-D-(-)-3-hydroxipropionic acid. Such polymers as vinyl acetate and lactic acid-glycolic acid, provide for the release of molecules for over 100 days, while certain hydrogels release proteins for shorter time. It is possible to develop rational strategies for stabilization depending on the characteristic mechanisms. For example, if the aggregation mechanism is associated with the formation of intermolecular bonds S-S due ridiculing interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using necessary additions, substitutions of amino acids and the development of special formulations of polymer matrix.

The composition containing the antibody or variant will be formulated, dosed, and administered in a manner that is in accordance with the appropriate principles of medical practice. Factors that should be taken into account include the specific violation, subject to cure, the particular mammal or human being treated, the clinical condition of the individual patient, the cause of the disease, the area of the delivery agent, method of introduction, the scheme of introduction of the other factors, known to doctors. "Therapeutic effective amount" of the antibody or variant that will be entered is determined by the above considerations, it may represent the minimum amount necessary to prevent, suppress or treat diseases, conditions or disorders mediated by IL-4 or IL-13.

The antibody or its variant can be included in a composition together with one or more agents currently used to prevent or treat the disease. The effective amount of such other agents depends on the amount of antibody present in the composition, the type of disorder or treatment, and other factors discussed above. They usually are used in the same dosages and with the same methods of introduction, which was used in the above text, or approximately from 1 to 99% of the employed still dosages.

Used herein the term "effective amount" refers to the number of medications (e.g., prophylactic or therapeutic agent)sufficient to reduce the severity and / or duration of the disease, mediated by IL-4 or IL-13, suppression of one or more of its symptoms, prevent the development of diseases mediated by IL-4 or IL-13, or the beginning is agressie disease, or are sufficient to prevent the development, recurrence, start or progress of a disease mediated by IL-4 or IL-13, or one or more symptoms, or strengthening or enhancing the preventive and / or therapeutic effect (effects) of other medications (for example, another therapeutic agent)used to treat diseases mediated by IL-4 or IL-13.

The number of drug-antibody or its fragment, which will be effective when using or treatment of a specific disease or condition will depend on the nature of the disorder or disease and may be determined by standard clinical techniques. If possible, first in vitroyou can get the curve dose-response relationships and pharmaceutical compositions of the present invention. In the presence of a suitable animal model can re-examine the curve dose-response relationships, and use it to extrapolate the appropriate dose for humans based on practical techniques known to experts in this field. However, based on General knowledge in this area, the drug, effective at stimulating the reduction of the inflammatory effect, for example, can provide local drug concentration between 5 and 20 ng/ml, and preferably between about 10 and 20 ng/ml.

In preference the sustained fashion example implementation of an aqueous solution of a medicinal polypeptide, the antibody or its fragment may be administered by subcutaneous injection. Each dose can be changed in the range from about 0.5 mg to about 50 mg per kilogram of body weight, or, more preferably, from about 3 mg to about 30 mg per kilogram of body weight. The dosage can be determined empirically for a particular disease, patient groups, as a way of introduction, etc. using pharmaceutical methods known to experts in this field.

Scheme dosage for subcutaneous injection can be changed from once a week to once a day, depending on the set of clinical factors, including the nature of the disease, severity of disease and the patient's sensitivity to a therapeutic agent.

The present invention provides methods of cooking liquid preparations of antibodies or IL-4 (or IL-13-binding fragment, and the above-mentioned methods include concentrating the fractions purified antibody to a final concentration of approximately 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 200 mg/ml, about 250 mg/ml, about 300 mg/ml or more, using, for example, semi-permeable membrane with an appropriate cut-off molecular weight (mV) (for example a cut-off of 30 KD d the I F (ab')2fragments; and a cut-off of 10 KD for Fabfragments).

In addition, the present invention encompasses stable liquid preparations of the product, which increased the half-life in vivo. So, the half-life period under consideration antibodies from the body, preferably human, more than 3 days, greater than 7 days, greater than 10 days, greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months more than 4 months more than 5 months or more.

To increase the duration of a finding of antibodies in serum in vivocan be used a variety of techniques. For example, inert polymer molecules such as polyethylene glycol (PEG) with high molecular weight, can be contacted with the antibody via a multifunctional linker or without either through site-specific conjugation of the PEG to the N-terminal or C-terminal end of the antibodies or via the ε-amino groups present in the lysine residues. It is possible to form such derivatives of linear or branched polymer that ensure minimal loss of biological activity. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules with antibodies. Unreacted PEG can be separated from conjug the tov antibody-PEG by gel filtration or ion exchange chromatography. Derived from PEG antibodies can also be tested for binding activity and efficacy in vivo, which can be used in methods known to experts in this field, for example, described in this document immunoassay methods.

The antibody, which has a higher level of half-life in vivo can also be obtained by performing one or more modifications of amino acids (such as substitutions, insertions or deletions) in the constant domain of IgG or FcR-binding fragment (e.g. Feor a fragment of the hinge region domain Fesee, for example, WO 98/23289; WO 97/34631; and U.S. patent No. 6277375.

In addition, the antibody can be konjugierte to albumin with the formation of antibodies, more stable in vivo or have a longer half-life in vivo. Methods well known to specialists in this field, see, for example, WO 93/15199, WO 93/15200 and WO 01/77137; and EPO 413622. The antibody can also be modified, for example by glycosylation, acetylation, phosphorylation, amidation, introduction of the famous defensive/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.

In one example implementation of the product are in accordance with standard procedures for drug preparato is, prepared for intravenous administration in humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include solubilizers agent and a local anesthetic such as lidocaine or other "kainic" anesthetic to reduce pain at the injection site. Typically, the ingredients are supplied either separately or mixed together in the form of a unit dosage, for example in the form of dry liofilizirovannogo powder or water-free concentrate in a hermetically sealed container such as an ampoule or in the package indicating the quantity of active agent. In cases where the composition must be administered by infusion, it can be supplied with a vial for infusion containing sterile water or saline solution, pharmaceutical grade purity. If the composition is injected through the injection can be supplied in vials with sterile water or saline for injection, for example in the set, so that the ingredients can be mixed prior to introduction.

