Anti-mif antibodies

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

 

The technical FIELD

The present invention relates to monoclonal antibodies and their antigen-binding portions that specifically bind To C-terminal part or the Central region of factor inhibiting macrophage migration (MIF). These anti-MIF antibodies and their antigen-binding portion additionally inhibit the biological function of MIF person. The invention also relates to selected heavy and light chains of immunoglobulins derived from anti-MIF antibodies, and molecules of nucleic acids encoding such immunoglobulins. The present invention also relates to a method of identifying anti-MIF antibodies, pharmaceutical compositions containing these antibodies, and use of these antibodies and compositions for treatment of diseases associated with MIF.

BACKGROUND of the INVENTION

Factor inhibiting macrophage migration (MIF) is a cytokine originally selected due to its ability to inhibit the random migration of macrophages in vitro (Bloom et al. Science, 1966, 153, 80-2; David et al. PNAS 1966, 56, 72-7). Although MIF was known since 1966, its exact function in most cells was not known, but, apparently, MIF is a key regulator operating at the beginning of the cascade of the innate and acquired immune response.

MIF cDNA human was cloned in 1989 (Weiser et al., PNAS 1989, 86, 7522-6), and positioning is of its gene was mapped on chromosome 22. The MIF gene product is an amino-acid protein with a molecular mass of 12.5 KD. Protein is a highly conservative: homology in sequence between MIF human, mouse, rat and ox is 90-96%. However, other proteins MIF has no significant homology. Three dimensional structure of the MIF is not similar to any other cytokine or pituitary hormone. The protein crystallizes in the form of a trimer of identical subunits. Each monomer contains two antiparallel alpha-helix, which is Packed against four parallel beta-layers. The monomer has two additional beta layer that interact with beta-layers adjacent subunits, forming the boundary between the monomers. Three beta-layer are arranged such that they form a barrel, including accessible to solvent channel passing through the center of the protein along the molecular symmetry axis of the third order (Sun et al. PNAS 1996, 93, 5191-5196).

It was reported that the secretion of MIF macrophages induce very low concentrations of glucocorticoids (Calandra et al. Nature 1995, 377, 68-71). However, as the Pro-inflammatory cytokine MIF also counteracts the effects is a counter-regulator of glucocorticoid and stimulates the secretion of other cytokines, such as tumor necrosis factor TNF-α and interleukin IL-1β (Baugh et al. Crit Care Med 2002, 30, S27-35), suggesting its involvement in Pato is enese inflammatory and immune diseases. MIF is also directly associated with the growth of lymphoma, melanoma and colon cancer (Nishihira et al. J Interferon Cytokine Res. 2000, 20:751-62).

MIF is a mediator of many pathological conditions, and, therefore, is associated with many diseases, including inflammatory bowel disease (IBD), rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), asthma, glomerulonephritis, IgA nephropathy, cancer, myocardial infarction (MI) and sepsis.

Against recombinant human MIF were obtained polyclonal and monoclonal anti-MIF antibodies (Shimizu et al., FEBS Lett. 1996, 381, 199-202; Kawaguchi et al., J. Leukoc. Biol. 1986, 39, 223-232; Weiser et al., Cell. Immunol. 1985, 90, 167-78).

In therapy, it was suggested to use anti-MIF antibodies to inhibit the release of TNF-α. It was published that the group Calandra et al. (J. Inflamm. 1995. 47, 39-51) used antibodies to MIF for the protection of animals from septic shock, experimentally caused by gram-positive and gram-negative bacteria. It was suggested to use anti-MIF antibodies as a therapeutic agent to modulate the production of cytokines in septic shock and other inflammatory diseases.

In U.S. patent No. 6645493 disclosed monoclonal anti-MIF antibodies obtained from cell hybridoma that neutralize the biological activity of MIF. In animal models it can be shown that these murine anti-MIF ant the body have a useful effect in the treatment of endotoxin shock. Some of the described antibodies to MIF (III. D.9 XIV.14.3 and XIV.15.5) was used in the present invention for comparative experiments.

In the US 2003/0235584 disclosed methods of obtaining high-affinity antibodies to MIF with animals that have held homozygous knockout MIF gene.

In 1994, the group Galat et al. was described factor, which inhibits glycosylation (GIF) (Eur. J. Biochem. 1994, 224, 417-21). At the moment it is known that MIF and GIF are identical. Group Watarai et al. (PNAS 2000, 97, 13251-6) were described polyclonal antibodies that bind different epitopes GIF, to identify the biochemical nature of post-translational modification GIF in Ts cells. Group Watarai et al. (PNAS 2000, 97, 13251-6) was published that GIF is present in different conformational isoforms in vitro. One type of isomer arises due to the chemical modification of a single cysteine residue. Chemical modification leads to conformational changes in the protein GIF and changes its biological function.

Taking into account the complex role of MIF in various diseases, it is highly desirable to clarify the functions of the epitope-specific antibodies to MIF and their use in therapy. Thus, for the treatment of diseases and conditions mediated MIF required epitope-specific anti-MIF antibodies that inhibit the biological function of MIF person.

A SUMMARY of IZABERETE THE OIA

The present invention relates to antibodies and antigen-binding portions that have been specifically bind C-terminal or Central region of the factor inhibiting macrophage migration (MIF).

The invention additionally relates to nucleic acid molecules coding for these antibodies or their antigen-binding part, and to vectors comprising such nucleic acid, and cell host comprising a vector, and recombinant methods of obtaining polypeptides encoded by nucleic acid molecules.

The invention also relates to pharmaceutical compositions containing anti-MIF antibody or antigen-binding portion. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier or other therapeutic agent.

The invention also relates to the use of anti-MIF antibodies or antigen-binding portion in the manufacture of medicinal products for the treatment of immunological diseases, such as inflammatory diseases and hyperproliferative disorders.

The invention additionally relates to an anti-MIF antibody or antigen-binding portion for use in the treatment of immunological diseases, such as inflammatory diseases and hyperproliferative disorders.

The invention also relates to a method of the treatment of a number of immunological diseases and conditions, such as inflammatory diseases and hyperproliferative disorders, using an effective amount of anti-MIF antibodies or antigen-binding part.

The invention also relates to methods of diagnosis. Anti-MIF antibody or antigen-binding portion can be used for the detection of MIF in a biological sample.

The invention additionally relates to a method of identifying anti-MIF antibodies capable of inhibiting the active MIF and cause beneficial effects in animal models.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the amino acid sequence of the variable region of the light chain of anti-MIF antibodies man according to the invention.

Figure 2 shows the amino acid sequence of the variable region of the heavy chain of anti-MIF antibodies man according to the invention.

Figure 3 shows the DNA sequence and its translation variable region of the light chain of anti-MIF antibodies man according to the invention.

Figure 4 shows the DNA sequence and its translation variable regions of the heavy chain of anti-MIF antibodies man according to the invention.

Figure 5 shows the competitive binding of mouse antibodies III. D.9 relative to a reference antibody (C3) and anti-MIF antibodies, Bax94. When the number of antibodies Wah you can clearly see the competition.

Figure 6 shows that the antibody VH(dotted line) and antibody VH (dotted line) increases survival and delayed time to death in an animal model of peritonitis compared with the control antibody (C3).

7 shows differential binding of antibodies Wah with active and inactive MIF. Antibody VH links active MIF in direct ELISA, while not binding inactive MIF.

On Fig table, which summarizes in vitro properties of anti-MIF antibodies.

Figure 9 shows the proapoptotic effects of the anti-MIF antibodies on the cells. Shows the activity of cellular caspase-3 (effector caspase) after treatment of cells PC-3 antibodies. The analysis was carried out in three repetitions, and data are presented as mean values ± SD (standard deviation).

