Humanised human osteopontin antibody

FIELD: medicine.

SUBSTANCE: substance of the invention involves a humanised human osteopontin antibody containing a variable region of a heavy chain consisting of the amino acid sequence SEQ ID NO:1 and a variable region of a light chain consisting of the amino acid sequence SEQ ID NO:3. Furthermore, the invention involves a polynucleotide containing a sequence coding the variable region of the respective light and heavy chains of the humanised antibody, an expression vector containing polynucleotide, a host cell, a medicine, a method of producing the humanised antibody, a medicine for treating an autoimmune disease, a method of treating, and application of the humanised antibody for producing a pharmaceutical agent.

EFFECT: advantage of the invention consists in creation of the humanised antibody exhibiting improved activity or stability, than activity and stability of standard human osteopontin antibodies.

13 cl, 14 ex, 1 tbl, 16 dwg

 

The technical field to which the invention relates

The present invention relates to humanitarianlaw antibody against osteopontin person with very high activity and stability and to a method of therapeutic and diagnostic use of antibodies for various diseases.

The level of technology

Osteopontin (hereinafter referred to in this document as "OPN) is an acidic calcium-binding glycoprotein found in large amounts in bone and, in men, as it is known, due to differences in mRNA splicing, presents at least three isoforms: osteopontin-a (hereafter in this document referred to as "OPN-a"), osteopontin-b (hereafter in this document referred to as "OPN-b"), osteopontin-c (hereafter in this document referred to as "OPN-c) (non-patent document 1). In particular, the predecessor of OPN-a has the amino acid sequence specified in the list below sequence as SEQ ID NO:23, and believed, after secretion from the sequence cleaved signal peptide with the formation of the Mature form of OPN-a, consisting of I17-N314. The Mature form of OPN in vivo cleaved by thrombin at 168 (in the case of OPN-a) the arginine residue at the C-end with the formation of the N-terminal fragment and C-terminal fragment.

The above OPN is responsible for a large RA is a variety of physiologically and pathologically important functions and features, for example, cell adhesion, cell migration, tumorigenesis, immune response, inhibition mediated by complement cytolysis, etc. These various functions are mediated by a wide range of receptors located on the cell surface. OPN contains the RGD sequence (for example, OPN-a, 159 to 161 residues); integrins that recognize this RGD sequence, such as αVβ3, αVβ1 and αVβ5, are the main receptors OPN, of which the integrins αVβ3, αVβ1 and αVβ5 mediate cell adhesion in the smooth muscle cells of blood vessels; in addition, αVβ3 is associated with migration of macrophages, lymphocytes, endothelial cells, smooth muscle cells, etc.

In addition, to date the study also showed that OPN binds to α9β1 integrins, α4β1 and α4β7 through the sequence SVVYGLR (SEQ ID NO:10), and the difference in the method of linking is that α4β1 binds to both OPN: unsplit thrombin (unsplit type OPN) and otdalennym thrombin N-terminal fragment (split type OPN), whereas α9β1 is associated only with split type OPN (non-patent documents 2 to 4). These subunits of integrins α9 and α4 and β1 and β7 are very similar to each other in amino acid sequence. The integrins α4β1 and α4β7 are mostly in lymphocytes and monocytes, but very low Express and observed in neutrophils. On the other hand, α9β1 selectively expressed at high levels in neutrophils and is responsible for the vital functions of neutrophil migration by VCAM-1, Tenascin-C, etc. Option α9β1 is widely expressed in myocytes, epithelial cells, hepatocytes, etc. Therefore, I believe that the cytoplasmic domains of the subunits α4 and α9 integrins through the respective slightly different intracellular routes of transmission of a signal involved in various inflammatory response through co-stimulation of migration and aggregation of leukocytes to the inflammation to enhance their infiltration activity.

As described above, due to the fact that a great variety of integrins stimulate leukocyte migration and involved in the inflammatory response, believe that drugs that inhibit these activities integrins can very effectively be used as anti-inflammatory drugs. For example, the integrin αVβ3 is expressed in osteoclasts, endothelial cells of blood vessels, smooth muscle cells and the like; as I believe that the inhibition of binding of the integrin with different binding ligands exerts a suppressive effect on the destruction of the joints, such as joints, continues to develop antibodies against αVβ3.

However, as the expression of Retz is Perov, belonging to the integrin family, is a versatile presented in a large number of tissues and is responsible for vital functions to maintain various kinds of biological activity, the use of antibodies against integrin in the treatment of rheumatoid arthritis or osteoarthritis can cause a similar inhibition in other areas, as well as interest from the point of view of adverse reactions.

From this point of view, to the present time, attempts were made to clarify the etiology of rheumatoid arthritis, osteoarthritis and the like, and to provide us with the best therapeutic approach.

For example, in WO 02/081522 (patent document 1) found that patients with rheumatoid arthritis and patients with osteoarthritis there is a high concentration of OPN in fluid articular cavity, and in patients with rheumatism observed increased ratio tsepliaeva thrombin-type N-terminal fragment compared with the total OPN, and confirmed that OPN directly associated with the emergence of these diseases. In patent document 1 is obtained antibodies, which individually recognize the N-terminal fragment and C-terminal fragment resulting from the cleavage of OPN by thrombin, respectively, in the study with their use shows that patients with rheumatoid arthritis have a high concentration of tsepliaeva thrombin N-terminal fragment in the glenoid cavity, in particular. In this N-terminal fragment simultaneously present the RGD sequence and the sequence SVVYGLR (SEQ ID NO:10), both recognized by the types of integrins person; confirmed that the antibody that blocks simultaneously these two sequences, significantly inhibits the binding of OPN and integrin and effective for the treatment of rheumatoid arthritis, osteoarthritis, etc.

In particular, in patent document 1, the obtained antibody that inhibits the binding sequence RGD OPN person and integrin binding of sequence SVVYGLR OPN human (SEQ ID NO:10) and integrin, and its effect was confirmed in experiments on cell adhesion, cell migration, etc. in Addition, the antibody against a synthetic peptide corresponding to internal sequence OPN mice, and its effect as a therapeutic drug is confirmed with the use of pathological model of arthritis in mice.

Thus, since the mouse OPN contains the RGD sequence and the SLAYGLR sequence (SEQ ID NO:12), each of which is recognized by the integrin mouse, the provisions on amino acid sequence homology to the provisions of OPN person, the antibody M5 received as an antibody, simultaneously blocking these sequences. Confirmed that the binding of this antibody M5 with OPN is Ishi and his otdalennym thrombin product is inhibited by peptide GRGDSP, which contains the RGD sequence, and that this antibody M5 inhibits the migration of activated TNF-α from monocytes isolated from the spleen of a mouse. When using the system organ culture of vault of skull mouse researched this antibody M5 observed suppressive effect on bone destruction. In addition, when the above-described antibody used in models of collagen arthritis in mice was confirmed by a pronounced therapeutic effect (patent document 1 and non-patent document 5).

These results strongly indicate that the antibody which blocks the binding sequence RGD and type integrin person, and the sequence SVVYGLR (SEQ ID NO:10) and the type of human integrin, inhibits the binding of OPN and integrin and is effective for the treatment of rheumatoid arthritis and the like, and, furthermore, show that it can be expected that the antibody will be effective not only in the treatment of such forms of rheumatism, as juvenile rheumatoid arthritis and chronic rheumatism, but also in the treatment of psoriatic arthritis and psoriasis. Chronic graft rejection after organ transplantation is characterized by obstructive lesions of blood vessels and bronchioles; on the basis of their histological study an assumption was made that since the activation of T-cells, macropha what s causing the production of cytokines and growth factors and damage to endothelial cells of blood vessels, and based on the fact that the growth of vascular smooth muscle causes fibrosis and the like, the condition progresses to vascular obstruction (non-patent documents 6 to 8).

Published that OPN acts as a critical protein for activation of macrophages and vascular smooth muscle fibrosis (non-patent document 9); OPN inhibitory antibody can supressive process leading to fibrosis, inhibiting the migration of monocytes and neutrophils. Thus, assume that the antibody suppresses chronic graft rejection after organ transplantation, promoting engraftment of bodies, and is effective in the treatment of autoimmune diseases such as systemic autoimmune disease, erythematous, uveitis, Behcet's disease, dermatomyositis, glomerulonephritides jade and sarcoidosis. Also confirmed that the expression level of OPN is increased in various malignant tumors and that OPN contributes to the development of malignant tumors and metastasis (non-patent documents 10 to 12) and that the growth and metastasis of malignant cells cupressinum antibody against OPN (patent document 3, non-patent document 13). Thus, suppose that the antibody against OPN can also be effective in the treatment of various malignant tumors.

In WO 03/027151 (patent document 2) describes him the RNA antibody against osteopontin person, containing the variable region of the antibody 2K1 mouse against osteopontin person described in patent document 1, and a constant region of human antibodies; and humanitariannet antibody against osteopontin person containing the complementarity determining region 2K1 antibody and framework region and constant region of human antibodies.

Meanwhile, the market is available a large number of monoclonal antibodies for treatment, including antibodies for the treatment of malignant tumors (e.g., rituximab, trastuzumab, bevacizumab), antibodies for the treatment of rheumatism (e.g., infliximab, adalimumab), antibodies for the suppression of transplant rejection (e.g., muromonab, basiliximab), etc.

Because of their basic properties, high specificity, and security believe that the research and development of drugs, monoclonal antibodies, in particular, intended for a wide range of diseases for which hindered the creation of a low-molecular-weight therapeutic drugs, will be accelerated.

On the other hand, the greatest challenge in the development of pharmaceuticals based on antibody is antibody production. Typically, the clinical dose of monoclonal antibodies that are released on the market, represent the magnitudes of the order of several mg/kg, what is needed is considerable production costs.