The invention also contemplates that the liquid preparation of the present invention is placed in an airtight container such as a vial or package with the number of the product in question. Liquid preparations of the present invention can be hermetically sealed container indicating the quantity and concentration of the antibody or antibody fragment. Liquid preparations of the present invention can be supplied in an airtight container, containing at least 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml or 300 mg/ml of antibody to IL-4 or IL-13, for example in an amount equal 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml

Produced product, comprising means for treatment of the above disorders. The product is a container and label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers can be made of various materials, for example glass or plastic. The container contains a composition that is suitable for the diagnosis, prevention or treatment of a condition or disease mediated by IL-4 or IL-13, and may have a sterile hole for selection (for example, as the container may be used a bag of intravenous solution or bottle with stopper, sharp needle for subcutaneous injection). On the label, which is located on the container or attached to it, indicates that the compound is used for treatment of a particular condition. The product may also include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline solution Ring the RA and dextrose. The product may also include other materials desirable from the point of view of commercial applications and user, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Below for information specialists in this field are illustrations of the present invention in the following, not exhaustive by this enumeration, the examples describing some of the embodiments by which the present invention can be implemented in practice.

EXAMPLES

EXAMPLE 1: the SEQUENCING of the Fv DOMAIN of CLONE B-B13 MONOCLONAL ANTIBODY MOUSE ANTI-HUMAN IL-13

In the above method as a reagent used clone B-B13 monoclonal antibody mouse anti-IL-13 acquired in Cell Sciences, Inc. (Canton, mA, USA). Cell Sciences is a distributor in the U.S. the company's products Diaclone (besançon, France), which produces the antibody B-B13.

Amino acid sequence of clone B-B13 monoclonal antibody mouse anti-IL-13 were determined simultaneously by N-terminal sequencing by Admino and mass spectrometric analysis. To obtain a polypeptide or peptide fragments of the antibody was subjected to the following treatments, and the resulting peptides were fractionally using different methods for sample preparation, which the ZAT is subjected to N-terminal sequencing by Admino and were analyzed by liquid chromatography/mass spectrometry (LC-MS/MS) in combination with the mapping of peptides with a database of protein sequences.

After electrophoresis in SDS page with DDS-Na antibodies, treated or untreated Pyroglutamate, to separate the heavy and light chains were blotting on PVDF membrane (PVDF) with subsequent N-terminal sequencing of the bands on Admino.

After a limited partial proteolysis of specific protease antibody electrophoresis was performed in SDS page with DDS-Na and blotting on PVDF membrane and N-terminal sequencing of the bands on Admino.

After a limited chemical cleavage strips gel electrophoresis in SDS page with DDS-Na full antibodies or heavy and light chains was performed electrophoresis in SDS page with DDS-Na and blotting on PVDF membrane and N-terminal sequencing of the bands on Admino.

Proteolysis strips gel electrophoresis in SDS page with DDS-Na full antibodies or heavy and light chain specific proteases and analysis of LC/MS/MS.

After proteolysis strips gel electrophoresis in SDS page with DDS-Na heavy and light chains specific proteases was performed chromatographic fractionation of high-pressure reversed-phase (RP HPLC) with subsequent N-terminal sequencing by Admino and analysis of fractions LC/MS/MS.

After limited proteolysis of antibodies by the protease papain, fractionation strips gel electrophoresis in SDS page with DDS-Na Fd fragment (fragment VH-CH1 heavy chain antibodies), FR is Alisa specific proteases conducted high-performance chromatographic fractionation by reversed phase (RP HPLC) with subsequent N-terminal sequencing by Admino and analysis of fractions LC/MS/MS.

EXAMPLE 2: SEQUENCING DOMAIN Fv CLONE 8D4-8 MONOCLONAL ANTIBODY MOUSE ANTI-HUMAN IL-4

As the reagent used clone 8D4-8 monoclonal antibody mouse anti-IL-4, which was acquired in Biozol diagnostica Vertrieb GmbH, Eching, Germany). Biozol is a distributor in a Germany company BioLegend (San Diego, CA, USA), which produces the antibody 8D4-8.

Amino acid sequence of monoclonal antibody mouse anti-IL-4 (clone 8D4-8) were determined simultaneously by N-terminal sequencing by Admino and mass spectrometric analysis (Pham et al., 2006, Anal. Biochem. 352: 77-86; Roberts et al., 2005, Anal. Chem. 67: 3613-25). Briefly, the antibody was initially divided into light and heavy chains, then every chain was digested with sequence speficically proteases or chemical means. The resulting peptides were separated obremenitve chromatography and analyzed by spectroscopy of matrix laser desorption/ionization (MALDI) and/or LC-MS/MS. Then for unambiguous sequencing of individual protein peptides and intact heavy and light chains were subjected to sequencing by Admino.

EXAMPLE 3: the HUMANIZATION of the Fv DOMAIN of CLONE B-B13 MONOCLONAL ANTIBODY MOUSE ANTI-HUMAN IL-13

For humanization of clone B-B13 used the humanization Protocol described above in this document. Proposed W is here humanized versions which included mutations in the CDR to account for the problematic residues (site deliciouse, solvent-accessible methionine, acid-labile positions).

Sequences of VL and VH for B-B13 were compared using the BLAST program on the database Protein Data Bank (PDB), version July 2007. Selected the most similar amino acid sequences of light and heavy chains. The closest homolog of the variable light chain was the structure 1EGJ. The closest homolog of the variable heavy chain was the structure 1FNS. Patterns 1EGJ and 1FNS used to build a homology model of the variable domains, which are then minimized energy using standard procedures implemented in the Molecular Operating Environment (MOE). MOE is a versatile software package for computational drug development, which is supplied by a group of Chemical Computing. Calculations of molecular dynamics (MD) for the three-dimensional homology model B-B13 spent time 1.7 nanoseconds using generalized method-born implicit solvent. Received 1700 mappings MD trajectory is then used to calculate for each amino acid B-B13 distribution of its mean-square deviation (rmsd) compared to a median position. A statistical test comparing the distribution of the rmsd of each amino acid with a global rmsd of RA the distribution is then used to install, sufficiently flexible one or another amino acid, as was seen in MD, and whether it can, therefore, interact with b-cell receptors and responsible for activation of the immune response. Flexible position variable regions of murine B-B13 were compared with the relevant provisions of the sequences of human antibodies according to the version of the database ImMunoGeneTics in January 2007, which was loaded into a local network. Search used only those residues that were shown flexibility, more than three times the average, and several flanking residues that were conserved three-dimensional structure of these flexible residues. Variable region of the antibodies of the person with the closest flexible residues, where special attention was paid to the positions located in the vicinity of 5.0 Å from the CDR, was selected to replace the flexible residues of the variable region of mouse antibody B-B13. To eliminate the problem remains, some mutations in the CDR also included in the proposed options. Considered the following sequence motifs: Asp-Pro (acid-labile bond), Asn-X-Ser/Thr (glycosylation), Asn-Gly/Ser/Thr (site deliciouse available in the area), Met (oxidation of the available space). To confirm the validity of the basic assumptions derived humanized sequence FOSS is toulali program BLAST for similarity with database information UniProtKB/Swiss-Prot. Ustanovili that all sequences show a high degree of similarity with several antibodies person. In addition, none of the sequences contained no known epitope - or T-cells contained in the database immune epitopes and analysis resource (IEDB database).