Figure 10 shows the anti-invasive effects of the anti-MIF antibodies. Explored the invasion of prostate cancer cells PC-3 through the pores coated with Matrigel Transwell inserts™. Counted the number of infiltrated cells in the visible field (microscope at 400-fold magnification). Data are presented as mean ± SD from 3 to 10 counts of cells in the visible field, and also shows significant differences.

DETAILED description of the INVENTION

Definitions and General methods

If this document is not specified, the scientific and technical terms used in relation to the present invention, will have the meaning commonly understood by average experts in this field. Usually described herein items used in relation to the cultivation of cells and tissues,molecular biology, immunology, Microbiology, genetics and chemistry of proteins and nucleic acids (and methods) are well known and commonly used in this field. Ways and methods of the present invention is usually carried out by standard methods, well known in this field and described in a number of General and more specific sources, referenced and described in this specification, unless otherwise specified. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992); and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1990); which is incorporated into this description by reference.

"MIF" or "factor inhibiting macrophage migration" refers to a protein, which is known as a key mediator in the immune and inflammatory response, especially as pin-regulator actions of glucocorticoids. MIF includes MIF mammals, in particular human MIF (primary access number in the database Swiss-Prot: P14174), and whose Monomeric form encoded in the form of a 115-amino acid protein and is produced in the form of a 114-amino acid protein due to the removal of the first methionine. "MIF" also includes "GIF" (factor inhibiting glycosylation) and other forms of MIF, such as fused proteins MIF. Numbering MIF begins with N-terminal methionine (amino acid 1) and ends With-terminal alanine (amino acid 115).

The term "active MIF" refers to the natural conformational isoforms MIF, which are important because of their biological functions. Active MIF include isoforms that are found on the cell surface (such as TNR etc). Active MIF also includes isoforms MIF found in the serum of mammals after bacterial infection.

"Antibody" refers to intact antibody or antigen-binding portion that competes with the intact antibody for specific binding. In General, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference). The term "antibody" includes received genetic engineering methods forms such as chimeric or humanized antibodies.

The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., MIF). The antigen-binding part can be obtained using the techniques of recombinant DNA or enzymatic or chemical cleavage of intact antibodies. The antigen-binding parts include Fab, Fab', F(ab')2, Fv and complementarity determining region (CDR), single-chain antibodies (scFv), chimeric antibodies, diately and polypeptides that contain at least part of the antibody is sufficient for specific binding of the antigen with poly is aptidon. Starting from the N-Terminus and up To the end of both Mature variable domain of the light and heavy chains contain region FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The distribution of amino acids in domains given in accordance with the definitions set forth in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia et. al. J. Mol. Biol. 196:901-917 (1987), or Chothia et al., Nature 342:878-883 (1989). The antibody or antigen-binding portion can be modified or linked to another functional molecule (e.g., another peptide or protein). For example, the antibody or antigen-binding portion may be functionally associated with one or more molecules, such as another antibody (e.g. an antibody with dual specificity or diatel), or detektivami agent, a cytotoxic agent, a pharmaceutical agent and/or linker molecule.

The term "human antibody" refers to an antibody in which the sequence of variable and constant domains are sequences of human rights. The term encompasses antibodies with sequences derived from human genes, but which have been modified, for example, to reduce potential immunogenicity, increasing the affinity removal cysteines that may cause undesired folding of proteins, etc. the Term covers such antibodies, obtained by recombinant method in ledah organisms other than man, which may be a typical human cell glycosylation.

The term "humanitariannet antibody" refers to immunoglobulin chains of immunoglobulins or their fragments, such as Fv, Fab, Fab', F(ab')2or other antigen-binding portion of an antibody)that contain sequences of immunoglobulins organisms other than human.

The term "chimeric antibody" refers to an antibody that contains regions of antibodies of one or more different biological species.

The term "isolated antibody" or "dedicated its antigen-binding portion" refers to the antibody or antigen-binding parts, which have been identified and isolated from the source of the antibody, such as a library of phage display or b-cell repertoire.

The term "KD" refers to the equilibrium constant of dissociation of the Fab-specific antibody with the corresponding antigen.

The terms "Central region" and "C-terminal region MIF relate to the field of human MIF, including amino acids 35-68 and 86-115, respectively.

The term "epitope" includes any protein determinant capable of specifically bind the immunoglobulin or the fragment of the antibody. Epitope determinants usually consist of chemically active surface groupings of molecules such as exposed amine is acid, the amino sugar or other carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

The term "vector" refers to a nucleic acid molecule that can carry another nucleic acid to which it is associated. In some embodiments of the invention the vector is a plasmid, i.e. a circular double-stranded loop of DNA that can be ligitamate additional DNA fragments.

The term "a host cell" refers to a cell line capable of producing recombinant protein after introduction of the expression vector. The term "recombinant cell line" refers to a cell line in which you have entered a recombinant expression vector. It should be understood that "recombinant cell line" means not only the specific cell line, but also the progeny of such a cell line. Because in subsequent generations may experience some modifications due to either mutation or influence of the external environment, such offspring in reality can be neodenticula the parent cell, but are still included in the scope of the term "recombinant cell line"as used in this description.

The term "pharmaceutically acceptable carrier" refers to any and all of rest is ritely, dispersive environments, shells, antibacterial and antifungal agents, isotonic and delaying absorption agents, and the like that are physiologically compatible.

Anti-MIF antibodies

In one embodiment, the invention relates to the selected monoclonal antibody, or antigen-binding portions that specifically bind To C-terminal or Central region MIF person and additionally inhibit the biological function of MIF. In some embodiments the invention, the monoclonal antibody is a monoclonal human antibodies. In other embodiments of the invention, monoclonal antibodies are humanized monoclonal antibodies.

In some embodiments the invention, the light chain of anti-MIF antibody contains amino acid sequence that is identical to the amino acid sequence of the VLantibodies Bax8 (SEQ ID NO: 1), antibodies Bax69 (SEQ ID NO: 2), antibodies Bax74 (SEQ ID NO: 3), antibodies Bax94 (SEQ ID NO: 4), antibodies Bax152 (SEQ ID NO: 5), antibodies BaxA10 (SEQ ID NO: 6), or amino acid sequence that has 85%, preferably 90% homology over a sequence with the specified amino acid sequences. In some embodiments the invention, the light chain contains the amino acid series is here from the beginning of the CDR1 to the end of the CDR3 of any one of these antibodies. In some embodiments the invention, the light chain of anti-MIF antibody contains at least CDR1, CDR2 or CDR3 light chain amino acid sequence shown in figure 1.

In some embodiments the invention, the heavy chain contains the amino acid sequence of the variable domain (VH) antibodies Bax8 (SEQ ID NO: 7), antibodies Bax69 (SEQ ID NO: 8), antibodies Bax74 (SEQ ID NO: 9), antibodies Bax94 (SEQ ID NO: 10), antibodies Bax152 (SEQ ID NO: 12), antibodies BaxA10 (SEQ ID NO: 12), or amino acid sequence that has 85%, preferably 90% homology over a sequence with the specified amino acid sequences. In some embodiments the invention, the heavy chain contains the amino acid sequence from the beginning of the CDR1 to the end of the CDR3 of any one of these antibodies. In some embodiments the invention, the heavy chain of anti-MIF antibody contains at least CDR1, CDR2 or CDR3 of the heavy chain amino acid sequence shown in figure 2.

The class and subclass of anti-MIF antibodies

Anti-MIF antibody according to the invention represents a selected monoclonal antibody. Anti-MIF antibody may be a molecule of IgG, IgM, IgE, IgA or IgD. In other embodiments the invention, the anti-MIF antibody is an IgG and is a subclass IgG1, IgG2, IgG3 or IgG4. In other embodiments implementing the tvline of the invention the antibody belongs to the subclass IgG1 or, or IgG4. In other embodiments of the invention the antibody belongs to the subclass IgG4. In some embodiments of the invention the antibody IgG4 has a single mutation that changes a serine (serine 228, according to the scheme of Kabat numbering) on Proline. Accordingly, suppositionally CPSC in the Fc-region of IgG4 becomes SRRS, which corresponds to the sequence in IgG1 (Angal et al. Mol. Immunol. 1993, 30, 105-108).