For this reason, selection of antibodies that exhibit very high activity of the antibodies with similar activity, antibodies with high expression level and high stability is an extremely important requirement for actual use as pharmaceutical agents based on antibodies.

Patent document 1: Technical instructions for the international patent publication No. WO 02/081522.

Patent document 2: Technical instructions for the international patent publication No. WO 03/027151.

Patent document 3: Technical instructions for the international patent publication No. WO 06/043954.

Non-patent document 1: Y. Saitoh et al., (1995): Laboratory Investigation, 72, 55 to 63.

Non-patent document 2: Y. Yokosaki et al., (1999): The Journal of Biological Chemistry 274, 36328-36334.

Non-patent document 3: P.M. Green et al., (2001): FEBS Letters 503, 75-79.

Non-patent document 4: S.T. Barry et al., (2000): Experimental Cell Research 258, 342-351.

Non-patent document 5: Yamamoto et al., (2003): The Journal of Clinical Investigation, 112, 181-188.

Non-patent document 6: P. Freese et al., (2001): Nephrology, dialysis, transplantation, 16, 2401-2406.

Non-patent document 7: J.R. Waller et al., (2001): British Journal of Surgery, 88, 1429-1441.

Non-patent document 8: S.R. Lehtonen et al., (2001): Transplantation, 72, 1138-1144.

Non-patent document 9: A. O'regan et al., (2000): International Journal of Experimental Pathology, 81, 373-390.

Non-patent document 10: G. F. Weber, (2001): Biochimica et Biophysica Acta, 1552, 61-85.

Non-patent document 11: H. Rangaswami et al., (206): TRENDS in Cell Biology 16, 79-87.

Non-patent document 12: S.S. Forootan et al., (2006): Int. J. Cancer: 118, 2255-2261.

Non-patent document 13: Z. Hu et al., (2005): Clin. Cancer Res. 11 4646-4652.

Description of the invention

Objectives of the invention

The present invention was created in consideration of the above circumstances, and refers to humanitarianlaw antibody against osteopontin person with improved activity (antigennegative activity, inhibiting leukocyte migration activity and the like) and/or stability (resistance to heat, low pH, denaturing means and the like), in comparison with the activity and stability of standard antibodies against osteopontin person. The authors of the present invention conducted in-depth research to improve object and they managed to create humanitariannet antibody against osteopontin person with such characteristics.

Solutions to problems

Thus, objects of the present invention are:

(1) Humanitariannet antibody against osteopontin person containing the variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO:1 and variable region light chain comprising amino acid sequence SEQ ID NO:3.

(2) Humanitariannet antibody against osteopontin person described in (1) above, where the intercept is ntna region of the heavy chain antibody is a Igγ1 person.

(3) Humanitariannet antibody against osteopontin person described in (1) above, where the constant region of the light chain of the antibody is a human Igκ.

(4) Humanitariannet antibody against osteopontin person described in (1) above, where the constant region of the heavy chain antibody is a Igγ1 man, and the constant region of the light chain of the antibody is a human Igκ.

(5) Humanitariannet antibody against osteopontin person, containing a heavy chain consisting of the amino acid sequence of SEQ ID NO:25 and a light chain consisting of the amino acid sequence of SEQ ID NO:27.

(6) Polynucleotide containing a sequence that encodes the variable region of the heavy chain gumanitarnogo antibodies against osteopontin person listed above in (1).

(7) Polynucleotide containing a sequence that encodes a variable region light chain gumanitarnogo antibodies against osteopontin person listed above in (1).

(8) Expressing the vector containing polynucleotide described above in (6) and/or (7).

(9) a host Cell that contains expressing vector described above in (8).

(10) the Method of obtaining gumanitarnogo antibodies against osteopontin person, including the stage of culturing the host cell described above in (9), the call is allowing the cell to Express humanitariannet antibody against osteopontin person.

(11) therapeutic drug for autoimmune of zabolevaniya, rheumatism, rheumatoid arthritis or osteoarthritis, containing humanitariannet antibody against osteopontin person described in any of paragraphs (1) through (5).

(12) a Method for prevention or treatment of autoimmune diseases, rheumatism, rheumatoid arthritis or osteoarthritis, comprising the stage of introducing a therapeutically effective amount gumanitarnogo antibodies against osteopontin person described in any of paragraphs (1) through (5).

(13) Application gumanitarnogo antibodies against osteopontin person described in any of paragraphs (1) through (5), to obtain a pharmaceutical for the prevention or treatment of autoimmune disease, rheumatism, rheumatoid arthritis or osteoarthritis.

The result of invention

The present invention relates to humanitarianlaw antibody against osteopontin person, having improved activities (antigennegative activity, inhibiting leukocyte migration activity and the like) and/or stability (resistance to heat, low pH, denaturing tools etc) in comparison with the activity and stability of standard antibodies against osteopontin person. With these properties, the antibody according to the present invention is suitable for the prevention or treatment of various inflammatory diseases, including autoimmune disease, rheumatism, rheumatoid arthritis and osteoarthritis.

Brief description of figures

Figure 1 presents the nucleotide sequence (top row: SEQ ID NO:15) and amino acid sequence (bottom row: SEQ ID NO:16) corresponding DNA containing the coding region R2K1-VH1.7, built-in vector (the underlined part is a leader sequence for secretion of the expression).

Figure 2 presents the nucleotide sequence (top row: SEQ ID NO:17) and amino acid sequence (bottom row: SEQ ID NO:18) corresponding DNA containing the coding region R2K1-VH1.8, built-in vector (the underlined part is a leader sequence for secretion of the expression).

Figure 3 presents the nucleotide sequence (top row: SEQ ID NO:19) and amino acid sequence (bottom row: SEQ ID NO:20) corresponding DNA containing the coding region R2K1-VL1.7, built-in vector (the underlined part is a leader sequence for secretion of the expression).

4 shows the nucleotide sequence (top row: SEQ ID NO:21) and amino acid sequence (bottom row: SEQ ID NO:22) corresponding DNA containing the coding region R2K1-VL1.8, built-in vector (the underlined section PR is dstanley a leader sequence for secretion of the expression).

Figure 5 presents the results of the analysis of the binding of the Chimera 2K1 antibody and gumanitarnogo 2K1 antibody with the peptide hOPN5 by ELISA method.

Figure 6 presents the results of the analysis of the binding of the Chimera 2K1 antibody and gumanitarnogo 2K1 antibody, subjected to heat treatment at 70°C with peptide hOPN5 by ELISA method. Ratio to the capacity of binding without heat treatment was taken as 100%.

Figure 7 presents the results of the analysis of the binding of the Chimera 2K1 antibody and gumanitarnogo 2K1 antibody, treated with buffer at pH 5, with peptide hOPN5 by ELISA method. Ratio to the capacity of binding without processing buffer at pH 5 was taken as 100%.

On Fig presents the data for the spectral peak wavelength of fluorescence of the Chimera 2K1 antibody and gumanitarnogo 2K1 antibody, treated with buffer containing different concentrations of guanidine hydrochloride.

Figure 9 presents the results of the measurement of the random structures in the Chimera 2K1 antibody and humanitariannet 2K1 antibody-treated buffer with different pH levels by CD.

Figure 10 is an illustration representing the results of the analysis of thermal stability of the Chimera 2K1 antibody and gumanitarnogo 2K1 antibody using ultrasensitive differential scanning calorimeter. The dashed arrow and splashin the arrow I mean Tm Chimera 2K1 antibody and antibodies R2K1v1.7, respectively.

Figure 11 presents inhibiting cell adhesion action R2K1v1.7 and R2K1v0 on OPN person.

On Fig presents the impact R2K1v1.7 on swollen joints induced by collagen arthritis in monkeys. Data are presented as mean ± SE for 8 animals, 7 animals and 5 animals in the control group, the group with the dosage of 25 mg/kg and groups with a dosage of 50 mg/kg, respectively. *p<0,05, **p<0,01: significantly different from control group that is defined using the criterion of multiple comparisons of Dunnet.

On Fig presents the results of the analysis of the purified HPLC R2K1v1.7-scFv.

On Fig presents the results of the analysis of the binding of purified R2K1v1.7-scFv with peptide hOPN5 by ELISA method.

On Fig presents the results of electrophoresis of a full-sized molecules antibodies type R2K1v1.7 and F(ab')2and purified F(ab')2-PEG antibodies R2K1v1.7 in polyacrylamide gel in the presence of sodium dodecyl sulfate.

On Fig presents the results of the analysis of the ability of binding of F(ab')2-PEG R2K1v1.7 with peptide hOPN5 by BIAcore.

The best way of carrying out the invention

The following steps detail the present invention.

The authors of the present invention conducted in-depth studies to solve the above described problems with respect to conventional antibodies against osteopontin man, and they managed to create humanitariannet antibody against osteopontin person having improved activity and/or stability than the activity and stability of the Chimera 2K1 antibody and gumanitarnogo 2K1 antibody described in WO 03/027151 (patent document 2).

The basic structure of antibody molecules common to all the classes and consists of a heavy chain having a molecular weight of from 50000 to 70000, and a light chain having a molecular weight of from 20000 to 30000. Heavy chain usually consists of a polypeptide chain containing approximately 440 amino acids; heavy chains have the structural characteristics of different classes and are called γ, µ, α, δ and ε chains corresponding to IgG, IgM, IgA, IgD and IgE. In addition, IgG occurs as IgG1, IgG2, IgG3 and IgG4, and the corresponding chain called γ1, γ2, γ3 and γ4, respectively. Light chain usually consists of a polypeptide chain containing approximately 220 amino acids; there are two types, type L and type K and is called the chain of λ and κ, respectively. With regard to the configuration of the primary peptide structure of a molecule antibodies, two homologous heavy chains and two homologous to the light chain linked by a disulfide bond (S-S bond) and non-covalent bonds and molecular weight is 150,000 to about 190,000. Two types of light chains can form a pair with any heavy chain. Each antibody molecule is always soteitis two identical light chains and two identical heavy chains.