Offered three versions of the heavy chain (H1, H2, H3) and three versions of the light chain (L1, L2, L3). Three versions of the light chain received from CAA83271.1 (Genebank access number CAA83271). Version L1 contains 4 mutations. The L2 version includes additional mutation to remove site DP (Pro99) in CDR3. L3 includes two additional mutations in CDR, compared to L2, which represent two hypothetical site deliciouse (N34Q, N96A). H1, H2 and H3 versions of the heavy chain received from CAC39364.1 (Genebank access number CAC39364). This matrix was not the best, but it was the most appropriate matrix that do not contain sequences with the highest homology (>70%) with a known immunogenic sequence. Version H1 contains 6 mutations and sequence H2 includes two additional mutations to account for the three sites deliciouse (N60A, N73T and N83T). Sequential numbering of amino acids reflects their natural order in the protein (N-terminal end to C-terminal end). H3 contain two additional mutations (Y100R and D106K), which, presumably, about specialt higher potential. For obtaining humanized antibodies recommended six combinations of variants of the VL and VH: VL1×VH1, VL2×VH2, VL1×VH3, VL3×VH1, VL3×VH2 and VL3×VH3. As shown in table 1, changes in amino acid sequence was performed in humanized variants of the VL and VH for In-B13 applying the methodology changes the surface (resurfacing), which is set out in the section of this application with a detailed description of the invention. In the left column lists the original amino acids and their position in murine B-mAb B13.

Table 1
Light chain
(sequential numbering)
(VL1)(VL2)(VL3)
Asn1AspAspAsp
Asn34AsnAsnGln
Pro44AlaAlaAla
Glu83GlnGlnGln
Asp85 GluGluGlu
Asn96AsnAsnAla
Pro99ProSerSer
4 mutation5 mutations7 mutations
Heavy chain(VH1)(VH2)(VH3)
Gln1GluGluGlu
Ser15GlyGlyGly
Gln16GlyGlyGly
Asn60AsnAlaAla
Ser61AspAspAsp
Asn73Asn SerSer
Lys81GluGluGlu
Asn83AsnThrThr
Gln86ArgArgArg
Tyr100TyrTyrArg
Asp106AspAspLys
6 mutations9 mutation11 mutations

EXAMPLE 4: the HUMANIZATION of the Fv DOMAIN of CLONE 8D4-8 MONOCLONAL ANTIBODY MOUSE ANTI-HUMAN IL-4

For humanization clone 8D4-8 used technology humanization (resurfacing), described earlier in this document. Prepared two humanized versions. One version includes one mutation of the CDR regions of the heavy chain, which, as expected, was replaced potentially problematic balance (available acid-labile position).

Sequences of VL and VH for 8D4-8 were compared BLST program database PDB, version July 2007. Selected the most similar amino acid sequences of light and heavy chains. The closest homolog of the variable light chain is a structure 1YDJ. The closest homolog of the variable heavy chain was the structure 1IQW. Patterns 1YDJ and 1IQW used to build a homology model of the variable domains, which are then minimized energy using standard procedures implemented in the MOE. Calculations of molecular dynamics (MD) for the three-dimensional homology model 8D4-8 was performed for time 1.7 nanoseconds using generalized method-born implicit solvent. Received 1700 mappings MD trajectory is then used to calculate for each amino acid 8D4 distribution of its mean-square deviation (rmsd) compared to a median position. A statistical test comparing the distribution of the rmsd of each amino acid with a global rmsd distribution is then used to determine whether there is enough flexible one or another amino acid, as was seen in MD, and whether it can, therefore, interact with b-cell receptors and responsible for activation of the immune response. Flexible position variable regions of murine 8D4-8 were compared with the relevant provisions of the sequences of human antibodies according to the version of the database ImMunoGeneTics in January 2007,which was loaded into a local network. Search used only those residues that were shown flexibility, more than three times the average, and several flanking residues that were conserved three-dimensional structure of these flexible residues. To replace the flexible residues of the variable segment of murine antibodies 8D4-8 chose the variable region of human antibodies with the highest degree of identity of flexible residues, paying particular attention to provisions within a 5.0 Å from the hypervariable sites. Then also prepared additional mutations to avoid problematic residues. Considered the following sequence motifs: Asp-Pro (acid-labile bond), Asn-X-Ser/Thr (glycosylation), Asn-Gly/Ser/Thr (site deliciouse available in the area), Met (oxidation of the available space). The only problematic balance was the site of DP in the region CDR2 of the heavy chain. To confirm the validity of the basic assumptions derived humanized sequences were compared by BLAST program for similarity with database information UniProtKB/Swiss-Prot. All sequences obtained a high degree of homology with a number of human antibodies. In addition, none of the sequences did not contain any of the known epitopes for B-cells or T-cells that are listed in the IEDB database.

PR is daily two variants of the heavy chain (H1, H2) and one variant light chain (L1). Version L1 light chain received from BAC01676.1 (Genebank access number BAC01676). Version L1 contains 3 mutations. Version H1 and H2 of the heavy chain received from BAC02418.1 (Genebank access number BAC02418). Version H1 includes 9 mutations, and the H2 version includes additional mutation to remove site DP (Pro53) in CDR2. Prepared two connections, VL1×VH1 and VL1×VH2.