Epitopes MIF, recognizable anti-MIF antibodies

In some embodiments implementing the invention relates to anti-MIF antibody or antigen-binding parts, specifically binding region 35-68 th or 86-115-th amino acid human MIF, respectively, preferably, anti-MIF antibodies specifically bind to the region from 50 to 68 or from the 86th to the 102nd amino acids, respectively, and inhibit the biological function of MIF person.

In other embodiments implementing the invention relates to anti-MIF antibodies that specifically bind active MIF and additionally inhibit the biological function of MIF. In some embodiments of the invention the active MIF is membrane-bound.

It was unexpectedly found that in the study of binding to MIF human anti-MIF antibodies according to the invention is able to compete with anti-MIF antibody III.D.9. Competition with III.D.9 can be determined as described in example 5.

The affinity of the binding is of anti-MIF antibodies with human MIF

The invention relates to anti-MIF antibody or antigen-binding portions that bind MIF person with KD5×10-7M or less. In other embodiments the invention, the antibodies bind MIF person with KD5×10-8M or less, 5×10-9M or less, or 5×10-10M or less.

The binding affinity of anti-MIF antibodies or their antigen-binding parts with MIF person can be determined by methods known in this field. For example, the affinity of binding can be measured using surface plasma resonance (BIACORE). In example 10 shows the method of determining the affinity constants of anti-MIF antibodies using BIACORE methodology.

In some embodiments implementing the invention additionally relates to an anti-MIF antibody or antigen-binding portions, which connect the active MIF with KDless than 500 nm, and optionally inhibit the biological function of MIF person. In some embodiments of the invention anti-MIF antibodies or their antigen-binding part of the link active MIF with KDless than 50 nm.

Getting anti-MIF antibodies

Anti-MIF antibodies or their antigen-binding portion according to the present invention can be obtained by many methods known to the person skilled in the art, such as the screening of libraries of phage display or fragments of antibodies. M is tenderly to use different formats libraries phage display, for example, libraries of scFv or Fab fragments, etc. Screening libraries of phage display is performed to search for fragments of the antibodies with the desired affinity to certain epitopes, and genetic material extracted from the corresponding clone. In consecutive rounds of creating and screening libraries you can select a range of antibodies with increased affinity compared to the affinity of the original selection of antibodies. The affinity of the identified anti-MIF fragment can be further improved by means of affinity maturation.

Nucleic acids, vectors, cells of the host and recombinant methods for the production of anti-MIF antibodies

The invention additionally relates to nucleic acid molecules coding for anti-MIF antibodies or their antigen-binding portion according to the present invention, and vectors comprising such nucleic acid, and cell host containing the vector, and recombinant methods of obtaining the polypeptide encoded by the nucleic acid molecule.

In some embodiments of the invention a DNA sequence encoding a VL-the anti-MIF antibody comprises a nucleotide sequence that is identical to the sequence VLantibodies Bax8 (SEQ ID NO: 13), antibodies Bax69 (SEQ ID NO: 14), antibodies Bax74 (SEQ ID NO: 15), antibodies Bax94 (SEQ ID NO:16), antibodies Bax152 (SEQ ID NO: 17), antibodies BaxA10 (SEQ ID NO: 18), shown in Figure 3, or the sequence that has 85%, preferably 90% homology in sequence with any of these nucleotide sequences.

In some embodiments of the invention a DNA sequence encoding a VH-the anti-MIF antibody comprises a nucleotide sequence that is identical to the sequence VHantibodies Bax8 (SEQ ID NO: 19), antibodies Bax69 (SEQ ID NO: 20), antibodies Bax74 (SEQ ID NO: 21), antibodies Bax94 (SEQ ID NO: 22), antibodies Bax152 (SEQ ID NO: 23), antibodies BaxA10 (SEQ ID NO: 24), shown in figure 4, or a sequence that has 85%, preferably 90% homology in sequence with any of these nucleotide sequences.

Getting anti-MIF antibody of the present invention includes any method of production of recombinant DNA by genetic engineering, e.g. via reverse transcription of RNA and/or DNA amplification and cloning in the expression vector.

In some embodiments of the invention the vector is a viral vector, into which additional DNA segments can be ligitamate in the viral genome. In some embodiments of the invention a vector capable of Autonomous replication in a cell-master, in which he introduced (e.g., bacterial vectors, is within the bacterial point of replication initiation, or episomal vectors mammals). In other embodiments of the invention, the vector (for example, episomal vectors mammals) can be incorporated into the genome of a host cell upon introduction into the cell of the host, and thus can be replicated together with the genome of the host. Moreover, certain vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are referred to in this description as "recombinant expression vectors" (or simply, "expression vectors").

Anti-MIF antibodies can be obtained using a conventional expression vectors, such as bacterial vectors (e.g., pBR322 and its derivatives) or eukaryotic vectors. These sequences encoding the antibody can be represented with the regulatory sequences that regulate replication, expression and/or secretion from the host cell. These regulatory sequences include, for example, promoters (e.g., promoters CMV or SV40) and signal sequence. Expression vectors can also include selection and amplication markers, such as gene digidrofolatreduktazy (DHFR), hygromycin-B-phosphotransferase and timedancing. Components of vectors, such as selection markers, replicons, enhancers can either be purchased in prod is the same either be obtained using standard methods. Vectors can be designed for expression in various cell cultures, for example, mammalian cells such as CHO, COS, HEK293, NS0, fibroblasts, insect cells, yeast and bacteria, such as E. coli. In some cases, using cells that allow optimal glycosylation of expressed protein.

The gene for the light chain of the anti-MIF antibody gene and the heavy chain of anti-MIF antibodies can be embedded in a separate vectors, or you can embed both genes in a single expression vector. Genes of the antibody is inserted into the expression vector by standard methods, for example, legirovaniem complementary restriction sites on the fragment of the antibody gene and the vector, or legirovaniem "blunt" ends, if the restriction sites are not available.

Getting anti-MIF antibodies or their antigen-binding parts can include any known in the field a way of introducing recombinant DNA into eukaryotic cells by transfection, e.g. via electroporation or microinjection. For example, recombinant expression of anti-MIF antibodies can be obtained by introducing the expression plasmid containing the coding anti-MIF antibody DNA sequence under the control of one or more regulatory sequences, such as a strong promoter in approach is asuu cell line host using the appropriate method of transfection, that results in cells that have entered the sequence stably integrated into the genome. Lipofection method is an example of a method of transfection, which can be used according to the present invention.

Getting anti-MIF antibodies may also include any known in the field the method of cultivation of these transformed cells, for example, a continuous or periodic manner, and the expression of anti-MIF antibodies, for example, constitutive or induced.

The host-cell according to the present invention can be any eukaryotic cell. In one embodiment of the invention the cell is a cell of the mammal with the ability to carry out post-translational modification of anti-MIF antibodies. For example, the specified mammal cells derived from cell lines of a mammal, such as, for example, cell lines selected from the group consisting of cells SkHep, CHO, HEK293 and KSS. In one embodiment of the invention the anti-MIF antibody is expressed in a Cho cell line, lacking DHFR, for example, DXB11, with the addition of G418 as a selection marker. With the introduction of recombinant expression vectors encoding antibody genes, in cells of the host mammal antibodies are produced by culturing the host cells for a time sufficient to expire is these antibodies in the cells of the host or the secretion of antibodies into the culture medium, in which growing cells of the host.

Anti-MIF antibodies can be isolated from culture medium using standard methods of protein purification.