In the heavy chain are four (five for µ and ε chains), and in light two intramolecular S-S links; one loop formed by amino acid residues in an amount of from 100 to 110, and this steric structure is the same for all loops is called a structural unit or domain. Amino acid sequence of the domain for both heavy chains and light chains, based on their N-ends, is impermanent, even in the standard sample of the same class (subclass) in animals of the same species, and this domain is called the variable region (V-region, variable region) domains (denoted as VHand VL, respectively). Amino acid sequence at its C-end of each class or subclass is practically constant and is called the constant region (C-region, constant region) domains (denoted as CH1, CH2, CH3 and CL, respectively).

Antipersonnel plot antibodies consists of VHand VLand the binding specificity depends on the amino acid sequence of this area. On the other hand, these types of biological activity, as binding to components of the complement or different cells reflects differences in the structure of the P-region among different classes of Ig. It is revealed that the variability of the variable regions of the light chain and heavy the ETUI is practically limited to three small hypervariable regions, available in both circuits, and these areas are called CDR (complementarity determining region). The remaining part of the variable region is called frame region and is relatively constant. As a rule, antigennegative plot form only 5-10 amino acids or complementarity determining region of each of the variable fields.

In the present description the antibody with a variable region derived from a mouse antibodies (also referred to as heterologous donor antibody) as reactive antigen variable region and a constant region derived from human antibodies as a constant region that is designated as a chimeric antibody; a chimeric antibody, which recognizes osteopontin and its fragments, termed chimeric antibody against osteopontin. Recombinant antibody obtained by replacing all areas, except complementarity determining region (antigennegative plot) antigen molecule antibodies mammalian, non-human (e.g., mouse) is designated as humanitariannet antibody. Included in humanized antibodies are antibodies with amino acid modifications (replacement, insertion, deletion, addition)made in its frame region, like the antibody of the present invention.

In the main, it is known that when receiving gumanitarnogo antibodies, when the amino acid sequence of complementarity determining region only grafted on the matrix skeleton human antibodies, in many cases antigennegative activity is reduced compared with antigennegative activity of the original antibody mouse. Confirmed that described above humanitariannet 2K1 antibody has a very low inhibitory cell adhesion to OPN activity and, as a result, thus, is not suitable for use as pharmaceutical agents based on antibodies, despite the fact that it is associated with OPN peptides (example 9 below).

The authors of the present invention conducted in-depth studies on the reduction of a slowdown in humanized antibodies and receiving gumanitarnogo antibodies with improved stability, which can be used as pharmaceutical agents based on antibodies, and found that humanitariannet antibody against one of osteopontin containing the variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO:1, and the variable region of the light chain consisting of the amino acid sequence of SEQ ID NO:3, and had a significantly improved activity and/or improved stability from the point of view of the hypoxia index stability, compared to the standard chimeric and humanitarianism antibodies against osteopontin person. Essentially, humanitariannet antibody against osteopontin man according to the present invention was obtained by making modifications for some amino acids in the framework regions of the heavy chain and light chain matrix human antibodies, and contains a sequence of frame areas other than the standard sequence gumanitarnogo antibodies against osteopontin person obtained only by including the complementarity determining region (patent document 2).

Specialists in this field can easily get humanitariannet antibody against osteopontin man of the present invention on the basis of the information provided in this document information about the sequence of its variable regions of the heavy chain and the variable region of the light chain with the well-known in this field. In particular, get a gene fragment of the variable region of the heavy chain with a sequence of bases that encodes the amino acid sequence of the variable region of the heavy chain of the antibody of the present invention (SEQ ID NO:1), and the gene fragment of the variable region of the light chain sequence of bases that encodes amino the PCI-e slot sequence of the variable region of the light chain of the antibody of the present invention (SEQ ID NO:3). Then, to obtain gene gumanitarnogo antibody, the genes of the variable regions are combined with the gene constant region of a corresponding class of human antibodies. After that, this gene gumanitarnogo antibodies embed expressing in a suitable vector and introduced into cultured cells. In conclusion, cultured cell is cultivated, resulting in humanitariannet antibody can be isolated from the supernatant of the culture.

Each of the above-described gene fragments of variable regions, which encodes amino acids of the variable region of the heavy chain and light chain of the present invention antibody (SEQ ID NO:1 and SEQ ID NO:3), can be obtained, for example, obtaining the gene fragment that encodes the variable region of the heavy chain or light chain, respectively, gumanitarnogo 2K1 antibody described in WO 03/027151 described in the document by the way, and inducyruya mutations in the specified area of the gene fragment that encodes the frame region gumanitarnogo 2K1 antibody. For the induction of mutations in the specified area in the frame area, the experts in this field can use various obvious ways, such as site-specific mutagenesis (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. Jhon Wiley & Sons Section 8.1-8.5). Alternatively, the gene fragments of variable regions of heavy chain and light chain and is Titel of the present invention can also be synthesized based on the sequence of bases, designed based on the amino acid sequences of the variable regions of the heavy chain and light chain (SEQ ID NO:1 and SEQ ID NO:3), or on the basis of the sequence of bases in the variable regions of the heavy chain and light chain of the antibody of the present invention, is presented in SEQ ID NO:5 and SEQ ID NO:7, well known in this field by the method of gene synthesis. As a method of gene synthesis antibodies can be used in various ways known to specialists in this field, such as methods of gene fusion antibodies described in WO 90/07861.

Then, to obtain gene gumanitarnogo antibodies combine the above-described gene fragments of variable region gene and the constant region of human antibodies. Although, as the constant region used human antibodies can choose any subclass of the constant region, preferably, the constant region of the heavy chain can be used Igγ1 man, and as the constant region of the light chain can be used Igκ person.

After receiving this gene gumanitarnogo antibodies can be embedding gene gumanitarnogo antibodies in expressing vector, introduction of expressing the vector in cultured cells, culturing the cultured cells, purification of antibodies and other various well-known given in the second field methods or with reference to the methods of obtaining chimeric antibodies against osteopontin person or gumanitarnogo antibodies against osteopontin person, described in WO 02/081522 or WO 03/027151. As the expressing vector to embed thus obtained gene gumanitarnogo antibodies can be used expressing vectors, described in International Patent Publication Official Gazette WO94/20632, such as AG-γ1 and AG-κ, but expressing vector is not a limitation, provided that it is able to Express the gene gumanitarnogo antibodies. It is preferable to use expressing vector, which already has a gene constant region Ig man, such as AG-γ1 or AG-κ, as it may be expressing the gene gumanitarnogo antibodies vector only by embedding it in a gene variable regions gumanitarnogo antibodies.

Above expressing the vector is introduced into cultured cells, for example, a method using calcium phosphate, etc.

As examples of cultured cells, which enter expressing vector, you can use these cultured cells, as cells CHO-DG44, and can be cultivated in a standard way.

After the above-described cultivation, the antibody accumulated in the culture supernatant, can be cleaned, for example, different ways chromatography using columns with protein A.

Antigenic activity of the thus obtained gumanitarnogo antibodies against the OST is pontina person, can be measured, for example, by ELISA using the peptide OPN, etc. as described in the example below, BIACore (BIAcore Company), etc. Inhibitory leukocyte migration activity gumanitarnogo antibodies against osteopontin person can be measured, for example, cultivating monocytes in human peripheral blood in the presence of the test antibody and OPN or tsepliaeva thrombin-type OPN, as described in the example below. Humanitariannet antibody against osteopontin man according to the present invention has a biological activity of inhibition of activated cytokine (such as TNF-α) migration of monocytes in human peripheral blood towards tsepliaeva thrombin-type OPN.

Then thus obtained humanitariannet antibody against osteopontin man is tested in respect to the various indices of stability. Humanitariannet antibody against osteopontin man of the present invention demonstrates the following indexes stability (A) to (D)):

A) Shows thermostability, where the activity of binding to a peptide containing the sequence SVVYGLR (SEQ ID NO:10), after the heat treatment in PBS at 70°C for 2 hours is not less than 90% of the binding activity without heat treatment.

B) the Average transition temperature (Tm) of at least 5°C higher than the average temperature is ur transition chimeric antibodies with variable scope derived from a heterologous donor antibody and a constant region derived from human antibodies.

C) is resistant to guanidine hydrochloride at concentrations above at least 0.5 M than the concentration of the chimeric antibody with a variable region derived from a donor gaetanomakino antibody and a constant region derived from human antibodies.

D) resistant to pH of at least 0.3 less than the levels of pH for chimeric antibody with a variable region derived from a heterologous donor antibody and a constant region derived from human antibodies.

In this case, the above indexes (A) and (B) are indices of resistance to heat; if antibodies improved the performance of these indices, it is more important from the standpoint of stability during long-term storage and dosage forms. That is, drug antibodies often have difficulty in terms of stability during storage, as it is a protein, so sometimes it is obtained by obtaining freeze-dried (it is difficult from the point of view of applicability in terms of medical practice, because during use it should be dissolved; in particular, the protein drug to dissolve often takes more than 30 seconds, which, in turn, in terms medicinalplants is often difficult); however, any antibody with good resistance to heat can be stored even in solution, providing long-term stability after freezing for 2 or more years. In fact, R2K1v1.7, humanitarianlaw antibody against osteopontin man of the present invention, described in the example below, are stable even at room temperature (25°C) for about 1 year. If it is possible to obtain a solution, it makes it possible to obtain more appropriate preparations in the form of pre-filled syringes, etc. Antibody with high heat resistance, which satisfies the above described indices, suggests a wider variability in obtaining the drug and makes it possible to design drugs that meet the more important therapeutic needs, and increases the possibility of choice.