Table 2 shows changes in the amino acid sequence given in humanized variants of the VL and VH for 8D4-8 technology humanization (resurfacing). In the left column lists the original amino acids and their position in murine 8D4-8 mAb.

Table 2
Light chain
(sequential numbering)
(VL1)
Asn5Thr
Leu15Val
Ser39Lys
3 mutation
Heavy chain(VH1) (VH2)
Gln10GluGlu
Arg13LysLys
Thr16AlaAla
Pro53ProAla
Lys65GlnGln
Asp66GlyGly
Arg74GluGlu
Ser76ThrThr
Leu93ValVal
Thr118LeuLeu
9 mutation10 mutations

EXAMPLE 5: CLONING AND GENERATION of CHIMERIC CLONE B-B13 MONOCLONAL ANTI-IL-13 ANTIBODIES, CHIMERIC CLONE 8D4-8 MONOCLONAL ANTIBODIES ANTI-IL-4 AND HUMANIZED VARIANTS

Amino acid sequence variablenature and light chain of clone B-B13 anti-IL-13 and clone 8D4-8 anti-IL-4 was broadcast back in the nucleotide sequence was generated, accordingly, using a modified PCR Protocol extension overlapping (OE-PCR)described by Young L. and Dong Q. (Nucl. Acids Res. (2004), 32(7), e59). The PCR products were cloned in pCR®4-TOPO using cloning kit Invitrogen TOPO TA (cat. No. 45-0641) and sequenced using a direct M13 and M13 reverse primers. The variable domains were merged in constant heavy (IGHG1, catalog number Genebank Q569F4) or light (IGKC, catalog number Genebank Q502W4) chain, respectively, was treated with NheI and HindIII and each of them is ligated to the provisions NheI/HindIII vector episomal expression pXL, analog pTT vector described by Durocher et al. (2002), Nucl. Acids Res. 30(2), E9, creating plasmids for the expression of mammalian chimeric heavy and light chains B-B13 and chimeric heavy and light chains 8D4-8.

The expression clones encoding the humanized variants of clone B-B13 anti-IL-13 and clone 8D4-8 anti-IL-4 also synthetically generated by PCR extension overlapping (OE-PCR) based on the proposed exchange of the amino acids of the original sequence.

The expression plasmids encoding the heavy and light chain antibodies, reproduce inE. coliDH5a. Plasmids used for transfection were obtained fromE. coliusing the kit Qiagen EndoFree Plasmid Mega Kit.

For transfection cells HEK293FreeStyle (Invitrogen) were seeded at 3×105cells per ml in 100-ml volume containing no serum media is FreeStyle (Invitrogen) in 500-ml shaker flask. Cells were cultured in an incubator at 37°C with humidified atmosphere containing 8% CO2on rotary shaker rotating at a speed of 110 rpm.

Three days after seeding determined the number of viable cells and total number of cells using electronic cytometer CASY (Schärfe System GmbH). Cells with a viability higher than 90% were used for transfection with the cellular density of 1-1 .5×106cells per ml 100 ml cells were transfectional in shaker flask 500 ml with a mixture of plasmids for the expression of heavy and light chain (5×10-7µg DNA per cell) using FugeneHD (Roche) at a ratio of DNA:FugeneHD 2:7 in the conditions described by the manufacturer. Transfection cells were cultured for 7 days in an incubator at 37°C (8% CO2)on a rotary shaker with a speed of 110 rpm.

The tablet Nunc F96-MaxiSorp-Measurement were covered with goat anti-human IgG (Fc-specific) [NatuTec A80-104A]. The antibody was diluted to 10 μg/ml in carbonate buffer coating (50 mm sodium carbonate, pH 9,6) was added at 50 μl per well. The plate was sealed with tape and stored overnight at a temperature of 4ºC. The plate washed three times with wash buffer (PBS pH 7,4 0,1% Tween20). 150 μl of blocking solution (1% BSA/PBS) was added to each well of the plates. After 1 hour at room temperature the plate is amywali 3 times with wash buffer. Added 100 μl of sample or standards (in the range from 1500 ng/ml to 120 ng/ml) and left for 1 hour at room temperature. Tablet 3 times washed with wash buffer. 100 μl of goat anticheleccami IgG-FC - HRP conjugate [NatuTec A80-104P-60], diluted in a ratio of 1:10000 was added with incubation solution (0.1%BSA, PBS pH of 7.4, 0.05% of Tween20). After 1 hour incubation at room temperature the plate was washed 3 times with wash buffer. 100 μl of ABTS substrate (10 mg tablet ABTS (Pierce 34026) in ml of 0.1 M Na2HPO4, 0.05 M citric acid, pH 5.0. Added 10 μl of 30% H2O2/10 ml buffer substrate before use) into each of the holes before painting. After the appearance of the painting (approximately 10-15 minutes) added 50 μl of 1% SDS solution to stop the reaction. The tablet looked at A405.

Proteins were purified by affinity chromatography with Protein environment A (HiTrap™ Protein A HP Columns, GE Life Sciences). After elution from the column acetate buffer 100 mm with 100 mm NaCl pH of 3.5 was obtained monoclonal antibodies in PBS and filtered through a 0.22 μm filter. The protein concentration was determined by absorption at a wavelength of 280 nm. Each batch was analyzed using a set of Protein 200 Plus LabChip on the Agilent Bioanalyzer 2100 in the recovery and nevosstanovlenie conditions to determine the purity and molecular weight of each subunit and monomer.

EXAMPLE 6: the CHARACTERISTIC IS CARITATE HUMANIZED VARIANTS of CLONE B-B13 ANTI-IL-13 HUMANIZED VARIANTS of CLONE 8D4-8 ANTI-IL-4

Reagents recombinant IL-13 and IL-4 person acquired the company Chemicon (USA). Biacore kinetic analysis was performed as follows.