In addition, obtaining anti-MIF antibodies may include any known in the field, the method of purification of antibodies, for example, by using anion-exchange chromatography or affinity chromatography. In one embodiment of the invention the anti-MIF antibody can be cleaned from supernatant culture cells using gel filtration.

Properties anti-MIF antibodies

The invention relates to anti-MIF antibody or antigen-binding parts that have at least one of the following properties:

a) contact C-terminal or Central region of the MIF person;

b) inhibit the inhibitory activity of glucocorticoids (GCO);

C) inhibit the proliferation of cell lines such as fibroblasts or tumor cells (e.g., NIH/3T3 or PC-3);

g) associated with the active MIF;

d) are not associated with inactive MIF;

e) compete with murine anti-MIF antibody III.D.9.

In some embodiments of the invention the active MIF isoform is active MIF, which occurs as a result of processing human MIF mild oxidizing agents, such as cystine, or as a result of immobilization MIF man on the substrate, such as a tablet for ELISA or balls. In other is the option of carrying out the invention the active MIF isoform is active MIF, which occurs in vivo after bacterial infection in animals. In other embodiments of the invention the active MIF isoform is active MIF, which occurs in vivo on the surface of cells (for example, THP1, CFB).

In some embodiments of the invention inactive MIF is restored MIF (e.g., described in example 7) or intracellular MIF.

In other embodiments of the invention anti-MIF antibodies or their antigen-binding portion of the associated active MIF with KDless than 500 nm.

Pharmaceutical compositions of anti-MIF antibodies and methods of treatment

The invention also relates to compositions containing anti-MIF antibody or antigen-binding part, for the treatment of a subject in need of treatment associated with MIF diseases, namely, immunological diseases, such as inflammatory diseases and hyperproliferative disorders.

In some embodiments of the invention a subject in need of treatment, is the man. Hyperproliferative disorders such as cancer that can be treated with anti-MIF antibodies according to the invention can occur in any tissue or organ and include, but are not limited to, cancers of the brain, lung, squamous cell, bladder, gastric, pancreatic, mammary, eles, head, neck, liver, kidney, ovarian, prostate, colon and rectal, esophageal, gynecological cancer, nasopharynx cancer, or thyroid cancer, melanoma, lymphoma, leukemia or multiple myeloma. In particular, anti-MIF antibodies according to the invention is suitable for treatment of carcinomas of breast, prostate, colon and lungs.

The invention also encompasses methods of treatment of a subject, including humans, inflammatory diseases such as vasculitis, arthritis, sepsis, septic shock, endotoxin shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic dermatitis, asthma, conjunctivitis, fever, malaria, or psoriasis, which include the stage of introduction of a specified subject in need, a therapeutically effective amount of anti-MIF antibodies or antigen-binding part.

In other embodiments of the invention a composition containing a specified anti-MIF antibody according to the invention, used for the treatment of inflammatory diseases selected from the group consisting of glomerulonephritis, inflammatory diseases of the stomach, jade and peritonitis.

Treatment may also include the introduction of one or more anti-MIF antibodies according to the invention or their and the of then-binding parts, alone or with a pharmaceutically acceptable carrier. Some examples of pharmaceutically acceptable carriers include water, saline, phosphate-saline buffer, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases, the composition is preferably isotonic agents, for example, sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride. Additional examples of pharmaceutically acceptable substances are moisturizers or minor amounts of auxiliary substances such as humidifiers or emulsifying agents, preservatives or buffer substances, which enhance the shelf life or effectiveness of the antibody.

Anti-MIF antibodies according to the invention or containing pharmaceutical compositions can be introduced in combination with one or more other therapeutic, diagnostic or prophylactic agents. Additional therapeutic agents depending on the disease include other anti-neoplastic, antitumor, antiangiogenic, chemotherapeutic drugs or steroids.

The pharmaceutical compositions according to this invention can be in various forms, for example, liquid, soft or solid dosage forms, such as liquid solutions (e.g., solutions for injection or infusion), dispersions or is Uspenskii, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended route of administration and therapeutic application. The usual preferred compositions are solutions for injection or infusion, such as compositions similar to those used for passive immunization in humans. The preferred route of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneally, intramuscular). In a preferred embodiment of the invention the antibody is administered via intravenous infusion or injection. In another preferred embodiment of the invention the antibody is administered via intramuscular or subcutaneous injection. For an experienced specialist in this field will be obvious that the path and/or type of injection will vary depending on the desired results.

Anti-MIF antibody can be entered once, but more preferably repeated introduction. For example, the antibody can be entered from three times daily to once in six months or a larger time interval. The introduction can be performed according to the scheme, such as the introduction of three times a day, twice a day, once a day, once in two days once in three days, once a week, once in two weeks once a month once in two months once in three months and od is n times in six months.

The invention also encompasses the use of anti-MIF antibodies or antigen-binding fragment in the manufacture of medicinal products for the treatment of immunological diseases, such as inflammatory diseases and hyperproliferative disorders.

The invention additionally encompasses the use of anti-MIF antibodies or antigen-binding fragment in the treatment of immunological diseases, such as inflammatory diseases and hyperproliferative disorders.

The invention also encompasses the use of anti-MIF antibodies or antigen-binding fragment in the diagnostic methods. In one embodiment of the invention the anti-MIF antibody or antigen-binding portion can be used for the detection of MIF person in a biological sample.

Anti-MIF antibodies and their antigen-binding portion can also be used to determine the level of MIF on the surface of cells in the tissue or cells obtained from a tissue. In some embodiments of the invention, the tissue affected by the disease. The fabric can then be used in immunological analysis to determine, for example, the General level of MIF, MIF level on the cell surface or localization of MIF.

The invention additionally relates to kits containing anti-MIF antibody or antigen-binding portion according to the invention or FA the pharmaceutical composition, containing such antibody or portion. In addition to the antibody or pharmaceutical composition, the kit may include diagnostic or therapeutic agents. The kit can also include instructions for use in diagnostic or therapeutic method.

The invention additionally relates to a method of identifying anti-MIF antibodies capable of inhibiting the biological function of MIF person and cause beneficial effects in an animal model, by carrying out the following stages:

a) selecting the antibody that binds to the active MIF inactive and does not bind MIF;

b) testing the specified antibodies in in vitro methods of analysis, such as analysis method suppressive activity of glucocorticoids (GCO) or the method of analysis of cell proliferation;

C) selecting an antibody that inhibits the GCO and/or cell proliferation.

The results showed that anti-MIF antibody, which binds only the active MIF inactive and does not bind MIF, and also inhibits the GCO and/or cell proliferation, causes a beneficial effect in animal models (e.g., see example 6).

The present invention will be further illustrated in the following examples, not limiting the invention.

EXPERIMENTAL PART

Example 1: Selection of antibodies

To obtain fragments of anti-MIF antibodies person used the technique of phage is the first display

Starting with a library of phage display was performed various screening options, three of them used a full-sized MIF (MIF man on the substrate/MIF man in the solution/the alternation of MIF human and mouse). Other used six peptides of MIF, alternating with full-MIF. These six peptides were constructed by separation of protein MIF six peptides with a length of approximately 30 amino acids overlapping sequences of length approximately 15 amino acids. After several rounds of selection were found unique sequences, which were associated with targeted screening, all binding sequences were freexpression and purified in the form of IgG4 antibodies person. These antibodies were tested in a series of tests to demonstrate the in vitro inhibition of MIF. To determine the portion of binding to MIF protein was performed epitope mapping. 193 antibodies were tested and distributed in accordance with their activity inhibition of MIF in vitro. In vitro methods of analysis are described below. As control was used three mouse anti-MIF antibodies (III.D.9, XIV.14.3 and XIV.15.5).

Example 2: Inhibition of MIF activity by suppressing effect of glucocorticoids (GCO).