The above index C) is an index of resistance to the action of the salts; the antibody with such resistance to the action of salts allows to conduct the study is more preferred formula to create a pharmaceutical drug. In particular, this index is suitable for pre-filled syringes, as high concentrations of salts are often used when obtaining the drug protein substances with a high concentration, such as from 100 to 200 µg/m is.

The above index (D) is an index related to the sustainability of antibodies to pH; antibody with such resistance to pH can be treated at lower pH levels at the stage of viral inactivation method of obtaining and purification of antibodies, and thus, is suitable. For this reason, the main advantage may be the presence of resistance to a pH of approximately 0.3 is less than the resistance to pH standard antibodies.

The following describes the method of testing for index A). First test humanitariannet antibody against osteopontin man was diluted in PBS (preferably 50 μg/ml) and subjected to heat treatment at 70°C for 2 hours. After that, the temperature of the solution was returned to room and measured the binding activity of the antibody with the peptide containing the sequence SVVYGLR (SEQ ID NO:10) using, for example, the ELISA method Kon et al. (Journal of Cellular Biology, 88: 420-432 (2002)). The binding activity of a given subjected to a heat treatment of the antibody compared to the binding activity of the same antibody, but measured without heat treatment. Humanitariannet antibody against osteopontin man of the present invention, subjected to such heat treatment, demonstrates the activity of binding to a peptide containing the sequence SVVYGLR (SEQ ID NO:10), not less than 90% of AK is Yunosti binding of the same, but raw antibodies. Preferably, the peptide containing the sequence SVVYGLR (SEQ ID NO:10), used in this testing index, is a peptide of osteopontin sequence CVDTYDGRGDSVVYGLRS (SEQ ID NO:13).

The following describes the method of testing for index B). First test humanitariannet antibody against osteopontin human and Chimera 2K1 antibody described in WO 03/027151 (C2K1), balance using a suitable buffer solution (preferably 20 mm citrate buffer + 120 mm NaCl (pH 6,0)), and evaluate the heat resistance using a differential scanning calorimeter (preferably VP capillary DSC platform of MicroCal Company). The average transition temperature (Tm), which shows the temperature of the destruction gumanitarnogo antibodies against osteopontin man of the present invention, at least 5°C higher than the average temperature of the transition C2K1.

The following describes the method of testing for index C). First test humanitariannet antibody against osteopontin person and the above-described chimeric antibody 2K1 (C2K1) is dissolved in a buffer solution containing guanidine hydrochloride in different concentrations from 0 to 5 M, preferably 20 mm phosphate + 120 mm NaCl (pH 7.0)), and the solution was adjusted to a suitable concentration, preferably 50 μg/ml). Then, each sample R is the target allow you to be at 10°C during the night, then measure the fluorescence spectrum of each sample. In particular, the fluorescence emitted by tryptophan with exciting light of 280 nm, is scanned in the wavelength range from 320 nm to 370 nm. The peak wavelength is shifted due to the loss of spatial structure of the protein under the action of guanidine hydrochloride. The concentration of guanidine hydrochloride to shift the peak wavelength is measured for each of the tested antibodies and chimeric antibodies. For gumanitarnogo antibodies against osteopontin man of the present invention, the concentration of guanidine hydrochloride to shift the peak wavelength, as described above, at least about 0.5 M higher than the concentration of guanidine hydrochloride to shift the peak wavelength C2K1.

The following describes the method of testing for index D). First, test humanitariannet antibody against osteopontin person and the above-described chimeric antibody 2K1 (C2K1) was adjusted using a suitable buffer solution (preferably 20 mm citrate buffer + 120 mm NaCl (pH 6,0)) (preferably 2 mg/ml), and adding thereto an acidic solution (preferably 0,1N. HCl) and water, get a sample of each of the low pH at the indicated concentrations (1 mg/ml). After this sample is supported at room temperature for 1 hour, measure the spectrum of the circular is Araiza (CD). Range of CD measured in the wavelength range from 205 nm to 260 nm, and for each sample of each antibody treated to changes in pH, measure the percentage of random structures, on the basis of spectral and analytical method CD Yang et al. (Methods in Enzymology, 130, 208-269 (1986)). the pH at which the percentage content of random structures in humanitariannet the antibody of the present invention begins to increase, at least about 0.3 less than pH C2K1.

The authors of the present invention on the basis of gumanitarnogo antibodies described in WO 03/027151, has conducted extensive research using a combination of modifications in the gene frame region by site-specific mutagenesis and the like, and stability studies using the above indices from A) to D), and for the first time successfully received humanitariannet antibody against osteopontin person with improved activity (antigennegative activity, inhibiting leukocyte migration activity and the like) and/or stability (resistance to heat, low pH, denaturing means and the like) than the activity and stability of standard antibodies against osteopontin person, by presenting parts of the skeleton of a human antibodies (FR 1 to 4) in the form of amino acid sequence SEQ ID NO:1 (amino acid is t from 1 to 30, from 36 to 49, from 67 to 98 and from 106 to 116, respectively) and amino acid sequence of SEQ ID NO:3 (amino acids 1 to 23, 40 to 54, 62 to 93 and from 103 to 113, respectively). Humanitariannet antibody against osteopontin man according to the present invention was tested on the above antigennegative activity, inhibitory leukocyte migration activity and various indices of stability, and found that it has these kinds of activity and in the quality of its properties shows all of the indices of A) to D).

Humanitariannet antibody against osteopontin man of the present invention, containing the variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO:1, and the variable region of the light chain consisting of the amino acid sequence of SEQ ID NO:3, you can easily get synthesizing DNA, which encodes the amino acid sequence represented in SEQ ID NO:1, and the DNA that encodes the amino acid sequence represented in SEQ ID NO:3 in a manner well known in this area, connecting them with the gene constant region of a corresponding class of human antibodies, preferably gene constant region for Igγ1 the heavy chain and the gene constant region for Igκ light chain, for constructing gene gumanitarnogo antibodies, embedding gene humans is fragmented antibodies in expressing vector various well-known in this field means or ways, described in WO 02/081522 or WO 03/027151 etc. by entering expressing the vector in cultured cells expressing cultured cells and purifying the antibody from the resulting culture. The preferred heavy chain gene gumanitarnogo antibodies of the present invention, obtained by the connection of the gene variable regions of the heavy chain, represented in SEQ ID NO:1, and the gene constant region of the heavy chain Igγ1 person, you can specify a gene containing a sequence of bases that encodes the amino acid sequence represented in SEQ ID NO:25, more preferably a gene containing a sequence of bases represented in SEQ ID NO:24. As a preferred light chain gene gumanitarnogo antibodies of the present invention, obtained by the connection of the gene variable region of the light chain, represented in SEQ ID NO:3 and the gene constant region of the light chain of human Igκ, you can specify a gene containing a sequence of bases that encodes the amino acid sequence represented in SEQ ID NO:27, more preferably a gene containing a sequence of bases represented in SEQ ID NO:26. As gumanitarnogo antibodies against osteopontin of the present invention, encoded by the gene of the heavy chain containing a sequence of bases represented by the SEQ ID NO:24, and light chain gene containing the sequence of bases represented in SEQ ID NO:26, you can specify R2K1v1.7 described below in the example.

Alternatively, humanitariannet antibody against osteopontin of the present invention, containing the variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO:1, and the variable region of the light chain consisting of the amino acid sequence of SEQ ID NO:3, can also be synthesized using a DNA, which encodes the above-described amino acid sequence represented in SEQ ID NO:1, and the gene constant region heavy chains of human antibodies, and the DNA that encodes the above-described amino acid sequence represented in SEQ ID NO:3, and the gene constant region of the light chain of human antibodies as matrices, using a cell-free system transcriptio/broadcast. Used cell-free system transcriptio/broadcast may be a commercially available cell-free system transcriptio/broadcast and you can get essentially known manner, in particular, the method described in Pratt J.M. et al., "Reduced and Translation", B.D. Hames and S.J. Higgins edt., IRL Press, Oxford 179-209 (1984), etc. to extract Escherichia coli. As commercially available cell lysate, you can specify the system extracts of E. coli S30 (produced by Promega Comany), a quick broadcast RTS 500 Rapid Translation System (produced by Roche Company), etc. derived from Escherichia coli, you can specify the system lysate of rabbit reticulocytes Rabbit Reticulocyte Lysate System (produced by Promega Company), etc. derived from rabbit reticulocytes, and you can specify PROTEIOSTM (produced by TOYOBO Company), etc. obtained from a lysate of wheat germ. Among them are suitable for use are those that use a lysate of wheat germ. As a method of obtaining a lysate of a germ of wheat, for example, you can use the method described in F.B. Johnston et al., Nature, 179, 160-161 (1957) or Erickson A.H. et al., Meth. Enzymol., 96, 38-50 (1996), etc.

The present invention also relates to fragments of humanized antibodies (antibody fragments) against osteopontin person, such as single-chain fragments of variable regions (scFv), Fab, Fab' and F (ab')2containing the variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO:1, and the variable region of the light chain consisting of the amino acid sequence of SEQ ID NO:3, and stores activities.

The linker for connecting variable region (VH) of the heavy chain and the variable region (VL) light chain, which can be used to obtain scFv, is not subject to restrictions, provided that the fragment of the antibody of the present invention may have the above-described x is perform; for example, you can specify a peptide consisting of the amino acid sequence represented by GGGGSGGGGSGGGGS (SEQ ID NO:14). On the basis of the present invention, the experts in this field can get merged antibody from gumanitarnogo antibodies against osteopontin person or fragment of the antibody and another peptide or protein, and get the modified antibody with its associated modifier tool. Another peptide or protein used for the merge, is not subject to restrictions, provided that it does not reduce the activity of the antibody binding sites; for example, you can specify serum albumin human, different peptide tags, artificial peptides spiral motifs, maltose binding protein, glutathione-S-transferase, various toxins, other peptides or proteins capable of activating polymerization etc. Modifying the tool used for the modification is not subject to restrictions, provided that it does not reduce the binding activity of antibodies, for example, you can specify the glycol chains of sugars, phospholipids, liposomes, low molecular weight compounds, etc.