For detailed kinetic characterization of purified antibodies used a surface plasmon resonance on a Biacore 3000 (GE Healthcare). Applied analysis method traps using antibodies specific to the species (e.g. human Fc-specific MAB 1302, Chemicon) to capture and orientation of the investigated antibodies. Antibody-trap was forced not to move the primary amine groups (10000 RU) on a chip research grade CM5 (GE Life Sciences) using standard methods. Analyzed the antibody was caught with the adjusted value of RU, leading to maximum binding of the analyte at 30 EN and flow rate 10 μl/min was Measured the binding kinetics depending on recombinant human IL-4 and IL-13 in the concentration range from 0 to 50 nm in HBS-EP (10 mm HEPES pH 7,4, 150 mm NaCl, 3 mm EDTA, 0.005 % of the surfactant P20) at a flow rate of 30 ál/min, the chip Surface was regenerated using 10 mm glycine pH of 2.5. Kinetic parameters were analyzed and calculated in BIAevaluation software package with flow-through cuvette without antibodies traps as a control sample. To study additive bind both antigens were used automatic method simultaneity is temporal injection, which was injectively one antigen, and immediately after that, the mixture of antigens IL-13/IL-4.

Antibodies that are the subject of the present invention, measured biological activity by measuring the inhibition of cell proliferation mediated by IL-4 or IL-13. In short, the applicants have used IL-4 or IL-13 to stimulate the growth of cells TF-1. TF-1 is a cell line-dependent cytokines for growth, which responds to many cytokines, including IL-4 and IL-13. Induced growth (compared with normal conditions in the absence of cytokine) represents the biological activity of IL-4 or IL-13. Anti-IL-4, anti-IL-13 and bespecifically antibodies anti-IL-4/IL-13 was found to block the growth of cells TF-1 induced IL-4 or IL-13. In addition, bespecifically antibodies anti-IL-4/IL-13 has been shown to block cell proliferation, TF-1, induced by combined stimulation with IL-4 and IL-13. The blocking effect was measured dependent dose-dependent manner with the receipt of IC50 (the concentration of antibody with 50% inhibition) as the ability to neutralize the antibody to its target, i.e. IL-4 or IL-13. Used methods are described in more detail below.

Cells TF-1 (ATCC, CRL-2003) were kept in complete medium (DMEM with high glucose, 25 mm Hepes buffer and glutamine, 10% FBS, 1×P/S, 1 mm sodium pyruvate)containing the newly added hGM-CSF at a final concentration of 4 ng/ml For 24 hours is up processing of IL-13 (15 ng/ml) or IL-4 (1 ng/ml). Cells were cultivated in 96-well tablet in the amount of 0.05×106/ml in complete medium without hGM-CSF. Serially diluted solutions of the antibodies with the corresponding cytokine pre-incubated for 30 minutes at 37°C before adding to the cells. Cells were cultured for 72 hours (37°C, 5% CO2). Was added an aqueous solution of MTS/PMS cellTiter 96. Then cells were incubated for 3 hours. After this period recorded the absorption at a wavelength of 490 nm using a device to read the tablet. IC50 values were calculated using the program Speed.

The kinetics of binding and neutralizing activity of humanized variants of the B-B13 shown in table 3. (nt=not tested).

Table 3
AntibodyThe rate of Association (M-1×S-1)The dissociation rate (S-1)KD
(M)
IC50
(M)
mouse B-B138,64X+053,73E-045,63E-10Nt
chB-B13 WT1,76E+064,61E-04 7,4E-9
huB-B13 VL1×VH11,74E+066,91E-043,96E-101,57E-8
huB-B13 VL1×VH31,93E+063,95E-042,05E-10Nt
huB-B13 VL2×VH21,13E+061,77E-041,57E-10Nt
huB-B13 VL3×VH11,93E+063,33E-041.72e-105,2E-9
huB-B13 VL3×VH22,55E+061,12E-044,39E-113,2E-9
huB-B13 VL3×VH32,14E+064,05E-041,89E-10Nt

One humanitarianly option B-B13, huB-B13 VL3xVH2, has a significantly higher affinity compared to the original murine B-B13 (13 times) and chimeric B-B13 (6 times). Increased affinity may lead to increased activity and efficiency, when those who humanitarian antibodies anti-IL-13 will be used for the treatment of asthma patients. Furthermore, humanized antibodies may have reduced immunogenicity compared to the murine antibody or chimeric antibody for use in the human body.

The kinetics of binding and neutralizing activity of humanized variants 8D4-8 shown in table 4.

One humanitarianly option 8D4-8, hu8D4-8 VL1xVH1, has a significantly higher affinity compared to the original mouse 8D4-8 (11 times) and chimeric 8D4-8 (2 times). Increased affinity may lead to increased activity and efficiency when it humanitariannet antibody anti-IL-4 will be used for the treatment of asthma patients. In addition, humanitariannet antibody may have reduced immunogenicity compared to the murine antibody or chimeric antibody for use in the human body.

EXAMPLE 7: CLONING AND GENERATION of HUMANIZED BESPECIFICALLY ANTIBODIES ANTI-IL-4/IL-13

The format used for the expression of bespecifically antibodies (BsAb), - the variant IgG dual-domain format with two heads described in U.S. patent 5989830. In this format, the IgG molecule is elongated in the limit N-residue corresponding heavy or light chain variable domain of the second antibody. Thus, the resulting IgG molecule is heterotetramer, consisting of two heavy and two light chains. The heavy chain consists of two variable heavy domain (VH1-VH2), derived from two different antibodies, connected by a bridge consisting of ten amino acids (G4S)2and fused with the constant domain of IgG4. Light chains consist of two variable light domain (VL1-VL2), PR is coming from two different antibodies, connected by a bridge consisting of ten amino acids (G4S)2and merged with the constant Kappa region.

The sequences for the variable heavy and light domains of options 8D4-8 generated PCR, with the introduction of the restriction site BamHI (GGA TCC) in the respective 5'-ends of the coding part (G4S)2-(GGA TCC)-8D4-8. The sequence of the 3' VH humanized variants 8D4-8 ended with the ApaI restriction site (encoding the first amino acids of the CH1 domain) for subsequent merging with the sequence IGHG4 (Q569F4, with detection limit Lys and S241P and L248E double mutation). 3'-end VL8D4-8 ended with BsiWI restriction site, encoding the first two amino acids of the constant Kappa-chain for subsequent merging with IGKC (catalog number Gene Bank Q502W4).

The sequences for the variable heavy and light domains of options B-B13 generated PCR with the introduction of the restriction site BamHI into the corresponding 3'-ends, encodes part of the (G4S)2-(B-B13)-(GGA GGC GGA GGG GGA TCC GGC GGAGGA TCC(SEQ ID NO: 7)). Both sequences for VH and VL variants B-B13 generated with restriction site NheI in the respective 5'-ends with the next start codon ATG and a sequence encoding a leader peptide.