This method is based on the inhibition of endogenous MIF, i.e. MIF produced used cell line. This method applies the ri screening of antibodies and to determine the curves of the effect of the doses.

GCO analysis for screening antibodies:

Suspension culture cells TNR besieged by centrifugation, and cells resuspendable in fresh complete medium to the density of cells 106cells in ml of This culture was transferred into wells of 96-well microplate (90 μl per well), and added anti-MIF antibody to a final concentration of 75 μg/ml of Each antibody was tested in three repetitions. After incubation over night at 37°C was added dexamethasone to a concentration of 2 nm and after 1-hour incubation at 37°C was added LPS to a final concentration of 3 ng/ml After 6-hour incubation at 37°C, collected the supernatant and determine the concentration of IL-6 using ELISA (set Cytoset, commercially available). Were averaged data for the three repetitions and determined the percentage of secretion of IL-6 compared to control antibodies. Antibodies that showed the secretion of IL-6 less than 75%were assessed as positive.

Method for determination of IC50 values

The experiment was performed as described for the screening analysis, except that used an increasing number of antibodies (usually 1-125 nm). The obtained curves of the effect of dose was expressed as % inhibition compared with the antibody used as a negative control. This curve was used to calculate the maximum inhibitory effect of the antibodies is (% inhibition of maximum) and the antibody concentration, which gave 50% of the maximum inhibitory effect (IC50).

The final results are shown in Fig in column 3 (IC50) and column 4 (maximal inhibition). For comparison murine antibody XIV.14.3 gives only a 36% inhibition of the GCO (data not shown).

Example 3: Inhibition of cell proliferation

We are in dormant cells NIH/3T3 serum stimulates the secretion of MIF and MIF, in turn, stimulates cell proliferation. Therefore, antibodies that inhibit this endogenous MIF, reduce the proliferation of resting cells NIH/3T3. The reduction of proliferation is determined by the inclusion of3H-thymidine.

1000 cells NIH/3T3 cells were then incubated in 96-well tablet during the weekend days at 37°C in a medium containing 10% serum. Then the cells were subjected to starvation over night at 37°C with incubation in medium containing 0.5% serum. Remove the medium containing 0.5% serum, and replaced with fresh medium containing 10% serum, 75 µg/ml antibody and 5 mccu/ml3H-thymidine. After 16-hour incubation in CO2-incubator at 37°C. cells are washed twice with 150 μl of cold PBS per well. Using a multichannel pipette was added 150 μl of 5% (wt/vol) trichloroacetic acid and incubated for 30 minutes at 4°C. the Tablets were washed with 150 μl of PBS. Added 75 μl of 0.5 M NaOH solution with 0.5% SDS per well, mixed and kept at room is the temperature. The level of radioactivity was measured on a β-counter mixing 75 ál of the sample solution with 5 ml of Ultima Gold (Packard). Spent three measurements for each image, and the values were compared with values for control antibodies using t-test. Antibodies that significantly reduced proliferation (P<0.05), and was evaluated as positive. The final results are shown in Fig, column 5.

Example 4: Study link: definition epitopes for anti-MIF antibodies

Each peptide was dissolved in buffer for sewing proteins to the substrate and the concentration of the peptide is typically 5 μg/ml, was added to the microplate (NUNC Immobilizer™ Amino Plate F96 Clear), and incubated overnight at 4°C (100 ál per well). As controls were used recombinant full-MIF and PBS. The tablet was washed 3 happy 200 μl PBST, was added to the antibody (4 μg/ml in PBS), 100 μl per well, incubated for 2 hours at room temperature with gentle rocking. The tablet was washed 3 times with 200 μl PBST and added the detecting antibody (e.g., labeled with HRP specific to the Fc-region of human IgG, Sigma) in an amount of 100 μl per well. After 1-hour incubation at room temperature with gentle rocking tablet was washed 3 times with 200 μl PBST. Each well was incubated with 100 μl of TMB solution (cat. # T-0440, Sigma) for 30 minutes in the dark. The reaction was stopped by adding 100 μl of 1.8 M solution of H2SO4on l the GCC. The samples were measured at 450 nm.

Example 5: Competition anti-MIF antibody human mouse anti-MIF antibody III.D.9

To compete with murine anti-MIF antibody III.D.9 used antibody VH.

96-well plates (NUNC Maxisorp) were coated with recombinant human MIF. Mouse anti-MIF antibody III.D.9 and anti-MIF antibodies person were diluted in TBST/2% BSA and mixed, and the final concentration III.D9 was maintained at a level of 2 µg/ml, and the concentration of anti-MIF antibodies person was increased from 0 µg/ml typically up to 32 mcg/ml After washing the microplate was applied antibodies, and incubated at room temperature usually within 2 hours. After washing tablet incubated with peroxidase-conjugated antibodies to mouse IgG (specific to the Fc-region) for 1 hour at room temperature. After washing tablet incubated with TMB solution, and the reaction was stopped by adding a solution of H2SO4. Approximation curve obtained competition allows us to calculate the maximum inhibition of binding III.D.9. The final results are shown in Fig, column 6.

Example 6: an Increase in survival with the use of anti-MIF antibodies in an animal model of peritonitis caused by infection of E. coli

The experiment was performed according to the methodology group Calandra et al. (Nature Immunology, 2000) using female NMRI mice (25-30 g, 6-10 weeks), which is introduced through intraperitoneally injection 6000 CFU suspension 0111:B4 E.coli in a solution of 15% mucin and 4% of hemoglobin. Two or three colonies (0111:B04 E.coli) from the culture on the Cup with a nutrient agar was inoculable in 10 ml of TSB and incubated overnight at 36°C with rocking. The culture was diluted in saline solution to the desired concentrations (night culture usually reaches a density of 2*109CFU/ml) and mixed with mucin and hemoglobin (1 volume of the diluted bacterial culture, volume 2 15% mucin, 2 volumes of 4% of hemoglobin). Because the bacteria in diluted mixtures tend to be precipitated, the mixture was stirred between injections. For injection used a wide needle (for example, the 23rd number) in order to avoid clogging of the needle sediment of the injection mixture. Antibody VH (IgG4) and matched isotype control antibody was administered intraperitoneally injected 2 hours before bacterial infection. The dosage of the antibodies was typically 800 μg/mouse, and each group took 20 mice. A statistically significant effect on the survival/death could be shown for the isotypes IgG1 and IgG4 anti-MIF antibodies person. Figure 6 shows the results obtained for antibodies Wah and antibodies Wah (IgG4). To estimate survival curves used statistical method of Kaplan-Meier.

Example 7: the binding Specificity with active MIF

Described in this invention anti-MIF antibodies able to distinguish between active and inactive MIF that education is described in the mild oxidation or restore respectively. The differences between these conformational variants was evaluated using ELISA or surface plasmon resonance.

ELISA for assessing differential binding of antibodies:

- Transformation of MIF in its active conformation with mild oxidation.

Recombinant human MIF (0.5 mg/ml in PBS) were incubated for 3 hours at 37°C with 3-fold excess (by volume) of a saturated solution of L-cysteine in PBS (~ 0.4-0.5 mm L-cystine). Then conducted twice a dialysis MIF against PBS in a dialysis cassette Slide-A-Lyzer® with cut-off molecular weight 7 KD (from Pierce).

- Transformation of MIF in its inactive conformation.

MIF was recovered at a concentration of 0.5 mg/ml overnight incubation with 8-16 mm dithiothreitol (final concentration) at 4°C.

- ELISA Protocol.