Humanitariannet antibody against osteopontin man of the present invention, thus obtained, or a fragment of an antibody that retain activity by antibody fused antibody resulting from the merger of the antibody or fragment of an antibody to a peptide or other protein or a modified antibody comprising the antibody or antibody fragment and associated modifying means further purified in accordance with the requirement, you can get as a pharmaceutical preparation in a standard way and can be used for the treatment of rheumatoid arthritis, such forms of rheumatism, as juvenile rheumatoid arthritis and chronic rheumatism, psoriatic arthritis, psoriasis, etc. for the suppression of malignant tumors and chronic transplant rejection after organ transplantation and for the treatment of autoimmune diseases, such as osteoarthritis, systemic autoimmune disease, erythematous, uveitis, Behcet's disease, dermatomyositis, glomerulonephritides jade and sarcoidosis.

Humanitariannet antibody against osteopontin man in the present invention preferably can be used as a therapeutic agent in the treatment of rheumatism, therapeutic agent for autoimmune disease, a therapeutic agent for osteoarthritis or rheumatoid arthritis, more preferably as a therapeutic agent for rheumatoid arthritis. As examples of dosage forms for therapeutic remedy for rheumatism and so on, you can get a parenteral preparation such as an injection or to pelna infusion, and preferably by intravenous injection, subcutaneous injection and the like (the same applies therapeutic agent for autoimmune disease). Upon receipt of a pharmaceutical product, you can use the media and additives that meet these medications within a pharmaceutically acceptable range.

The number gumanitarnogo antibodies against osteopontin person added to the above-described receiving the drug varies depending on the severity of symptoms and the patient's age, used the drug dosage form or title of the binding of recombinant inhibitory antibodies against OPN and the like; for example, you can use from about 0.1 mg/kg to 100 mg/kg

Regarding therapeutic agent thus obtained, the active ingredient gumanitarnogo antibodies against osteopontin man of the present invention strongly binds to the RGD sequence and the sequence SVVYGLR OPN (SEQ ID NO:10), inhibiting the binding between this plot OPN and integrin, leading to the suppression of acute symptoms of rheumatism and rheumatoid arthritis and other autoimmune diseases.

As humanitariannet antibody against osteopontin man in the present invention binds specifically with Stour is Noah OPN, and not with the party integrin, very unlikely that it inhibits any other important function of integrin, and expect to avoid the consequences of adverse reactions.

In addition, humanitariannet antibody against osteopontin man in the present invention can also be used as a diagnostic reagent in rheumatoid arthritis. As stated above, it is proved that in the joints of patients with rheumatoid arthritis find high concentrations tsepliaeva thrombin N-terminal fragment of OPN. Thus, there may be measuring in the sample the number of OPN or its N-terminal fragment using this gumanitarnogo antibodies against osteopontin man in the diagnosis of rheumatoid arthritis. As a way you can use different methods used for standard immunochemical assays, such as the way radioisotope immunoassay (method RIA), ELISA method (E. Engvall et al., (1980): Methods in Enzymol., 70, 419-439), a method using a fluorescent antibody method plaques way spots, the way agglutination and method according to Ouchterlony ("Hybridoma Method and Monoclonal Antibodies", published by R&D Planning, pages 30-53, March 5, 1982).

Although among the above described ways, from different points of view, you can choose one suitable, the ELISA method is preferred in terms of sensitivity, Udo the CTB, etc. As an example of a more preferred method, for example, humanitariannet antibody against osteopontin man of the present invention is fixed on the carrier, an antibody that recognizes a different site on OPN than that recognizes humanitariannet antibody against osteopontin man of the present invention, mark, resulting detect OPN or its N-terminal fragment, and this can be used as a diagnostic reagent in rheumatoid arthritis.

As aiming substance used for labeling the above-described antibodies, you can specify proteins/peptides for the formation of a chimeric protein/peptide, such as glutathione-S-transferase, enzymes such as horseradish peroxidase (hereinafter referred to in this document, referred to as "HRP) and alkaline phosphatase (hereinafter referred to in this document, referred to as "AP"), fluorescent compounds such as fluorescein isocyanate and rhodamine, radioactive substances, such as32P and125I, and modifying means, such as a chemiluminescent substance.

For example, in relation to the method detection OPN isoforms, detection, can be performed with well-known in this field means, such as a sandwich or, more specifically, by using the same method as the method of detection described in WO 02/081522 (patentlydecision 2) or WO 03/027151 (patent document 3).

The present invention also relates to a gene that encodes the antibody of the present invention or its fragment, containing his expressing vector. Expressing vector of the present invention is not subject to restrictions, provided that he is able to Express the gene that encodes the antibody of the present invention or its fragment in different cells of the host prokaryotic cells and/or eukaryotic cells, and to produce these polypeptides. For example, you can specify plasmid vectors, viral vectors (e.g. adenoviral, retroviral), etc.

Expressing vector of the present invention may contain a gene that encodes the antibody of the present invention or its fragment, and a promoter functionally linked to a gene. As a promoter for expression of the polypeptide of the present invention in bacteria, when the host is a bacterium of the genus Escherichia, for example, you can specify the Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, the tac promoter, etc. as a promoter for expression of the antibodies of the present invention or its fragment in yeast, for example, you can specify the PH05 promoter, PGK promoter, GAP promoter and ADH promoter; when the host is a bacterium of the genus Bacillus, you can specify the promoter SL01, SP02 promoter, penP promoter and the like, When is sainam is eukaryotic cell such as mammal cells, you can specify a promoter derived from SV40, retroviral promoter, the promoter of heat shock proteins, etc.

When the host cell using bacteria, especially Escherichia coli expressing vector of the present invention may further comprise initiating codon, stop codon, a termination region, and the unit of replication. When the owner uses yeast, animal cell or insect cell expressing vector of the present invention may contain the initiating codon and a stop codon. In this case, it may contain enhancer sequence, non-coding region at the 5'-end and 3'end of the gene that encodes the polypeptide of the present invention, portions of the connection splicing, polyadenylation site or unit of replication, etc. In accordance with the intended use may contain a selective marker (e.g., tetracycline, ampicillin, kanamycin).

The present invention also relates to transforminto, included gene of the present invention. Such a transformant can be obtained, for example, transformation of a host cell expressing vector of the present invention. A host cell used to obtain transformant, is not subject to restrictions, provided that it can match estolate above expressing the vector to be transformed; as examples, you can specify different cells, such as natural cells or artificially derived cell line commonly used in the technical field of the present invention (for example, bacteria (bacteria of the genus Escherichia, bacteria of the genus Bacillus), yeast (genus Saccharomyces, the genus Pichia, and the like), animal cells or insect cells (e.g., Sf9), and so on). The transformation can be known in a standard way.

The present invention also relates to a method of production of the antibodies of the present invention or its fragment, which includes ensuring the expression of the host-cell gene of the present invention, i.e. using a transformant.

Upon receipt of the antibodies of the present invention or its fragment, the transformant can be cultured in a nutrient medium. The culture medium preferably contains a carbon source and a source of inorganic nitrogen or a source of organic nitrogen for growth transformant. As examples of the carbon source, you can specify glucose, dextran, soluble starch, sucrose and the like; as examples of the inorganic source of nitrogen or organic source of nitrogen, you can specify ammonium salts, nitrates, amino acids, aqueous extract of wheat peptone, casein, meat extract, soybean cake, potato extract, etc. Etc is desired, can contain other nutrients (for example, inorganic salts (e.g. calcium chloride, sodium dihydrophosphate, magnesium chloride), vitamins, antibiotics (such as tetracycline, neomycin, ampicillin, kanamycin and the like) and the like).

The cultivation of transformant can be known in a standard way. Culturing conditions, such as temperature, pH of the medium and time of cultivation choose accordingly. For example, when the host is a cell of the animal, as the environment you can use the MEM medium containing about 5 to 20% fetal calf serum (Science, vol.122, p.501, 1952), DMEM medium (Virology, vol.8, p.396, 1959), medium RPMI-1640 (J. Am. Med. Assoc., vol.199, p.519, 1967), medium 199 (Proc. Soc. Exp. Biol. Med., vol.73, p.1, 1950), etc. pH of the medium is preferably about 6 to 8, cultivation generally can be conducted at a temperature from about 30°to 40 ° C for about 15 to 72 hours, and the culture can be subjected to aeration or stirring as necessary. When the host is an insect cell, for example, you can specify an environment of Grace, containing fetal calf serum (Proc. Natl. Acad. Sci. USA, Vol.82, p.8404, 1985), and the like, and its pH is preferably from about 5 to 8. Cultivation generally can be conducted at a temperature of from about 20 to 40°C for 15 to 100 hours, and the culture can be subjected to aeration or paramesh the VAT as appropriate. For example, when the host is a bacterium, actinomyces, yeast or filamentous fungus, is suitable liquid medium containing the above-described power sources. It is preferable environment with a pH from 5 to 8. When the host is E. coli, as the preferred environment, you can specify the LB medium, M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p.431, 1972), etc. In this case, the cultivation generally can be conducted at temperatures from 14 to 43°C for about 3 to 24 hours, by aeration or mixing of the culture as needed. When the host is a bacterium of the genus Bacillus, cultivation generally can be conducted at a temperature of from 30°to 40 ° C for about 16 to 96 hours by aeration or mixing of the culture as needed. When the host is yeast, as examples environment, you can specify a minimal environment of Burkholder (Bostian, Proc. Natl. Acad. Sci. USA, vol.77, p.4505, 1980), and the pH is preferably from 5 to 8. Cultivation generally can be conducted at a temperature of from 20°to 35 ° C for approximately 14 to 144 hours, and the culture can be subjected to aeration or shaking as necessary.