VH B-B13 and 8D4-8 was poured on the provisions of BamHI in the linker (G4S)2. VL B-B13 and 8D4-8

poured on the provisions of BamHI in the linker (G4S)2. Thus, the generated tandems t the heavy and light chains had the following composition.

Heavy chain especifismo antibodies: NheI-leader peptide-VH-B-B13 - (G4S)2- VH 8D4-8-ApaI.

Light chain especifismo antibodies: NheI-leader peptide-VL-B-B13 - (G4S)2- VL 8D4-8-BsiWI.

All intermediate PCR fragments were cloned into pCR®4-TOPO using a kit for cloning Invitrogen TOPO TA (cat. No.: 45-0641) and sequenced using direct M13 and M13 reverse primers.

After checking sequence tandem with the heavy chain was poured on the status ApaI sequence IGHG4, and variables tandems with a light chain poured on the situation with BsiWI IGKC. Received dual-domain heavy chain and light chain was treated with NheI and HindIII and ligated to the provisions NheI/HindIII

vector episomal expression pXL, creating plasmids for the expression of mammalian TBTI-heavy and light chains, respectively.

Four humanized bespecifically design anti-IL-4/anti-IL-13 were generated on the basis of the following combinations of humanized versions of VH and VL B-B13 and 8D4-8, as shown in table 5.

Table 4
AntibodySpeed Association
(M-1×S-1)
The dissociation rate
(S-1)
KD
(M)
IC50
(M)
mouse 8D4-85,57E+062,17E-043,77E-119,7E-11
ch8D4-8 WT2,49E+071,95E-047,83E-128,4E-11
Hu8D4-8 VL1×VH14,72E+071,55E-043,29E-124,1E-11
Hu8D4-8 VL1×VH22,57E+073,48E-041,39E-111,35E-10
Table 5
Bespecifically antibody anti-IL-4/anti-IL-13Anti-IL-13 FvAnti-IL-4 Fv
huTBTI3_1_1B-B13 VL3xVH2huTBTI3_2_1B-B13 VL3xVH28D4-8 VL1xVH1
huTBTI3_1_2B-B13 VL2xVH28D4-8 VL1xVH2
huTBTI3_2_2B-B13 VL2xVH28D4-8 VL1xVH1

EXAMPLE 8: CHARACTERIZATION of HUMANIZED BESPECIFICALLY ANTIBODIES

Analysis of the activity of binding and neutralization were carried out in accordance with the description given in the previous examples.

Table 6 shows the kinetics of binding four humanized variants of the antibodies anti-IL-4/IL-13. All four designs bespecifically antibodies are associated with IL-4 and IL-13 with high affinity.

Table 6
The affinity of IL-13The affinity of IL-4
BsABSpeed Association
(M-1×S-1)
The dissociation rate
(S-1)
KD (M)Speed Association
(M-1×S-1)
The dissociation rate
(S-1)/td>
KD (M)
huTBTI3-1_12,27E+061,70E-047,47E-112,55E+063,78E-041,48E-10
huTBTI3-2_12,17E+061,69E-047,80E-114,00E+061,39E-043,47E-11
huTBTI3-1_28,50E+051.64e-041,93E-102,23E+063,08E-041,38E-10
huTBTI3-2_28,20E+052,12E-042,59E-103,96E+061,32E-043,32E-11

Data on the activity of neutralizing humanized variants especifismo antibodies anti-IL-4/IL-13 are shown in table 7. And huTBTI3-1_1 and huTBTI3-2_1 completely neutralized cell proliferation, TF-1 induced IL-13 or IL-4, with IC50 values below.

Table 7
AntibodyIC50 (nm) in the analysis of IL-13IC50 (nm) in the analysis of IL-4
huTBTI3-1_13,71,7
huTBTI3-2_14,10,32

It is well known that the mutant allele of IL-13 is often associated with asthma (Heinzmann A. et al., 2000, Hum Mol Genet 9, 4, p549-559). Therefore, it was studied the kinetics of binding bespecifically antibodies mutant protein IL-13 (variant IL-13 human R112Q, PeproTech, Rocky Hill, NJ, USA). The results showed that huTBTI3-1_1 and huTBTI3-2_1 contact with the variant IL-13 as well as with IL-13 wild-type.

Table 8 shows the kinetics of binding of humanized molecules anti-IL-4/IL-13 mutant protein IL-13.

Table 8
The affinity of the variant IL13
BsABSpeed Association
(M-1×S-1)
The dissociation rate
(S-1)
KD
(M)
huTBTI3-1_19,74E+051,18E-04 1.21e-10
huTBTI3-2_19,48E+052,00E-042,11E-10

1. Bespecifically antibody or functional fragment of the antibody that specifically binds to IL-13 and IL-4, where bespecifically antibody or functional fragment contains:
a) a variable domain of the light chain containing the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:3, and the variable domain of the heavy chain containing the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:4; or
b) the variable domain of the light chain containing the amino acid sequence of SEQ ID NO:1 and SEQ ID NO:3, and the variable domain of the heavy chain containing the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:5.

2. Bespecifically antibody or functional fragment according to claim 1, where
a) amino acid sequence of SEQ ID NO:1 and SEQ ID NO:3 linked together by peptide linker and the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:4 linked together by peptide linker, or
b) amino acid sequence of SEQ ID NO:1 and SEQ ID NO:3 linked together by peptide linker and the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:5 linked together by peptide linker.

3. Bespecifically antibody or functional fragment according to claim 2, where the peptide linker comprises the amino acid serial is a major SEQ ID NO:6.

4. Bespecifically antibody or functional fragment that specifically binds to IL-13 and IL-4, where bespecifically antibody or functional fragment contains:
a) amino acid sequence RASESVDSYGQSYMH,LASNLES, QQNAEDSRT, HASQNIDVWLS, KASNLHTG, QQAHSYPFT,GFSLTDSSIN, DGRID, DGYFPYAMDF, GYSFTSYWIH, IDPSDGETR and LKEYGNYDSFYFDV or
b) amino acid sequences RASESVDSYGQSYMH,LASNLES, QQNAEDSRT, HASQNIDVWLS, KASNLHTG, QQAHSYPFT, GFSLTDSSIN, DGRID, DGYFPYAMDF, GYSFTSYWIH, IDASDGETR and LKEYGNYDSFYFDV.