96-well microplates (NUNC Maxisorp™) were coated with anti-MIF antibodies at a concentration of 5 μg/ml (diluted in buffer for sewing proteins to the substrate). After washing tablet with TBST (Tris-saline buffer with 0.1 % Tween-20 (by volume)) and blocking with 2% solution (W/W) BSA (TBST/2%BSA) per well) was added serial dilution of either active or inactive MIF, and incubated at room temperature for 1-2 hours. Associated MIF was detected using rabbit polyclonal anti-MIF antibody and horseradish peroxidase is Asiago antibodies to rabbit antibodies (Biorad). In order to reduce nonspecific binding for cultivation MIF, rabbit anti-MIF antibody and peroxidase conjugate used TBST/2%BSA. 7 shows the ELISA results obtained with the antibody Bax94.

The evaluation of the differential binding of antibodies using Biacore

The binding kinetics of active and inactive MIF with the antibody VH was investigated using surface plasmon resonance using Biacore 3000. This was immobilized 10,000 units resonance Bax 94 on the sensor chip with SM matrix (carboxyethylgermanium dextran), and incubated with active or inactive MIF, huMIF, preostanovlena and procyclical glutathione redox buffers in the range of 4.8 mm GSH/0.2 mm GSSG (GSSG = exelency glutathione) to 5 mm GSSG buffer HBS-EP (GE Healthcare). As the control used MIF for analysis of binding in the second flow cell containing immobilized matched isotype control antibody. To evaluate read response unit binding of the control antibody and antibodies Wah 94.

Example 8: Detection of active MIF on the cell surface TNR-1

Cells were incubated with anti-MIF antibody Wah 94. Cells were washed in ice-cold PBS and resuspendable in cold buffer for lysis of the cells (Cell Signaling Technology®). Magnetic beads protein G Magnetic Protein G Dynabeads® (Invitrogen), blocked with TBST with 5% skim su the CSOs milk (weight/volume), washed and added to the lysed cells. Immunoassay was performed over night at 4°C. Then the beads were washed with buffer for lysis of cells and TBST and boiled in buffer for sediment samples for LTO-page (without reducing agents). Conducted LTO-PAGE samples in non conditions for Western blotting.

Example 9: Binding of anti-MIF antibodies with membrane-bound MIF

Cells TNR-1 were washed in ice-cold PBS and resuspendable in cold buffer for staining cells (Biolegend) with 200 μg/ml mouse IgG. Added FITC - or TRITC-labeled anti-MIF antibody to a final concentration typically 200-500 nm, and incubated at 4°C. Cells are then washed with ice buffer for staining cells and resuspendable in the buffer for staining cells with a solution Via Prohe™ Cell Viability Solution (BD Biosciences). The fluorescence intensity of the cells was measured on cytoprotection fluorimetry FACS Canto™ II Flow Cytometry System (BD Biosciences) and secondary FITC-/TRITC-shift of populations of living cells was compared with data obtained using dye-labeled control antibody of the same isotype.

Example 10: Determination of the affinity of Fab fragments of anti-MIF antibodies using Biacore analysis

Usually 40 EN (units resonance) recombinant human MIF was immobilized on the sensor chip with SM matrix (carboxyethylgermanium dextran) (Biacore). Razvedennyi HBS-EP Fab fragments were injected in the concentration range usually 6-100 nm. After each cycle, the chip was regenerated with 50 mm NaOH + 1 M NaCl. The affinity was calculated based on the model of Langmore 1:1. The final results are shown in Fig, column 7.

Example 11: Beneficial effect of anti-MIF antibodies in an animal model of sickle glomerulonephritis

Anti-MIF antibodies were tested in a rat model of sickle glomerulonephritis described Frederick W.K. Tarn et. al. (Nephrol Dial Transplant, 1999, 1658-1666).

Nephrotoxic nephritis caused the male rats of Wistar Kyoto single intravenous injection of anti-serum to the glomerular basal membrane of rats. In the "warning" version of the experiment, the treatment with anti-MIF antibodies and matched isotype control antibody was begun during the induction of nephritis (day 0) using intraperitoneally injection. The treatment is usually repeated every other day, and the animals were penalized on the 7th day for histological analysis. Urine was collected before the experiment (baseline) and before the end of the experiment (day 7). In "therapeutic" version of the experiment, the treatment with anti-MIF antibody was started 4 days after induction of disease and repeat every other day. Rats usually penalized, on the 8th day. Urine was collected before the experiment (baseline), before treatment (day 4) and before culling of animals (day 8). The dosage of the antibodies was usually 1-20 mg/kg per injection, and for each group used 6 is about 8 rats. Disease severity was determined by measuring proteinuria, infiltration of macrophages in renal glomeruli and histological damage (education extracapillary proliferation, i.e. polloni). In the "warning" version of the experiment, the treatment with anti-MIF antibody VH (10 mg/kg per dose) for 7 days resulted in a 47%reduction in proteinuria compared to animals treated with control antibody. Treatment of existing diseases (therapeutic experiment) led to dose-dependent reduction of proteinuria by 16% (10 mg/kg Wah on the dose) and 34% (20 mg/kg UAH per dose) compared to animals treated with control antibody.

Example 12: Beneficial effect of anti-MIF antibodies in an animal model of ulcerative colitis (adaptive transfer of naive T cells to mice Rag -/-)

Mice C57BL/6 were killed and allocated CD45RBhi cells (naive T cells) by sorting on cytoprotection fluorimetry population of spleen cells. CD45RBhi cells (5×105) was administered using intraperitoneally injection 7-9 week old mice C57BL/6 Rag-/-, of which approximately 2 weeks developed ulcerative colitis (de Jong et al., Nature Immunology, 2001, 1061-1066). Anti-MIF antibodies and matched isotype control antibody was injected using intraperitoneally injections twice a week (dose of 1 mg per mouse). In the "warning" version of the experiment, treatment was started during injectii cells. In "therapeutic" version of the experiment, treatment was started 4 weeks after induction of the disease. In mice weekly checked the weight and the development of the disease. Usually eight weeks after transfer of cells CD4CD45RBhi recipients C57BL76 Rag-/- calculated index of disease activity (DAI) and gathered sections of the colon to assess histological index (HI). The index of disease activity (DAI) and histological index (HI) was determined at the end of the experiment on animal models (DAI based on four parameters: the stoop and depletion (score 0 or 1), thickening of the rectum (0-3) and stool consistency (0-3)). In a therapeutic experiment for treatment for existing diseases used anti-MIF antibodies Wah and Waha, and the average value of the DAI was significantly reduced by approximately 60% (UAH) and approximately 40% (Waha) compared with mice treated with matched isotype antibody. In addition, the average score HI was reduced by approximately 33% after treatment with Wah.

Example 13: Beneficial effect of anti-MIF antibodies in an animal model of ulcerative colitis (agonistic anti-CD40 model)

This model is based on the activation of macrophages and dendritic cells agonistic anti-CD40 antibody, which induces a pathological process in the intestine, resembling an inflammatory disease of the stomach in mice and Rag1-/-.

Showpad the marijuana by age and sex of the mouse Rag-1-/- (4-5 weeks) were acquired in the company's Jackson Laboratories and housed in the vivarium two weeks before the beginning of the experiment. Agonistic monoclonal antibody to CD40 (FGK45, IgG2a) or izotopicheskii control, rat IgG2a, was dissolved in PBS to a concentration of 1 mg/ml Five groups (10 mice per group) were injected with intraperitoneally injection of 200 µg of agonistic anti-CD40 monoclonal antibodies, and of these four groups were injected with anti-MIF antibodies at day 0 and 1 (2×1 mg per mouse). Sixth group (10 mice) were injected only izotopicheskii control (rat IgG2a, group of healthy animals). Mice were weighed in over the next 7 days. On the 7th day was calculated index of disease activity (DAI) and gathered sections of the colon to assess histological index (HI). Evaluation DAI based on: stoop (0-1), depletion (0-1), stool consistency (0-3) and thickening of the rectum (0-3). Histological evaluation based on thickening (0-3), crypt elongation, inflammation (0-3), and abscess (0-1). The treatment with anti-MIF antibodies Wah, Waha and Wah significantly reduced assessment DAI (Waha: decrease by ~48%; VH: decrease by ~62%; VH: decrease by ~73%) compared with mice treated with izotopicheskii control. In addition, the average rating HI also decreased under the action of these antibodies.