The antibody of the present invention or its fragment can be obtained, preferably separating and purifying from the culture of transformant as described above. As examples of the JV is soba isolation and purification, you can specify the methods based on differences in solubility, such as salting out and the deposition solvent; methods based on differences in molecular weight such as dialysis, ultrafiltration, gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; methods based on differences in electric charge, such as ionoobmennaya chromatography and chromatography on hydroxyapatite; methods based on specific binding, such as affinity chromatography; methods based on differences in hydrophobicity, such as high performance liquid chromatography with reversed phase; methods based on differences in isoelectric point such as isoelectric focusing and so

In General, the present invention described above; the following examples are listed to facilitate understanding of the invention, which, however, is provided only as an illustration and in no case do not limit the scope of the invention.

EXAMPLES

Below are examples. If not stated otherwise, the methods, including the use of set and so on, carried out in accordance with the designated Protocol.

1.Getting gumanitarnogo antibodies 2K1

In the present invention have two kinds gumanitarnogo antibodies against osteopontin through humanizes and antibodies 2K1, which is an antibody against osteopontin person obtained from the mice described in International Patent Publication Official Gazette WO 2003/027151 (hereinafter referred to in this document also referred to as humanitariannet 2K1 antibody or antibody R2K1).

Since each humanitariannet 2K1 antibody was obtained, as a rule, in accordance with the method described in the above official Gazette, a brief description is presented below.

First by PCR using synthetic oligonucleotides were obtained DNA, which encodes the variable region of the heavy chain (VH) 2 types of humanized antibodies against OPN, having the base sequence represented in figure 1 and figure 2, and the DNA that encodes the variable region of the light chain (VL) 2 types of humanized antibodies against OPN, having the base sequence represented in figure 3 and figure 4. Below in the description, in order to distinguish them, VH gumanitarnogo antibodies against OPN person presented on figure 1 and figure 2 indicate how R2K1-VH1.7 and R2K1-VH1.8, respectively. Similarly, VL gumanitarnogo antibodies against OPN man presented in figure 3 and figure 4, is designated as R2K1-VL1.7 and R2K1-VL1.8, respectively.

Further, each of the above-described DNA, which encodes the VH gumanitarnogo antibodies against OPN person, embedded in the AG-γ1, which is expr shiroudi vector, contains the gene for the constant region γ1-chain of human immunoglobulin, using a plot of recognition restriction endonuclease HindIII and the site of the BamHI recognition, through which he obtained a plasmid expressing a heavy chain having R2K1-VH1.7, and a plasmid expressing a heavy chain having R2K1-VH1.8. Similarly, each of the above-described DNA, which encodes the VL gumanitarnogo antibodies against OPN person embedded in the AG-κ, which is expressing a vector containing the gene for the κ chain constant region of human immunoglobulin, whereby a received plasmid expressing the light chain with R2K1-VL1.8, and a plasmid expressing the light chain with R2K1-VL1.7. These expressing plasmids were introduced and amplified in Escherichia coli and purified using a commercially available kit for plasmid purification (QIAGEN Company).

As a result, various combinations of the above-described purified expressing plasmids were subjected to transfection into cells CHO-DG44 method using calcium phosphate, and the cells were subjected to selection on medium MEM (Invitrogen Company), containing geneticin (Invitrogen Company) and detalizirovannoi fetal calf serum (Invitrogen Company), thus, the received cells expressing two types gumanitarnogo 2K1 antibody. That is expressionlist antibody R2K1v1.8 to the / establishment, which is humanitariannet 2K1 antibody, consisting of a heavy chain having R2K1-VH1.8 and light chain with R2K1-VL1.8, and antibody R2K1v1.7, which is humanitariannet 2K1 antibody comprising a heavy chain having R2K1-VH1.7 and light chain with R2K1-VL1.7.

Cells producing each antibody R2K1, obtained by the methods described above were allowed to grow in MEM medium, supplemented with 10% detalizirovannoi fetal calf serum, were sown in roller flasks (BD Biosciences Company) and were cultured in 37°C and the rotation speed of 1 rpm a Few days later, it was found that cells adhere and grow on the wall of the vessel, the culture fluid was decanted, changed the environment in 500 ml of free serum medium MEM, and granulosa cells under the above conditions. After about 2 weeks, when many cells are separated from the wall of the vessel suspendirovanie, the cultivation was stopped and filtered culture supernatant through a 0.22 μm filter and got the culture supernatant containing each antibody R2K1.

With these supernatants culture, as starting materials, and using columns with protein A (MILLIPORE Company) and anion-exchange columns (Amersham Company), received a few milligrams of each of the two types of purified gumanitarnogo antibodies, i.e. antibodies R2K1v1.8 and antibodies R2K1v1.7.

In various experiments, op the toboggan below, used purified antibodies obtained as described above. Used Chimera 2K1 antibody (hereinafter referred to in this document also referred to as the C2K1 antibody) was obtained by a method described in the above International Patent Publication Official Gazette WO 2003/027151.

2.The confirmation of the ability of binding to the peptide of osteopontin human ELISA method

Binding activity of each antibody R2K1 and C2K1 antibody with the peptide of osteopontin person (CVDTYDGRGDSVVYGLRS: SEQ ID NO:13) were compared in accordance with the method ELISA Kon et al. (Journal of Cellular Biology, 88:420-432 (2002)). The basic principle is presented below.

The peptide having the above-described sequence (hereinafter referred to in this document also referred to as hOPN5 peptide) was reacted with BSA, including maleimido group entered using Sulfo-EMCS (Dojindo Laboratories), to obtain the conjugate hOPN5-BSA. Conjugate hOPN5-BSA was detected at 200 ng/100 μl/well on a tablet for ELISA (Nunc Company) at 4°C overnight and washed the tablet, after which he was assigned blocking PBS, supplemented with 1% BSA at 4°C over night. Sample antibodies, diluted in PBS, supplemented with 1% BSA, was added to the tablet 100 μl/well, and they were reacted at 37°C for 1 hour. Detection was performed using labeled peroxidase (HRP) antibody against IgG (H + L) human (Wako Pure Chemical Industries, Ltd.). The optical density was measured at a wavelength of 450 nm, using citymouse device for microplate (Molecular Devices Company).

The result confirmed that the ability of antibodies R2K1v1.7 and antibodies R2K1v1.8 to contact the peptide hOPN5 equivalent to the ability of the C2K1 antibody binding sites (figure 5).

3.Inhibiting the migration of monocytes in human peripheral blood activity R2K1 antibodies

Inhibitory activity of the purified antibodies against activated by cytokines migration of monocytes in the peripheral blood was examined as described below.

First 2 times were diluted heparinized blood from a healthy person, the medium RPMI-1640. Diluted blood was layered on Fikkal-PAC (Pharmacia K.K.) and centrifuged at 400×g and room temperature for 30 minutes. The white layer located at the interface between the plasma and Fikkal-Pak, was extracted and used as monocytes. Monocytes, thus obtained, were cultured and activated TNF-α, human (20 ng/ml) over night, and used in experiments on migration.

Experiments on the migration was performed using a 48-hole camera for microhematuria (Neuro Probe Inc.). OPN person uncoupled reaction with bovine thrombin (Sigma) at 37°C for 2 hours. Was added to each antibody R2K1 and the C2K1 antibody at various concentrations and the mixture was allowed to remain at 37°C for 15 minutes, after which it was added in the lower chamber (final concentration of OPN person was 10 μg/ml). In ve is hnwu camera was added 50 μl of cell suspension (2×10 6cells/ml) and fixed on her polycarbonate filter (pore size 5 μm).

After cultivation at 37°C in the presence of 5% CO2within 2 hours, the polycarbonate filter was removed, cells on the upper surface of the filter were removed, after which the dyed cells Diff-Quick (Baxter Company). At ×40 magnification was counted the number of cells on the upper surface of the filter and the results were expressed as the average number of cells (cells/mm3) ± SE for six holes (table 1). Based on these results, both antibodies R2K1v1.7 and antibody R2K1v1.8 to tsepliaeva thrombin to osteopontin person, inhibited the migration of activated TNF-α monocytes in human peripheral blood, as in the case of the C2K1 antibody.

Table 1
R2K1v1.7 & R2K1v1.8
The average number of cellsSEM
Wednesday701,724,8
Thr-0PN881,724,0
R2K1v1.7 50 mg/ml723,343,0
R2K1v1.8 0 mcg/ml 688,316,6
C2K1
The average number of cellsSEM
Wednesday686,715,9
Thr-0PN860,030,7
C2K1 50 mg/ml671,748,5

4.Evaluation of heat resistance by ELISA method

Each of the C2K1 antibody and two types of antibodies R2K1 was diluted to 50 μg/ml of PBS and kept in a water bath at 70°C for 2 hours. Thereafter, each solution was brought to room temperature and was depicted as a graph of the ratio of the absorption coefficient obtained by the method described above ELISA, the absorbance of the control sample as a residual activity. The residual activity was calculated using the values of the absorption coefficient lying in the range from 0.2 to 2.0 with linearization (the same applies below). As a result, found that the residual activity after the above treatment was higher for antibodies R2K1v1.7 and antibodies R2K1v1.8 than for the C2K1 antibody (6). In particular, the antibody R2K1v1.7 showed residual AK is Yunosti, greater than 90%. This serves as proof that the antibody R2K1v1.7 and antibody R2K1v1.8 have improved heat resistance compared with the C2K1 antibody.