5. Bespecifically antibody or functional fragment according to claim 1 or 4, which optionally contains a constant domains.

6. Bespecifically antibody or functional fragment according to claim 5, where the constant domains consist of CN, CH2, CH3, and CL.

7. Bespecifically antibody or functional fragment according to claim 1 or 4, which is optionally conjugated with an effector molecule.

8. Bespecifically antibody or functional fragment according to claim 7, where the effector molecule is selected from the group consisting of a heterologous polypeptides, drugs, radionucleotides and toxins.

9. Pharmaceutical composition for treating diseases or disorders associated with the function or metabolism of IL-4 and/or IL-13, containing bespecifically antibody or functional fragment according to any one of claims 1 or 4 and a pharmaceutically acceptable carrier.

10. A method of treating allergies the CSOs disease in a mammal, providing stage introduction to the mammal a therapeutically effective amount especifismo antibody or functional fragment according to any one of claims 1 or 4.

11. A method of treating cancer in a mammal to which the stages of introduction to the mammal a therapeutically effective amount especifismo antibody or functional fragment according to any one of claims 1 or 4.

12. A method of treating asthma in a mammal which the stages of introduction to the mammal a therapeutically effective amount especifismo antibody or functional fragment according to any one of claims 1 or 4.

13. A method of treating diseases associated with abnormal production of IL-4 and/or IL-13 in a mammal which the stages of introduction to the mammal a therapeutically effective amount especifismo antibody or functional fragment according to any one of claims 1 or 4.

14. A method of suppressing T-2 mediated response in a mammal to which the stages of introduction to the mammal a therapeutically effective amount especifismo antibody or functional fragment according to any one of claims 1 or 4.

15. The nucleic acid molecule encoding bespecifically antibody or functional fragment according to any one of claims 1 or 4.

16. The expression vector, the content is in which the nucleic acid molecule according to item 15.

17. Prokaryotic, yeast or fungal a host cell containing a vector according to item 16, producing bespecifically antibody or functional fragment according to any one of claims 1 or 4.



 

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14 cl, 23 dwg, 17 tbl, 27 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a human antibody or an antigen-binding antibody fragment which specifically binds to human CD20 and contains CDR1-3 of a light chain and CDR1-3 of a heavy chain. What is described is a version of the antibody or antigen-binding antibody fragment characterised by amino acid sequences of the light and heavy chains. There are presented: a coding nucleic acid molecule, a based expression vector, as well as a method for producing the antibody or antigen-binding antibody fragment with using the vector. What is disclosed is a pharmaceutical composition based on the antibody. What is described is a method for reducing a severity or suppressing a CD20-caused disease or condition, and using the antibody or antigen-binding antibody fragment for preparing a medicine for reducing a severity or suppressing the CD20-mediated disease or condition.

EFFECT: invention provides the antibodies that extend asymptomatic survival from approximately 2 to approximately 9 times or more, relative to animals control in human lymphoma simulated in mice, have high affinity, that can be find application in medicine.

15 cl, 1 dwg, 14 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. What is presented is an antibody specifically bound to human CD26. There are described versions of coding NC, expression vector, and versions of a host cell for producing the antibodies. There are disclosed methods of inhibiting (cell proliferation, tumour growth) and a method of treating a condition caused by CD26 expression, as well as a pharmaceutical composition using the antibody of cl. 1. There are described methods of inhibiting (tumour growth, metastasis proliferation) using the pharmaceutical composition. What is disclosed is E.coli for producing the antibody, deposited under accession number PTA-7695 in ATCC (American typical culture collection).

EFFECT: use of the invention can find application in treating the immune disorders associated with CD26 expression, including cancer.

29 cl, 42 dwg, 14 tbl, 14 ex

FIELD: biotechnology.

SUBSTANCE: polypeptides for the prevention or treatment of myocardial hypertrophy correspond to sequences of amino acids SEQ ID NO:2-3, 5-8. The composition for prevention or treatment of myocardial hypertrophy comprises a polypeptide corresponding to sequences of amino acids SEQ ID NO:2-3, 5-8, and pharmaceutically acceptable additives. The method of production of the said polypeptides comprises a step of synthesis of the polypeptide with the amino acid sequence SEQ ID NO:2-3, 5-8 in a synthesizer of polypeptides or ligation of the corresponding nucleotide sequence with the vector to form a recombinant vector, transformation of the said recombinant vector into a host cell, induction of expression of the said polypeptide in the said host cell, and isolating of the said polypeptide.

EFFECT: invention enables to produce new polypeptides for prevention or treatment of myocardial hypertrophy.

12 cl, 6 dwg, 9 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: peptide is presented, which is produced from protein WT1, which is able to bind with a molecule HLA-A*1101 and to induce CTL, having a sequence SEQ ID NO:6 or SEQ ID NO:7, presented in the description. Besides, a peptide dimer is described, which is used for the same purposes and comprises two peptide monomers selected from a group of peptides that includes SEQ ID NO:7, SEQ ID NO:3, SEQ ID NO:8 and SEQ ID NO:9, available in the description. A nucleic acid is presented, which codes the specified peptide and the expression vector, which contains the specified nucleic acid. A pharmaceutical composition is described for treatment or prevention of cancer of an individual, positive by HLA-A*1101, which contains the specified peptide, nucleic acid or vector. A WT1-specific CTL is described, which is induced by the specified peptide or dimer, and an antigen-presenting cell that presents the peptide. There is data on the method and set for induction of the WT1-specific CTL and for induction of the antigen-presenting cell. The method of in vitro diagnostics of cancer in an individual positive by HLA-A*1101 is presented, which includes incubation of the specified CTL or the antigen-presenting cell with a sample received from an individual positive by HLA-A*1101, and detection of the quantity of the specified CTL or antigen-presenting cell.

EFFECT: invention makes it possible to expand assortment of peptides that bind with HLA-A*1101 and are produced from the antigen WT1.