Example 14: Inhibition of tumor growth in Nude mice Mf1 using anti-MIF antibodies

Cells of BPH man (PC-3) were collected from exponentially growing cultures and mixed with Matrigel deprived grew the new factors. 2×106cells in 0.25 ml of Matrigel were inoculated subcutaneously in the right groin area bare Mf1 mice. The treatment with anti-MIF antibody VH and ezotericheskim control C3 was started one day after inoculation of tumor (0.6 mg antibody per mouse per day) and was repeated every other day. The size of the tumor is usually started to measure two weeks after injection of cells and carried out through the day. Tumor volume was calculated using the formula V=0,5×a×b2(where "a" is the largest diameter and b is the smallest diameter). Tumor growth in mice treated Wah was significantly reduced, and the average tumor volume after 28 days after induction of the tumor was 4.3 times higher in the group treated izotopicheskii control, compared with the group treated with the antibody VH.

In "therapeutic" version of the experiment, treatment with antibodies was started one week after inoculation of tumor. Using intraperitoneally the injection was administered through day 50 mg/kg dose izotopicheskogo control antibody C3 and anti-MIF antibodies Wah. After 22 days after the start of injection of the antibodies was determined by the mean tumor volume, which was 2.7 times higher in the group treated with C3, compared with the group treated with Wah 69.

Example 15: Proapoptotic effect of anti-MIF antibodies

Proapoptotic effect of anti-MIF antibodies Wah was shown in cell analysis of the activity of the caspase-3 cancer cell lines of human prostate PC-3. Cells PC-3 were sown on cultural cups with a diameter of 10 cm (~106cells per Cup) in the presence of 10% fetal bovine serum. After 24 hours was added to fresh medium containing the antibody VH (100 nm) or control antibody C3 (100 nm). After another incubation for 48 hours to prepare cell lysates, and measured the activity of caspase-3 by adding fluorescently-labeled kaspesky substrate (Fig.9).

Example 16: Inhibition of invasion of tumor cells

Anti-MIF antibodies Wah and Wah were tested using the method of analysis of invasive activity of the cells in Transwell™using cancer cell line human prostate PC-3.

Sown 5×104cells PC-3 per well of 24-well plates Transwell™ (pore size 8 μm)coated with poly-D-lysine on the bottom surface of the polycarbonate membrane and having deprived of growth factors Matrigel on the surface of the Transwell inserts™. The cells were allowed to attach for 4 hours in the presence of 10% fetal bovine serum. Then the medium was replaced with serum-free medium, and cells were left to starve during the night (16 hours). Next, in the lower compartment was added compound (10 nm MIF, 500 nm antibody). The cells were allowed to migrate through the porous membrane within 24 hours. After incubation attached migrated cells on the lower membrane surface were stained with a solution GIMS is. The number of cells attached to the lower surface of the membrane were counted in independent fields of view at 400-fold increase (Figure 10).

1. Monoclonal antibody or its antigennegative part that specifically binds a C-terminal or Central region of MIF and inhibits the biological function of MIF human coding VL-the region of the specified antibody comprises a nucleotide sequence that is identical to the sequence VLantibodies Bax8 (SEQ ID NO: 13), antibodies Bax69 (SEQ ID NO: 14), antibodies Bax74 (SEQ ID NO: 15), antibodies Bax94 (SEQ ID NO: 16), antibodies Bax152 (SEQ ID NO: 17), antibodies BaxA10 (SEQ ID NO: 18) or a sequence that has at least 85% homology in sequence with any of these nucleotide sequences, and a DNA sequence encoding a VH-region antibody comprises a nucleotide sequence that is identical to the sequence VHantibodies Bax8 (SEQ ID NO: 19), antibodies Bax69 (SEQ ID NO: 20), antibodies Bax74 (SEQ ID NO: 21), antibodies Bax94 (SEQ ID NO: 22), antibodies Bax152 (SEQ ID NO: 23), antibodies BaxA10 (SEQ ID NO: 24) or a sequence that has at least 85% homology in sequence with any of these nucleotide sequences.

2. Monoclonal antibody or its antigennegative part according to claim 1, where the aforementioned antibody or antigennegative part has at least one of the following properties:
a) inhibits inhibitory activity of glucocorticoids (GCO);
b) inhibits proliferation of cancer cells or fibroblasts;
C) binds to the active MIF;
d) is not associated with inactive MIF;
d) competes with murine anti-MIF antibody III.D.9.

3. Monoclonal antibody or its antigennegative part according to claim 1 or 2, where the aforementioned antibody or antigennegative part binds MIF person with KDless than 500 nm.

4. Monoclonal antibody or its antigennegative part according to claim 1, where the aforementioned antibody or antigennegative part binds the active MIF.

5. Monoclonal antibody according to claim 1, where the aforementioned antibody is an antibody of subformat IgG4.

6. Monoclonal antibody according to claim 5, where the specified subformat IgG4 has one mutation, which resulted in suppositionally CPSC in the Fc-region of IgG4 becomes SRRS.

7. Monoclonal antibody or its antigennegative part according to claim 1, where the aforementioned antibody or antigennegative part contains:
a) CDR1, CDR2 and CDR3 of the heavy chain, is independently selected from the heavy chain antibody is selected from the group consisting of antibodies Bax8, antibodies Bax69, antibodies Bax74, antibodies Bax94, antibodies Bax152 and antibodies BaxA10;
b) CDR1, CDR2 and CDR3 of light chain, independently selected from the light chain of the antibodies selected from the group consisting of antibodies Bax8, anti who ate Bax69, antibodies Bax74, antibodies Bax94, antibodies Bax152 and antibodies BaxA10.

8. Monoclonal antibody according to claim 1, where the antibody contains:
C) amino acid sequence of heavy chain that is at least 90% identical to the amino acid sequence of heavy chain antibodies Bax8, antibodies Bax69, antibodies Bax74, antibodies Bax94, antibodies Bax152 and antibodies BaxA10;
g) the amino acid sequence of a light chain that is at least 90% identical to the amino acid sequence of the light chain of the antibody Bax8, antibodies Bax69, antibodies Bax74, antibodies Bax94, antibodies Bax152 and antibodies BaxA10.

9. Monoclonal antibody or antigennegative part according to claim 1 for use in the treatment of immunological diseases, and specified immunological disease is an inflammatory disease or a hyperproliferative disorder.

10. Monoclonal antibody or antigennegative part according to claim 9, where the specified inflammatory disease selected from the group consisting of vasculitis, arthritis, sepsis, septic shock, endotoxin shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis and psoriasis.

11. Pharmaceutical composition for the treatment of immunological diseases, in the alarm of inflammatory diseases or hyperproliferative disorders, containing monoclonal antibody or its antigennegative part according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, in which a monoclonal antibody is an antibody selected from the group consisting of antibodies Bax8, antibodies Bax69, antibodies Bax74, antibodies Bax94, antibodies Bax152 and antibodies BaxA10.

13. The pharmaceutical composition according to claim 11, where the specified inflammatory disease selected from the group consisting of vasculitis, arthritis, sepsis, septic shock, endotoxin shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis and psoriasis.

14. The method of treatment of a subject, including humans, immunologic disease selected from inflammatory diseases or hyperproliferative disorders, including the stage of introduction of a specified subject in need, a therapeutically effective amount of monoclonal antibody or antigennegative part according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 11 or 12, with the indicated antibody or specified antigennegative part of the further inhibits the biological function of MIF person.