5. Evaluation of resistance to low pH method ELISA

Each of the purified received the C2K1 antibody and two types of antibodies R2K1 was diluted with PBS to 50 μg/ml of Each solution using 1H. HCl using a pH meter (HORIBA Company), brought to pH 5 and maintained at 25°C for 2 hours. Then, using 1M Tris-HCl (pH 9,5), brought the pH up to 7 and depicted in the form of a graph of the relationship of the absorption coefficient obtained by the method described above ELISA, the absorbance of the control sample as a residual activity. As a result, it was found that after the above processing, the residual activity was significantly higher for antibodies R2K1v1.7 than for the C2K1 antibody and antibodies R2K1v1.8 (Fig.7). This serves as proof that the antibody R2K1v1.7 have improved resistance to conditions with low pH levels, compared with the antibody R2K1v1.8 and the C2K1 antibody.

6.Evaluation of resistance to guanidine hydrochloride by fluorescence spectrometry

Each of the C2K1 antibody and two types of antibodies R2K1, using 20 mm sodium phosphate buffer + 120 mm NaCl (pH 7)containing different concentrations of guanidine hydrochloride (control guanidine hydrochloride not add recipients who do), brought to 50 μg/ml and allowed to remain at 10°C during the night, and then measured the fluorescence spectrum for each sample. Measurement of the fluorescence spectrum was performed using a spectrophotometer FP-6500 (JASCO Company). Using the cell with the length of the light path 3 mm, scanned fluorescence emitted by the tryptophan excited by light of 280 nm, in the wavelength range from 320 nm to 370 nm. Among antibodies compared the relationship between the concentration of guanidine hydrochloride and peak wavelength. As a result, observed the shift of the peak wavelength due to the loss of spatial structure of a protein from a point of time where the concentration of guanidine hydrochloride has just exceeded 1 M for C2K1 or 2 M for R2K1v1.8, whereas the peak wavelength shifted to 3.8 M for R2K1v1.7 (Fig). This serves as proof that the antibody R2K1v1.7 has improved resistance to guanidine hydrochloride compared with the antibody R2K1v1.8 and the C2K1 antibody.

7.Evaluation of resistance to low pH by CD

Each of the C2K1 antibody and antibody R2K1v1.7 with 20 mm citrate buffer + 120 mm NaCl (pH 6) was brought to 2 mg/ml There was added 0.1 G. of HCl and distilled water to prepare samples with different pH, with the concentration of antibody 1 mg/ml; after processing was measured CD spectrum of each sample at room temperature is PE within 1 hour.

Dimension CD (circular dichroism) was performed using spectropolarimeter J-820 (JASCO Company). Using the cell with the length of the light path of 0.1 mm, was measured CD spectrum in the wavelength range from 205 nm to 260 nm. When spectral analysis software has been used to model the secondary structure of the protein JWSSE-480 (JASCO Company), which is based on spectral analytical method CD Yang et al. (Methods in Enzymology, 130, 208-269 (1986)). Among antibodies compared the relationship between the ratio of the content of random structures, calculated by this method and by processing pH. As a result, the content of random structures was increased from pH 3 to C2K1 antibody, where not observed to increase the content of random structures, to pH 2.7 for R2K1v1.7 (Fig.9). This confirms that the antibody R2K1v1.7 resistant to pH 0.3 compared with the resistance to pH C2K1 antibody.

8.Evaluation of thermal stability using differential scanning calorimeter

Each of the C2K1 antibody and antibody R2K1v1.7 was dissolved in 20 mm citrate buffer + 120 mm NaCl (pH of 6.0) to a concentration of 1 mg/ml and investigated its thermal stability using MicroCal Company ultracentrally differential scanning calorimeter (VP capillary DSC platform). The results are shown in figure 10. The average transition temperature (Tm), which shows the temperature of denato the promotion of a more complex structure, was 76,0°C for C2K1 antibody and 82.8°C for antibodies R2K1v1.7; confirmed an increase of approximately 6°C. This is evidence that the antibody R2K1v1.7 has a remarkably improved heat resistance.

9.Inhibition of cell adhesion action R2K1v1.7 on OPN

For comparison of the pharmacological effects R2K1v1.7 of the present invention of this application and known gumanitarnogo antibodies against OPN (see WO 03/027151; hereafter in this document referred to as R2K1v0), investigated the inhibiting cellular adhesion effects of these two antibodies to OPN person.

1. Cultivation and the passage of cells

Cells Jurkat E6.1 purchased from Dainippon Pharmaceutical Co., Ltd. and passively and were cultured using RPMI-1640 (10% FCS, penicillin-streptomycin).

2. The preparation of reagents

Buffer for adhesion (medium L-15, 1% BSA, 50 mm HEPES, pH 7,4)

a solution of PMA (40 ng/ml phorbol-12-myristate-13-acetate (PMA) [SIGMA] in the buffer for adhesion)

a coloring solution CV (with 0.5% crystal violet, 1% formamide, 20% methanol)

the solution GST (5 µg/ml of glutathione-S-transferase (GST) [SIGMA] in PBS (-))

A solution of IgG1man (400 μg/ml in PBS (-)) [CALBIOCHEM]

3. Getting tsepliaeva thrombin N-terminal of osteopontin (OPN) man fused with GST ottsepleny thrombin N-terminal of osteopontin person (GST - N-OPN person, 1.6 mg/ml) prepared as described in WO 02/081522, and using the in experiments after dilution in PBS (-) to 5 μg/ml

4. Getting tested medicines

Each of R2K1v1.7 (18.6 mg/ml) and R2K1v0 (4,39 mg/ml) diluted in PBS (-) to 4, 12, 40, 120, and 400 (μg/ml); to obtain total protein at a concentration of 400 μg/ml in all of these diluted solutions were added IgG1person.

5. Grouping

The only group with a token (GST)

The control group

The group tested medicines R2K1v1.7(1, 3, 10, 30, 100 µg/ml)

R2K1v0(1, 3, 10, 30, 100 µg/ml)

6. Experiments with cell adhesion

To all wells of 96-hole microplate, in addition to holes only with marker, was added 25 μl of a solution of GST-N-OPN person, or added 25 μl of a solution of GST to the group only with the token, and the plate is incubated at 37°C for 1 hour, after which the tablet twice washed with PBS (-). Added 50 μl of a solution of PMA and the plate is incubated at 37°C for 30 minutes, after which was added 25 μl of a solution of the test drug (group test drug) or a solution of IgG1man (the only group with a token and a control group). Cells Jurkat E6.1 suspended in the buffer for adhesion to obtain the density of cells 2×106cells/ml in each well was added 25 μl. The suspension was centrifuged at 15×g for 1 minute to precipitate the cells on the bottom of the tablet, then the tablet incubated at 37°C for 1 hour. After the reaction PLA is Shet turned and was centrifuged at 47×g for 2 minutes and remove supernatant (not adherent cells). To count the number of non-adherent cells, was added 25 μl of staining solution CV, allow the tablet to remain at room temperature for 10 minutes for staining and fixation of the cells, after which the tablet three times washed with clean water, into each well was added 25 μl of 1% solution of Triton-X100 and after solubilization of the cells was assessed a result, the ratio of the absorption wavelength was measured at 595 nm) was measured using a card reader for microplates (SPECTRAmax250, Molecular Devices).

7. Analysis

In the experiments used 5 holes on the group. Calculate the average value of the absorption coefficient and the degree of suppression for each group was calculated IC50values (concentration of test drug for 50% of the degree of suppression). The degree of suppression for a free group was defined as 100%, and for the control group was defined as 0%. The value of the IC50was calculated by plotting the logarithmic concentrations of the tested drugs on the X-axis and the degree of suppression on the Y-axis, and the method of least squares gave the data to the linear regression equation. Calculate the values of the IC50using data obtained by testing concentrations of drugs, showed a linear dose-effect. Based on the results presented figure 11, was the clear that inhibits cell adhesion effect is commonly known gumanitarnogo antibodies against OPN person is extremely low, whereas R2K1v1.7 has a very high inhibiting cellular adhesion effect (IC50size: 6,4).

10.Effects R2K1v1.7 on collagen-induced arthritis in cynomolgus macaques

Female cynomolgus macaques for 36 days before treatment were immunized in the backs and tails of bovine collagen type II (collagen Gijyutsu Kenshukai) in the emulsion in complete Freund's adjuvant (Becton Dickinson and Company), and injected with a booster dose for 15 days prior to administration of the drug. Animals were randomly divided into three treated with the medication group (n=10) based on the percentage change in body weight and oblong areas proximal interphalangeal joints compared with levels before immunization. R2K1v1.7 or solvent, as a control, was injected at a dose of 25 mg/kg or 50 mg/kg intravenous injecting once a week for a total of eight times. The first day of injection was defined as day 0. A day 0, 6, 13, 20, 27, 34, 41, 48 and 55 during the period of introduction, as a characteristic swelling of the joint, watched elongated region of the proximal interphalangeal joint. The major and minor axes of articulation of the proximal interphalangeal joints of the front and hind legs was measured using calipers, Merali oblong region, and the average oblong areas 16 of the fingers was used as the value of the oblong region of the proximal interphalangeal joint. The percentage change in an elongated region of the proximal interphalangeal joint was calculated by taking the value before administration of the drug as 100. At day 0 and day 6, 13, 20, 27, 34, 41, 48 and 55 (6 days after administration of the drug), was collected and plasma was measured R2K1v1.7 and antibody against R2K1v1.7. These measured plasma concentration R2K1v1.7 relate to the minimum level. Analysis of data was performed after removal of data about anti-R2K1v1.7 antibody-positive animals and animals that died during the study period.