14 cl, 1 tbl, 14 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically preparing a tumour necrosis factor receptor and may be used in medicine. Genetic engineering technique is used to produce mutant TNFRp75 bound to a tumour necrosis factor and lymphotoxins substantially consisting of N-terminal 257 amino acid residues TNFRp75 wherein the N-terminal residue Glu92 is substituted by Asn, His, Ser or Ala and wherein the N-terminal residue Trp89 is optionally substituted by Tyr or Phe. The produced mutant is used to construct a fused protein with an additional amino acid fragment specified in a constant area of human immunoglobulin and one of five functional areas of albumin found on C-terminal of the soluble mutant TNFRp75. The produced mutant TNFRp75 and the fused protein is used as a part of a pharmaceutical composition for treating the diseases associated with TNFα overexpression which involve rheumatoid arthritis, psoriasis, sclerodermatitis, Sjogren syndrome, Strumpell-Marie disease, lupus erythematosus, acute disseminated myositis and syndrome similar to systemic lupus erythematosus.

EFFECT: invention enables producing soluble TNFRp75 mutant able to be bound to tumour necrosis factor and lymphotoxins at a high affinity degree.

9 cl, 12 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: invention relates to genetics and sports medicine. Claimed is method of determining predisposition to long physical effort. Method is based on genotyping polymorphism rs2070744 (C786T) of NOS3 gene and polymorphism rs5370 (G925T) of EDN1 gene by method of polymerase chain reaction. In case of determining genotype TT (rs2070744) and GG (rs5370), genetic predisposition to long physical effort is diagnosed. Technical result of claimed invention is increased efficiency, accuracy and reliability of diagnostics of predisposition to long physical effort.

EFFECT: increase of efficiency, accuracy and reliability of diagnostics of predisposition to long physical effort.

4 ex

Silk proteins // 2467015

FIELD: medicine.

SUBSTANCE: there are produced silk polypeptides having a double-spiral structure and originated from honey bee, bumble bee, bulldog ant, green tree ant and golden-eyed fly. A recombinant method is used to produce a host cell, transgenic plant and animal which produce silk polypeptide. Antibodies are produced for prepared polypeptides.

EFFECT: invention enables using producing silk polypeptides in various fields of industry.

21 cl, 14 dwg, 14 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry and represents versions of antibodies binding human anaplastic lymphoma kinase (ALK) protein. There are also presented: versions of radiolabelled or toxin antibodies; a DNA sequence coding the antibody, an expression vector containing the DNA sequence; a host cell transformed by the vector, a method for producing the antibody by cell cultivation, as well as using the antibody for preparing a drug for treating cancer or tumours.

EFFECT: invention may be effectively used for treating tumour or cancer.

32 cl, 5 dwg, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention is an isolated nucleic acid comprising a canine RNA polymerase I regulatory sequence and containing (i) at least 250 or at least 350 or at least 450 adjoining nucleotides or an entire nucleotide sequence, which is in form of SEQ ID NO:26, (ii) a nucleotide which is at least 80% identical to said nucleotide sequence (i) or includes a complementary or reverse complementary (i) or (ii) sequence. The nucleotide sequence (i) or (ii) is operably linked to cDNA which encodes influenza virus vRNA, and when produced in MDCK cells, is capable of directing expression of said influenza virus vRNA. The present invention also describes expression vectors and cells containing such nucleic acids, as well as methods of using such nucleic acids to obtain influenza viruses, including infectious influenza viruses.

EFFECT: canine plasmid rescue system pol I enables to obtain recombinant influenza viruses in a canine cell culture with high titre.

25 cl, 16 dwg, 7 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention may be used in medicine. DNA coding a peptide able to bind with TGF-β1 and to inhibit its biological activity in vitro and in vivo is used as an agent inhibiting biological activity of TGF-β1.

EFFECT: invention enables the effective treatment of diseases or pathological disorders associated with hyperexpression or dysregulated expression of TGF-β1 due to inhibition of biological activity of TGF-β1.

4 cl, 6 dwg, 4 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of producing recombinant spider-web proteins of orb-weaving spiders in yeast cells, fused proteins containing spider-web recombinant protein sequences of orb-weaving spiders, recombinant DNA encoding fused proteins, yeast host cells and expression vectors used to realise the method, as well as producer strains of recombinant proteins of orb-weaving spiders. The method involves constructing an expression vector containing a DNA sequence encoding recombinant spider-web proteins of orb-weaving proteins, fused with a sequence which encodes ubiquitin or an ubiquitin-lie protein SUMO of yeast Saccharomyces cerevisiae, which occupies the N-terminal position in the fused protein relative the recombinant spider-web protein, transformation of yeast cells with the obtained expression vector and expression of orb-weaving spider web protein in the transformed cells.

EFFECT: method increases production of recombinant spider-web protein during accumulation thereof in yeast cells in a water-soluble fraction in form of a protein which does not contain a hybrid component.

24 cl, 6 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: there is constructed a recombinant plasmid DNA pAP271 containing a rhFVII protein gene, a MAR matrix attachment region of an avian lysozyme gene, CMV virus transcription amplifier, a promoter of translational factor of human EF-1α elongation, an internal site of translational initiation of encephalomyocarditis IRES virus, a mouse DHFR gene, a SV40 virus polyadenylation signal, a cartridge comprising all elements required for aminoglycoside-3'-phosphotransferase (APH) gene expression, a cartridge for expression in bacterial cells of β-lactamase gene, as well as unique recognition sites of the following restriction endonucleases: Agel (850 base pairs), BbvCI (1657 base pairs), BmgBI (4202 base pairs), BssHII (6672 base pairs), Eco47III (11269 base pairs), EcoRI (10929 base pairs), EcoRV (11863 base pairs), Fsel (1455 base pairs), NotI (4812 base pairs), RsrII (6790 base pairs), Sbfl (2330 base pairs), Sfil (6027 base pairs), Tthllll (6390 base pairs), Xcml (2404 base pairs).

EFFECT: presented invention provides producing stably high-yield recombinant human blood coagulability factor VII.

2 cl, 4 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is an anti-factor D antibody and an antigen-binding fragment thereof, as well as a pharmaceutical composition, a commercial product and methods for treating the complement-binding diseases. What is considered is a polynucleotide, an expression vector, a host cell and a method for preparing the antibody according to the invention or the antigen-binding fragment thereof.

EFFECT: invention can find application in treating, preventing and diagnosing the diseases and disorders associated with excessive and uncontrolled complement activation, particularly alternative pathway of complement.

70 cl, 5 ex, 3 tbl, 13 dwg

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