15. The method according to 14, in which the specified inflammatory is s disease selected from the group consisting of vasculitis, arthritis, sepsis, septic shock, endotoxin shock, toxic shock syndrome, acquired respiratory distress syndrome, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, peritonitis, nephritis and psoriasis.

16. The selected nucleic acid molecule containing the nucleotide sequence encoding a heavy chain or light chain of the monoclonal antibody or antigennegative part according to any one of claims 1 to 8.

17. The expression vector containing the nucleic acid molecule according to clause 16, where the vector optionally includes controlling the expression of the sequence is functionally associated with the specified nucleic acid molecule.

18. A host cell to obtain the antibody according to claim 1, containing a vector of 17 or a nucleic acid molecule according to item 16.

19. A host cell according p containing a nucleic acid molecule encoding a heavy chain and a nucleic acid molecule encoding a light chain of the monoclonal antibody or antigennegative part according to any one of claims 1 to 8.

20. A host cell which produces a monoclonal antibody or antigennegative part according to any one of claims 1 to 8, containing a vector of 17 or a nucleic acid molecule according to item 16.

21. The method of obtaining monoclonal antibody or its antigen is waywayway part, comprising culturing the host cell according p or 20 in suitable conditions, and the selection of the indicated antibodies or antigennegative part.

22. The method of identifying anti-MIF antibodies capable of inhibiting the biological function of MIF person and cause beneficial effects in an animal model, by carrying out the following stages:
a) selection of an antibody that specifically binds a C-terminal or Central region of MIF;
b) testing the specified antibodies in in vitro methods of analysis;
C) selecting an antibody that inhibits the activity of glucocorticoids (GCO) and/or cell proliferation.



 

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1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: method provides sampling of the material under study. Inoculation of the material under study on nutrient medium sabouraud followed by incubation on this medium at 37°C for 24 hours. A quantitative assessment of growth of yeast-like fungi of the genus Candida, and with a value exceeding 10*1 CFU/swab the candidiasis of upper respiratory tract is diagnosed. The obtained colonies are examined under the microscope and the isolated colonies of yeast-like fungi of the genus Candida are separated from these colonies. The isolated colonies of yeast-like fungi of the genus Candida are suspended in five test tubes containing liquid medium sabouraud with the phenol red indicator, where the discs with carbohydrates are added, and in the first test tube - a disc containing maltose, in the second - sucrose, the third - lactose, the fourth-galactose, the fifth - trehalose, and are incubated at 37°C for 24 hours, followed by assessment of colour change of the indicator. The colour change of the indicator to yellow is taken as one, lack of colour change is taken as zero, the sum of the values obtained in assessment of the indicator colour in five test tubes is calculated. If the value of the sum is 0 the candidiasis of upper respiratory tract caused by Candida kruzei is diagnosed, if it is 1 - the candidiasis of the upper respiratory tract caused by Candida glabrata is diagnosed, if it is 3 - the candidiasis of the upper respiratory tract caused by Candida albicans is diagnosed, if it is 4 - the candidiasis of the upper respiratory tract caused by Candida tropicalis is diagnosed, if it is 5 - the rest.

EFFECT: invention enables to identify several types of yeast-like fungi of the genus Candida, causing candidiasis, and enables to take into account the quantitative growth of yeast-like fungi of the genus Candida, according to which the presence of candidiasis is judged.

6 ex

FIELD: biotechnology.

SUBSTANCE: as an object for indirect estimation apparently healthy leaves of the plant Peireskia aculeate are used, on which sterile surface the notches are made, the infecting doses of the strains Burkholderia pseudomallei and Burkholderia mallei under study in different concentrations are applied, placed on a wet swab and incubated at 32°C for 24-48 h. The extent of their cytopathogenicity is evaluated visually according to the damage to the leaf blade - maceration, ulceration and blackening. Simultaneously the level of virulence of strains of pathogens for laboratory animals is determined, and the extent of cytopathogenicity for P. aculeata is correlated with virulence for laboratory animals.

EFFECT: invention provides high sensitivity, reproducibility, versatility, ease and accessibility of the study.

5 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: methods are described to prevent a virus flu infection and detection of efficiency of a flu virus vaccine using a molecule of hemagglutinin (HA) of a flu virus A of subtype H5, immunogenicity of which is increased by replacement of amino acids in the HA sequence.

EFFECT: replacement of specific remains in HA, such as introduction of asparagin into the position 223 in HA H5, makes it possible to increase sensitivity of hemagglutination moderation reaction as a result of variation of receptor specificity or ability to binding of antibody-antigen.

24 cl, 7 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.

2 cl, 1 dwg, 4 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: biotechnologies.

SUBSTANCE: invention describes polynucleotide, expression vector, host cell and production method of humanised antibody together with their use, as well as medical preparation against rheumatoid arthritis, prophylaxis or treatment method of rheumatoid arthritis and use of humanised antibody at production of pharmaceutical preparation for prophylaxis or treatment of rheumatoid arthritis. This invention can be used in therapy of human diseases associated with α9 integrin.

EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

Organic compounds // 2502802

FIELD: biotechnologies.

SUBSTANCE: invention refers to eucariotic vector for expression of target recombinant product in a mammal cell and to its use, to a mammal cell for production of target recombinant product and to a method for its production, a method of a mammal cell selection and a method for obtaining a target recombinant product. Vector includes the first polynucleotide coding a functional folate receptor bound to a membrane as a selective marker and the second polynucleotide coding the target product that is expressed in a recombinant manner. Target product represents a pharmaceutically active, therapeutically active or diagnostic polypeptide. Functional folate receptor bound to the membrane and target product are expressed from the above expression vector. Sampling system is based on introduction of a gene of exogenic functional folate receptor bound to the membrane to a mammal cell.

EFFECT: invention allows effective selection of transformed cells and high yield of target product.

26 cl, 3 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: expression vector includes: (a) replication origin OriP obtained from Epstein-Barr virus (EBV), where replication origin contains: 1) symmetry element of the second order (DS); and 2) duplication section (FR) that contains fixation point EBNA; (b) replication origin SV40; (c) insertion section for inserting a gene of concern; (d) promoter EF-1b functionally bound to the insertion section; (e) poly-A signal; (f) bacterial replication origin; (g) selected marker; and unnecessarily containing (h) sequence of nucleic acid, which codes constant area of heavy or light chain of antibody, which is functionally bound to the insertion section. With that, replication origin OriP is bound to an initiation factor of replication EBNA 1, which acts from outside and is not coded with an expression vector.

EFFECT: use of an expression vector in an extracted host cell, a set and a method for obtaining recombinant protein provides production of abundant protein expression.

26 cl, 25 dwg, 3 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant human blood coagulability factor VIII with deletion of B-domain (hFVIII-BDD). Recombinant plasmid DNA pAP227 coding polypeptide with sequence hFVIII-BDD also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 2H5 producing recombinant hFVIII-BDD with highly stable yield at the level of about 20 IU/ml/24 h. Cultivation of cells-producers is performed in medium DME/F12 containing 2-4% of Fetal Bovine Serum, 1% of dimethylsulphoxide and 50 IU/l of insulin.

EFFECT: improvement of the method.

4 cl, 5 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

EFFECT: improvement of the method.

5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically connects segment M1' IgE and that induces apoptosis in IgE-expressing B-cells and its antigen-binding fragment. Besides, compositions and curing methods of IgE-mediated abnormalitiy, an item, a specific elimination method of IgE-producing B-cells, methods for prophylaxis and reduction of IgE products induced with an allergen, as well as isolated nucleic acid, an expression vector, a host cell and a method for obtaining an antibody as per the invention together with their use are considered.

EFFECT: invention can be further used in therapy of diseases associated with IgE.

46 cl, 19 dwg, 5 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.

2 cl, 1 dwg, 4 ex

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