In 1 animal in the group dosing R2K1v1.7 25 mg/kg and 4 animals in group dosing R2K1v1.7 50 mg/kg, produced antibody against R2K1v1.7. Two animals in the control group with the solvent, 2 animals in group dosing R2K1v1.7 25 mg/kg and 1 animal in the group dosing R2K1v1.7 50 mg/kg died after drug administration. Deaths attributed to the General depletion due to severe inflammation. Treatment with 50 mg/kg R2K1v1.7 significantly reduced the swelling of the foot, as measured by the percentage change in an elongated region of the proximal interphalangeal joint in comparison with the control group with the solvent, from 27 days to 55 days (Fig). R2K1v1.7 at a dose of 25 mg/kg not been the al to have a significant influence on the change in an elongated region of the proximal interphalangeal joint. The minimum concentration R2K1v1.7 plasma at doses of 25 mg/kg and 50 mg/kg ranged from 38,41 to 76,13 µg/ml and from 73,91 to 125.3 mg/ml, respectively. The sequence SVVYGLR OPN person, unlike the corresponding sequence OPN monkeys (SVAYGLR) (SEQ ID NO:11), the binding affinity of R2K1v1.7 with this peptide OPN person more than 100 times higher than the affinity of binding with the corresponding peptide OPN monkeys. Given these data, the effective concentration R2K1v1.7 plasma in the treatment of arthritis presumably not more than 100 μg/ml.

11.Getting scFv R2K1v1.7

By PCR with the above described plasmid expressing a heavy chain having R2K1-VH1.7, and a plasmid expressing the light chain with R2K1-VL1.7 as the matrix obtained DNA fragment, which encodes a single-chain fragment variable region (scFv)having the structure VH1.7-linker-VL1.7 (linker was the sequence of bases that encodes the amino acid sequence represented by GGGGSGGGGSGGGGS (SEQ ID NO:14)). Built in the end of this DNA fragment is the sequence recognized by the restriction endonuclease SfiI and NotI. This DNA fragment was digested with restriction endonuclease SfiI and NotI and was built in plot SfiI and NotI site of the vector pCANTAB5E (Marks, J.D., et. al., J. Mol. Biol., vol.222, p581-97, 1991), also pre-split with what omashu SfiI and NotI, resulting received expressyour R2K1-VH1.7 scFv plasmid. In expressing this plasmid, the sequence of bases that encodes E-Tag, enter the following coding region of scFv. This plasmid accepted way is introduced into the strain HB2151 E. coli and plated on the tablet agar SOBAG (tablet SOB containing 2% glucose and 100 μg/ml ampicillin) to obtain a clone of transformants. From the obtained clone was isolated plasmid DNA; sequence coding region of scFv was confirmed by sequence analysis of DNA bases using plasmid DNA as template. When analyzing the sequence of DNA bases used set DTCS-Quick Start and system analysis DNA CEQ2000XL (both from Beckman Coulter, K.K.). The resulting sequence of bases is presented in SEQ ID NO:9.

After the clone of Escherichia coli, whose sequence of bases was confirmed to be cultivated with the use of 2xYT medium containing 2% glucose and 100 μg/ml ampicillin, part of it suspended in 2xYT medium, supplemented with 1 mm IPTG and 100 μg/ml ampicillin, and cultured overnight for the induction of expression of scFv. After cultivation, the cells were isolated by centrifugation, suspended in PBS containing 1 mm EDTA, and allowed to remain on ice for 30 minutes. Next, the suspension was centrifuged at 10,000 rpm for 15 minutes, and superna the ant was collected and filtered through a 0.45 µm filter, what has been the faction of periplasm containing scFv. scFv R2K1v1.7 (hereafter in this document referred to as R2K1v1.7-scFv) was purified from this fraction periplasm by affinity chromatography using an antibody against E-Tag.

Thus obtained R2K1v1.7-scFv were subjected gelfiltration chromatography; on the basis of pattern separation, presented at Fig confirmed that almost all were monomers.

12.The confirmation of the ability to bind R2K1v1.7-scFv with the peptide of osteopontin person

Binding activity of purified R2K1v1.7-scFv with peptide hOPN5 was measured by ELISA method. In General, the method was the same as described above; in this dimension as labeled antibodies used HRP-labeled antibody against E-Tag. The results are presented on Fig. Confirmed that the purified R2K1v1.7-scFv was not associated with negative control BSA, but was associated in particular with the peptide hOPN5.

13.Receiving a modified polyethylene glycol fragment antibodies

After antibody R2K1v1.7 was ipsilaterally standard way, has been purified F(ab')2using HP column with protein G (both from Amersham Biosciences K.K.) and high-resolution column Hi prep 16/60 with sephacryl S-200 (Amersham Biosciences K.K.). Further purified F(ab')2restored 0.1 M DTT to activate Tilney group, and then to remove DTT Prov the Dili gel-filtration, using a column with Sephadex G-25 (Amersham Biosciences K.K.). Fab'obtained in this way was mixed with multimediaand polyethylene glycol SUNBRIGHT ME-120MA (NOF Corporation) in a molar ratio of 1:10 and allowed to remain at 4°C over night for the induction of binding assays. After adding iodoacetamide (Nacalai Tesque) to stop binding assays, has been modified with polyethylene glycol F(ab')2(later in this document also referred to as F(ab')2-PEG (F(ab')2-PEG)) by gel filtration using high-resolution column Hi prep 16/60 with sephacryl S-200. The results of SDS-PAGE presented on Fig. Compared to the unmodified F(ab')2subjected to electrophoresis for standard control, confirmed the increase of molecular weight by modifying polyethylene glycol.

14.Confirmation of binding activity of the F(ab')2-PEG with the peptide of osteopontin

Binding activity of purified F(ab')2-PEG R2K1v1.7 with peptide hOPN5 confirmed using the method of surface plasmon resonance. Biokinesiology peptide hOPN5 recorded on the sensor chip SA (BIAcore Company), and its binding activity was confirmed using F(ab')2-PEG, pre-diluted HBS-EP buffer(BIAcore Company) to 5 μg/ml; the results are presented on Fig. On the basis of ascending signal, confirmed that this F(ab') 2-PEG has the same activity of binding to the peptide hOPN5 that the activity of binding R2K1v1.7 with peptide hOPN5.

Industrial application

As humanitariannet antibody against osteopontin man according to the present invention has a very high activity (antigennegative activity, inhibiting leukocyte migration activity and the like) and/or stability (resistance to heat, low pH, denaturing means and the like), it is applicable as a more effective drug than standard antibody against osteopontin man for the prophylaxis or treatment of various inflammatory diseases, including autoimmune disease, rheumatism, rheumatoid arthritis and osteoarthritis.

Although the present invention is described by means of preferred embodiments, the experts in this field it is obvious that the preferred options for implementation can be modified. The present invention is intended that the present invention can be implemented in ways different from the ways, described in detail in the present description. Thus, the present invention applies to all modifications included in the scope not changing the essence of the claimed in the claims.

This application is based on patent for which WCA No. 2006-152892 registered in Japan, and thus its contents are fully incorporated herein by reference. In addition, the content of any information provided in this description publications, including patents and patent applications, thus, fully incorporated by reference in full.

1. Humanitariannet antibody against osteopontin person containing the variable region of the heavy chain consisting of the amino acid sequence of SEQ ID N0:1 and variable region light chain comprising amino acid sequence SEQ ID NO:3.

2. Humanitariannet antibody against osteopontin man according to claim 1, where the constant region of the heavy chain antibody is a Igγ1 person.

3. Humanitariannet antibody against osteopontin man according to claim 1, where the constant region of the light chain of the antibody is a human Igκ.

4. Humanitariannet antibody against osteopontin man according to claim 1, where the constant region of the heavy chain antibody is a Igγ1 man, and the constant region of the light chain of the antibody is a human Igκ.

5. Humanitariannet antibody against osteopontin person, containing a heavy chain consisting of the amino acid sequence of SEQ ID NO:25 and a light chain consisting of the amino acid sequence of SEQ ID NO:27.

6. Paul is a nucleotide, containing a sequence that encodes the variable region of the heavy chain gumanitarnogo antibodies against osteopontin man according to claim 1.

7. Polynucleotide containing a sequence that encodes a variable region light chain gumanitarnogo antibodies against osteopontin man according to claim 1.

8. Expressing the vector containing polynucleotide described in claim 6 and/or 7.

9. A host cell containing expressing the vector of claim 8.

10. The method of obtaining gumanitarnogo antibodies against osteopontin person, including the stage of culturing the host cell according to claim 9, and, thus, the expression of cell gumanitarnogo antibodies against osteopontin person.

11. Drug for the treatment of autoimmune diseases, rheumatism, rheumatoid arthritis or osteoarthritis, containing humanitariannet antibody against osteopontin man according to any one of claims 1 to 5.

12. The method of prevention or treatment of autoimmune diseases, rheumatism, rheumatoid arthritis or osteoarthritis, comprising the stage of introducing a therapeutically effective amount gumanitarnogo antibodies against osteopontin man according to any one of claims 1 to 5.

13. Application gumanitarnogo antibodies against osteopontin man according to any one of claims 1 to 5 to obtain pharmaceuticals, despropylation or treatment of autoimmune diseases, rheumatism, rheumatoid arthritis or osteoarthritis.



 

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6 cl, 5 dwg, 2 tbl, 8 ex

FIELD: food industry.

SUBSTANCE: mixture for baby feeding contains a source of proteins based on milk whey, casein and their mixture as well as based on soya in an amount of no more than 2.0 g/100 kcal, a source of lipids, a source of carbohydrates and a probiotic or a mixture of probiotics. The probiotic or the mixture of probiotics is/are represented by strain(s) preferably chosen from Lactobacillus and/or Bifidobacterium genus in an amount equivalent to 102-105 CFU/g of the dry mixture. The source of lipids and carbohydrates is suitable for use in baby mixtures.

EFFECT: possibility for application of the mixture for newborn baby immune system modulation for initiation of development of useful digestive microbiota (in the initial few weeks of baby life) comparable to microbiota of babies fed with breast milk as well as for stimulation of maturation of newborn baby immune system in the initial few weeks of baby life.

12 cl, 1 tbl, 2 ex

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